Chronic Liver Diseases 2017

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HEPATOLOGY RESEARCH AND CLINICAL DEVELOPMENTS
CHRONIC LIVER DISEASE

FROM MOLECULAR BIOLOGY TO THERAPY
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HEPATOLOGY RESEARCH AND CLINICAL DEVELOPMENTS
CHRONIC LIVER DISEASE

FROM MOLECULAR BIOLOGY TO THERAPY
J. C. PERAZZO,
FRANCISCO EIZAYAGA,
SALVADOR ROMAY,
CARLOS E. BRODERSEN,
ALBERTO E. MUÑOZ
AND NÉSTOR R. LAGO
EDITORS
New York

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CONTENTS
List of Contributors vii

Preface xiii
Acknowledgments xv
Abstract xvii
Chapter 1 Cell Death Issue for Researchers and Clinicians 1
Pamela Valva, Mario Alejandro Lorenzetti
and María Victoria Preciado
Chapter 2 Hepatic Stellate Cells in Liver Sinusoid Function 43
Luís Perea, Júlia Vallverdú, Alberto E. Muñoz and Pau Sancho-Bru
Chapter 3 Liver Sinusoidal Endothelial Cells: From Liver Function
to Liver Cirrhosis 59
Alberto E. Muñoz and Luís Perea
Chapter 4 Hyperammonemia and Hyperammonemic Conditions 79
Pablo A. Souto and Lisandro Orbea
Chapter 5 Cannabinoids: Implications in Liver Disease 97
Laura Caltana and Alicia Brusco
Chapter 6 Extracellular Matrix-Liver Relationship 105
Néstor R. Lago, Ariel Marcotegui, Lisandro Orbea, Pablo A. Souto
and Juan C. Perazzo
Chapter 7 Endothelins in Liver Health and Disease 123
Myrian R. Rodriguez, María Julia Guil, Luis Cassinotti,
Yanina Bota, Mercedes Scholler, Marcelo S. Vatta
and Liliana G. Bianciotti
Chapter 8 Hepatitis B Virus 149
Diego Flichman

vi Contents
Chapter 9 Chronic Hepatitis B: Current Treatment and New

Therapeutic Options 169
Maria Gimena Fernandez 169
Chapter 10 Hepatitis C Virus 179
Chapter 11 Mechanism Lipotoxicity in the Progression from Fatty Liver
to Non-Alcoholic Steatohepatitis 203
Fernando J. Barreyro and Juan José Poderoso
Chapter 12 Portal Hypertension 231
Francisco Eizayaga, Leandro Steinberg, Carlos Brodersen
and Salvador Romay
Chapter 13 Pathophysiology of Portal Hypertension 239
Salvador Romay, Leandro Steinberg and Francisco Eizayaga
Chapter 14 Hepatic Encephalopathy 257
Silvina Tallis
Chapter 15 Hepatic Encephalopathy Therapy 275
Sara Beatriz Chao
Chapter 16 The Role of Systemic Inflammation in Encephalopathy
Consequence of Chronic Liver Failure 289
Ariel R. Marcotegui, Soraya Wilke Saliba, Nizar Yousif
and Bernd L. Fiebich
Chapter 17 Hyperammonia and Skeletal Muscle 307
Lisandro Orbea, Ariel R. Marcotegui, Pablo A. Souto,
Juan Skerl and Juan C. Perazzo
Chapter 18 Cellular Therapies to Approach Severus Liver Pathologies 319
Gustavo A. Moviglia
Index 331

LIST OF CONTRIBUTORS
Fernando J. Barreyro, MD, Ph.D

Laboratory of Microbiology, Faculty of Chemical and Natural Sciences,
National University of Misiones, Argentina.
Member of the National Council of Scientific and Technological Research (CONICET),
Argentina
Liliana Bianciotti, Biochem. Ph.D

Instituto de Inmunología, Genética y Metabolismo (INIGEM-CONICET-UBA),
Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires.
Professor of Pathophysiology, Faculty of Pharmacy and Biochemistry,
University of Buenos Aires, Buenos Aires, Argentina.
Yanina Bota
Instituto de Inmunología, Genética y Metabolismo (INIGEM-CONICET-UBA),
Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires.
Carlos Brodersen, MD, Ph.D

Chair of the Gastroentherology Unit Durand Hospital, Ciudad de Buenos Aires, Argentina.
Professor of Internal Medicine, University of Buenos Aires, Buenos Aires, Argentina.
Alicia Brusco, Biochem. PhD

Director of the Instituto de Biología Celular y Neurociencia
“Prof. E. De Robertis” (UBA-CONICET).
Facultad de Medicina, Universidad de Buenos Aires, Argentina

viii Contributors
Laura Caltana, MD, PhD

Instituto de Biología Celular y Neurociencia “Prof. E. De Robertis” (UBA-CONICET).
Facultad de Medicina, Universidad de Buenos Aires, Argentina
Luis Cassinotti, Msc

Doctoral fellow at catedra de Fisiología-Instituto de Química y Metabolismo del Fármaco
(IQUIMEFA-CONICET-UBA)
Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires
Sara Chao, MD
Gastroenterology Division in President Perón Hospital. Avellaneda, Buenos Aires, Argentina.
Intensive Care Specialist at the Critical Care Division, Marie Curie Hospital,
Buenos Aires, Argentina.
Francisco Eizayaga, MD, Ph.D

Senior Researcher of the Laboratory of Hepatic Encephalopathy and Portal Hypertension
Department of Pathophysiology, University of Buenos Aires,
Ciudad de Buenos Aires, Argentina.
Professor, Maimonides University, Buenos Aires, Argentina.
Maria Gimena Fernandez, MD.

Hepatologist of the Churruca Hospital, Buenos Aires, Argentina
Bernd L. Fiebich, Ph.D

Department of Psychiatry. Director of the Neurochemistry Lab,
University of Freiburg Medical School, Freiburg, Germany
Diego Flichman, Biochem. Ph.D

Professor of Virology, Facultad de Farmacia y Bioquímica,
Universidad de Buenos Aires, Buenos Aires, Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)
María Julia Guil, Biochem. Ph.D

(IQUIMEFA-CONICET-UBA)

Contributors ix
Néstor R. Lago, MD
Director of the Center of Experimental and Applied Pathology, Department of Pathology,
Faculty of Medicine, University of Buenos Aires, Argentina.
Full Professor of Pathology. Department of Pathology, Faculty of Medicine,
University of Buenos Aires, Argentina
Mario Alejandro Lorenzetti, Ph.D

Instituto Multidisciplinario de Investigaciones en Patologías Pediátricas
(IMIPP- CONICET-GCBA)
Laboratorio de Biología Molecular, División Patología,
Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina
Ariel R. Marcotegui, MD, MSc

Researcher at the Laboratory of Hepatic Encephalopathy and Portal Hypertension,
Gustavo A. Moviglia, MD, Ph.D

Director of the Center of Investigation and Engineering of Tissues and Cellular Therapy.
Maimonides University. Buenos Aires. Argentina
Alberto E. Muñoz, MD
Hepatologist at the Hospital de Gastroenterología
Dr. Carlos B. Udaondo, Buenos Aires Argentina.
Lisandro Orbea, MD. MSc.

Researcher at the Laboratory of Hepatic Encephalopathy and Portal Hypertension.
Faculty of Pharmacy and Biochemistry,
University of Buenos Aires. Buenos Aires, Argentina.
Member of the Center of Experimental and Applied Pathology (CPEA).
Faculty of Medicine, University of Buenos Aires, Buenos Aires, Argentina.
Juan C Perazzo, MD
Director of the Laboratory of Hepatic Encephalopathy and Portal Hypertension
Faculty of Pharmacy and Biochemistry, University of Buenos Aires, Argentina.
Professor of Pathophysiology, Faculty of Pharmacy and Biochemistry,
University of Buenos Aires, Argentina.

x Contributors
Luís Perea, MD.

Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS),
Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas
(CIBERehd), Barcelona, Spain.
Juan José Poderoso, MD, Ph.D

Director of the Laboratory of Oxygen Metabolism, University Hospital,
School of Medicine, University of Buenos Aires. Argentina
Member of the National Council of Scientific and Technological Research
(CONICET), Argentina
Full Professor of Internal Medicine, Faculty of Medicine,
University of Buenos Aires, Buenos Aires, Argentina
María Victoria Preciado, Ph.D

Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina.
Myrian R. Rodríguez, Biol. Ph.D

Assistant Researcher at Instituto de Inmunología,
Genética y Metabolismo (INIGEM-CONICET-UBA),
Salvador Romay, MD
Senior Researcher at the Laboratory of Hepatic Encephalopathy and Portal Hypertension,
Chair of Internal Medicine II, Hospital Pirovano, Ciudad de Buenos Aires, Argentina.
Pau Sancho-Bru, MD. Ph.D

Mercedes Scholler, Msc

(IQUIMEFA-CONICET-UBA),

Contributors xi
Juan Skerl, MD, MSc

Pablo A Souto, Biochem. MSc

Researcher at the Laboratory of Hepatic Encephalopathy and Portal Hypertension.
Faculty of Pharmacy and Biochemistry. University of Buenos Aires.
Leandro Steinberg, MD
Gastroentherology Unit Durand Hospital, Ciudad de Buenos Aires, Argentina
Silvina Tallis, Biochem. MSc, Ph.D

Júlia Vallverdú
Pamela Valva, MD, Ph.D

Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina.
Marcelo S. Vatta, Biochem. Ph.D

Professor of Physiology, Physiology Department, Faculty of Pharmacy and Biochemistry,
University of Buenos Aires. Buenos Aires, Argentina.
Principal researcher at Catedra de Fisiología-Instituto De Química Y
Metabolismo Del Fármaco (IQUIMEFA-CONICET-UBA),
Faculty of Pharmacy and Biochemistry, University of Buenos Aires,

xii Contributors
Soraya Wilke Saliba, Pharm.

Department of Psychiatry, Neurochemistry Lab,
University of Freiburg Medical School, Freiburg, Germany.
Nizar Yousif, MSc

Department of Psychiatry, Neurochemistry Lab,
University of Freiburg Medical School, Freiburg, Germany.

PREFACE
Liver Cirrhosis, similar to congestive heart failure, chronic obstructive pulmonary

disease, or chronic kidney disease, represents the final stage of chronic disease which results
in the severe and permanent damage of a vital organ. Furthermore, cirrhosis has the
peculiarity of being an important risk factor in the development of liver cancer. In recent
years liver cirrhosis has emerged as being an enormous health problem worldwide. Global
epidemiology studies have shown that the cirrhosis is one of the principal causes of mortality
in both men and women. In 2015, the number of deaths attributed to cirrhosis worldwide was
almost 1.3 million people. If we then add the 800.000 people who died of liver cancer, which
almost always occurs with cirrhosis, the total number of deaths due to a consequence of end
stage liver disease surpasses 2 million people. What is more, liver cirrhosis is one of the most
important causes of disability-adjusted life years, has a markedly negative impact on patients’
quality of life and is also responsible for a high consumption of health resources. For this
reason if we are to reduce the impact and mortality of liver diseases in the world, there is a
need to increase knowledge and understanding of the pathogenic mechanisms of liver injury
and disease progression of chronic liver diseases. This will bring about without doubt an
improvement in the early diagnostic methods and treatment of diseases.
In this context, this book by Juan Carlos Perazzo and his co-authors has a special
meaning, as it represents a substantial effort in the dissemination of understanding of the
biological mechanisms that drive the development and progression of chronic liver diseases.
The book covers the different areas from cell biology in the different types of liver cells to the
description of the most frequent syndromes in Hepatology. The authors of the chapters have
done an excellent job of in-depth reviewing of the various issues without losing sight of the
whole picture. For this reason the book is useful for investigators in both experimental and
clinical research, as well as clinicians who care for patients with liver disease and want to
improve their knowledge and understanding. In this day and age, much of the information is
available on the internet but many times it is difficult to select and apply this information.
The compilation of this information in a book format is much more attractive as is this book
when you hold it in your hands.
Pere Ginès, MD, PhD

Professor and Chairman
Liver Unit, Hospital Clinic of Barcelona
University of Barcelona.

ACKNOWLEDGMENTS
Broadening our state of consciousness improves human welfare.
This book is an attempt to complete a vision of the chronic liver disease. We focus on the
basic molecular mechanisms as a rational basis for the steps that follow, the accurate
diagnosis and therapy. In this way, we pretend to achieve a global vision of the organ disease.
Many pathophysiological aspects are not included, not because we think they lack of
importance, but because we believe that the chosen ones include comprehensive
pathophysiological aspects. We emphasize in the interrelationship of the numerous medical
disciplines. The impact on an organic system is inextricably linked to its effects on others, but
it could be considered that the basic pathophysiological pathways are shared by different
pathologies, and each one of them with its own characteristic response. This book is an
attempt to expose these shared pathways.
A substantial aspect is that currently the most productible working way is as a team. This
book is the result of a team work plus the personal experience that attests it. A broad view
also teaches that there are so many people involved in each chapter that is difficult to give a
true recognition list. Anyway, we all agree that families and friendship are essential to treat
this type of work and that both concepts should be expanded. We recognize and thank all who
explicitly and implicitly worked in this presentation.
With the hope that this book could be of some help to researchers and clinicians, we will
keep working with the new arriving challenges.
We dedicate this book to our families, whose love, tolerance and support sustained us; to
our colleagues, from whom we have learned; to the chapter authors, who have given so much
of themselves and, finally, to the readers.

ABSTRACT
Chronic liver disease (CLD) is one of the most prevalent pathologies in developed
countries. The large amount of new knowledge drove to subspecialities, even in issues such as
hepatology. Every day is more difficult to access all the relevant information that is being
published. And even more, biomolecular techniques lead to a complexity level that except of
the specific professional working area, becomes so complicated that excludes other
professionals.
This book attempts to give a broad overview of the molecular biology of the liver,
emphasizing in how this knowledge supports the rationale for treatments. Thus,
pathophysiology and therapies are updated in viral hepatitis, hepatic encephalopathy and
Portal hypertension, among others. However appended issues, which might look less relevant,
as stem cells and endocannabinoids are included. These two issues will be soon relevant due
to their close relationship with the liver tissue and especially in liver disease.
New paradigms such as cell death and involvement of the extracellular matrix, are also
contemplated. In addition, important issues, such as stellate cells and their intimate
relationship with liver function and fibrogenesis are covered in depth.
The basic role of endothelins in CLD is presented in-depth manner. Because of its
prevalence, NASH is discussed with special interest, with emphasis in fatty liver process, its
molecular relation with liver cells and its metabolism in CLD. We believe this is a broad
vision, although it does not cover all issues, which describes the basic pathophysiological
mechanisms shared by many liver diseases, giving a rational support for specific therapies.

In: Chronic Liver Disease: From Molecular Biology to Therapy ISBN: 978-1-53610-237-6
Editors: J. C. Perazzo, F. Eizayaga, S. Romay et al. © 2017 Nova Science Publishers, Inc.
Chapter 1
CELL DEATH ISSUE FOR RESEARCHERS

AND CLINICIANS

ABSTRACT
Hepatocyte cell death is a central mechanism involved in liver injury and is present
in almost all types of human liver diseases. Indeed, excessive cell death has been
identified as a central mechanism of liver damage in conditions such as acute and chronic
viral hepatitis, alcoholic and non-alcoholic steatohepatitis (ASH and NASH), and drug-
induced liver injury (DILI). Different mechanisms of cell death such as apoptosis,
necrosis, necroptosis and autophagy, which may vary substantially amongst liver
diseases, can trigger specific cell death responses and promote disease progression. In
this chapter, we first describe the molecular mechanisms of different forms of liver cell
death and then we discuss how cell death contributes to the development of liver disease.
INTRODUCTION
Cell death is likely to be one of the most widely studied topics among cell biologist. It is
a well-known fact that tissue homeostasis is dependent on the perfect balance between cell
proliferation and cell death. This balance is finely regulated by positive and negative signals,
which determine the final fate between life and death; any imbalance in this process may
result in disease linked with unwanted cell death or cell growth. Since the initial description
of cell death in the 1960s, a number of different death mechanisms have been described
(Favaloro et al., 2012). Cell death is defined by the Nomenclature Committee on Cell Death
as a process by which the cell ceases to carry out its physiological functions, a process that
remains reversible until an irreversible phase or ‘point-of-no-return’ is trespassed. It can be
classified according to: 1) the cells morphological appearance (which may be apoptotic,
necrotic, autophagic or associated with mitosis), 2) an enzymological criteria (with and
without the involvement of nucleases or of distinct classes of proteases, such as caspases,

2 P. Valva, M. Alejandro Lorenzetti and M. Victoria Preciado
calpains, cathepsins and transglutaminases), 3) according to functional aspects (programmed

or accidental, physiological or pathological) or 4) in relation to immunological outcome
(immunogenic or non-immunogenic) (Kroemer et al., 2009). Hepatocyte cell death is a
central mechanism involved in liver injury and is present in almost all types of human liver
disease. Indeed, excessive cell death has been identified as a central mechanism of liver
damage in conditions such as acute and chronic viral hepatitis, alcoholic and non-alcoholic
steatohepatitis (ASH and NASH), and drug-induced liver injury (DILI) (Eguchi et al., 2014;
Dolganiuc et al., 2012; Bantel and Schulze-Osthoff, 2012; Jaeschke et al., 2012). Clinical data
and animal models also suggest that hepatocyte death is the key trigger in liver disease
progression, manifested by the subsequent development of inflammation, fibrosis, cirrhosis,
and hepatocellular carcinoma (HCC). Different mechanisms of cell death such as apoptosis,
necrosis, necroptosis and autophagy, which may vary substantially amongst liver diseases,
can trigger specific cell death responses and promote its progression (Luedde et al., 2014).
Classically, hepatocyte death has been presented in two mutually exclusive forms:
programmed cell death (PCD), or apoptotic cell death, vs. accidental or necrotic cell death,
based on morphological criteria. However, a growing understanding of the signalling events
involved in triggering cell death has allowed for the dissection of the different molecular
pathways that result in hepatocyte death and has even identified a number of cell death modes
that present with crosstalk and cooperation in the execution of cell death (Eguchi et al., 2014).
In the healthy liver, cell death controls organ homeostasis, with a tight equilibrium
between the loss and replacement of hepatocytes (Michalopoulos and DeFrances, 2005).
Turnover is low, with approximately 0.05% of hepatocytes at any given time being removed
by apoptosis, mostly in zone 3 (Benedetti et al., 1988). The presence of hepatocyte death,
reflected by increased levels of serum alanine aminotransferase (ALT) and aspartate
aminotransferase (AST), is the most widely used parameter to screen for and monitor patients
with liver disease. Moreover, these markers drive therapeutic decisions, have prognostic value
for patients with hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, NASH, and
autoimmune hepatitis and correlate with liver-specific mortality in the general population
(Feld et al., 2005; Amarapurka et al., 2006; Fassio et al., 2004; Angulo et al., 1999; Hui et al.,
2003; Ghany et al., 2009; Lee et al., 2008; Kim et al., 2004; Ruhl and Everhart, 2009). These
well-established facts emphasize the importance of cell death as the ultimate driver of liver
disease progression and the development of liver fibrosis, cirrhosis, and HCC (Eguchi et al.,
2014; Luedde et al., 2014). Notably, the contribution of cell death to liver disease is cell-,
stage- and context-specific for instance; whereas increased cell death in hepatocytes
contributes to fibrogenesis, cell death in fibrogenic cells is an important mechanism for
resolution of liver fibrosis (Luedde et al., 2014; Iredale et al., 1998; Friedman, 2008).
In view of the fundamental role of cell death in virtually all hepatic diseases, precise
knowledge on the mechanisms regulating cell death and cell death responses is essential to
understand the pathophysiology of liver diseases and to develop new therapeutic approaches.
FORMS OF LIVER CELL DEATH

Most forms of liver cell death have been extensively characterized in vitro in primary
hepatocytes cultures or in several immortalized hepatocyte cell lines, but only a few have

Cell Death Issue for Researchers and Clinicians 3
been well defined in vivo using various experimental animal models or patients with liver
diseases. The complexity of studying cellular demise in either ex vivo (explanted liver tissue
from animal models or liver biopsy tissue from humans) or in vivo (model organism and/or
humans) comes from the recognition that, in many instances, hepatic cell death represents a
highly heterogeneous process. Moreover, frequent overlap and crosstalk between involved
pathways may result in molecular transitions between different cell death modalities. In the
damaged liver, forms of cell death include apoptosis, necrosis, necroptosis, and autophagy
(Figure 1). These medical terms have clear definitions in pathology; however, different modes
of cell death are intermingled as a continuous process during liver injury. Therefore, cells
initiating a specific form of PCD during an acute or chronic insult could finally evolve into a
different form of demise, resulting in a mixed pattern of cell death (Eguchi et al., 2014).
Apoptosis
The term “apoptosis” derives from the Greek meaning “dropping off” and refers to the
falling of leaves from the trees in autumn. It is used, in contrast to necrosis, to describe the
situation in which a cell actively pursues a course towards its death upon receiving certain
stimuli (Kerr et al., 1972). Ever since it was described by Kerr et al. in the 1970’s, apoptosis
remains one of the most investigated processes in biologic research (Kerr et al., 1972).
Apoptosis is a typical form of PCD, an ordered and orchestrated cellular process that occurs
in both physiological and pathological conditions. The mechanism of apoptosis is complex
and involves many pathways, where any fault, at any given point, may play a pivotal role in
the pathogenesis of many diseases.
Under physiological conditions, cell death by apoptosis regulates the sculpting of tissues
during embryonic development such as the removal of interdigital webs, shaping of the inner
ear or in cardiac morphogenesis. Moreover, in the adult organism, apoptosis regulates
involution processes such as shedding of the endometrium, regression of the post-lactating
mammary gland, and normal destruction of cells before their replacement (Duprez et al.,
2009). Pathological conditions involve for example some forms of virus-induced cell death,
such HCV and HBV; pathologic atrophy of organs and tissues such as prostatic atrophy after
radiation or hypoxia; degenerative diseases such as Alzheimer’s and Parkinson’s disease, in
immune graft rejection; depletion of CD4+ cells in AIDs; and cell death that occurs in heart
diseases such as myocardial infarction (Wong, 2011). Moreover, cancer is one of the
scenarios where too little apoptosis occurs, resulting in malignant cells that will not die.
In the liver, apoptosis is particularly important, as this is an organ that is naturally
exposed to toxins, drugs, and virus; however, excessive apoptosis can result in tissue
destruction and organ failure. Moreover, hepatic apoptosis is considered a prominent
pathological feature in most forms of liver injury and usually, an excess in apoptosis of
hepatocytes accompanies chronic disease. However, apoptosis may act as a double-edged
sword, being the cause of the problem as well as its solution. This fact has impelled many
investigations which have now ventured into the quest for new drugs targeting various aspects
of apoptosis (Wong, 2011). Interventions in hepatic apoptosis can delay disease progression
and reduce the morbidity of liver diseases. Nevertheless, to date, no therapeutic approach has
proven successful in clinical practice and those mechanisms responsible for hepatic apoptosis
in different liver diseases are still under investigation (Wang, 2015).

Figure 1. Highlight: Forms of liver cell death.

Morphological and Biochemical Changes in Apoptosis
Apoptosis occurs following the activation of specific pathways, which result in a series of
well-defined morphological events, both in the nucleus and cytoplasm, and are conserved
across cell types and even across species (Saraste and Pulkki, 2000; Hacker, 2000).
Morphological hallmarks of apoptosis are chromatin condensation and nuclear fragmentation,
which are accompanied by rounding up of the cell, reduction in cellular volume (pyknosis)
and retraction of pseudopods (Kroemer et al., 2009; Majno and Joris, 1995). Chromatin
condensation starts at the periphery of the nuclear membrane, forming a crescent or ring-like
structure. Then, chromatin further condenses until it breaks up inside a cell with an intact
membrane, a feature described as karyorrhexis (Majno and Joris, 1995). At the later stage,
some of the morphological features include membrane blebbing, ultrastrutural modification of
cytoplasmic organelles and a loss of membrane integrity (Kroemer et al., 2009). Blebbing of
the plasma membrane results in the release of small membrane-enclosed particles containing
cellular components known as apoptotic bodies, which are rapidly identified by neighbouring
cells or professional phagocytes and are generally disposed without induction of
inflammation or tissue scarring. In the liver, hepatocellular apoptotic bodies have long been
referred to as acidophilic bodies or Councilman bodies. However, phagocytic cells usually
engulf apoptotic cells before apoptotic bodies occur. This is the reason why apoptosis was not
discovered until quite late in the history of cell biology, and apoptotic bodies are mainly seen
in vitro under special conditions. If the remnants of apoptotic cells are not phagocytized, these
will undergo a degradation process that resembles of necrosis and that is termed secondary
necrosis (Ziegler and Groscurth, 2004).
Concerning biochemical changes, three main characteristics define an apoptotic cells: 1)
membrane changes and recognition by phagocytic cells, 2) activation of caspases and 3) DNA
and protein breakdown. During an early apoptotic phase, phosphatidylserine (PS) is exposed
in the outer layers of the cell membrane, which has been “flipped out” from the inner layers.
It acts as an “eat me” signal and allows early recognition of dying cells by macrophages,
resulting in phagocytosis without the release of pro-inflammatory cellular components
(Hengartner, 2001). Another specific feature of apoptosis is the activation of a group of
enzymes belonging to the cysteine protease family named caspases. Activated caspases cleave
vital cellular proteins and break up the nuclear scaffold and cytoskeleton. They also activate
DNAase, which degrade nuclear DNA (Wong, 2011; Lavrik et al., 2005) into large 50 to 300
kilobase pieces. Later, further internucleosomal endonuclease cleavage of DNA into multiple
oligonucleosomes of 180 to 200 base pairs produce the typical DNA ladder observed in
agarose gel electrophoresis ((Wong, 2011) and references herein).
Mechanism of Apoptosis
As mentioned above, apoptosis results from the collapse of cellular infrastructures

through internal proteolytic digestion, which leads to cytoskeletal disintegration, metabolic
derangement, and genomic fragmentation. Members of the caspase family of proteases form
the core engine of apoptosis and are involved in initiation, execution, and regulatory phases of
the pathway (Figure 2). Caspases are cysteine proteases that cleave substrates after aspartate

residues within specific peptide recognition sequences. To preclude unwarranted cell death,
caspases are expressed as inactive zymogens which consists of a pro-domain followed by two
subunits which compose the catalytic domain. Full activation is achieved through the
cleavage of the pro-domain, generally by other caspases which operate the hierarchical
cascade that serves to amplify the apoptotic signal (Los et al., 1999). Based on their structure
and order in the cell death pathways, caspases can be classified as initiators or effectors of
apoptosis. Effector caspases such as caspase-3, -6, and -7 cleave diverse cellular substrates. In
contrast, initiator caspases, such as caspase-8, -9, and -10, exert regulatory roles by activating
downstream effector caspases ((Bantel and Schulze-Osthoff, 2012) and references herein).
From a functional point of view, we can distinguish two types of caspases: up-stream and
down-stream caspases. Up-stream caspases become active when they come into close
proximity with other equal molecules. This interaction leads to conformational changes upon
binding with activation complexes and ultimately results in their cleavage and full activation.
Once activated they can activate additional molecules of the same enzyme as well as down-
stream caspases. Down-stream caspases on the other hand can only be activated by cleavage
of the pro-domain by up-stream caspase ((Favaloro et al., 2012) and references herein).
Besides, there are other schemes for human caspases classification, based on their
phylogenetic relationships, their pro-domain length and substrate preference; as well as their
non-apoptotic functions (such as cytokine maturation, inflammation and differentiation) (Man
and Kanneganti, 2016).
Figure 2. Members of the caspase family of proteases. A) caspases structure and classification. Note
that both inflammatory and initiator caspases carry at their N-termini a caspase activation and
recruitment domain (CARD) or a death effector domain (DED) allowing them to interact with other
molecules that regulate their activation. DED in caspase-8 binds to a homologous domain in the adaptor
proteins to form DISC. Meanwhile, the CARD domain of procaspase-9 allows the interaction with
Apaf-1 and promotes apoptosome formation. B) Scheme of procaspase activation for both initiator and
effector caspases.

Two major signalling routes, namely the extrinsic death receptor and the intrinsic
mitochondrial pathway, activate caspases. Both pathways eventually lead to a common point
before the execution phase of apoptosis, the activation of caspase-3. As mentioned above,
caspases induce cleavage of protein kinases, cytoskeletal proteins (such as vimentin, laminin,
actin and cytokeratin), as well as DNA repair proteins and inhibitory subunits of
endonucleases family. It is well known that caspase-3 cleaves PARP-1 [Poly (ADP-ribose)
polymerase-1, involved in DNA repair process] a as well as the inhibitor of the caspase-
activated deoxyribonuclease (ICAD), which are the ultimate responsible for DNA cleavage
and nuclear apoptosis. Hence, effector caspases exert their effect over the cytoskeleton, cell
cycle and signalling pathways, which together contribute to the typical morphological
changes that characterize apoptosis (Wong, 2011; Bantel and Schulze-Osthoff, 2012) (Figure
3).
Extrinsic Death Receptor Pathway
Extrinsic apoptosis defines a form of death induced by extracellular signals that result in
the binding of ligands to specific transmembrane receptors, collectively known as death
receptors (DR) and which belong to the TNF/NGF family. These receptors possess three
domains: an extracellular ligand-interacting domain, a transmembrane domain, and an
intracellular death domain. Death receptors of importance in the liver include Fas
(CD95/Apo-1), tumor necrosis factor receptor 1 (TNFR1), tumor necrosis factor-related
apoptosis inducing ligand (TRAIL) receptor 1 (TRAIL-R1/Death receptor 4 [DR 4]); TRAIL
receptor 2 (TRAIL-R2/DR5/Killer/TRICK2), death receptor 3 (DR3/Apo-3/ TRAMP/WSL-
1/LARD), and death receptor 6 (DR6) (Hengartner, 2001; Malhi et al., 2006). All death
receptors function in a similar way: upon ligand binding, several receptor molecules are
mobilized and brought together. Once stacked, they undergo conformational changes that
allow the assembly of a large multi-protein complex known as death initiation signaling
complex (DISC), which ultimately leads to the activation of the caspase cascade. In a
simplified model, binding of death ligands such as Fas ligand (FasL) or tumor necrosis factor
(TNF)-α or TRAIL to their respective death receptors leads to receptor oligomerization.
These death receptors have an intracellular death domain (DD) that recruits adaptor proteins
such as TNF receptor-associated death domain (TRADD) and Fas-associated death domain
(FADD) (Schneider and Tschopp, 2000) (Figure 3). At the same time, adaptor proteins
contain another conserved protein interaction domain known as death effector domain (DED)
that binds to a homologous domain in caspase-8. Upon death receptor activation, DISC
initiates the assembly and activation of pro-caspase-8, which is the key mediator of the
extrinsic pathway. Active caspase-8 will then activate additional caspase-8 molecules by
dimerization and auto-proteolytic cleavage, as well as down-stream caspases such as caspase-
3, culminating in the demise of the so-called type I cells (Lavrik and Krammer, 2012;
Favaloro et al., 2012; Reed, 2000). However, in most cells, including hepatocytes, only low
amounts of initiator caspases, insufficient to trigger the extrinsic pathway on their own, are
activated by DISC. In these cells, type II cells, the extrinsic receptor pathway needs to be
amplified by the joint activation of the intrinsic mitochondrial pathway through caspase-8-
mediated cleavage of Bid. Bid is a pro-apoptotic Bcl-2 family protein, which subsequently

initiates together with other Bcl-2 family members, Bak and Bax, the release of mitochondrial
pro-apoptotic mediators (Scaffidi et al., 1998; Zimmermann et al., 2001).
The extrinsic cell death pathway is typically triggered by cells of the immune system,
which express death receptor ligands, including TNF itself, Fas ligand and TRAIL. Moreover,
cytotoxic T lymphocytes (CTL) and natural killer (NK) cells secrete FasL. Apoptosis
stimulated by FasL provides an efficient means to remove unwanted hepatocytes in various
hepatic disorders, for example the removal of virus-infected hepatocytes and cancer cells by T
lymphocytes (Malhi et al., 2006).
Figure 3. Schematic overview of extrinsic death receptor and intrinsic mitochondrial pathway of
apoptosis. The death receptor pathway is activated when a specific ligand binds to a death receptor.
This binding leads to the formation of a signaling complex resulting in activation of initiator caspases
such as pro-caspase-8. Active caspase-8 can activate two ways. The first path involves large amounts of
caspase-8 which directly activate the effector caspases such as caspase-3, -6 and -7, responsible for
most of the changes that result in cell death. The second involves the mitochondrial pathway of
apoptosis activation, where by cleavage of the pro-apoptotic Bid molecule cytochrome-c is released
from mitochondria. While anti-apoptotic molecules such as Bcl-2 and Bcl-xL can block this stage and
inhibit the process by blocking the mitochondrial release of cytochrome-c, the pro-apoptotic proteins,
such as Bax, act by promoting its release. Cytochrome-c binds to Apaf-1, forming a complex that binds
to caspase-9 constituting the apoptosome. This activates effector caspases. Cleavage of BID
permeabilize the mitochondrion, resulting in release mitochondrial apoptogenic factor [including
Apoptosis Inducing Factor (AIF), second mitochondria-derived activator of caspase (Smac),
endonuclease G (endo G), direct IAP Binding protein with Low pI (DIABLO) and Omi/high
temperature requirement protein A (HtrA2). All of them directly induce apoptosis by different
mechanisms, in a caspase-dependent or –independent manner. Moreover, the cytotoxic cells can
activate the apoptotic process through perforin and granzyme B. Perforin produces pores in the plasma
membrane to allow entry of granzyme B into hepatocytes. Granzyme B can directly activate effector
caspases. All internal cellular stimuli such as DNA damage, oxidative stress, endoplasmic reticulum
stress, can activate the apoptotic process through the mitochondrial pathway. Finally, due to the
lethality, the system is subject to a number of controls such as the balance of Bcl-2 family of proteins,
the presence of IAPs (Inhibitor of Apoptosis Protein) and cFLIC, etc (for more detail see the text).

Intrinsic Mitochondrial Pathway
The intrinsic pathway is activated in response to a number of internal stimuli such as

irreparable genetic damage, hypoxia, extremely high concentrations of cytosolic Ca2+ and
severe oxidative stress. In all cases, these multiple forms of stress converge on the
mitochondria and determine mitochondrial outer membrane permeabilization (MOMP). This
results in the dissipation of the mitochondrial membrane potential and therefore in the
cessation of ATP production, the release of pro-apoptotic molecules such as cytochrome-c
into the cytoplasm, and the release of a number of proteins that contribute to caspase
activation (Wong, 2011). Cytochrome-c normally functions in the electron transport processes
in the respiratory chain to generate ATP. However, once it is released to the cytosol of
apoptotic cells, it serves as a cofactor for the adaptor protein Apaf-1 (apoptotic peptidase
activating factor-1). Upon binding of cytochrome-c and dATP, Apaf-1 oligomerizes and
recruits the initiator caspase-9 to trigger the formation of the apoptosome. Both caspase-9 and
Apaf-1 contain homologous caspase activation and recruitment domain (CARD) and interacts
through these homophilic domines. Thus, similar to DISC, the apoptosome is a high
molecular weight complex that serves as a caspase activation platform. Once assembled into
the apoptosome, caspase-9 becomes activated and subsequently triggers the caspase cascade
(Zimmermann et al., 2001).
This pathway is meticulously regulated by a group of proteins that belong to the Bcl-2
family, which are characterized by the presence of at least one Bcl-2 Homology (BH) domain
(Figure 4). From a functional point of view, they can be classified in anti-apoptotic members
containing four BH domains (such as Bcl-2, Bcl-xl, Bcl-w, Mcl-1) and pro-apoptotic
members with two or three BH domains (such as Bax, Bak, Bcl-xs, Bok) or with just one
(such as Bad, Bik, Bid, Bim, Noxa, Puma), termed BH3-only proteins. On the one hand, pro-
apoptotic members of this family mediate apoptosis by disrupting membrane integrity, either
by directly forming pores or by binding to mitochondrial channel proteins such as adenine
nucleotide transporter (ANT) and the voltage dependent anion channel (VDAC). On the other
hand, anti-apoptotic members prevent apoptosis by interfering with pro-apoptotic member
aggregation. The different apoptotic signals are sensed by BH3 only proteins (Bad, Bik, Bid,
Bim, Noxa, Puma) which are induced or activated and migrate to the mitochondria. Once
there, they bind and suppress anti-apoptotic members of the family or bind to the pro-
apoptotic ones, promoting their aggregation and activation, and are therefore named pro-
apoptotic ligands. Consequently, while the anti-apoptotic proteins regulate apoptosis by
blocking the mitochondrial release of cytochrome-c, the pro-apoptotic proteins act by
promoting its release. It is not the absolute quantity but rather the balance between the pro-
and anti-apoptotic proteins that determines whether apoptosis will be initiated ((Zimmermann
et al., 2001; Rust and Gores, 2000; Tsujimoto, 1998; Reed, 1997) and references herein).
As mentioned above, the intrinsic and extrinsic pathways are not mutually independent
since, the activation of caspase-8 results in the activation of the mitochondrial pathway by
cleavage of the BH3 only protein BID. Cleavage of BID generates a truncated fragment
known as truncated BID (tBID) that can permeabilize the mitochondrion, resulting in MOMP
and the release mitochondrial pro-apoptotic mediators (Kaufmann et al., 2012). These
components, named mitochondrial apoptogenic factors, include apoptosis-inducing factor
(AIF), second mitochondria-derived activator of caspase (Smac), endonuclease G (endo G),
direct IAP Binding protein with Low pI (DIABLO) and Omi/high temperature requirement

protein A (HtrA2) (Kroemer et al., 2007). All of them directly induce apoptosis by different
mechanisms, in a caspase-dependent or –independent manner. For example, it is well known
that when endonuclease G is released from the mitochondria, it translocates to the nucleus
and acts as a DNaseI in DNA fragmentation. Moreover, AIF induces chromatin condensation
and large-scale DNA fragmentation (50kbp) when released into the cytosol. Meanwhile,
DIABLO/SMAC bind to inhibitors of apoptosis proteins (IAPs), removing their inhibition
and allowing apoptosis to occur (Shoshan-Barmatz et al., 2010) (Figure 3).
Figure 4. The Bcl-2 protein family. The Bcl-2 protein family is characterized by the presence of at least
one Bcl-2 Homology (BH) domain, the BH3 domain (dotted box). From a functional point of view,
they can be classified in anti-apoptotic members containing three or four BH domains (Bcl-2, Bcl-xl,
Bcl-w, Mcl-1) and pro-apoptotic members with two or three BH domains (Bax, Bak, Bcl-xs, Bok) or
with just one (such as Bad, Bik, Bid, Bim, Noxa, Puma). While the anti-apoptotic proteins regulate
apoptosis by blocking the mitochondrial release of cytochrome-c, the pro-apoptotic proteins act by
promoting its release. It is not the absolute quantity but rather the balance between the pro- and anti-
apoptotic proteins that determines whether apoptosis will be initiated.
Endoplasmic reticulum (ER) stress and p53 activation also trigger the intrinsic
mitochondrial pathway (Figure 3). ER stress is typically the result of accumulating unfolded
or misfolded proteins in the ER, leading to the so-called unfolded protein response. Whereas
mild ER stress is cytoprotective, profound or prolonged ER stress promotes cell death by
activation of c-Jun N-terminal kinases (JNK) and the CCAAT/enhancer-binding protein
homologous protein (CHOP). Notably, JNKs are a subfamily within the mitogen activated
protein kinases (MAPKs) family and they are master protein kinases that regulate different
physiological processes, including morphogenesis, differentiation, inflammatory responses,
cell proliferation, survival and death. JNKs are activated by a series of phosphorylation events
in response to multiple stimuli such as cytokines, growth factors, pathogens, stress, toxins or
drugs. Upon activation, either by autophosphorylation or by phosphorylation by other

kinases, they catalyze the transfer of the terminal phosphate group of ATP to specific amino
acid residues in target proteins. Phospholylation modifies the functions of the target protein
either by affecting its activity or by controlling subcellular localization, degradation and
association with other binding partners. Eventually, this phosphorylation cascade leads to the
regulation of a wide variety of cellular responses, such as proliferation, differentiation, cell
death and survival (Bubici and Papa, 2014). In the context of ER stress, JNKs activate
apoptotic signaling by up-regulating pro-apoptotic genes via the transactivation of specific
transcription factors or by directly modulating the activities of mitochondrial pro- and anti-
apoptotic proteins through distinct phosphorylation events (Dhanasekaran and Reddy, 2008).
The exact contribution of CHOP to the induction of apoptosis remains still poorly understood,
although CHOP-deficient cells are resistant to apoptosis triggered by ER stress. It was
suggested that CHOP is crucial for the induction of caspase-11, which plays an important role
in the processing of pro-IL-1β through caspase-1 activation under LPS-induced inflammatory
stress. In addition, it was also suggested that CHOP could induce the depletion of cellular
glutathione and increases oxygen reactive species in the ER ((Nishitoh, 2012) and references
herein). Meanwhile, p53 is another important regulator of the intrinsic death pathway and the
central component of a continuously operative cell-fate program that determines whether a
cell should initiate DNA damage repair or die by apoptosis. This fate mainly depends on its
sub-cellular localization, nuclear or cytoplasmic. In response to oncogene activation, DNA
damage, and senescence, nuclear p53 becomes active and induces apoptosis through mostly
transcriptional regulation of specific target genes such as Bax, Bid, Noxa, Puma. On the other
hand, and under certain condition, p53 can also promote apoptosis by a transcription-
independent mechanism, acting directly over the mitochondria (Haupt et al., 2003).
Localization of p53 to the mitochondria occurs in response to apoptotic signal and precedes
cytochrome-c release and pro-caspase-3 activation; thus, p53 promotes outer mitochondrial
membrane permeabilization by forming a complex with the protective Bcl-xL and BCL-2
proteins. Notably, p53 can also activate the extrinsic pathway through the induction of genes
encoding transmembrane protein such as Fas and DR5 (a receptor for TNF-related apoptosis
inducing ligand, TRAIL) ((Luedde et al., 2014) and references herein). In contrast, when
damage is less severe, p53 induces cell cycle arrest through the interaction with p21, allowing
for cellular repair. Hence, p53 functions as the “guardian of the genome,” preventing
malignant transformation. For example, hepatocytes, which escape from this control
mechanism by acquiring p53 mutations, commonly render HCC (Haupt et al., 2003).
Finally, as mentioned above, although the immune system typically triggers the extrinsic
cell death pathway, CTL and NK cells may also induce apoptosis through a process that
involves perforin and granzyme B. While perforin makes holes in the plasma membrane of
the target cell, granzyme B uses them to penetrate into the cytoplasm of the target cell and
directly cleaves intracellular proteins such as pro-caspases, particularly pro-caspase-3 which
is necessary for apoptosis in this pathway. Moreover, granzyme B can induce many of the
features of apoptosis, including DNA fragmentation (Trapani and Smyth, 2002) (Figure 3).

Apoptosis Regulation
Due to its lethality, the system is subject to numerous controls exerted at different levels,
in particular at the cell death receptor level and at the effector phase of cell death. Concerning
the first, cellular FLICE-like inhibitory protein (cFLIP) is an important regulator of the
extrinsic pathway of apoptosis at the DISC level. The cFLIP shares sequence homology with
pro-caspase-8, and therefore, binds to FADD interfering with binding and activation of pro-
caspase-8 (Safa et al., 2008). On the other hand, decoy receptors are a non-signalling subset
of the TNFR superfamily that attenuates death receptor function. Structurally, they are not
able to form signaling complexes and initiate the signaling cascade because they do not
possess the essential death domain, but are capable of binding the same ligands as normal
DR, thereby attenuating or inhibiting their function by competition (Ashkenazi and Dixit,
1999). TNF and Fas ligand-induced cell death share initial components of signal transduction.
However, while the outcome of Fas activation is solely cell death induction, TNF receptor
activation affects multiple cellular responses that also include survival, inflammation, and
proliferation. Transcription factor nuclear factor kB (NF-kB) represents a key cytoprotective
pathway that up-regulates anti-apoptotic genes including anti-apoptotic Bcl-2 family
members such as Bcl-xl, IAP (Inhibitor of Apoptosis Protein) family members and c-FLIP; as
well as it blocks prolonged activation of JNK, another key pathway through which TNF
induces cell death. Activation of NF-κB is mediated by TRAF-2, RIP-1, and other signaling
molecules that lead to activation of IκB kinase and subsequent activation of NF-κB target
genes (Bantel and Schulze-Osthoff, 2012) (Figure 5).
Related to the regulation of the effector phase of cell death, IAPs can bind the active sites
of caspases in the cytoplasm, and either promote the degradation of active caspases or keep
them away from their substrates. To date eight IAPs have been identified, namely, NAIP
(BIRC1), c-IAP1 (BIRC2), c-IAP2 (BIRC3), X-linked IAP (XIAP, BIRC4), Survivin
(BIRC5), Apollon (BRUCE, BIRC6), Livin/MLIAP (BIRC7) and IAP-like protein 2 (BIRC8)
(Wong, 2011; Vucic and Fairbrother, 2007; de Almagro and Vucic, 2012; Kocab and
Duckett, 2015; Budhidarmo and Day, 2015).
Finally, it is important to note that there are other forms of regulation that were described
under specific conditions and in different diseases which involving alternative splicing of
mRNA encoding death receptors, receptor protease digestion, regulation of expression,
receptor internalization, mRNA degradation by miRNA, protein degradation by
ubiquitination and proteasomes digestion. Consequently, like a double-edge sword every
regulatory step along the apoptotic pathway may also be an interesting target of treatment in
diseases where apoptosis is altered (Wong, 2011).
Necrosis
Necrosis is generally considered an accidental and uncontrolled type of cell death, which
lacks genetic control of specific signaling pathways. Necrotic cell death may arise as a
consequence of acute perturbations such as physical injury, physiochemical stress, infection
or ischemia. However, recent studies have suggested the existence of signaling regulation in
the necrosis process. Necrosis is mediated by the opening of the mitochondrial membrane
permeability transition (MPT) pore, which initiates the collapse of the membrane potential

and cessation of ATP formation. As a result, mitochondrial swelling leads to the rupture of
the outer mitochondrial membrane and the release of intermembrane proteins. Other
prominent features preceding necrotic death include massive energy depletion, formation of
reactive oxygen species (ROS) and nitric oxide, activation of non-apoptotic proteases (e.g.,
calpains and cathepsins) and loss of osmotic regulation. Moreover, there are lysosomal
changes (ROS production by Fenton reactions, lysosomal membrane permeabilization),
lipid degradation (following the activation of phospholipases, lipoxygenases and
sphingomyelinases), nuclear changes (hyperactivation of PARP-1 and concomitant hydrolysis
of NAD+) and subsequent nuclear DNA fragmentation (Kroemer et al., 2009). Furthermore,
during necrosis a strong increase of intracellular calcium is observed. The elevated calcium
levels in the cytosol trigger mitochondrial calcium overload, leading to the depolarization of
the inner mitochondrial membrane and a shut-down of ATP production. While depletion of
ATP impedes the function of membrane channels, increased calcium activates calcium
dependent proteases, such as calpains. Calcium fluxes, ATP depletion and oxidative stress
involve complex and interactive feedback loops, which self-amplify and potentiate each other
leading to an exaggerated cell death. The relative amount of ATP might be an important
factor that determines whether cells die by apoptosis or necrosis (Bantel and Schulze-Osthoff,
2012; Ferrari et al., 1998; Agarwal et al., 2011; Kroemer et al., 2009). These changes in
mitochondrial function lead to the morphological characteristics of necrosis: rapid swelling of
the cell and organelles, membrane blebbing and cell rupture (Luedde et al., 2014). Following
cell rupture, the constituents of the cell are dumped into the surrounding extracellular space
where a pro-inflammatory response is initiated, mainly due to the release of damage-
associated molecular patterns (DAMPs), rendering necrosis an “immunogenic" form of cell
death (Fadok et al., 2001).
Necroptosis
Necroptosis is described as a programmed form of necrosis which combines aspects of

apoptosis and necrosis and can function as a back-up for apoptotic cell death when caspases
are inhibited, e.g., by viruses expressing anti-apoptotic genes (Vanden Berghe et al., 2014)
(Figure 5). Necroptosis is triggered in a similar fashion as the apoptotic extrinsic pathway, by
ligands binding to death receptors, as well as by a variety of extracellular and intracellular
stimuli that induce the expression and/or activation of ligands of death receptors (Holler et al.,
2000; Aggarwal, 2003). As previously mentioned, the activation of death receptors by their
ligands recruit and activate caspase-8 through death domain and death effector domain-
containing adaptors and trigger apoptosis in the absence of the NF-κB survival pathway
(Thorburn, 2004). Under conditions where caspases fail to be activated, cells undergo
necroptosis as an alternative means of cell death. TNFR1, FAS (CD95), and DR4/5 (TRAIL-
R1/2) have been reported to mediate necroptosis in the presence of caspase inhibitor
zVAD.fmk (zVAD) (Holler et al., 2000; Degterev et al., 2005). The decision whether death
receptor activation induces apoptosis or necroptosis depends on 2 kinases: receptor
interacting protein (RIP) 1 and RIP3 which are essential cell stress sensors (Saeed and Jun,
2014; Zhou and Yuan, 2014).

RIP1 is thought be a crucial kinase that takes the decision between cell survival and cell
death (Festjens et al., 2007). RIP1 has three domains; a serine/threonine kinase domain
essential for necroptosis, an intermediate domain that contains a homotypic interaction motif
for NF-κB and a death domain for apoptosis activation (Holler et al., 2000). RIP1
ubiquitination promotes cell survival pathways while de-ubiquitination promotes kinase
dependent cell death pathways (Wu et al., 2012). As RIP1 has multiple domains, its activation
can result in multiple outcomes such as activation of NF-κB and MAPKs, apoptosis or
necrosis; however, only the kinase activity of RIP1 was reported to be essential for
necroptosis execution but not for other pathways ((Cho et al., 2009; Saeed and Jun, 2014)
and references herein).
In a simplified model, the trimerization of TNFR1 triggered by the interaction with TNF
initiates the assembly of a transient molecular complex associated with the intracellular
domain of TNFR1, named complex I, which consists of TRADD, TRAF2 and RIP1 (Figure
5). Once complex I is assembled, RIP1 is rapidly modified by multiple forms of
ubiquitination in the presence of cIAP1 and cIAP2. Ubiquitination of RIP1 functions as the
scaffold for the recruitment of NEMO and TAK1, critical mediators of the TNF-activated NF-
κB survival pathway. In the absence of apoptosis inhibitors such as cIAP1 or cFLIP, the
interaction of RIP1, FADD and caspase-8 form a complex, cytosolic complex IIa that
activates the caspase cascade and induces apoptosis. Activation of caspase-8 shifts the
balance towards apoptosis by cleaving RIP1 and RIP3, while the inhibition of caspase-8,
leads to assembly of RIP1/RIP3 complexes. So, under conditions where caspase-8 activity is
inhibited, RIP1 interacts with RIP3 and mixed lineage kinase domain-like protein (MLKL) to
form complex IIb. This complex, known as the “necrosome,” triggers necroptotic cell death
(Galluzzi et al., 2009). The kinase activity of RIP1 is essential to complex IIb and RIP1
kinase-inhibitor necrostatin-1 (Nec-1) prevents necroptosis. RIP3 and MLKL are
phosphorylated in complex IIb and translocated to the plasma membrane, where the complex
mediates cell membrane permeabilization (Wang et al., 2014). Activated MLKL may
translocate to intracellular membranes in addition to the plasma membrane, possibly leading
to the permeabilization of ER, mitochondria, and lysosomes (Zhou and Yuan, 2014; Wang et
al., 2014). Moreover, it has been suggested that MLKL increases the production of
mitochondrial ROS (Wang et al., 2014; Wang et al., 2012). Recent studies have shown that
MLKL triggers a cytotoxic influx of either calcium or sodium ions into the cell and this
requires the translocation of MLKL to the plasma membrane (Chen et al., 2014; Cai et al.,
2014). The final stages of necroptotic cell death resemble those of necrosis, where cell
constituents and DAMPs are released to the extracellular space, after cell swelling and
rupture, with a consequent inflammatory response. Necroptosis is best characterized in the
setting of TNF-induced cell death, which has high relevance for many types of liver diseases
but may also occur in other conditions, including ischemia-reperfusion injury (Luedde et al.,
2014; Chen et al., 2014). Evidence on necroptosis and the protective effect achieved by
blocking necroptosis has been extensively reported in the recent past; however, only few
studies have been conducted in liver related diseases (Vanden Berghe et al., 2014; Zhou and
Yuan, 2014; Saeed and Jun, 2014).

Figure 5. TNFR1 mediated pathways: cell survival, apoptosis and necroptosis. Upon stimulation by
tumor necrosis factor (TNF), TNF receptor 1 (TNFR1) recruits TNF receptor-associated death domain
(TRADD), which in turn attracts receptor-interacting protein kinase 1 (RIP1), TNF receptor-associated
factor 2 (TRAF2). Simultaneously, cellular inhibitor of apoptosis protein 1 (cIAP1) and cIAP2 are also
attracted and promote Lys63-linked polyubiquitylation of RIP1, which allows docking of TAK1
(transforming growth factor-β-activated kinase 1) in complex with TAB2 (TAK1 binding protein 2) or
TAB2, as well as of the IKK (inhibitor of NF-κB kinase) complex. The assembly of the IKK complex
activates the NF-κB (nuclear factor-κB) pathway and the resulting upregulation of antiapoptotic genes
lead to cell survival. Subsequently, Lys63-linked polyubiquitins is removed from RIP1, rendering
complex I unstable and allowing RIP1 to dissociate from the plasma membrane and to interact with
FAS-associated death domain (FADD) and pro-caspase-8, resulting in apoptosis. However, when
caspase-8 is inhibited the domains of RIP1 and RIP3 associate in microfilament-like complexes called
necrosomes. The auto- and transphosphorylation of RIP1 and RIP3 and the recruitment of mixed
lineage kinase domain-like (MLKL) initiate necroptosis. Dotted boxes indicate the formation of a
complex. Blue dotted boxes indicate the dissociation of a complex from TNFR and plasma membrane.
Even though the reported amount of necroptosis initiators continues to increase, it is still
not clear which ones can actually trigger necroptosis in different liver pathologies or whether
each specific liver disease has its own necroptosis initiator and down-stream signaling
molecules. Understanding the precise mechanisms involved in necroptosis, as well as
counteracting other cell death pathways in liver diseases, could provide a useful insight
towards achieving extensive therapeutic significance ((Saeed and Jun, 2014; Zhou and Yuan,
2014) and references herein).
Autophagic Cell Death
A great part of our current understanding of mammalian macroautophagy derives from

studies in the liver. The term “autophagy” was introduced by Christian de Duve, in part based
on ultrastructural changes in rat liver following glucagon injection (De Duve and Wattiaux,
1966). Subsequent morphological, biochemical, and kinetic studies on autophagy in the liver
defined the basic process of autophagosome formation, maturation, and degradation as well as

the regulation of autophagy by hormones, phosphoinositide 3-kinases, and mammalian target

of rapamycin (De Duve and Wattiaux, 1966; Yin et al., 2008).
Autophagy, or cellular self-digestion, is a cellular pathway crucial for development,
differentiation, survival, and homeostasis. Autophagy is a regulated catabolic process by
which the cell disassembles and recycles unnecessary or dysfunctional components
(Kobayashi, 2015). It is now clear that macroautophagy is important in 1) energy and nutrient
balance for basic cell functions, 2) the removal of misfolded proteins resulting from genetic
mutations or pathophysiological stimulations, and 3) the turnover of major subcellular
organelles such as mitochondria, endoplasmic reticulum, and peroxisomes under both normal
and pathophysiological conditions. So, its implication in human diseases has been highlighted
during the last decade. Thus, the disturbance of the autophagic function in the liver could
have some major impact on liver physiology and liver disease (Yin et al., 2008). Recent data
showed that autophagy is involved in major fields of hepatology. In liver ischemia-
reperfusion injury, autophagy mainly acts as a pro-survival mechanism, allowing the cell to
cope with nutrient starvation and anoxia; during hepatitis B or C infection, autophagy is also
increased but subverted by the virus to fulfil its own benefit; finally in HCC, the level of
autophagy is decreased in comparison with the healthy liver (Yin et al., 2008; Rautou et al.,
2010).
Molecular Machinery of Autophagy
Autophagy (Greek for ‘‘self-eating”) is a general term for processes by which

cytoplasmic materials, including organelles, fuse to lysosomes for degradation. Two major
degradation systems exist in eukaryotic cells: the proteasome and the lysosome. These differ
in their functional significance and in the type of substrates they take in for degradation. The
proteasome is a multi-subunit enzyme complex that degrades unneeded or damaged proteins
by proteolysis (Adams, 2003). On the other hand, the lysosome system degrades either
extracellular material in an endocytosis-mediated fashion (heterophagy), or intracellular
components, such as deteriorated organelles, through autophagy. Commonly, three forms of
autophagic processes have been described: 1) macroautophagy, 2) microautophagy and 3)
chaperone-mediated autophagy, which differ with respect to their physiological functions and
mode of cargo delivery to the lysosome (Kobayashi, 2015) (Figure 6).
In macroautophagy (hereafter referred to as autophagy), an isolation membrane or
phagophore elongates and encloses a portion of cytoplasm, which results in the formation of a
double membrane structure called autophagosome, which subsequently fuses with the
lysosome (Rautou et al., 2010). In mammals, autophagy can be activated by various stimuli,
some of which are physiological and some pathological. The best known signaling pathways
regulating this process are those related to phosphatidylinositol 3 Kinase (PI3K) and
mammalian target of rapamycin (mTOR). Although other stimuli are thought to activate
autophagy, their mechanism of action is still not known. Recent data show that the outer
membrane of mitochondria participates in autophagosome biogenesis (Hailey et al., 2010).
Then, the outer membrane of the autophagosome fuses with a lysosome to form an
autolysosome, leading to the degradation of the enclosed material together with the inner
autophagosomal membrane. Amino acids and other small molecules that are generated by
autophagic degradation are delivered back to the cytoplasm for recycling and/or energy

production. Autophagy occurs at low basal levels in virtually all cells, mainly as a
homeostatic process, particularly in protein and organelle turnover. It is rapidly up-regulated
through the inhibition of mTOR when cells need to generate intracellular nutrients and
energy, for example, during starvation, growth factor withdrawal, or high bioenergetic
demanding situations (Mizushima et al., 2008; Mehrpour et al., 2010; Rautou et al., 2010).
Subsequently, prolonged starvation reactivates mTOR signaling which both attenuates
autophagy and generates proto-lysosomal tubules and vesicles that extrude from
autolysosomes and ultimately mature into functional lysosomes, thereby restoring the full
complement of lysosomes in the cell (Rautou et al., 2010; Yu et al., 2010). The execution of
autophagy involves a set of evolutionarily conserved gene products known as the Atg proteins
that are required for the formation of the isolation membrane and the autophagosome.
Currently, 31 autophagy-related genes (ATGs) have been identified since the first gene, Atg1,
was discovered through genetic screening in yeast (Yin et al., 2008; Klionsky, 2007).
Figure 6. Type of autophagy.
In microautophagy the lysosome can directly take in cytosolic components including

cytosol, organelles, and even a piece of nuclear material through membrane invagination.
This leads to the formation of single-membrane intralumenal vesicles that contain the
engulfed material. The content is then processed by lysosomal hydrolases. In chaperone-
mediated autophagy, the proteic substrates to be degraded contain a KFERQ motif. They are
associated with Hsc70 and its co-chaperones including Hip, Hop, Bag-1, Hsp40, and Hsp90.
The complex binds to the multiunit LAMP-2a on the lysosomal membrane. The substrate
protein, but not the chaperones, is transported into the lysosome through the putative
translocon formed by the LAMP-2a transmembrane domains. An intralysosomal hsc70 (lyso-
hsc70) is also required for substrate translocation. The substrate protein is then degraded by
lysosomal proteases. Chaperone-mediated autophagy differs from the other 2 types of

autophagy in the lack of vesicle formation, the type of substrates, and the absence of
organelle degradation ((Yin et al., 2008) and references herein).
There are at least three steps in the formation of autophagosomes: initiation, nucleation,
and elongation/closure (Figure 7). Autophagy is initiated by the uncoordinated 51-like kinase
1 (ULK1, homologous in mammals of Atg1) complex. This complex is formed by ULK1
Ser/Thr protein kinase, Atg13, and FIP200 (200-kDa focal adhesion kinase family-interacting
protein). Subsequently, the activation of class III PI3K (Vps34) is necessary to generate
phosphatidylinositol 3-phosphate, required for vesicle nucleation. This activation depends on
the formation of a multiprotein complex that includes Beclin-1, Vps15, Atg14L (Atg14-like
protein), and Ambra1. Since Beclin-1 constitutively interacts with its inhibitors, Bcl-2 or its
close homolog Bcl-xL, the induction of autophagy requires the dissociation of Beclin-1 from
Bcl-2 or Bcl-xL (He and Levine, 2010). Thereafter during the vesicle elongation step, the
membrane is formed, elongated and closed on itself to form the autophagosome. Two
conjugation systems are successively involved in order to facilitate the elongation step. The
first one involves the covalent conjugation of Atg12 to Atg5, with the aid of Atg7 and Atg10.
This conjugate is organized into a bigger structure by associating with Atg16 to form the
Atg16–Atg5–Atg12 complex. Thus, this complex appears to provide the necessary
platform for autophagy activation. The second one involves the conjugation of
phosphatidylethanolamine (PE) to LC3 (microtubule-associated protein light chain 3) by the
sequential action of Atg4, Atg7, and Atg3. This lipid conjugation leads to the conversion of
the soluble form of LC3 (named LC3-I) to the autophagic vesicle associated form (LC3-II),
allowing for the closure of the autophagic vacuole. The assembly and maturation of the
autophagosome is regulated by the action of ATG proteins, beclin-1 and LC3 ((Yin et al.,
2008; Rautou et al., 2010; Rockenfeller et al., 2015) and references herein). After maturation,
the autophagosome fuses its outer membrane with the lysosome to promote the final
degradation of its content. Under normal physiological conditions autophagy occurs at low
basal levels, it is an essential process during starvation, in which context it contributes to
maintain energy homeostasis and cell survival (Wang, 2015). However, an excess in
autophagy can lead to a form of cell death known as “autophagic cell death” (Choi et al.,
2013). Since the catabolized material inside the autophagosome is not released into the
extracellular space, this type of process does not elicit an immflamatory response.
Autophagy is considered a predominantly cytoprotective pathway that protects from
alcoholic liver disease (ALD) (Ding et al., 2010), TNF-induced liver injury (Amir et al.,
2013), acetaminophen-induced liver injury (Ni et al., 2012), ischemia-reperfusion injury
(Czaja et al., 2013), and a high fat diet-induced lipid accumulation (Singh et al., 2009; Luedde
et al., 2014). However, dual roles of autophagy are obviously observed in cancer development
and liver fibrosis. Autophagy can serve either to promote cell/tumor survival at certain stages,
or to stimulate its elimination at other stages. Autophagy activation is beneficial for
hepatocyte proliferation and liver repair, but the up-regulation of autophagy in hepatic stellate
cells favors their activation and consequently initiates fibrogenesis (Wang, 2015).

Figure 7. Basic molecular machinery and signaling pathway in autophagy. A) autophagy process b)
Autophagosome formation steps (initiation, nucleation, maturation and elongation/closure).
Cross-Talk between Autophagy and Apoptosis
It is considered that there are significant cross-talks between autophagy and apoptosis.
The evidence that suggests that autophagy may regulate, at least to some extent, apoptosis
includes: (i) mitophagy; (ii) morphological similarity of final products; (iii) interaction
between Beclin-1 and anti-apoptotic Bcl-2 family members; (iv) p53 can co-regulate
autophagy and apoptosis. Briefly, since autophagy contributes to bulk degradation of
cytoplasm and mitochondrion, it influences mitochondrial recycle and can thus modulate
hepatic mitochondrial apoptosis pathway (Wang, 2015). There is also a similarity between
autophagosomes (autophagic cell death) and apoptotic bodies (general apoptosis) in
morphology. Moreover, Bcl-2 proteins not only counteract the activity of pro-apoptotic
proteins to down-regulate apoptosis, but interact with Beclin-1 to impede autophagy as well.
Thus, autophagy and apoptosis can be coordinately regulated by the Bcl-2 family proteins.
Furthermore, Beclin-1 is cleaved and inactivated by caspases during activation of apoptosis;
thereby apoptosis-effector molecules may suppress autophagy. Finally, p53 modulates the
expression of apoptosis-related genes [i.e., Bcl-2 family protein (Bax, Bid, Noxa, Puma) and
Apaf1], autophagy related pathways (i.e., AMPK/mTOR and Bmf/Beclin-1) and targets the
expression of DRAM (damage-regulated autophagy modulator, a gene encoding a lysosomal
protein that induces macroautophagy), which can stimulate both autophagy and apoptosis
(Ouyang et al., 2012). Only partial chromatin condensation is found in autophagy mediated
cell death, but DNA fragmentation is identified in apoptotic cell death. Since a clear

distinction between autophagy and apoptosis is still not completely defined, it is plausible that
both processes may occur simultaneously in the same cell. Under some circumstances,
apoptosis and autophagy could exert synergetic effects, whereas in other situations autophagy
can be triggered only when apoptosis is suppressed. What remains to be elucidated is if both
mechanisms can act as independent parallel pathways or one may influence, or serve as a
back-up, for each other ((Wang, 2015; Ouyang et al., 2012) and references herein).
CELL DEATH IN CLINICAL LIVER DISEASE
Ischemia-Reperfusion Injury
Highly aerobic tissues are sensitive to suffer damage after the loss of blood supply
(ischemia), and the liver is no exception. Hepatic hemodynamic perturbations can lead to no-
flow ischemia, in which blood supply is totally blocked, or to low-flow hypoxia, in which
blood flow is inadequate to meet oxygen demand (Malhi et al., 2006). In no-flow ischemia the
entire liver becomes anoxic, whereas in low-flow hypoxia pericentral (centrolobular) regions,
but not periportal regions, of individual liver lobules become anoxic and subject to injury
(Lemasters et al., 1981; Lemasters et al., 1983; Malhi et al., 2006). Ischemia-reperfusion
occurs during hepatic resection, liver transplantation, and hypotensive shock followed by
recovery (Hubsche et al., 2012). Ischemia-reperfusion in liver has two phases. The first phase
reflects the immediate cellular events after ischemia and subsequent reperfusion, whereas the
resultant second phase involves the activation of the innate immune system. More
specifically, it involves the activation of Kupffer cells and infiltration by circulating
neutrophils and lymphocytes into the post-ischemic liver (Malhi et al., 2006; Jaeschke, 2003).
Necrosis is the predominant mode of cellular death in ischemia states since there is extreme
ATP depletion and oxidative stress with formation of reactive oxygen species; although
apoptosis also may contribute to cell death in the latter pathology (Hubsche et al., 2012)
(Table 1). The depletion of intracellular ATP during the ischemic period is follow by the
increase in intracellular Ca1+, owing to decreased active extrusion of Ca1+ from the cell by
Ca1+-ATPase. Moreover, intracellular Ca1+ levels are also increased by the opening of
voltage-dependent Ca1+ channels due to membrane depolarization caused by decreased
activity of the Na+-K+ ATPase (the latter of which consumes approximately 25% of cellular
ATP under normal conditions) (Hubsche et al., 2012). The increase in intracellular Ca1+
destroys the cytoskeleton, and plays a critical role in the opening of the mitochondrial
permeability transition pore, thereby stimulating the mitochondrial pathway of apoptotic cell
death (Hubsche et al., 2012). The second key event in ischemia-reperfusion injury is the
generation of oxygen radicals following reperfusion (Hubsche et al., 2012). During the
ischemic interval, cytosolic xanthine dehydrogenase is converted to xanthine oxidase.
Meanwhile, the degradation of adenine nucleotides leads to the accumulation of hypoxanthine
in the ischemic organ. Upon reperfusion and introduction of oxygen, xanthine oxidase
generates massive amounts of superoxide anion. The resultant oxidative damage disrupts
cytoplasmic and nuclear proteins, organelle membranes, and DNA. The cytoskeleton also
undergoes proteolysis and mitochondrial oxygen reduction is also impaired. The reactive

oxygen species also act as intracellular second messengers, inducing cascade reactions
through the transcriptional factor NF-κB. It is important to mention that the liver has the
greatest amount of macrophages, in relation to any other organ in the body, and that these
macrophages can also secrete oxygen radicals as well as tissue-toxic cytokines such as TNFα
and IL-1; the recruitment of inflammatory cells further exacerbates tissue injury. Nitric oxide
(NO) potentiates hepatocellular necrosis by a NO-induced decrease in intracellular ATP. As
NO is generated by endothelial cells, which become activated during tissue injury and
inflammation, the sinusoidal endothelium can be yet another contributor to hepatic injury. In
fact, necrosis frequently exhibits a zonal distribution. The necrotic focus of hepatocytes
immediately around the terminal hepatic vein becomes usually more apparent and is a
characteristic sign of ischemic injury related to a number of drugs and toxic reactions.
Necrosis of entire lobules (sub-massive necrosis) or of most of the liver (massive necrosis) is
usually accompanied by hepatic failure (Hubsche et al., 2012).
In relation to authophagy, it has been observed that anoxia decreases autophagy protein
levels. Indeed, anoxia induces mitochondrial dysfunction but, due to the decrease in
autophagy proteins, autophagy fails to remove dysfunctional mitochondria. As a
consequence, the mitochondria ladens with reactive oxygen species and calcium and
undergoes mitochondrial permeability transition, which in turn leads to uncoupling of
oxidative phosphorylation, energetic failure, ATP depletion, and ultimately cell death (Rautou
et al., 2010). However, more studies should be conducted to clarify the role of autophagy in
this pathology since other authors observed an increase in liver cell autophagy, suggesting
that autophagy enhancement could allow for decreasing liver cell death and, in turn
promoting cell survival (Domart et al., 2009; Rautou et al., 2010).
Drug-Induced Liver Injury (DILI)
DILI is the major cause of acute liver failure in western countries and an important cause
of acute hepatitis or cholestasis in clinical practice. The most common cause of DILI is
acetaminophen toxicity. Acetaminophen (paracetamol, N-acetyl-p-aminophenol; APAP)
overdose represents one of the most common causes of acute liver failure in developed
countries (Larson et al., 2005).
The pathogenesis of DILI includes cell stress, mitochondrial impairment, and specific
immune reactions. The liver, as the central place for detoxification, is constantly exposed to
cell stress which usually breaks the balance between inflammatory cytokines that promote
(e.g., IL-12) or prevent (e.g., IL-4, IL-10, IL-13, MCP-1) injury. Consequently, liver cells
become more susceptible to lethal effects of TNFα, FasL, and IFNγ (Wang, 2014). APAP-
induced hepatotoxicity is due to the formation of the toxic metabolite N-acetyl-p-
benzoquinone imine by the cytochrome P450 system. This reaction causes glutathione
depletion, oxidative stress and alterations in calcium homeostasis, which finally result in
MPT, loss of mitochondrial membrane potential, and ATP depletion (Hinson et al., 2010).
Although necrosis has been thought to be the predominant mode of cell death in APAP-
induced liver injury, conflicting in vitro and animal data have emerged suggesting a potential
role of apoptosis through the mitochondrial pathway (Schulze-Osthoff and Bantel, 2011; Hu
and Colletti, 2010; El-Hassan et al., 2003; Bantel and Schulze-Osthoff, 2012). When APAP
causes profound ATP depletion, ATP depletion-dependent necrotic cell killing ensues.

However, when fructose and glycine are used to prevent ATP depletion, necrosis is blocked;
on the contrary, caspase-dependent apoptosis increases. Indeed, several studies demonstrated
that APAP induces mitochondrial dysfunction with ATP depletion, which even interrupts
initial Fas-induced mitochondrial signaling pathways (Lawson et al., 1999; Knight and
Jaeschke, 2002).
Table 1.
LIVER DISEASE CELL DEATH

↑↑ Necrosis.
Ischaemia-
↑ Apoptosis through the mitochondrial pathway.
reperfusion injury
? Autophagy.
↑↑ Apoptosis.
↑ Necrosis (in cases of profound ATP depletion).
Drug-induced
Necroptosis.
liver injury (DILI)
? Autophagy (experimental activation of autophagy protect from APAP-
induce hepatotoxicity).
Cholestatic Liver ↑↑↑ Apoptosis mediated by Fas and TRAIL.
Injury ↑ Necrosis.
Viral Hepatitis ↑↑↑ Apoptosis.
(HCV and HBV) ↑ Autophagy.
↑ Apoptosis through the mitochondrial pathway (lipid accumulation
Nonalcoholic
induces inflammatory pathway).
steatohepatitis
? Necroptosis (high expression of RIP3).
(NASH)
↓ Autophagy through the insulin amino acid-mTOR signaling pathway.
↑↑ Apoptosis through the mitochondrial pathway (over-production of
Alcoholic liver
ROS).
disease
↓ Autophagy through a reduction in AMPk-mediated mTOR pathway.
Alpha-1- ↑ Autophagy (probably to degrade protein aggregates and damage
antitrypsin organelles).
deficiency
↓ Apoptosis (down-regulation of pro-apoptotic Bax and Bcl-xs and up-
Hepatocellular
regulation of anti-apoptotic proteins such as Bcl-xl).
carcinoma (HCC)
↓ Autophagy?
Conflicting data also exists about the role of TNFα in APAP induced liver injury.
Increased expression of TNFα, both in the liver and in circulation, has been observed after
APAP poisoning. However, the role of TNFα in APAP-induced necrosis remains
controversial, as TNF-α inhibitors exerted either protection against or no effect over cell
death (Blazka et al., 1996; Blazka et al., 1995; Simpson et al., 2000). Although, apoptotic
alterations can occur, profound energy depletion and mitochondrial failure presumably diverts
cell death to necrosis as the principal mode of APAP-induced liver toxicity. However, APAP
toxicity is one more example of necroptosis in which necrosis and apoptosis represent
alternate outcomes of the same mitochondrial death pathway.
Autophagy represents another process that might influence the outcome of APAP-
induced liver toxicity. Interestingly, Ni HM et al. using a series of morphological and
biochemical autophagic assays, found that APAP induced autophagy both in primary cultured
hepatocytes and in in vivo mice livers. They also found that treatment with N-acetylcysteine,
against APAP, might suppress mTOR in hepatocytes. Furthermore, pharmacological

inhibition of autophagy by 3-methyladenine or chloroquine further exacerbated APAP-

induced hepatotoxicity. In contrast, induction of autophagy by rapamycin inhibited APAP-
induced hepatotoxicity. Altogether, these results suggest that autophagy plays an important
role against APAP in the liver (Ni et al., 2012; Bantel and Schulze-Osthoff, 2012) (Table 1).
Cholestatic Liver Injury
In cholestatic liver disease, bile acids accumulate and cause damage by triggering
different cell death pathways. Bile acids are normally secreted rapidly from hepatocytes by
transporters located in the canalicular membrane. In the context of cholestasis this secretion is
impaired and leads to elevated concentrations of toxic bile acids within hepatocytes. In cell
culture, toxic bile salts cause hepatocyte apoptosis in a Fas and TRAIL-dependent manner
(Table 1) (Faubion et al., 1999; Higuchi et al., 2003). Fas activation in cholestasis occurs by
both FasL ligation-dependent and ligand-independent mechanisms. In the absence of FasL,
bile acids enhance cellular trafficking of Fas receptor, leading to an increase in plasma
membrane density of the receptor (Sodeman et al., 2000). As a consequence, spontaneous
receptor oligomerization occurs with the subsequent recruitment of FADD and activation of
caspase-8. These signals then converge to produce mitochondrial permeabilization, release of
cytochrome-c and activation of down-stream caspases (Canbay et al., 2003). Moreover, acute
cholestasis is associated with oxidative stress as a consequence of bile acid-induced activation
of NADPH oxidase via signaling through sphingomyelinase, ceramide and protein kinase zeta
(Reinehr et al., 2005). Bile acids also activate the mitochondrial apoptotic machinery by
induction and translocation of Bax to the mitochondria (Miyoshi et al., 1999). It has been
reported, that caspase inhibition protects against cholestatic hepatocyte injury and decreases
stellate cell activation and fibrosis (Canbay et al., 2004).
Despite evidence of Fas and TRAIL-dependent apoptotic signaling, recent in vitro studies
have suggested that necrosis is the predominant form of cholestatic cell death, instead of
apoptosis, since severe mitochondrial dysfunction was observed. This mitochondrial
dysfunction may also be a consequence of Fas signaling which will finally result in ATP
depletion. Such ATP depletion blocks the activation of down-stream caspases while
simultaneously promotes necrosis. However, experimental in vitro studies or in vivo bile duct
ligation procedure do not mimic the exact processes that occur in human cholestatic disease.
Bile duct ligation produces a sudden and complete blockage of bile flow, whereas human
cholestatic disease is typically slower in its onset. In the slow onset context, death receptor-
induced mitochondrial dysfunction is likely less severe and ATP is at least partially
preserved; hence, necrosis would be prevented and caspase activation may proceed to an
apoptotic form of cell killing (Malhi et al., 2006) (Table 1).
Viral Hepatitis
Viruses can only replicate inside the host cells but these, in turn have developed several
defense mechanisms to limit viral infection, including cell-mediated immune response,
inflammation and programmed cell death. In order to accomplish their replication and spread-
out, viruses have as well developed several strategies to inhibit or delay cell death. On the

contrary, some viruses can induce apoptosis to facilitate viral spreading and/or to kill
uninfected cells of the immune system.
Hepatotropic viruses such as hepatitis A virus, hepatitis E virus and cytomegalovirus,
often cause acute liver injury, whereas HCV and HBV lead to chronic liver injury. While
acute liver injury involves much of necrosis, chronic liver injury promoted by HCV or HBV
infection generally exhibits abundant apoptosis (Wang, 2014).
HCV pathogenesis is a very complex phenomenon that still requires further investigation
in order to determine the actual role of each contributing factors. Increasing evidence suggests
that the damage of liver cells in chronic HCV infection is mediated, at least in part, by
apoptosis induction (Table 1) (Bantel and Schulze-Osthoff, 2003; Que and Gores, 1996;
Bantel et al., 2004; Kerr et al., 1979). However, the relative contribution of apoptosis or
necrosis as well as the functional role of caspase in liver damage is largely unknown.
Apoptosis of infected cells may be viewed as a cellular defense mechanism to prevent viral
propagation; however, to circumvent this defense and diminish the apoptotic response, HCV
has developed mechanisms to disrupt the normal regulation of death within the infected cell.
Among cell death receptors mediating apoptosis, the Fas/FasL system has already been
described in a previous section. With regards to this system, it was postulated that the
interaction between Fas receptor in the hepatocyte and FasL in the lymphocyte leads to
apoptosis of the infected hepatocyte; therefore, cell injury could result from deregulation of
this mechanism (Kanto and Hayashi, 2006; Macias et al., 2005). Moreover, cytotoxic T
lymphocytes also secrete FasL, which can initiate apoptosis both on infected and uninfected
cells (Mengshol et al., 2007; Fischer et al., 2007). In this way, over-expression of Fas in HCV
infected hepatocytes from adult patients, as well as induction of FasL expression in cytotoxic
T lymphocyte in the inflammatory infiltrate, were reported to correlate with the severity of
liver damage (Bantel and Schulze-Osthoff, 2003; Hiramatsu et al., 1994). Additionally,
increased FasL expression was also reported in infected hepatocytes. Accordingly, Fas-FasL
interaction could also facilitate viral persistence by inducing apoptosis of activated T
lymphocytes (Iken et al., 2006). Furthermore, some studies including adult patients also
indicated that hepatocyte apoptosis plays a significant role in the pathogenesis of HCV
infection, which is clinically recognized as liver inflammation and fibrosis (Albertoni et al.,
2012; Bantel and Schulze-Osthoff, 2002; Calabrese et al., 2000; Fischer et al., 2007;
Kountouras et al., 2003; Rust and Gores, 2000; Valva et al., 2014). Finally, high levels of
caspases activation and its association with liver damage, both in serum and liver samples
from HCV pediatric an adult patients were also described (Bantel et al., 2004; Bantel et al.,
2001; Valva et al., 2010; Valva et al., 2014).
Both in vitro studies and in vivo models have demonstrated that several viral proteins
display either apoptotic or anti-apoptotic features according to the HCV model under study
(Fischer et al., 2007; Mengshol et al., 2007). Furthermore, the ability of HCV to induce
apoptosis was also reported (Deng et al., 2008; Fischer et al., 2007; Joyce et al., 2009;
Mengshol et al., 2007). Hence, a logical assumption is that HCV may directly inhibit
hepatocyte apoptosis to promote cell survival and allow the virus to proliferate. In fact, it was
postulated that apoptosis inhibition is the key step in the pathogenesis of HCC (Baskin-Bey
and Gores, 2005). However, multiple molecular interactions have been described between
HCV proteins and components of the apoptotic process that led to conflicting conclusions
(Benali-Furet et al., 2005; Berg et al., 2009; Jahan et al., 2011; Lai, 2002; Lerat et al., 2002;
Moriya et al., 1998; Moriya et al., 1997; Pazienza et al., 2009). Some studies showed that

HCV-encoded Core interacts with Fas receptor, c-FLIP, and p53 and leads to the release of
cytochrome-c from the mitochondria; events that guide towards apoptosis (Benali-Furet et al.,
2005; Berg et al., 2009). Moreover, it has been described that Core participates in steatosis,
by increasing oxidative stress and mitochondrial damage (Moriya et al., 1997; Moriya et al.,
1998; Mengshol et al., 2007; Jahan et al., 2011; Lai, 2002; Lerat et al., 2002). However,
others do not share these observations, since it was shown that the expression of Core in
transgenic mice reduces the susceptibility to apoptosis (Lai, 2002; Lerat et al., 2002).
Interestingly, results often differ in relation to which viral genotype encodes Core protein
(Jahan et al., 2011; Pazienza et al., 2009). Similar controversies exist regarding the E2 and
NS3 HCV-encoded proteins (Chiou et al., 2006; Lee et al., 2005; Prikhod'ko et al., 2004;
Tanaka et al., 2006). These disparities amongst reported results may be the cause why it is
still not possible to conclude unequivocally about the plausible regulation of HCV proteins
over apoptosis. However, using an efficient viral infection system that uses a particular clone
of HCV genotype 2a (J6/JFH1) and a permissive cell line derived from a human hepatoma
cells (Huh7.5), Deng et al. demonstrated the capability of HCV to induce apoptosis through
the mitochondrial pathway (Deng et al., 2008). This result was also observed by Joyce et al.
in an in vivo model of HCV infection in humanized SCID/Alb-uPA mice (Joyce et al., 2009).
In parallel, HBV can also induce several liver diseases, including asymptomatic
infections, acute or fulminant hepatitis, and chronic hepatitis with progression to cirrhosis and
hepatocellular carcinoma. Since apoptosis plays an important role in the progression of HBV
infection, viral proteins can trigger apoptosis through various processes. Mainly, four HBV-
encoded proteins are known to modulate the apoptotic pathways: the large surface protein
(LHBs), a truncated form of the middle surface protein [C-terminally truncated middle
hepatitis B surface protein, MHBs(t)], the Hepatitis B X protein (HBx) and the HBV splice-
generated protein (HBSP) (Assrir et al., 2010). HBV codes for three forms of the surface
(envelope) protein, known as large, middle and small surface proteins (termed LHBs, MHBs
and SHBs, respectively) (Foo et al., 2002). LHBs induce apoptosis in cultured hepatoma cells
by activating the ER stress pathway (Foo et al., 2002). MHBs(t) is a potent regulator of
TRAIL-induced apoptosis and also increases the cleavage of caspases-3 and -9 (Liang et al.,
2007). HBx is a small regulatory protein, involved in the establishment and/or maintenance of
the chronic state of the infection, which can also induce apoptosis through the inhibition of
cFLIP, resulting in hyper-activation of caspases-8 and -3 by death signals (Bouchard and
Schneider, 2004). However, different HBx-dependent effects on apoptosis have been
reported; none the less, caution should be taken since these differences are likely to be a
consequence of the experimental conditions and/or cell types used in each study (Rawat et al.,
2012). In a particularly relevant study, the effects of HBx on the apoptotic pathways were
analyzed in primary cultured rat hepatocytes. This study demonstrated that HBx could act
either in a pro- or anti-apoptotic manner depending on the status of NF-kB, since HBx could
stimulate NF-kB and inhibit apoptosis as well as it could induced apoptosis when HBx-
induced activation of NF-kB was blocked (Clippinger et al., 2009). HBSP is a splice variant
of the HBV DNA polymerase, with a conserved BH3 domain in the N-terminus. It is
expressed during viral replication and can induce apoptosis functioning as a BH3 only protein
(Table 1) (Lu et al., 2006; Favaloro et al., 2012).
Related to autophagy, as mentioned above, certain viruses such as HCV and HBV have
developed strategies to subvert or use autophagy for their own benefit. Several studies have
assessed the autophagic pathway in hepatocytes infected with HCV both in vitro and in liver

biopsies from chronic HCV patients and concluded that autophagy was inefficient. Moreover,
in vitro studies using HCV JFH1 (genotype 2a) observed that although HCV induced the
production of autophagosomes, it was not able to enhance autophagic protein degradation. In
line with these results, three other pieces of evidences suggest that HCV avoids and subverts
autophagy: 1) HCV seems to avoid its recognition by the autophagic machinery since no or
rare co-localizationof HCV proteins with autophagic vacuoles has been observed; 2) HCV
appears to prevent the maturation of the autophagosome into an autolysosome based on the
absence of late autophagic vesicles in hepatocytes from chronic HCV patients as compared to
controls, while a strong augmentation in the number of early autophagic vesicles is observed;
and 3) HCV seems to utilize functions or components of autophagy to enhance its
intracellular replication. It has been recently shown that autophagy proteins are required for
translation and/or delivery of incoming HCV RNA to the cell translation apparatus; the
autophagy proteins or autophagic vesicles might provide an initial membranous support for
the transport of the incoming RNA, prior to the accumulation of viral proteins and the
eventual establishment of virus-induced cellular modifications. (For more detail read (Rautou
et al., 2010) and reference herein).
HBV also induces autophagosomes in liver cells, as demonstrated both in vitro, in several
liver derived cell lines, and in vivo, in the liver of transgenic mouse lines harboring low and
high replication levels of HBV DNA. Importantly, this induction was also observed in the
liver of a HBV infected patient but not in a non-infected one (Sir et al., 2010). In contrast to
HCV, HBV can enhance the autophagic flux, as demonstrated by the presence of late
autophagic vacuoles; however, no significant increase in protein degradation was observed in
HBV DNA-transfected cells (Sir et al., 2010). In particular, the HBx protein, seems to play a
crucial role in HBV-induced autophagy (Sir et al., 2010; Tang et al., 2009), at least in part,
given its ability to bind to class III PI3K, a regulatory molecule that controls autophagy.
Although conflicting, HBx may also up regulate the transcription of beclin-1, a protein that
forms a complex with class III PI3K and thus sensitizes the cells to starvation-induced
autophagy (Sir et al., 2010; Tang et al., 2009). Although our knowledge at this point is still
incomplete, some reports suggested that autophagy enhances HBV replication mostly at the
step of viral DNA replication, slightly at the step of RNA transcription, but not at other levels
(Table 1) (for more detail read (Rautou et al., 2010) and reference herein).
NASH
Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver
disease both in children and adults. It encompasses a wide spectrum of conditions associated
with over accumulation of fat in the liver, ranging from simple steatosis to nonalcoholic
steatohepatitis (NASH) and cirrhosis (Angulo and Lindor, 2002; Wieckowska et al., 2006).
Simple steatosis is the most common form of NAFLD and typically follows a benign non
progressive clinical course. In contrast, NASH is a potentially serious condition, since as
many as 25% of these patients may progress to cirrhosis and experience complications of
portal hypertension, liver failure, and hepatocellular carcinoma (Adams et al., 2005).
Emerging data suggest that hepatocyte apoptosis, may also play an important role in liver
injury and disease progression in NAFLD. Even though, the pathophysiological pathways
involved in liver damage and the progression from simple steatosis to NASH remain largely

unknown. Results from the comparison amongst simple steatosis, alcoholic steatohepatitis
and NASH displayed that there is enhanced hepatocyte apoptosis in NASH vs. the others,
suggesting that additional pro-apoptotic forces or diminished anti-apoptotic defenses are
involved in NASH pathophysiology (Valva et al., 2008; FeldsteinCanbayAngulo et al., 2003).
Moreover, apoptosis degree correlated with the severity of steatohepatitis and the stage of
fibrosis. Several experimental models of NAFLD, as well as subsequent human studies
supported these initial observations (Feldstein et al., 2004; Ribeiro et al., 2004; Ramalho et
al., 2006).
NASH is considered to be induced by two consecutive steps, excess fat accumulation and
subsequent liver necro-inflammation, the so-called “two-hit hypothesis” (Day and James,
1998). Further in vivo an in vitro studies on hepatocytes steatosis are now providing
additional evidence that free fatty acids accumulation in liver cells may lead to an increase in
apoptotic cell death (Table 1) (Feldstein et al., 2003). Briefly, excess lipid accumulation
activates inflammatory pathways and induces insulin resistance. Extracellular free fatty acids
(FFA) activate toll-like receptors (TLR), causing down-stream activation of the NF-κB
pathway and in turn allowing the transcriptional expression of multiple pro-inflammatory
chemokines (e.g., macrophage chemotactic protein 1 [MCP-1]), cytokines, and adhesion
molecules (e.g., vascular cell adhesion molecule-1). In addition to TLR activation, some
intracellular lipid molecules may also stimulate JNK/NF-κB activation by forming reactive
oxygen species (ROS). In turn, ROS may arise from excessive β-oxidation of FFA,
uncoupling of oxidative phosphorylation and mitochondrial damage caused by free
cholesterol accumulation and crystallization. Alternatively, some intracellular lipids may
induce endoplasmic reticulum (ER) stress, leading to the intrinsic mitochondrial pathway of
apoptosis (Farrell et al., 2012). In contrast, the functional regulation and the participation
extent of the extrinsic (death receptor–mediated) apoptotic pathway in NASH is less clear.
Hepatocyte apoptosis has also been linked to liver fibrogenesis (Canbay et al., 2004).
Engulfment of apoptotic bodies by hepatic stellate cells stimulates the fibrogenic activity of
these cells and may be one mechanism through which hepatocyte apoptosis promotes fibrosis
(Canbay et al., 2003). Moreover, beyond the role of apoptosis in NASH pathogenesis, it has
been recently shown that human NASH livers express high levels of RIP3 (Gautheron et al.,
2014), suggesting that necroptosis might represent an emerging target for future clinical
studies in NASH.
The implication of autophagy in hepatocyte lipid metabolism has been recently
demonstrated (Singh et al., 2009). Besides cytosolic lipases, autophagy regulates intracellular
lipid stores through a process called macrolipophagy, in which portions of lipid droplets, or
even whole droplets, become trapped inside double-membrane autolipophagosome vesicles.
These lipid vesicles are then fused to lysosomes, where the lipid droplets are degraded to fatty
acids. The existence of this alternative lipid degradation pathway in hepatocytes may explain
their ability to rapidly mobilize large amounts of lipids, despite their low levels of cytosolic
lipases in comparison with adipocytes (Czaja, 2010). Under physiological conditions,
autophagy functions in the basal turnover of lipids by engulfing and degrading lipid droplets;
however, in obesity, autophagy level is decreased in hepatocytes. Several mechanisms may
account for this decline. First, obesity-induced increase in the calcium-dependent protease
calpain-2 leads to down-regulation of Atg7 and hence, defective autophagy. Second, in
obesity, the autophagy inhibitor mTOR is over activated in the liver, presumably as the result
of increased amino acid concentrations following overnutrition (Codogno and Meijer, 2010).

Third, and given that both lipolysis and macrolipophagy are inhibited by the hormone insulin
(Levine and Kroemer, 2008; Yin et al., 2008; Singh et al., 2009), hyperinsulinemia may also
contribute to down-regulating autophagy since this is inhibited by the insulin amino acid-
mTOR signaling pathway. Autophagy down-regulation impacts on other cellular functions,
such as the associated decrease in lysosomal degradation rate, which further contributes to
increase ER stress induced by nutrient overload. It is also important to mention that the
efficiency of macrolipophagy varies with the nutritional status. Together, autophagy decline
and ER stress finally lead to insulin resistance (Table 1) ((Rautou et al., 2010) and reference
herein).
Alcoholic Liver Disease
Long-term alcohol abuse leads to the development of alcoholic liver disease (ALD).
Acute and chronic alcohol consumption induces production of ROS, lowers cellular
antioxidant levels, and enhances oxidative stress in the liver tissue. During the body’s
metabolic reactions, ROS is naturally generated in small amounts; however, following
alcohol exposure, massive ROS actively reacts with and damages complex cellular molecules
such as lipids, proteins, or nucleotides. Alcohol-induced oxidative stress has a major role in
the mechanism by which alcohol causes liver injury. Many signaling molecules take part in
alcohol-induced oxidative stress. Cytochrome P4502E1 (CYP2E1), as an effective generator
of ROS, produces superoxide anion radical, hydrogen peroxide, and powerful oxidants such
as the hydroxyl radical in the presence of iron as a catalyst. Although CYP2E1 levels can be
elevated under a variety of physiological and pathophysiological conditions, alcohol is one of
the strongest inducers of CYP2E1. Consequently, alcohol abuse stimulates an over-
production of ROS, which leads to hepatic apoptosis via mechanism of oxidative stress
mitochondrial dysfunction, decreased methylation capacity, endoplasmic reticulum stress,
impaired vesicular trafficking and altered proteasome function. Liver injury, following
alcohol abuse, involves both parenchymal and non-parenchymal cells. The occurrence of
apoptotic bodies generated from hepatocytes activates Kupffer cells resulting in an enhanced
production of inflammatory cytokines. Moreover, the expression of endotoxin receptors and
intracellular signaling molecules are also altered following alcohol exposure. This
phenomenon causes both tolerance and sensitization of KCs to endotoxins, which enhances
progression of alcoholic liver injury by promoting the production of TNFα and ROS. These
inflammatory mediators contribute to hepatocyte dysfunction, apoptosis, necrosis, and
fibrogenesis. Inflammatory and innate immune responses in KC, due to elevated
lipopolysaccharides, increased oxidative stress, and profibrogenic factors such as
acetaldehyde or lipid peroxidation products, all contribute to the activation of hepatic stellate
cells. These cells then differentiate into collagen-producing cells. This triggers the wound-
healing response to recurrent liver injury and finally leads to fibrogenesis ((Wang, 2014) and
references herein). (Table 1)
Several arguments suggest that alcohol consumption suppresses liver cell autophagy: (a)
rats chronically fed with ethanol have a reduced number of autophagic vacuoles in liver cells,
as determined morphometrically; (b) chronic ethanol consumption slows down the catabolism
of long-lived proteins in the rat liver; (c) alcohol abuse is associated with protein
accumulation in the liver, as demonstrated in ethanol fed rats; (d) hepatocytes from patients

with alcoholic steatohepatitis contain protein aggregates called Mallory-Denk bodies; (e) loss
of autophagy in transgenic mice induces the formation of protein aggregates in hepatocytes,
resembling Mallory-Denk bodies; (f) ethanol also suppresses autophagy in cortical
neuroepithelial progenitors, suggesting that this effect is not restricted solely to liver cells (for
more detail read (Rautou et al., 2010) and reference herein). The mechanisms responsible for
the decrease in autophagy are not clear; however, it is well known that ethanol consumption
significantly reduces adenosine monophosphate-activated protein kinase (AMPk) activity in
the liver and that AMPk suppression reduces autophagy via the mTOR pathway (Table 1).
Moreover, ethanol is known to alter vesicle transport in hepatocytes and autophagy requires
the action of cytoskeletal elements, including microtubules and microfilaments necessary for
autophagosome formation ((Rautou et al., 2010) and reference herein). This decline in the
autophagic pathway must contribute to the pathological consequences of alcohol ingestion
since, as mentioned above ethanol causes mitochondrial damage. It is crucial that such
damaged mitochondria be removed from the cell by engulfment in autophagic vacuoles, since
the absence of mitochondrial autophagy leads to uncoupling of oxidative phosphorylation and
cell death (Rautou et al., 2010; Donohue, 2009).
Alpha-1-Antitrypsin Deficiency
Alpha-1-antitrypsin (AT), the archetype of the Serpin supergene family, is the principal
blood-borne inhibitor of destructive neutrophil proteases. The normal AT protein is secreted
from hepatocytes into the bloodstream, where it inhibits neutrophil proteases. However,
mutations in the AT gene can result in misfolding of the molecule, which leads to its
degradation and AT deficiency. The classical form of AT deficiency is caused by the z
mutation (α1-antitrypsin z mutant, ATZ), which confers an abnormal conformation on the
nascent polypeptide. This results in an accumulation of the mutant protein within the
endoplasmic reticulum of the hepatocyte rather than the appropriate and highly efficient
secretion of the wild-type protein. Therefore, homozygous ATZ mutation causes the early
onset of pulmonary emphysema, chronic liver inflammation, and HCC (Rautou et al., 2010;
Yin et al., 2008).
As a secretory protein, AT matures in the ER; however, when ATZ accumulates in the
ER, it can be degraded by two major mechanisms, the proteasomal and autophagic pathways.
The proteasome is probably specialized for the soluble forms of ATZ, presumably bound
to multiple chaperones; meanwhile the autophagic pathway is specialized for the
polymerized/aggregated forms of ATZ. In the liver of patients homozygous for ATZ, as well
as in those from ATZ transgenic mice, an increasing number of autophagosomes can be
observed, probably there for degrading protein aggregates and damage organelles (Teckman
and Perlmutter, 2000). However, the precise mechanism by which the misfolded proteins are
recognized and removed by the autophagosome is yet not completely understood (Rautou et
al., 2010; Yin et al., 2008) (Table 1).

HCC
Hepatocellular carcinoma (HCC) is the sixth most common malignant disease worldwide
and the third greatest cause of cancer-related death. The etiology of HCC has been reported to
be related to a variety of diseases such as viral hepatitis, alcoholic hepatitis, NAFLD, and
metabolic syndromes including diabetes mellitus (Luedde et al., 2014). It is a multifactorial
pathology, and the relative contribution of cell death to its development depends on the
underlying disease. Moreover, different genes have been implicated in the pathogenesis of
HCC, and may be divided into four major groups: genes regulating DNA damage response;
those involved in cell cycle control; those involved in growth inhibition and apoptosis, and
genes responsible for cell-cell interaction and signal transduction (Rocken and Carl-McGrath,
2001).
Cancer is one of the scenarios where too little cell death occurs, resulting in malignant
cells that will not die. Mainly apoptosis, or its failure, is the primary driver of HCC
development. As apoptosis mechanism is complex and involves many intracellular pathways,
many defects can occur at any point along these pathways, leading to malignant
transformation of the affected cells, to tumor metastasis and resistance to anticancer drugs.
Down-regulation of pro-apoptotic Bax and Bcl-xs and up-regulation of anti-apoptotic
proteins such as Bcl-xl endows transformed hepatocytes with survival properties (Rocken and
Carl-McGrath, 2001; Takehara et al., 2001). Recently, survivin deregulation and
overexpression have been described in a very wide variety of tumors, including HCC. As
mentioned above, Survivin is the smallest member of the IAP family. Survivin expression is
essential for cell division during embryonic and fetal development, particularly for the G2/M
transition, but its expression is almost undetectable in normal differentiated adult tissue. In
the normal context, survivin is expressed in myeloid stem cells, CD34+ umbilical cord blood
cells, peripheral blood mononuclear cells and T lymphocytes and normal dividing cells
(Boidot et al., 2014; Khan et al., 2015; Soleimanpour and Babaei, 2015; Kanwar et al., 2013;
Su, 2015). Transcription of survivin mRNA is regulated by several cellular factors including
NF-κB, GATA-1 or STAT3 and signaling pathways like PI3K/AKT and MAPK.
Alternatively, P53 is a key regulator in the repression of survivin mRNA transcription (for
extended reading refer to (Boidot et al., 2014)). Alterations in these cellular mediators, often
described in cancer, may lead to the overexpression of survivin; in this context, its main role
is the inhibition of apoptosis. While the nuclear localization of survivin is essential to enable
the formation of the chromosomal passenger complex during mitosis, its translocation to the
cytoplasm is associated with the inhibition of apoptosis (Kanwar et al., 2013). The exact
mechanism of apoptosis inhibition is still a matter of research; however, it has been proposed
that within the mitochondrial compartment, survivin may promote mitochondrial membrane
stability in a similar fashion as bcl-2 family members (Dohi et al., 2004). When released to
the cytosol, upon apoptotic stimuli, surviving was shown to block the activation of initiator
caspase-9 [6]. Subsequent studies suggested that this inhibition could be enhanced in
association with hepatitis B X-interacting protein (HBXIP), by forming a complex with pro-
caspase-9 and inhibiting the formation of the apoptosome (Marusawa et al., 2003).
Moreover, in vitro studies suggested that cytoplasmic survivin, in association with XIAP,
could promote drug resistance by directly inhibiting terminal effector caspases. More
specifically, the formation of a survivin–XIAP complex promotes increased XIAP stability,

protecting XIAP from proteasomal degradation, resulting in a facilitated inhibition of

caspase-dependent cell death (Khan et al., 2015).
Furthermore, alternative splicing of birc5 gene results in different functional transcripts:
survivin, survivin-2B, Survivin-delta-Ex-3, survivin-3B and survivin 2α, which are
differentially expressed in different tumors types and differ in anti-apoptotic properties (for
further reading refer to (Soleimanpour and Babaei, 2015; Vargas and Vivas-Mejia, 2013) and
references herein).
Additionally, extracellular surviving has been detected in small membrane-bound
vesicles known as exosomes (Khan et al., 2009; Khan et al., 2011). Exosomal survivin can be
secreted by cancer cells and be taken up by surrounding cells, producing a field effect that
may confer a general stress-survival phenotype (Khan et al., 2015).
Particularly in HCC tissue, surviving has been detected in up to 70% of studied cases but
was undetectable in paracancerous tissues and cirrhotic liver tissues (Ito et al., 2000). In the
context of liver pathology, survivin could presumably alter the biological behavior of HCC
cells by inhibiting apoptosis, promoting their proliferation and enhancing their resistance to
chemotherapy. Histological tumor-specific over-expression of survivin suggests it could serve
as a broad-spectrum diagnostic marker for HCC. Moreover, in vitro studies have shown over-
expression of survivin in many HCC-derived cell lines (Cao et al., 2013). Even though
survivin is over-expressed in HCC, serum level of exosomal survivin has proven not to be an
adequate marker for the diagnosis of HCC (Jia et al., 2015).
Concerning autophagy, cancer has been genetically linked to autophagy malfunctions.
Moreover, the regulation of autophagy overlaps closely with signaling pathways that regulate
tumorigenesis. Additionally, several studies assessing autophagy in HCC have clearly
demonstrated that autophagy is a tumor suppressor mechanism. It has been observed that
mice with heterozygous disruption of beclin-1 have a higher frequency of spontaneous HCC.
Moreover, the most aggressive malignant HCC cell lines and HCC tissues with recurrent
disease display much lower autophagic levels than less aggressive cell lines or tissues,
especially when Bcl-xL protein is over-expressed. However, the mechanisms responsible for
the diminished autophagic activity are not elucidated ((Ding et al., 2008; Rautou et al., 2010)
and references herein) (Table 1).
CONCLUSION
Cell death is a basic biological phenomenon that is fundamental for development and
regulation of tissue homeostasis and whose alteration has important implications in
pathology. Since its initial description in the 1960s, a number of different death mechanisms
have been described. Nowadays, over 50 years of research in the field have clarified many
aspects of this process and brought to the attention of scientists its role in a large number of
diseases. Programmed cell death is a finely balanced process driven by positive and negative
signals, which determine the final fate between life and death; any imbalance in this process
may result in disease. Particularly, hepatocyte cell death is a central mechanism involved in
liver injury and is present in almost all types of human liver diseases by means of apoptosis,
necrosis, necroptosis and/or autophagy; all of which promote progression of liver disease

through distinct mechanisms. Even though our growing understanding on these triggering and
signalling mechanisms has allowed for the dissection of the different molecular pathways that
result in hepatocyte death, considerable crosstalk and cooperation between these pathways
was also described. In view of the fundamental role of cell death in virtually all hepatic
diseases, the identification of key molecules and the understanding of the precise biochemical
cascades that lead to cell death, are still important factors that impel the quest towards
developing and testing novel pharmacological and/or gene mediated therapies for patients
with liver diseases. Therefore, the knowledge of the basic aspect of each form of cell death
still represents a fundamental interest for both researchers and clinicians.
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Chapter 2
HEPATIC STELLATE CELLS IN LIVER

SINUSOID FUNCTION
Luís Perea, Júlia Vallverdú, Alberto E. Muñoz

and Pau Sancho-Bru
ABSTRACT
Hepatic stellate cells (HSCs) reside in the space of Disse, between hepatocytes and
liver sinusoidal endothelial cells. In the healthy liver, HSCs are mainly responsible for
vitamin A storage and the turnover of extracellular matrix (ECM) present in the space of
Disse. After injury, HSCs activate, participating in the tissue response to injury and the
healing response. If the injury persists, HSCs further activate acquiring myofibroblastic
characteristics and gaining proliferative and migration capacity, chemotaxis and
contractile activity. Moreover, during activation HSCs suffer a change in the expression
profile of ECM components increasing the production of fibrillar collagens, and reducing
matrix degradation, becoming the main cell type responsible for the accumulation of liver
scar and the development of fibrosis and cirrhosis. In this chapter, we review the main
characteristics and functions of HSCs in their quiescent phenotype and after activation.
Moreover, we describe the role of HSC in the regulation of the function of the sinusoid
and highlight its cross-talk with liver sinusoidal endothelial cells.
HEPATIC STELLATE CELLS PHENOTYPE AND ORIGIN

Hepatic stellate cells (HSCs) are cells of mesenchymal origin and reside in the space of
Disse, between hepatocytes and liver sinusoidal endothelial cells (LSEC). HSCs are lining the
endothelial cells of the sinusoids and represent between 5-8% of the total liver cells. HSCs are
named for the long cellular processes they have, which mostly are subendothelial, embracing
the sinusoid but also some may be interparenchymal, extending between more than one
sinusoid. This location and stellate shape allows the HSC to have important cross talk among
several cell types by direct cell contact and also by soluble factors (Reynaert et al. 2002) (A.
Geerts 2001). In the healthy liver, HSC are responsible for vitamin A storage and the turnover

44 Luís Perea, Júlia Vallverdú, Alberto E. Muñoz et al.
of extracellular matrix (ECM) present in the space of Disse. However, after injury, HSCs
activate, acquiring myofibroblastic characteristics and participating in tissue healing response.
If the injury persists, HSCs further activate and become the main cell type responsible for the
accumulation of ECM forming the liver scar and eventually the development of fibrosis and
cirrhosis.
The embryonic origin of HSCs has long been debated. This controversy was raised by the
fact that HSCs express a number of mesodermal markers such as vimentin, different
collagens, several actin family members as well as neural markers such as desmin,
synaptophysin, glial fibrillary acidic protein, nestin, and p75Ntr (Schmitt-Gräff et al. 1991)
(A. Geerts 2001). Based on this combination of markers, it was suggested that HSCs derived
from the embryonic neural crest. However, over the last few years, well-performed lineage
tracing studies have found that HSCs have a mesodermal embryo rather than a neural crest
origin, and have suggested that HSCs arise from the septum transversum. Indeed, lineage
tracing studies with the neural crest marker Wnt-1 enhancer/promoter CRE/YFP mice failed
to detect neural crest-derived HSCs in the developing liver (Cassiman et al. 2006). On the
other hand, lineage tracing of Wilms tumor suppressor (Wt1) and MesP1 gene–expressing
cells in mice showed that HSCs develop from the septum transversum–derived mesothelium
lining the liver bud (Asahina et al. 2009) (Asahina et al. 2011), suggesting a mesodermal
origin. Interestingly, the septum transversum-derived mesothelial cells were found to give rise
not only to HSCs but also to portal fibroblasts, smooth muscle cells around the portal vein,
and fibroblasts around the central vein (Asahina et al. 2011).
HSC activation is a central event in hepatic response to injury and can be divided into
two phases: the first step, known as the initiation phase, in which the HSC phenotype changes
by reorganizing its cytoskeleton, expressing receptors for vasoactive substances as well as
cytokines, growth factors and vasoactive substances and gaining proliferative, chemotaxis and
contractile properties. Moreover, activating HSCs experience a shift in the expression profile
of ECM components. The second phase is known as the perpetuation phase, in which HSCs
acquire characteristics of myofibroblasts, become resistance to apoptosis and actively
participate in inflammatory response and fibrosis progression (Lindquist, Stefanovic, and
Brenner 2000). Mechanisms of HSC activation have been mainly studied in vitro. When
cultured on plastic and in the presence of serum, HSCs attach, proliferate and undergo
spontaneous activation, a process closely resembling that observed in chronic liver diseases
(Friedman et al. 1992) (Sancho-Bru et al. 2005). Due to the rapid activation of HSCs in
culture, most studies have focused on activated HSCs, however, much less is known
regarding mechanisms controlling HSC quiescence (Perea, Coll, and Sancho-Bru 2015).
Early activation of HSCs is still poorly understood, but it is mediated by paracrine stimuli
from activated or damaged neighboring cells. Damaged hepatocytes produce reactive oxygen
species which mediate HSC activation (Nieto, Friedman, and Cederbaum 2002). Moreover,
hepatocyte necrosis and apoptosis promote HSC activation by releasing DAMPS, the
activation of Fas and TNF-related apoptosis-inducing ligand (TRAIL) pathway and the
generation of apoptotic bodies, which are released and interact with HSC (Canbay, Friedman,
and Gores 2004) (Canbay, Taimr, et al. 2003). Products produced by damaged hepatocytes
not only interact directly with HSC, but also activate Kupffer cells and endothelial cells,
which promote HSC activation through pro-inflammatory mediators (Canbay, Feldstein, et al.
2003). Activated Kupffer cells and infiltrating macrophages contribute to HSC activation,

Hepatic Stellate Cells in Liver Sinusoid Function 45
stimulating ECM synthesis, cell proliferation and survival, and enhancing its response to
cytokines and reactive oxygen intermediates (Bilzer, Roggel, and Gerbes 2006). LSECs have
been found to play an important role in the regulation of HSC activation and the maintenance
of the quiescent phenotype through paracrine soluble factors such as nitric oxide (NO)
(Muñoz 2016), which is produced by fenestrated LSECs and contributes to maintaining the
HSC phenotype quiescent (DeLeve, Wang, and Guo 2008) (Marrone et al. 2013) (Figure 1).
Importantly, inhibition of eNOS by L-NAME in co-culture experiments of LSECs with
HSCs, capillarize LSECs and prevent the capability of LSECs to maintain HSCs quiescence
(DeLeve, Wang, and Guo 2008). Moreover, HSC produce vascular endothelial growth factor
(VEGF), which is involved in the maintenance of the LSEC phenotype and enhance the
expression of NO (DeLeve et al. 2004). Overall, all of these studies highlight the importance
of the cross-talk effect between HSC and LSEC in maintaining their phenotype. To the
contrary, during injury, damaged endothelial cells secrete pro-inflammatory and pro-
angiogenic factors and participate in the conversion of TGF-β from the latent to the active
form, thus mediating HSC activation (Li et al. 2008) (Figure 1).
Figure 1. Crosstalk between hepatic stellate cells (HSC) and liver endothelial cells (LSEC). Upper:
HSC in quiescent state are responsible for vitamin A storage and the homeostasis of extracellular matrix
(ECM). The maintaining of the differentiated LSEC phenotype is related with a correct ECM
homeostasis and with the production of vascular endothelial growth factor (VEGF) by HSC. Which is
involved in the production of NO by LSEC. Bottom: Activated HSC acquiring proliferative and
migratory capacity, a vasoactive phenotype, chemotaxis and contractile activity. HSC activation
induces an imbalance in ECM production and composition, the accumulation of collagens in ECM
induces loss of fenestrations in LSEC. The production of vasoactive substances has an impact on the
cross-talk between HSC and LSEC. Decreasing production of NO by LSEC, as a result of acquiring
capillarized phenotype, increase HSC activation and contractility in response to chemokines and
vasoactive factors.

Upon activation HSC proliferate and acquire chemotactic capacity, which enhances the
accumulation of activated HSCs in sites of injury. Several chemoattractants have been
described to mediate HSC migration, including PDGF, IGF-1, ET-1 or MCP-1 (Ikeda et al.
1999) among others. In liver fibrosis, HSC are believed to be the major source of
myofibroblasts. However, the population of liver portal fibroblasts and vascular
myofibroblasts can also become myofibroblasts and, depending on the etiology of the liver
injury, they may, to a major or lesser extent, contribute to fibrogenesis (Knittel et al. 1999)
(Iwaisako et al. 2014) (Mederacke et al. 2013).
LIVER FUNCTION IN RELATION TO THE HEPATIC STELLATE

CELL PHENOTYPE
Vitamin A Homeostasis
The main physiological and metabolic function of HSCs in the healthy liver is the storage
of Vitamin A. Vitamin A is primarily stored in the liver, which contains 90% of the total body
amount of vitamin A (Hendriks et al. 1988). In the liver, HSCs play a central role in the
uptake, storage and release of retinoids and are the main cell type storing vitamin A, mainly
in the form of retinyl esters, the storage form of retinol in cytoplasmic lipid droplets
(Ghyselinck et al. 1999).
Circulating retinyl esters are uptaken by hepatocytes and hydrolyzed to retinol, which can
then bind to the retinol binding protein (RBP) in order to be secreted and exported to HSCs
by the cellular retinol binding protein I (CRBP-I). Retinol uptaken by HSC is re-esterified to
retinyl esters by lecithine:retinolacetyltransferase (LRAT) or by acyl-CoA retinol transferase
(ARAT) which can then be stored in cytoplasmic lipid droplets. If the vitamin A status of the
body requires its circulation, retinyl esters are mobilized by a hydrolase that produces retinol.
This retinol can be oxidized to retinoic acid or can bind to RBP in order to secret it or
transport it back to the hepatocytes (Ghyselinck et al. 1999) (Andersen et al. 1992). In fact, in
vitamin A deficient conditions, retinoids are rapidly bound to the RBP in hepatocytes and
exported to the circulation without transfer to HSCs (Batres and Olson 1987).
One of the main features of HSC activation is the rapid loss of vitamin A lipid droplets.
Upon activation in vitro, when HSCs are cultured on plastic, they lose their lipid droplets
while gaining myofibroblastic characteristics. Interestingly, the addition of a combination of
palmitic acid and retinois in the medium reduces their activation and pro-fibrogenic
phenotype to a certain extent (Lee et al. 2010). In addition, in vivo, HSCs present in fibrotic
liver have a lower content of lipids as well as vitamin A in lipid droplets. Although, lipid
droplets are strongly associated with a quiescent HSC phenotype, experimental evidence
suggest that vitamin A content might not influence the HSC activation state, since LRAT-
deficient mice, which lack retinoids containing lipid droplets in HSCs did not promote HSC
activation and the studied animals did not show major differences in fibrosis development
(Kluwe et al. 2011).

Extracellular Matrix Homeostasis
HSCs play a central role in ECM regulation in the healthy, injured and diseased liver.
The healthy liver is an organ with a low ECM content that constitutes approximately 0.5% of
the total wet weight of the liver. ECM is crucial to maintain the architecture of the liver. Most
ECM in healthy liver can be found forming the capsule of Glisson, in the portal areas and
around the central vein. Albeit in a lower quantity, the space of Disse contains ECM that
constitutes the pseudo basal lamina of the liver sinusoids. This ECM in the space of Disse is
not only important as a structural component of the sinusoid but also as a regulator of liver
homeostasis and cell function. ECM is an important reservoir of soluble factors that
participate in cell-cell communication. In the healthy liver the space of Disse contains a
variety of non-fibrilar ECM components such as laminin, elastin, fibronectin, latent-
transforming growth factor beta-binding proteins 1 (LTBP1) and 4 (LTBP4) together with
proteoglycans, glycoproteins or glycosaminglycans (Baiocchini et al. 2016). The space of
Disse is surrounded by hepatocytes, endothelial cells and HSCs, all of which are able to
produce ECM components. Maher et al. isolated liver cells from control rats for analysis of
several ECM mRNAs in specific populations. In this regard, quiescent HSC express mainly
collagen type III, collagen type IV, laminin and low amounts of collagen I, and endothelial
cells are able to produce collagen type IV (Albert Geerts et al. 1993) (A. Geerts 2001).
Hepatocytes express both cellular fibronectin and plasma fibronectin, being the main cell
contributors to plasma fibronectin (To and Midwood 2011).
Extracellular matrix in the space of Disse is tightly controlled by the regulation of the
production of the different components as well as degrading enzymes such as matrix
metalloproteinase (MMPs), calcium-dependent enzymes that specifically degrade collagens
and non-collagenous substrates (Benyon and Arthur 2001), and their inhibitors, the tissue
inhibitors of MMPs (TIMPs). In this way, ECM content and amount is balanced in the space
of Disse thanks to the turnover of its components and their continuous synthesis and
degradation. In this process HSC in addition to endothelial cells, Kupffer cells and
hepatocytes participate in the regulation of ECM turnover. Quiescent HSCs express MMP-2
(gelatinase A), MMP-3 (transin/stromelysin 1), MMP-10 (stromelysin 2), MMP-13
(collagenase 3) and MMP-14 (membrane type 1-matrix metalloproteinase) among others [34].
The activity of MMPs is regulated by TIMP-1 and TIMP-2, which are produced by activated
but not by quiescent HSCs (Kristensen et al. 2000) (Iredale et al. 1992). Moreover, HSCs also
produce members of the A desintegrin and metalloproteinases (ADAMs) such as the ADAM9
and ADAM12-13 members which are involved in the degradation of different components of
the ECM such as gelatins and fibronectin, and they are also involved in the cleavage of
growth factors, adhesion molecules and cytokines (Niiya et al. 2006).
ECM accumulation is the result of an imbalance between ECM production and
degradation. As mentioned before, during activation HSCs experience a shift in the
composition of ECM products expressed and they can also change the level and type of
MMPs expressed. Moreover, there is an increased expression of TIMPs, which prevent matrix
degradation by inhibiting MMP. Therefore, this combination of factors results in a net
increase in matrix deposition, which in advanced liver disease may be six times higher than in
normal liver. This change in ECM composition and the increase in collagens (I, III, and IV),
fibronectin, undulin, elastin, laminin, hyaluronan, and proteoglycans not only affects the HSC
activation state but also the behavior of all the liver cell types [40]. For instance, the loss of

fenestrations is associated with hepatic fibrosis and may be related, in part, to an

accumulation of interstitial collagens in the space of Disse. In this respect, while LSEC
cultured on a matrix resembling healthy liver matrix maintain their fenestrated phenotype,
LSEC cultured on collagen-rich matrix do not maintain fenestration (McGuire et al. 1992)
(Muñoz 2016).
Liver Regeneration and Angiogenesis
In liver cell homeostasis HSCs play an important role in the regulation of hepatocyte and
endothelial cell growth. Most data regarding the interaction of HSCs with hepatocytes and
endothelial cells have been obtained from experimental studies of liver regeneration and
tumorigenesis. It is well documented that besides remodeling the ECM, during liver
regeneration HSCs produce soluble factors involved in hepatocyte proliferation and
angiogenesis, thereby playing an important role in liver regeneration (Roskams 2008).
HSCs express neutrophilin, epidermal growth factor, and HGF, all of which are well
known inducers of hepatocellular growth which participate in liver regeneration after injury
(Passino et al. 2007) (Mullhaupt et al. 1994). Moreover, epimorphin, expressed in quiescent
HSCs, and pleiotrophin are expressed after partial hepatectomy and are morphogen proteins
involved in liver regeneration and hepatocyte growth (Yoshino et al. 2006) (Asahina et al.
2002). Furthermore, HSCs closely interact with LSEC as HSCs produce angiogenic factors
such as vascular endothelial growth factor (VEGF) or angiopoietin-1 in response to platelet-
derived growth factor (PDGF), TGF-β, NO and other angiocrine factors released from the
endothelium (Thabut and Shah 2010) (Ding et al. 2014).
Sinusoidal Blood Flow Regulation
The anatomical location of HSCs suggests that they may play a role as pericytes of the
liver, regulating the blood flow along the sinusoids. While the role of HSCs in flow
circulation in injured liver is clearly demonstrated that of quiescent HSCs in the regulation of
sinusoidal flow is controversial. Quiescent HSCs have long processes surrounding endothelial
cells, express receptors for some vasoactive substances and are in contact with nervous
terminations; however, they do not express alpha-smooth muscle actin (ACTA2) or ionic
channels and are lacking most vasoconstrictor receptors. One of the few evidence suggesting
an active role of HSCs in the regulation of blood flow was obtained by infusion of
endothelin-1 in the portal vein of healthy liver rats. Intravital microscopy showed a contractile
response of the healthy liver to endothelin-1, and areas of contraction localized with HSCs,
suggesting their role in the regulation of sinusoidal blood flow (Zhang, Pegoli, and Clemens
1994) (Bauer et al. 1994).
HSC activation is characterized by the acquisition of a vasoactive phenotype, with the
expression of ACTA2 and that of several receptors for vasoactive substances as well as ionic
channels (Bataller et al. 2005). Activated HSCs have been shown to respond in vitro and in
vivo to vasoconstrictors such as angiotensin-II, endothelin-1, vasopressin or thrombin as well
as to vasodilator substances such as adrenomedullin, natriuretic peptide or NO (Bataller et al.
2005). Moreover, the effect of these vasoactive substances is also important to mediate their

activation and fibrogenic phenotype. Activated HSC express ionic channels typically
expressed in contractile cell types such as calcium voltage-operated channels and calcium-
dependent potassium channels, which regulate intracellular calcium levels in response to
vasoactive factors (Gasull et al. 2001). It is important to note that activated HSCs may also be
a source of vasoactive substances such as angiotensin II and endothelin-1, which have an
autocrine and paracrine effect to neighboring cells. Changes in the production of vasoactive
substances during liver injury have a great impact on the cross-talk between HSC and LSEC
(Muñoz 2016). Decreased bioavailability of NO as a result of LSEC dysfunction and
increased production of the constrictors such as endothelin-1 and angiotensin-II by HSCs
increase HSC activation and contractility, thereby increasing intrahepatic resistance (Muñoz
2016) (Figure1) (Iwakiri, Grisham, and Shah 2008). As a result, the contractile state of HSCs
depends on the balance between vasodilators and vasoconstrictor factors, in this regard
circulating levels of vasoactive substances may certainly play an important role, but most
importantly intrahepatic levels produced locally by liver cells such as endothelial cells and
activated may be of crucial importance (Iwakiri, Grisham, and Shah 2008) (Shao et al. 2003).
Overall, in liver fibrosis, activated HSCs increase intrahepatic resistance by first
producing excess ECM deposition and thereby contributing to the structural component of
flow regulation and vascular distortion, and second, by actively promoting sinusoidal
contraction and locally regulating the sinusoidal flow.
Liver Inflammation/Immunology
Little is known about the immunoregulatory capacity of quiescent HSCs and their role in
the regulation of inflammation in healthy liver. However, in the context of liver inflammation,
activated HSCs respond to a number of inflammatory mediators and are also able to produce
a wide range of inflammatory mediators. Moreover, they also physically interact with resident
Kupffer cells and leukocytes infiltrating the injured liver. The broad range of inflammatory
mediators produced by HSCs and the immunoregulatory properties of HSCs underline their
potential for establishing paracrine interaction with a diversity of liver cells and infiltrating
leukocytes. In this regard, the cross-talk of HSCs with immune cells is of key importance for
the inflammatory response of the liver to injury.
While most inflammatory mediators are reported to induce HSC activation, other such as
INF-g, IL-22, IL-10, CXCL9 are shown to reduce the pro-fibrogenic phenotype of HSC and
to modulate their activation state. Generally speaking, HSC activation is associated with the
acquisition of a pro-inflammatory phenotype (Marra et al. 1993; Maher, Lozier, and Scott
1998; Sprenger et al. 1999a). Once activated, pro-inflammatory mediators such as cytokines
and chemokines promote nuclear factor kappa B (NF-kB) activation and the synthesis of
inflammatory mediators (Affò et al. 2014) (R F Schwabe et al. 2001). Much attention has
been given to the expression of chemokines by activated HSCs and their potential role in
leukocyte recruitment. Several inflammatory mediators such as TNF or IL-1 have been shown
to promote the expression of CXC and CC family chemokines by HSCs, thus promoting
leukocyte recruitment (Sprenger et al. 1999b) (Affò et al. 2014), (Robert F Schwabe, Bataller,
and Brenner 2003). Moreover, during activation and as a response to pro-inflammatory
stimuli the expression of adhesion molecules such as intercellular adhesion molecule 1
(ICAM-1, CD54) and vascular adhesion molecule (VCAM) in HSCs is increased and has

been shown to play essential functions in liver inflammatory responses by promoting

leukocyte recruitment (Sligh et al. 1993) (Hellerbrand et al. 1996).
The interaction between HSCs and inflammatory cells is of great importance for the
pathogenesis of chronic liver diseases. Kupffer cells and inflammatory monocyte-derived
macrophages from peripheral blood produce inflammatory mediators such as TGF-β, TNF or
IL-1 that mediate HSC activation, enhance their pro-inflammatory phenotype and promote
HSC survival (Pradere et al. 2013). Interestingly, via their activating receptor NKp46, natural
killer (NK) cells selectively kill early or senescence activated HSCs and induce apoptosis in
activated HSCs in a TRAIL-, FasL- and NKG2D-dependent manner (Gur et al. 2012). HSC
can also interact with T lymphocytes and suppress adaptive immune responses. Activated
HSC express PDL-1, which inhibits T cell response by inducing T cell apoptosis via B7-
H1/PD-1 ligation in vitro and in vivo (Yu et al. 2004) (Charles et al. 2013). Moreover, it has
been shown that HSCs inhibit the activation of naïve CD8 T cells by expressing CD54 and
limiting the expression of IL-2 and IL-2R on T cells. In addition, HSCs may induce immune-
suppressive Treg in an IL2-dependent fashion (Feng et al. 2014).
Several reports have suggested that HSCs may also modulate adaptive inflammatory
response by exerting functions of antigen presenting cells. In the liver it is clearly shown that
professional antigen presenting cells are dendritic cells as well as macrophages, monocytes
and B lymphocytes. However, HSCs have also been reported to express members of the HLA
family (HLA-I and HLA-II), lipid-presenting molecules (CD1b and CD1c), molecules
involved in T cell activation (CD40 and CD80) and proteins involved in antigen trafficking
(Vinas et al. 2003). Moreover, pro-inflammatory mediators induce the upregulation of genes
related to antigen presentation, suggesting that their potential function of antigen presenting
cells may be linked to cell activation (Vinas et al. 2003) (Maubach et al. 2007). In this regard,
only cytokine-stimulated HSCs stimulate allogeneic T-lymphocyte response in an HLA-II-
dependent manner (Vinas et al. 2003). Functional studies on the actual capacity of HSCs to
present antigens are not conclusive; while, it has been shown that murine HSCs can present
antigens to CD1-, MHC-I-, and MHC-II-restricted T cells, other reports have suggested that
HSC could not efficiently present antigens (Winau et al. 2007) (Dunham et al. 2013).
The liver is exposed to many bacteria products through portal vein inflow and frequently
to damage-associated cell products; therefore many cell types in the liver express pattern
recognition receptors (PRRs), damage-associated molecular patterns (DAMPS) and pathogen-
associated molecular patterns (PAMPS). The best-studied family of PRR in HSCs is that of
the TLR family receptors. Activated HSCs express TLR2, TLR3, TLR4, TLR7 and TLR9,
which are able to sense bacterial products and cell products such as DNA fragments or single-
stranded RNA. An important receptor involved in the activation and perpetuation of the
activated state in HSCs is TLR4, which together with MD2 and CD14 form the LPS receptor
complex (Park et al. 2009). The LPS level is often increased in liver patients due to increased
gut permeability and bacterial product translocation (Yan and Schnabl 2012). In vitro, LPS
mediates HSC activation and is a powerful inducer of the expression of pro-inflammatory
mediators in HSC (Muhlbauer et al. 2004). In vivo, LPS have been shown to enhance HSC
activation and proliferation while promoting liver fibrosis (Seki et al. 2007; Yang and Seki
2012). Moreover, DAMPS such as HMGB1, heat shock proteins, S100s, hyaluronan or DNA
enhance HSC activation through TLRs signaling, promoting HSC proliferation and
recruitment to the site of injury (Nakamoto and Kanai 2014). Moreover, they are also
involved in the progression of liver inflammation and fibrosis (Brun et al. 2005).

REGRESSION OF LIVER FIBROSIS AND INACTIVATION

OF MYOFIBRIBLASTS
Increasing evidence indicate that the liver has an important capacity to recover even after
advanced liver fibrosis when the etiological source of chronic injury is removed. Although it
is still not clear to what extent cirrhosis can revert, reversion of fibrosis has been shown in
advanced chronic liver diseases of different etiologies including hepatitis C and B viruses
(HCV, HBV), alcoholic liver diseases and non-alcoholic steatohepatitis (NASH) (Tai et al.
2012) (Marcellin et al. 2013) (Pellicoro et al. 2014). In addition, a number of experimental
studies have shed light on the mechanisms underlying the regression of fibrosis.
The regression of liver fibrosis comprises both decreasing pro-fibrogenic genes such as
Collagen I, TIMP1 and pro-inflammatory cytokines and also increasing ECM degradation by
MMPs. Different cell populations have been shown to participate in this process. Immune
cells have been shown to play a central role in the regression of fibrosis, especially NK and
macrophages. Particularly, Ly-6C infiltrating macrophages have been shown to orchestrate
fibrosis resolution by activating fibrolytic properties and contributing to the breakdown of
ECM (Ramachandran et al. 2012). Moreover, regression of liver fibrosis is necessarily
accompanied by a reduction of the number of HSCs or their activation state. After removal of
the injury, HSCs may undergo apoptosis or senescence, thus reducing the HSC pool and
allowing regression of liver fibrosis. Several reports have shown that down-regulation of
survival genes (Bcl-22) and the up-regulation of apoptotic proteins such as p53, Bax and
caspase 9 together with the activation of cell death pathways (Fas or TNFR-1 receptor)
contribute to HSC apoptosis during resolution (Kisseleva and Brenner 2011) (Guicciardi and
Gores 2010; Fischer et al. 2002; Novo et al. 2006).
For many years, HSC activation was considered to be a unidirectional process. Cells with
a myofibroblast phenotype were not considered to be able to revert to a quiescent cell, and
apoptosis was considered to be the only mechanism by which the HSC pool was reduced.
However, recent studies using lineage tracing strategies allowed to follow HSC activation and
reversion during liver fibrosis and resolution (Troeger et al. 2012; Kisseleva et al. 2012). By
following vimentin and collagen-α1 expressing cells, these studies showed that after the
acquisition of a myofibroblast phenotype, HSCs could revert to a quiescent-like phenotype
called inactivated state. Inactivated HSC (iHSC) down-regulated fibrogenic genes and
overexpressed PPAR-ϒ dependent genes, re-acquiring vitamin A and quiescent features. Yet,
the expression level of key quiescent genes and the analysis of the global gene expression
profiles demonstrated that iHSC were different from qHSC (Kisseleva et al. 2012).
Interestingly, inactive HSCs were in a more active state than quiescent HSCs, allowing rapid
activation to a fibrogenic stimulus. Therefore, after a new insult iHSC re-acquired an active
myofibroblast phenotype faster than unprimed quiescent HSC (Kisseleva et al. 2012)
(Troeger et al. 2012). All these studies demonstrate that myofibroblasts may be cleared by
several mechanisms during fibrosis resolution, either undergoing apoptosis and senescence, or
inactivation, reverting to a non-fibrogenic quiescent-like phenotype.

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Chapter 3
LIVER SINUSOIDAL ENDOTHELIAL CELLS:

FROM LIVER FUNCTION TO LIVER CIRRHOSIS
Alberto E. Muñoz and Luís Perea
ABSTRACT
The liver is strategically located between the splanchnic and the systemic
circulations, interacting with both circulatory systems. Many hepatic functions depend on
the sinusoid. It is lined by the liver sinusoidal endothelial cells (LSECs). The fenestrated
phenotype allows LSECs work as a dynamic bidirectional filter between the sinusoidal
lumen and the space of Disse and as scavenger endothelial cells that mediates
endocytosis through receptors of a broad range of molecules. Fenestrated LSECs
maintains hepatic stellate cells (HSCs) quiescence through the nitric oxide (NO).
Decrease of NO bioavailability in liver injury would be the initial cause of defenestration
and subsequent capillarization of hepatic sinusoid, which lead to liver switch from an
open (sinusoid) to a closed (capillary) circulation. In this chapter we reviewed: the
relationship between phenotype of LSECs and liver function and their interaction with
HSCs; the age-related changes in the morphology of liver sinusoids; the effect of LSECs
and HSCs phenotypes changes on liver function in liver cirrhosis and the exciting
possibility of LSECs and HSCs phenotypes reversion and recovery of liver function in
human cirrhosis.
INTRODUCTION
The liver is strategically located between the splanchnic and the systemic circulation,
interacting with both circulatory systems. It receives substances from the splanchnic territory
via the portal vein and the systemic circulation via the hepatic artery (Gumucio J et al., 1991).
The liver parenchyma is composed of several cell types. Parenchymal cells or
hepatocytes represent approximately 60% of liver cells. The non-hepatocyte cells population,
encompasses liver sinusoid cells: endothelial, stellate and Kupffer cells and intrahepatic
lymphocytes (McCuskey R et al., 1992) (Maher J et al., 1993).

60 Alberto E. Muñoz and Luís Perea
HEPATIC SINUSOID AND ITS RELATIONSHIP TO LIVER FUNCTION

In 1981 Gumucio et al., coined the concept of “heterogeneity function of hepatocytes” to
attempt explain the main liver function: regulate the concentration of substances in hepatic
venules and in bile, in response to the demands of the moment, in different organs of human
body (Gumucio J et al., 1981).
Hepatocellular heterogeneity is established, and latter modulated, mainly by the
sequential portal (nutrient-rich) and arterial blood (oxygen-rich) unidirectional perfusion from
hepatocytes surrounding the portal vein to those surrounding hepatic vein, leading to a
differential expression of metabolic processes of substances in hepatocytes of zones 1, 2
(uptake, processing and secretion) and 3 (uptake-efflux, secretion) of liver acinus. Another
advantage of the sequential perfusion of hepatocytes is that it allows intercellular cycling, i.e.,
the secretion of substances by hepatocytes located upstream and the uptake of these
substances by hepatocytes located downstream (Traber P et al., 1988). For example, glucose-
release processes (gluconeogenesis and glycolysis) occur predominantly in hepatocytes
located in zone 1 of the hepatic acinus. On the other hand, the glucose-uptake processes
(glycolysis and glycogenesis) appear to occur predominantly in hepatocytes located in zone 3
of the hepatic acinus. In addition, lactate produced by hepatocytes located in zone 3 may be
taken up and converted to glycogen by hepatocytes of zone 1.
Many hepatic functions depend of capillary liver: the sinusoid. The sinusoidal
endothelium has intercellular and transcellular gaps (fenestrae) and lack diaphragms and
basement membrane. This unique human phenotype allows a rapid and bidirectional
exchange of substances between sinusoid plasma and hepatocytes: “liver filter” (Gumucio J
et al., 1991) (Martínez Hernández A et al., 1992).
LIVER SINUSOID CELLS

The hepatic sinusoids represent the capillary bed of the liver. They have a diameter of
approximately 5 to 10 μm (Wisse E et al., 1983) (Warren A et al., 2008), and are lined by the
liver sinusoidal endothelial cells, a special type of fenestrated endothelium that differs both
structurally and functionally from other endothelia in the body (Wisse E et al., 1985)
(Sorensen K et al., 2012). Stellate cells, or Ito cells, which are the pericytes of the sinusoids,
are located in the space of Disse, whereas Kupffer cells (resident macrophages) and different
types of resident lymphocytes, including natural killer (NK) cells, NK T-cells, naïve T-cells,
and B-cells are normally located on the luminal aspect of the sinusoidal lining (Wake K,
1980) (Wake K et al., 1989) (Wu J et al., 2010).
Liver Sinusoidal Endothelial Cells
Until 1990, most of the studies on liver sinusoidal endothelial cells (LSECs) were
performed by scientists that had a special interest in the biology of these cells. After about
1990 the general interest in LSECs increased, and the number of scientists that were not
raised in the tradition of cells of the liver sinusoid started to grow. With an increased number

Liver Sinusoidal Endothelial Cells 61
of scientists coming in from other disciplines (cancer, immunology, virology, pharmacology,

hematology, and more), showing an interest to include LSECs in their studies, there has been
an increasing tendency to find in the literature conflicting descriptions on LSECs (Elvevold K
et al., 2008).
The LSECs ultrastructure was first described in detail by Wisse in 1970, who introduced
improved methods of fixation (Wisse E, 1970) (Wisse E, 1972). Until then, it was debated in
the literature whether the LSECs and Kupffer cells represented distinct cell types (Aterman K,
1964). Wisse’s detailed electron microscopy study of perfusion fixed with osmium instead
with glutaraldehyde rat liver put an end to this discussion, and described the LSECs as a
unique cell type, clearly different from the Kupffer cell (Wisse E, 1972).
LSECs contain fenestrae, nondiaphragmed pores that traverse the cytoplasm. In general,
endothelial fenestrae measure 100 nm in diameter, occur at a frequency of 9–13 per μm2, and
occupy 6–8% of the endothelial surface in scanning electron microscopy (SEM) (Wisse E et
al., 1985). In addition, differences in fenestrae diameter and frequency in periportal and
centrilobular zones were demonstrated; in SEM the diameter decreases slightly from 110.7 ±
0.2 nm to 104.8 ± 0.2 nm, whereas the frequency increases from 9 to 13 per μm2, resulting in
an increase in porosity from 6 to 8% from periportal to centrilobular (Wisse E et al., 1983).
Fenestration is not unique to LSECs, but is seen in various endothelial cells in other organs.
However, in mammals only the glomerular endothelial cell and the LSECs have open
(nondiaphragmed) fenestrae, and the glomerular endothelial cell differs from the LSECs in
that it resides on an organized basement membrane. Thus, the LSECs have a morphologic
phenotype that is unique in the mammal, with the combination of open fenestrae and lack of a
basement membrane. This creates open access for solutes between the sinusoidal blood and
the space of Disse (Braet F et al., 2002).
Phenotype of LSECs
The maintenance of the LSECs differentiated phenotype (fenestrated) requires both

paracrine and autocrine cell signaling (DeLeve L et al., 2004). Vascular endothelial growth
factor (VEGF), derived from hepatocytes and hepatic stellate cells, is a key regulator of the
LSECs phenotype, as demonstrated both in vitro and in vivo (DeLeve L et al., 2004) (Xie Get
al., 2012). In vitro studies have demonstrated that maintenance of the fenestrated LSECs
phenotype requires VEGF, as paracrine cell signaling, working through two pathways, a nitric
oxide (NO)-dependent and an NO-independent pathway, as autocrine cell signaling (DeLeve
L et al., 2004) (Xie Get al., 2012). VEGF secreted by either hepatocytes or stellate cells
stimulates NO release from endothelial nitric oxide synthase (eNOS) in LSECs (Figure 1).
NO in turn acts through soluble guanylate cyclase, conversion of GTP to cGMP, and
stimulation of protein kinase G, which can then phosphorylate protein targets (Xie G et al.,
2012). An alternative possibility, i.e., that NO acts through protein S-nitrosylation, was found
not to be necessary to maintain the differentiated LSECs phenotype (Xie G et al., 2012). In
addition to the VEGF stimulated NO pathway, maintenance of the LSEC phenotype also
requires an NO-independent pathway, which remains to be characterized (Xie G et al., 2012).

Figure1. Crosstalk between liver endotelial cell (LSEC) and hepatic stellate cell (HSC). Upper: the
maintenance of the LSEC differentiated phenotype requires autocrine nitric oxide (NO) signaling. The
ability of fenestrated LSEC to maintain hepatic stellate cells (HSC) quiescence it is through the
paracrine NO signaling. The LSEC differentiated phenotype it is associated with their main functions:
fenestrated membrane / sinusoidal blood flow; expression of receptors / scavenger function. Bottom:
reduction of NO bioavailability is associated with capillarized LSECs and with loss of maintain HSC
quiescence (activated HSC), through vasoactive substances, cytokines and chemokines. The reduction
in NO production - LSEC capillarized phenotype it is associated with loss of their main functions:
fenestrae disappearance / bidirectional filter and decline in endocytic capacity.
Differing Effects of Differentiated and Capillarized LSECs on Hepatic

Stellate Cells Activation
The ability of fenestrated LSECs to maintain hepatic stellate cells (HSCs) quiescence it is
through the NO, a paracrine factor. Different authors have reported that NO produced by
fenestrated LSECs maintains HSCs quiescence (DeLeve LD et al., 2008) (Marrone G et al.,
2013). Instead, in coculture of LSECs with HSCs, inhibition of eNOS by L-NAME blocks the
ability of LSECs to maintain HSCs quiescence. LSECs cultured in the presence of L-NAME
capillarize and capillarized LSECs do not maintain HSCs quiescence (activated HSCs)
(DeLeve L et al., 2008) (Figure 1) (See Ch. xx Perea, Vallverdú, Muñoz, Sancho-Bru).

LIVER FUNCTION IN RELATION TO LIVER SINUSOIDAL

ENDOTHELIAL CELLS PHENOTYPE
Fenestrae
Fenestrae is a Latin word that means windows, and the English term may be more
appropriate for introducing this structure to scientists outside the field of hepatic sinusoidal
cells (Braet F, 2004).
In the last century, much attention has been paid on the structure and functional
peculiarities of the wall of the sinusoids in the liver, because of the biological role of this is
thought to play in the exchange material between the blood and the hepatocytes. Light
microscopical investigations were not fully able to resolve these structural problems (Wisse
E, 1970).
The fenestrae were described for the first time in 1970 by Wisse, using perfusion fixation
with osmium and transmission electron microscopy (Wisse E, 1970). Later scanning electron
microscopy showed that fenestrae are clustering in sieve plates.
On the basis of morphological and physiological evidence, it was reported that the
grouped fenestrae act as a dynamic filter (Fraser R et al., 1978). Fenestrae filter fluids, solutes
and particles that are exchanged between the sinusoidal lumen and the space of Disse,
allowing only particles smaller than the fenestrae to reach the parenchymal cells or to leave
the space of Disse.
The results of electron microscopic studies on LSECs have added the following new
insights in the structure and function of the cytoskeleton in fenestrated areas (Braet F et al.,
1995) (Braet F et al., 1996): I. Sieve plates and fenestrae are delineated by cytoskeleton rings
which form a network; II. Because the sieve plates and fenestrae associated cytoskeleton
network opens and closes in response to different treatments such as luminal blood pressure,
hormones, drugs, toxins, changes in extracellular matrix, disease, aging and exposure to
environmental pollutants, it is assumed that this network regulates the size changes of the
fenestrae; and III. Therefore, the fenestrae associated cytoskeleton probably controls the
important function of endothelial filtration.
A recent study suggested that actin disruption increases fenestration by its effects on
membrane rafts (Svistounov D et al., 2012). Sieve plates and plasma membrane rafts were
inversely distributed in freshly isolated LSECs, and it was hypothesized that fenestrations
form in non-raft lipid-disordered regions of the plasma membrane once the membrane
stabilizing effects of actin cytoskeleton and membrane rafts are diminished (Figure 2).
More than 100 years ago the German pathologist, Disse, indicated that the permeability
of the sinusoidal endothelium, is so critical for the transfer of nutrients both to and from the
liver (Schmid R, 1991).

Fgure 2. The Sieve-Raft hypothesis. It is proposed that fenestration form in non-raft microdomains of
the lipid bilayer and that rafts and actin engender membrane stability, while limiting fenestration
formation.
The liver has a rich network of vessels nourishing the hepatocytes, and among many
other functions, is ideally adapted for the metabolism of lipids (Fraser R et al., 1995). Dietary
fats, absorbed by the epithelium of the small intestine, are assembled with specific
apolipoproteins to form chylomicrons, which have a size between 100 and 1.000 nm. After
synthesis by the enterocytes, the triglyceride-rich chylomicrons are secreted into the
mesenteric lymph and extracellular fluid to eventually enter the systemic circulation via the
thoracic duct. Once into the circulation, triglycerides are hydrolysed to fatty acids in the
capillaries of adipose tissue and muscles through the action of lipoprotein lipase present on
the endothelium of capillaries. The resulting smaller particles have a mean diameter of 90–
250 nm and are called chylomicron remnants, which are taken up rapidly by the apo E
(remnant) receptors of the liver parenchyma. Before hepatic recognition and uptake of
chylomicron remnants can occur, these remnants must first enter the space of Disse through
the fenestrated sinusoidal endothelium (Wisse E et al., 1985) (Fraser R et al., 1986) (Fraser R
et al., 1995). Wisse (Wisse E, 1970) suggested at first that fenestrae might play an important
role in the exchange of lipid particles between the sinusoidal blood and the parenchymal cells.
The term “liver sieve” was first coined by Fraser and co-workers in 1978 (Fraser R et al.,
1978). They then demonstrated a filtration effect by comparing chylomicrons of different size
in the portal blood with those in the space of Disse, illustrating that the largest particles in the
sinusoidal blood were as large as fenestrae (>100 nm), compared with the smaller particles
(remnants) observed in the space of Disse (< 100 nm), supporting the sieving hypothesis
(Fraser R et al., 1995).
Scavenger Endothelial Cells
Every second, the animal body challenged with waste material, both self-made and
foreign. “Waste” is defined as material whose presence in the blood is incompatible with
homeostasis (Adachi H, et al., 2002). Most macromolecules in the body are highly dynamic
structures, with natural turnover rates spanning from minutes to months. This natural turnover

includes some limited extracellular degradation, but in fact, complete degradation to single
building blocks (amino acids, monosaccharides, fatty acids, and nucleotides) will primarily
take place after uptake and intralysosomal processing in specialized scavenger cells. In
addition to natural turnover mechanisms, chemical and mechanical wear-and-tear processes
constantly turn macromolecules into harmful waste substances, such as advanced glycation
end products and oxidized lipoproteins that are recognized and eliminated by the same
scavenger cells. Moreover, foreign material (primarily microbes and microbial products) that
constantly invade the body are removed by these scavenger cells.
Cells responsible for the removal of waste macromolecules have long been the subject of
research in immunology, metabolism, and physiology.
In his account on the waste-clearing cells of animals, Aschoff (Aschoff L, 1924)
launched the name “the reticuloendothelial system” (RES) to denote the cells that were most
actively taking part in the uptake of intravenously administered vital stains. He observed that
macrophages and sinusoidal cells of liver and certain other organs took up massive amounts
of vital stains. Over the years that followed after Aschoff’s work, it became common to think
that the RES consisted of macrophages only. This was partly due to the misconception that
the cells of the RES were professional phagocytes only and used phagocytosis to clear vital
stains from the circulation. In line with this view, van Furth et al., (van Furth R et al., 1972)
proposed in 1972 that the term RES be replaced with the mononuclear phagocyte system
(MPS).
Using modern technology and contemporary knowledge based on a reflective
understanding of historical findings, Kawai et al., (Kawai Y et al., 1998) showed that the
major site of uptake of vital stain in liver was LSECs rather than macrophages. What seems
clear today is that the major scavenger cell systems of vertebrates and invertebrates are based
on a dual-cell principle of waste clearance (Figure 3). In vertebrates, the scavenger
endothelial cells (SECs) represents the professional pinocyte, clearing the blood of a wide
range of soluble macromolecules, and small particles (< 200 nm) by clathrin-mediated
endocytosis, while the macrophage represents the professional phagocyte, eliminating larger
particles (Seternes T et al., 2002). A corresponding dual scavenger cell system is present in
invertebrates, with the nephrocyte and hemocyte being geared to clathrin-mediated
endocytosis and phagocytosis, respectively.
Figure 3. The dual-cell principle of waste clearence. A: Taken of macromolecules: insoluble are by
phagocytosis and soluble are by pinocitosis. B: Clearance of circulating waste: the major responsable
cells are macrophage and the scavenger endothelial cell.

While the cells of the MPS have been studied in great detail since Metchnikoff’s
discovery of the macrophage about 130 years ago (Metchnikoff E, 1884), our knowledge of
the SECs is the result of investigations over the last 40 years. The shift of paradigm from RES
= MPS to RES = MPS + SECs is a major achievement, allowing novel understanding of how
the blood is cleared of its own and foreign waste material. We now know that the cellular arm
of the innate immune system consists of both the MPS and the SECs. In spite of this fact,
textbooks in pathology and immunology still teach students that RES = MPS, thus giving the
false impression that the macrophage alone is responsible for the blood clearance of both
soluble macromolecules and most types of particles. To understand how viruses and an array
of pathogens and biopharmaceuticals are eliminated from the blood, we need a better
understanding about the blood clearance function of SECs.
SECs of vertebrates share some striking common traits: they 1) are extremely active in
clathrin-mediated endocytosis; 2) are rarely, if ever, observed to perform phagocytosis
(uptake of particles < 0.5 µm) under normal conditions; 3) have a well-developed intracellular
apparatus to process internalized substances; 4) have super high specific activity of lysosomal
enzymes; 5) carry out an overall anaerobic metabolism as reflected by the fact that the major
end products of degraded ligands are lactate and acetate; 6) are located at sites where
epithelial cells display a very high metabolic activity; and 7) express scavenger and mannose
receptors (Seternes T et al., 2001) (Laurent T et al., 1986).
LSECs as SECs
From his thorough studies on rat liver endothelial cells (Wisse E, 1970) (Wisse E, 1972),
Wisse concluded that the aforementioned characteristics of the LSECs, their cytoplasm
contains bristle-coated micropinocytic vesicles (0.18 um in diameter) and smooth
macropinocytic vesicles (0.7 um in diameter), both of which are part of the endocytic
apparatus and related to the selective uptake of proteins.
Although our tissues appear to exist as static structures, they are indeed the result of a
rather active yet balanced synthesis and degradation. Part of the degradation takes place
locally, but a large portion of the tissue structures are degraded in local lymph nodes (Laurent
T et al., 1986) (Ostgaard G et al., 1995). Tissue macromolecules that escape uptake and
degradation in lymph nodes, reach the blood circulation, from where they are efficiently
cleared by the LSECs/SECs () (Ostgaard G et al., 1995) (Elvevold K et al., 2008) (Smedsrod
B et al., 1985) (Smedsrod B, 1988). The active uptake mechanism in LSECs/SECs explains
why most connective tissue macromolecules and modified proteins are removed from the
circulation of rats and mice with the very rapid t1/2 of 30 to 60 s (Elvevold K et al., 2008)
(Smedsrod B et al., 1985) (Smedsrod B, 1988). Not all connective tissue waste has to pass
through lymph nodes to reach the circulation. Fragments of collagen and other molecules
released as the result of bone matrix turnover are passed directly into the blood circulation,
from where they are rapidly taken up in LSECs/SECs and degraded. This direct tissue-to-
blood path of waste is important since bone is the largest reservoir of collagen in the body. It
should be noted that in various pathologies, in particular in inflammatory episodes and during
anticancer treatments, the amount of waste macromolecules that enter the circulation, may
reach very high levels. The role of LSECs/SECs as waste eliminators in such situations is
pivotal, yet understudied.

Various other macromolecules that find their way to the circulation are also taken up by
the LSECs/SECs. Examples are oxidatively modified proteins, in particular oxidized low
density lipoproteins (OxLDLs) (Oteiza A et al., 2011), and advanced glycation end products
(AGEs) (Svistounov D et al., 2013) that are both categorized as atherogenic. Microbial
products that are constantly entering the portal vein from the gastrointestinal tract are also
scavenged by the LSECs/SECS. In that sense, the LSEC/SECs play an important role in host
defense. As will be discussed below, a unique feature of the LSEC/SECs is its ability to clear
soluble IgG-antigen complexes from the circulation. This uptake function assigns a
macrophage-like role to the LSEC/SECs as a functional link between the adaptive and innate
arms of the immune system.
The LSECs cytoplasm is packed with vesicles and organelles associated with the uptake,
transport, and degradation of endocytosed material (Wisse E et al., 1996). Although LSECs
represent only a small fraction (2.8% in rat) of the total liver cell volume, and 15% to 20% of
all liver cells they contribute to around 45% of the total mass of pinocytic vesicles, and
around 17% of the lysosomal volume of rat liver (Blouin A et al., 1977), indicating a high
endocytic activity (SECs). In accordance with this, the specific activity of several lysosomal
hydrolases is reported to be higher in LSECs than in hepatocytes and Kupffer cells (Elvevold
K et al., 2008), ensuring effective degradation of endocytosed material (Elvevold K et al.,
2008). Clathrin-mediated endocytosis is dominating in LSECs (Kjeken R et al., 2001). The
cells contain approximately twice as many clathrin-coated pits per membrane unit than
hepatocytes and Kupffer cells (Kjeken R et al., 2001), supporting the notion that they are
SECs.
To fulfill their role as true SECs, LSECs expressed three main waste-clearing receptors:
1) scavenger receptor; 2) mannose receptor, uptake of a wide range of endogenous
glycoproteins and microbial glycans and has a proposed role both in immunity and
glycoprotein homeostasis (Elvevold K et al., 2008); and 3) Fc gamma-receptor IIb2, mediated
clearance of circulating IgG immune complexes was previously assumed to be mediated by
Kupffer cells only, and also contributes importantly to the uptake of small soluble immune
complexes (Ganesan L et al., 2012). In addition, several other receptors are in these cells that
may be involved in scavenging activity: LRP-1, a multifunctional endocytic receptor
expressed in hepatocytes, neurons, astrocytes and fibroblasts (Willnow T et al., 1999), L-
SIGN, recognition and uptake of virus, including human immunodeficiency virus (Pohlmann
S et al., 2001) and hepatitis C virus (Gardner J et al., 2003), and LYVE-1, hyaluronic acid
binding protein (Mouta Carreira C et al., 2001). Then we will refer only on scavenger
receptors.
Scavenger Receptors
The term “scavenger receptors” (SRs) was originally coined to describe a macrophage
receptor that mediates endocytosis of a broad range of polyanionic molecules (Goldstein J et
al., 1979). Currently, there are ten known structurally unrelated SRs subclasses (SR-A to SR-
J) that recognize common ligands (Prabhudas M et al., 2014).
The LSECs expresses SR-A, also known as macrophage SR (Hughes D et al., 1995), SR-
B (SR-B1 and CD36) (Malerod L et al., 2002), and SR-H (stabilin-1/ FEEL-1 and stabilin-
2/FEEL-2/HARE) (Hansen B et al., 2005).

Studies over the last 10 to 15 years have established that the LSEC scavenger function
rests largely on the ability of stabilin-1 (SR-H1) and stabilin-2 (SR-H2) to mediate binding
and metabolism of a multitude of ligands (Hansen B et al., 2005).
Their expression pattern is highly overlapping; both are found in sinusoidal endothelia of
the liver (Politz O et al., 2002), bone-marrow (Qian H et al., 2009), spleen and lymph nodes
(Falkowski M et al., 2003), and eye choriocapillaris (Li R et al., 2009).
Stabilin-2 ligands are cellular and extracellular components: hyaluronic acid (Politz O et
al., 2002), chondroitin sulphate (Harris E et al., 2008), heparin (Harris E et al., 2008) and N-
terminal propeptides of procollagen types I and III (Melkko J et al., 1994).
Stabilin1 and 2 ligands are modified macromolecules of mammalian origin: oxidized
LDL (Li R et al., 2011) and formaldehyde-treated albumin (Blomhoff R et al., 1984).
SPARC (secreted protein acidic and rich in cysteine, also known as BM-40 and
osteonectin) that binds only to stabilin-1 (Kzhyshkowska J et al., 2006).
LOSS OF LIVER SINUSOIDAL ENDOTHELIAL CELLS

DIFFERENTIATED PHENOTYPE
LSECs with normal fenestration and function are referred as differentiated LSECs,
whereas LSECs that are defenestrated and lack functions are termed capillarized LSECs
(DeLeve L, 2015).
Capillarized LSECs in Cirrhosis
Studies in animals revealed that initial alteration of the fenestrae, to liver injury, is its
dilatation (Fraser R et al., 1980) (Mak K et al., 1984). This allows endotoxins
(lipopolysaccharides) coming from the digestive tract, come into contact with LSECs, which
promotes interaction between caveolin-1 and eNOS (Kamoun W et al., 2006) and decrease
eNOS activity, mechanism by which there is a reduction in the production of nitric oxide
(NO) (Kamoun W et all, 2006). Also, in rats and humans increased activity of Rho/ROCK
signaling pathway, is associated with a decrease in the expression and activity of eNOS, wich
enhances the reduction in NO production from LSECs (Zhou Q et al., 2006) (Rikitake Y et
al., 2005).
Differentiated LSECs function as a gatekeeper in the process of liver fibrosis (DeLeve L,
2015). Data obtained from animal models indicate that two events occur in liver sinusoid
preceding liver fibrosis: 1) capillarization, expressed by a change in the phenotype of LSECs
from differentiated to capillarized, characterized by fenestrae disappearance (Figure 1,
bottom). Defenestration is a reversible damage due to a reduction of NO production and
consequent reduction of its autocrine action and 2) activation of HSCs (See Ch. xx Perea,
Vallverdù, Muñoz, Sancho-Bru). LSECs by lowering NO production lose the ability to keep
paracrine on HSCs in their quiescent state, move to an activated phenotype and
producecollagen deposit in the space of Disse, under the capillarized LSECs with formation
of a basement membrane (irreversible damage) (DeLeve L, 2004) (DeLeve L, 2008). Finally,
capillarization not only precedes, also has an action at least permissive on fibrosis and

subsequent development of cirrhosis (Schaffner F et al., 1963) (Orrego H et al., 1979)

(DeLeve L, 2015).
Shift Open to Closed Hepatic Circulation and Hepatic Failure

in Cirrhosis
In humans decreased bioavailability of NO would initial cause of defenestration (Horn T

et al., 1987) and subsequent capillarization of hepatic sinusoid (Schaffner F et al., 1963)
which leads to liver switch from an open (sinusoid) to a closed (capillary) circulation. Also,
was observed in humans that there is a direct relationship between the degree of
capillarization and stage of alcoholic liver disease (Orrego H,et al., 1979). These changes
would make the liver loses its bidirectional filter function between the sinusoid and the
hepatocytes and vice versa (Martínez Hernández A et al., 1992). As a consequence,
capillarization of LSECs interfere with the transfer of nutrients between the intravascular
compartment and the space of Disse leading to perpetuating of liver injury and may therefore
be a major contributor to hepatic failure in cirrhosis (Schaffner F et al., 1963) (Schmid R et al.
1991) (Martínez Hernández A et al., 1992) (Braet F et al., 2002). These findings support the
“intact hepatocyte theory”: anomalies of hepatic microcirculation could result in impaired
exchanges between the hepatocyte and the blood perfusing the liver and thus contribute to
liver failure irrespective the metabolic capacity of hepatocytes (Wood A et al., 1979).
Reduced Clearance of Blood Borne Waste by Scavenger Receptors

in Cirrhosis
LSECs phenotype is related to functions thereof within liver microenvironment (DeLeve

L, 2015). A characteristic functional phenotypic feature of the LSECs is its high endocytic
capacity (Sorensen et al., 2012), which deteriorates with capillarization (Tamaki S et al.,
1996). There is not yet published evidence that the decline in endocytotic capacity precedes
fibrosis or a comprehensive description of which endocytotic receptors are affected in
capillarization.
Recently were described scavenger receptors on LSECs, responsible of blood waste
soluble macromolecular clearance (Murphy J et al., 2005). Two members of this protein
family are Stabilin 2 (Hansen B et al., 2005) (Politz O et al., 2002) and LYVE-1 (Banerji S et
al., 1999), which clears serum hyaluronic acid (AH) and thereby control the viscosity of the
blood. In human liver cirrhosis, was showed a decreased LYVE-1 receptor (Mouta Carreira C
et al., 2001), mechanism that may be responsible for increased serum AH observed in these
patients (Gudowska M et al., 2015).

Age-Related Changes and Clinical Implications
The specific change in morphology of the liver sinusoids due to high age alone has
therefore been termed age-related pseudocapillarization, to distinguish it from capillarization
associated with hepatic diseases (Le Couteur D et al., 2001).
In human (McLean A et al., 2003), baboon (Cogger V et al., 2003), mouse (Warren A et
al., 2005) and rat (Le Couteur D et al., 2001) aging is associated with a gradual thickening of
the sinusoidal endothelium, reduced LSECs porosity, and increased depositions of basal
lamina and collagens in the space of Disse. The HSCs become highly fat-engorged in normal
old liver, but in contrast to in liver fibrosis, they remain quiescent (Warren A et al., 2011).
Decreased LSEC porosity has implications for vascular disease in extrahepatic organs.
Reduced numbers and size of LSEC fenestrae have been linked to postprandial
hypertriglyceridemia (Fraser R et al., 2012), a common condition in old age (Krasinski S et
al., 1990), and a risk factor of atherosclerosis. Normally sized fenestrae allow passage of most
triglyceride rich chylomicron remnants, whereas reduced LSEC porosity impairs his passage
(Hilmer S et al., 2005), increasing the risk of lipid deposition in other vascular beds and
development of atherosclerosis (Le Couteur D et al., 2002). Although it was established
several years ago that OxLDL is cleared from the circulation by endocytosis in both LSECs
and Kupffer cells, recent studies have shown that the type of OxLDL that is mostly present in
the blood of individuals with atherosclerosis, that is, low-modified OxLDL, is taken up by
LSECs only (Li R et al., 2011).
Fenestration is also necessary to permit the passage of high-clearance drugs. It allows not
only free but also protein-bound drug to enter the space of Disse. In one pass through the
liver, free drug can be taken up by hepatocytes, the free and bound fractions can reequilibrate,
and more of the newly formed free drug can be cleared. Thus fenestration allows hepatic
clearance to exceed the free fraction for high-clearance drugs. In capillarization, protein-
bound drug is unable to enter the space of Disse, which impairs clearance of protein bound,
high-clearance drugs. Two consequences of pseudocapillarization are predicted to play an
important role in the age-related decrease in drug metabolism: the impairment to clearance of
high clearance, protein-bound drugs and the decrease in oxidative drug metabolism (e.g.,
P450 metabolism) (DeLeve L, 2007).
Finally, it has been suggested that the long-term effects of gut-derived toxins (i.e.,
ethanol and endotoxins), nicotine, and oxidants, which can all cause defenestration (Braet F,
2002), are involved in the development of pseudocapillarization (Le Courter D et al., 2002).
REVERSAL TO DIFFERENTIATED PHENOTYPE OF LSECS AND

QUIESCENT PHENOTYPE OF HSC
When differentiated LSECs are added to culture-activated HSCs and LSEC
differentiation is maintained with a soluble guanylate cyclase (sGC) activator, activated HSCs
revert to quiescence (Figure 4) (DeLeve L, 2015). The paracrine mediator for the paracrine
effect of LSECs on HSC activation has not been identified, but NO does not promote
reversion of activated HSCs to quiescence. When differentiated LSECs are added to activated
HSCs and no measures are taken to preserve LSEC differentiation, LSECs capillarize and

HSCs remain activated. Thus, differentiated LSECs promote reversion of activated HSC to
quiescence, but capillarized LSECs do not (DeLeve L, 2015).
Figure 4. Reversal to differentiated phenotype of liver sinusoidal endothelial cell (LSECs) and
quiescent phenotype of hepatic stellate cell (HSC). When differentiated LSEC are added to culture-
activated HSC and LSEC differentiation is maintained with a soluble guanylate cyclase (sGC) activator,
activated HSC revert to quiescence.
The effect of normalizing LSEC differentiation was also examined in a model in which
early cirrhosis (3 weeks of TAA) was induced, followed by treatment with an additional 3
weeks of thioacetamide plus an sGC activator (DeLeve L, 2015). Treatment with the sGC
activator completely normalized LSEC fenestration, which then promoted reversal of HSC
activation to quiescence and some degree of apoptosis, and completely prevented any further
progression of cirrhosis despite ongoing treatment with TAA.
To summarize, differentiated LSECs prevent HSCs activation and promote reversion to
quiescence, but capillarized LSECs do not (Figure 1, bottom). The mediator for this has not
been identified. Taken together with the observation that capillarization precedes fibrosis,
these findings suggest that capillarization is at least permissive for fibrosis.
These in vivo studies demonstrate that pharmacologic therapy that stimulates downstream
in the VEGF-NO-sGC-cGMP-protein kinase G pathway restores LSECs fenestration in
cirrhosis. Restoration of LSECs differentiation accelerates regression of fibrosis after
discontinuing a fibrotic stimulus and prevents progression of cirrhosis despite an ongoing
fibrotic stimulus.

RECOVERY OF LIVER FUNCTION IN HUMAN CIRRHOSIS?

In humans, defenestration (Horn T et al., 1987) and subsequent capillarization of hepatic
sinusoid (Schaffner F et al., 1963) lead to liver switch from an open (sinusoid) to a closed
(capillary) circulation. These changes would make the liver lose its bidirectional filter
function between the sinusoid and the hepatocytes and vis versa (Martínez Hernández A et
al., 1992) (Fraser R et al., 1995). As a consequence, capillarization of LSECs may therefore
be a major contributor to hepatic failure in cirrhosis (Schaffner F et al., 1963) (Schmid R,
1991) (Martínez Hernández A et al., 1992) (Braet F et al., 2002). Recovery of differentiated
LSECs and quiescence HSCs phenotypes, allow the restoration of bidirectional filter liver
(Martínez Hernández A et al., 1992) (Fraser R et al., 1995) and thus the liver function
(Schaffner F et al., 1963) (Schmid R, 1991) (Martínez Hernández A et al., 1992) (Braet F et
al., 2002) in human cirrhosis.
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Chapter 4
HYPERAMMONEMIA
AND HYPERAMMONEMIC CONDITIONS
Pablo A. Souto and Lisandro Orbea
ABSTRACT
Ammonia is considered a key molecule in altering normal homeostatic functions of
the liver with subsequent organ failure, leading to neurological and neuromuscular
disorders, neurotransmission with alterations in sleep patterns and cognition. The gut and
intestinal bacteria are the largest producers of ammonia, as a direct result of metabolizing
of amino acids, and have an active contribution to the development of hyperammonemia
complications worsen the condition in patients with acute or chronic liver disease.
Patients with liver injury concomitantly decrease the ability of detoxifying ammonia into
urea, shifting this metabolic pathway to muscular system and astrocytes in the central
nervous system (CNS). Bioenergetic alterations could be considered as the main reason
for ammonia toxicity that trigger cell damage, and ultimately leads to cell death. There
are many clues that ammonia interferes with many cellular processes such as ion
homeostasis, cytoskeletal organization, neurotransmitter release/absorption and energy
metabolism. However, it is unclear so far how ammonia affects these processes.
A controversial issue is whether astrocytes and/or neurons are the targets of
ammonia inducing sublethal or lethal cell damage. The focus of this chapter considers the
different sources of ammonia in the body and how the increase in ammonia systems leads
to cell injury and cell death in different tissues.
INTRODUCTION
As it is known, hyperammonemia and hyperammonemic conditions in vitro trigger
multiplicity of pathways involved in human pathology, both studied in animal models and in
cultured cells, respectively. Among the different pathways triggered by hyperammonemia that
could be cited, changes in energy metabolism leading to cell injury, oxidative/nitrosative state
and a neuroinflammatory condition were already demonstrated. All together have impact in
neurological and neuromuscular neurotransmission disorders, alterations in sleep patterns and

80 Pablo A. Souto and Lisandro Orbea
cognition. Even though the etiology varies grossly, the hepatic failure arises when there is a
dramatic reduction in the hepatic metabolic capacity, increasing plasma ammonia
concentration, among other substances. Ammonia, from the last 100 years has been
considered a key molecule in the derangement of the normal homeostatic functions of the
liver with subsequent organ failures, including among the major complications, acute or
chronic hepatic encephalopathy. This is a condition the poses an imminent threat of death. In
acute or fulminant liver failure, the central nervous system is injured, and astrocyte swelling
represents its basic feature. From a metabolic point of view, the prevalence of an oxidative
state, the possible inhibition of alpha-ketoglutarate dehydrogenase and the mitochondrial
functional impairment severely affects cerebral energy metabolism. In chronic HE, changes
can be observed in calcium signalling, mitochondrial membrane potential and long-term
potential expression, NMDA-cGMP and peripheral benzodiazepine receptor alterations,
changes in the mRNA and protein expression, and redistribution in the cerebral blood flow.
Recently a new broad way was opened, that is the hyperammonemia relation with cell injury
in both, sub lethal and lethal damage. This review will focus in that issue where, to the best of
our knowledge, more research is needed in order to clarify the role of hyperammonemia in
cell damage, especially in brain tissue.
Gut, the Main Ammonia Source
Virtually, all human tissues produce ammonia as a consequence of amino acid

metabolism. Its accumulation can exerts toxicity in different tissues, mainly the central
nervous system. In order to avoid ammonia toxicity, several detoxification mechanisms can
be seen in the evolution of biology, involving different animal species with wide ammonia
detoxification mechanisms. Gut is the major glutamine consumer and ammonia producer
organ in the body. Ammonia generation derives from the consumption of glutamine and
glutamate as the most important oxidative fuel for enterocytes and colonocytes (P.
Newsholme et al. 2003). In Gut epithelial cells, this amino acids have several roles, i.e.,
represents substrates for another amino acids synthesis (Wu 1998), oxidative fuel (Blachier et
al. 2009), nucleotide and nucleid acid synthesis (E. A. Newsholme and Carrié 1994),
glutathione production (Reeds et al. 1997), amino sugars, NAD+, N-acetylglucosamine and
N-acetylgalactosamine synthesis (Reeds and Burrin 2001), etc. It’s recognized that glutamine
administration in patients with different diseases, can improve gut function and reduce
bacterial translocation, reestablishing normal epithelial permeability (Panigrahi et al.) (Neu,
DeMarco, and Li 2002).
The main energy source for enterocytes is blood circulating glutamine. On the other
hand, colonocytes can take up metabolites from lumen (over apical pole) and from vascular
bed (on basal pole). In these cells (but not in enterocytes), short-chain fatty acids produced by
luminal bacterial metabolism are also a relevant metabolic fuel, as important as L-glutamine.
Once in epithelial cells, glutamine is substrate of the enzyme Glutaminase, generating
one molecule of ammonia and glutamate. At the same time, Glutamate can be substrate of
glutamate dehydrogenase or transaminases, depending on cell metabolic requirements. The
metabolic residues of glutamine-glutamate consumption by gut epithelial cells are carbon
dioxide, ammonia and L citrulline, a non-essential amino acid, which cannot be incorporated
by food. This is the reason why L-Citrulline is considered a specific marker of functional

Hyperammonemia and Hyperammonemic Conditions 81
absorptive mass of enterocytes (Vecino López et al. 2013). Once generated, these substances
are released into portal bed blood. Another extra source of ammonia is luminal bacteria
microbiota, mainly those urease positive bacteria.
The anatomy of gut-liver axis, at physiological conditions, favors ammonia metabolism
into urea, by urea cycle into hepatocytes, once Portal blood reaches liver circulation. In this
way, ammonemia is maintained under physiological levels (30-50 mg/dl) (Meijer, Lamers,
and Chamuleau 1990).
Hepatocellular dysfunction due to liver disease, results in an impaired clearance of
ammonium and in its inter-organ trafficking (Wright et al. 2011, Olde Damink, Jalan, and
Dejong 2009). Besides acute or chronic liver failure, specific genetic disorders are also
characterized by hyperammonemia accompanied by liver and brain disorders with different
degrees of severity.
Congenital Disorders That Trigger Conditions of Hyperammonemia
The urea cycle in humans comprises six enzymes and a deficiency in any one of them
causes a urea cycle disorder characterized by hyperammonemia, HE and respiratory alkalosis
(Flannery, Hsia, and Wolf). Clinical disorders have been described involving defective urea
cycle enzymes: ornithine transcarbamylase deficiency (OMIM 311250), carbamoyl phosphate
synthetase I deficiency (OMIM 237300), argininosuccinate synthetase deficiency (OMIM
215700), argininosuccinate lyase deficiency (OMIM 207900), and arginase deficiency
(OMIM 207800). A sixth cause of hyperammonemia is N-acetlyglutamate synthase (NAGS)
deficiency (Flannery, Hsia, and Wolf) (OMIM 237310). NAG is also a cofactor of N-
Carbamoyl Phosphate Synthetase I (CPS1), promoting the function of this enzyme.
Therefore, clinical signs of CPS1 and NAGS deficiencies are indistinguishable. Two clinical
presentations of NAGS deficiency are recognized: an acute neonatal hyperammonemia form
and a delayed onset form (Häberle et al. 2003). NAGS deficiency also presents with
irritability and hyperammonemia leading to coma and death if untreated. Hyperammonemia
due to NAGS deficiency is an autosomal recessive disorder, and its mutations are distributed
throughout the coding region (Caldovic, Morizono, and Tuchman 2007). The majority of
mutations are missense, however nonsense and splice site mutations are known as well. A
regulatory mutation (c.-3064C > A) in an enhancer region 3kb upstream of the NAGS coding
sequence has also been identified, and patients homozygous for c.-3064C > A responded well
to NCG treatment, showing enhanced ureagenesis (Heibel et al. 2011).
The hyperinsulinism–hyperammonemia syndrome is caused by mutations in the
glutamate dehydrogenase gene that impair the control of enzymes, namely NAGS, CPS 1,
OTC, argininosuccinate synthetase, argininosuccinate lyase, and arginase (Stanley et al.
1998).
At this point it is necessary to ask how hyperammonemia exerts its cell toxicity.
Adquired Disorders That Trigger Conditions of Hyperammonemia
The incomplete clearance of ammonium into urea presented in liver diseases (Adeva et
al. 2012) contributes to hyperammonemia. One of the most severe complications of acute or

chronic liver failure is hepatic encephalopathy (HE) and ammonia appears as a key factor in
its pathogenesis (Ferenci et al. 2002, Kundra et al. 2005).
The gravity of presentation can differ among patients, from almost asymptomatic to coma
and death. Also, several patients can be considered normal during a conventional
semiological evaluation, but obtaining abnormal performance during psychometric test, this
subclinical presentation is called “minimal HE” (MHE) (Kundra et al. 2005). This
neurological alteration can be persistent or intermittent (spontaneous or precipitated for
different circumstances), but the data indicates that between two clinical episodes, patients
don’t return to its previous neurological, presented a MHE. There are factors that can be
viewed with the ability to identify clinical situations with a higher possibility of developing
brain edema, such as deep encephalopathy, hyper acute liver failure (acetaminophen-induced
ALF), severe hyperammonemia, younger age and infection (Vaquero et al. 2003). Currently
an area of controversy is to establish conclusively whether the percentages of deaths could be
attributable to cerebral edema or to multiorgan failure. So, brain edema is a key concept for
understanding the pathophysiology of ALF (Bernal et al. 2007).
In acute liver failure (ALF), the progression of HE is associated with an increased risk of
brain edema that could lead to brain herniation, a major cause of death (Bernal et al. 2007).
Therefore it is clear that when the ALF is complicated by HE, the life risk is increased
because of the development of brain edema, an unique complication of ALF. The severity of
HE is also associated with difference in survival. The Trojan horse theory explains, at least in
part, the pathophysiology of brain edema. In order to reduce ammonia toxicity on neurons,
Astrocytes take up ammonia from blood by their feet, and conjugate it with glutamine by
glutamine synthase enzyme. This glutamine accumulated into astrocytes exerts osmotic
effect, establishing water entrance in cell.
On the other hand, chronic liver failure (CLF) shows minimal to mild edema located
surrounding the blood-brain barrier. The edema in CLF has very different consequences than
in ALF (Laleman et al. 2011). As astrocyte swelling is the neuropathological characteristic
change in acute HE, the hallmark in brain in CLF consists in the so-called Alzheimer type II
astrocytes. These astrocytes, were described in both human and experimental models of
advanced HE and recently in a model of the subclinical stage, minimal HE (mHE) (Tallis et
al. 2014).
Patients with liver cirrhosis, a chronic and inflammatory liver disease, may develop
Portal hypertension, splanchnic hyperdynamic syndrome, and collateral veins that shunt liver
circulation, diverting Portal blood with high gut derived ammonium content, directly to the
systemic circulation (Vorobioff, Bredfeldt, and Groszmann 1983, Møller, Bendtsen, and
Henriksen 2001).
As was mentioned above, the gut is the major glutamine consumer and ammonia
producer organ in the body (van de Poll et al. 2008). Ammonia production derives from the
consumption of glutamine and glutamate as the most important oxidative fuel for enterocytes
and colonocytes (Wasa et al. 2004). A direct consequence of this amino acids metabolism is
that the gut is the major ammonia source in whole body, once generated, is released into
portal bed. Intestinal bacteria, also can represent and extra source of ammonia production.
The most relevant bacteria are those with urease enzyme such as enterobacteriaceae. In
cirrhosis, small intestinal bacterial overgrowth (SIBO) (Steffen and Berg 1983) and dysbiosis
(Kakiyama et al. 2013) (Rogers et al. 2013) can be observed. This modification in gut
microbiota is produced for various circumstances (Figure 1).

Figure 1. Pathogenesis of bacterial translocation in cirrhosis. Bellot, P et al.
First, gut motility is reduced, and acid gastric secretion in stomach is reduced as a
consequence of gastric enteropathy, both phenomena influence SIBO (Bellot, Francés, and
Such 2013). Moreover, an impairment in bile acids production and secretion by the damaged
liver was demonstrated (Kakiyama et al. 2013). The amount and the profile modification of
bile acids are due a reduction in secondary fecal bile acids, a bacteriostatic compound.
Another factor involve on SIBO development in cirrhosis, is the alteration in local
immunological system, mediated by a low cellular and humoral components of gut immune
system (Bellot, Francés, and Such 2013). SIBO generates an increase in ammonia gut
production, generating an active role of gut in the predisposition in HE development. In
addition of ammonia generation, luminal bacteria can generate other substances such as
phenols, mercaptans, benzodiazepine-like compounds and short and medium chain fatty
acids, also implicated in pathogenesis of HE (F. J. Zieve et al. 1974) exerting a synergic effect
on central nervous system alteration, by modulating the synaptic processes. For example, the
neurosteroid allopregnenolone is elevated in patients with HE. This steroid can enhance the
effects of GABA on its specific receptors (mainly in GABA-A), increasing it neurodepressive
function (Williams 2007). On the other hand, endogenous benzodiazepines produced by
intestinal bacteria, can augment the opening time of GABA-A receptor after its interaction.
The levels of this benzodiazepine like compounds are also elevated in blood of HE patients,
exerting a potentiation effect with neurosteroids named before (Norenberg, Itzhak, and
Bender 1997). Other molecules involved in HE who are increased in serum mercaptans, short
and medium fatty acids (L. Zieve, Doizaki, and Zieve 1974), and aromatic amino acid and
manganese, while others are directly increased in CNS as octopamine (false
neurotransmitters), GABA (Zeneroli et al. 1982, Baraldi and Zeneroli 1982), indols and
oxindoles (Mousseau and Butterworth 1995, Fischer and Baldessarini 1971). Several of these

substances elevated in serum are generated by flora gut and increased in cirrhosis because a
decreased metabolisms of those molecules during chronic liver failure. These substances can
be toxics “per se” or can generate a neurological impairment by blocking or altering the
normal neurotransmission process. Another finding in cirrhosis, involves an increase in the
expression of Glutaminase in enterocytes (Romero-Gómez et al. 2004) increasing ammonia
generation, from glutamine. Also an increment in absorption rate of intestinal epithelia, was
observed, as a consequence of the tight junction’s disruption between enterocytes (Pascual et
al.), facilitating para cellular passage of substances. Hence, all these factors can contribute to
a major absorption of all substances generated into gut lumen, worsening even more the
prognosis of cirrhotic patients, due an increase in the absorption rate of ammonia and other
synergic molecules and also by an increase in bacterial translocation, generating a
predisposition for infection development, that can trigger HE by the synergic role of pro
inflammatory cytokines in the development of neurological impairment, as was mentioned
above. So, now it’s clear why there is an active contribution of the intestine in the
development of complications, in those patients with chronic liver disease, exerting a synergic
role with the reduction in liver detoxification capability.
Similar findings were described in a non-cirrhotic model of Portal Hypertension, by
partial portal vein ligature (PVL). In this situation, fourteen days after partial stenosis, distal
ileum showed impaired contractile response to Ach and KCl, and reduced expression of
proteins markers of occlusive and adherents junctions, such as Zonulin 1 (ZO-1) and β-
catenin (β-cat), respectively. Also, a reduction in number of cells related with mucosal
immune system was observed (data not published yet). All these findings named before
suggest an active role of gut in the development of complications in portal hypertension even
with liver parenchyma indemnity.
Those patients with liver injury concomitantly decrease the capability of detoxification of
ammonia to urea, shifting this metabolic pathway to the muscular system and astrocytes in the
central nervous system (CNS) (Olde Damink, Jalan, and Dejong 2009). Under
hyperammonemic conditions, skeletal muscle acquires a main role in ammonia detoxification,
generating glutamine from ammonia and glutamate. This capability is important due to it high
muscle mass in whole body, and its available glutamine synthase content. Somehow
surprisingly, the skeletal muscle, considered an ammonia metabolizing tissue, under liver
disease evolves to atrophy called sarcopenia. Histological analyses of striatal muscle cells
under hyperammonemic conditions undergo similar membrane and organelles pathological
changes than neurons in HE (data non-published yet), can trigger the death of muscle cells.
As it can be seen, hyperammonemia exerts a wide range of actions, from sub-lethal cell
damage to cell death in its different patterns.
So, astrocytes, skeletal muscle cells and kidney remain as the unique metabolic pathway
of ammonia and enterocytes as the main source. In this way these specific cells are the targets
to study cell ammonia metabolism in hyperammonemic conditions.
Hyperammonemia and Its Cytotoxic Effects
Multiple factors are involved in the hyperammonemia pathophysiology, such as central

and neuromuscular neurotransmission disorder, alterations in sleep patterns and cognition due
cellular damage. The aim in this chapter is to try to understand how under hyperammonemic

condition, the ammonia effects and its role in cellular injury. At the moment, many
publications have shown that ammonia affects many cellular process, such us ion
homeostasis, lysosome function, cytoskeleton organization, neurotransmitter release/uptake
and energy metabolism. The last mentioned, could be considered a key to understand how
ammonia triggers cell damage, and finally leads to cell death.
Alterations of energy metabolism due to mitochondrial dysfunctions have been
considered a pathogenic factor in different pathologies such as Hepatic Encephalopathy (HE)
and Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS). In
spite of many differences, these two pathologies share an important part of its
pathophysiology such as BBB abnormalities and alterations in brain mitochondrial respiratory
chain. MELAS is a common and widely studied maternally inherited mitochondrial disease
that is frequently associated with the m.3243AG point mutation in the mitochondrial tRNA
LeuUUR gene (Finsterer 2007). The clinical phenotype is multisystemic but the triad of lactic
acidosis, seizures and stroke-like episodes remains crucial to the diagnosis and reflects the
complex and unique pathogenesis of this syndrome (Kaufmann et al. 2004).
An alteration in brain bioenergetics has also been suggested as an important factor in
ammonia neurotoxicity (Cooper and Plum 1987). Many studies had shown that high levels of
ammonium induce mitochondrial morphological and functional changes, such as
mitochondrial permeability transition and dissipation of the mitochondrial membrane
potential (ΔΨm) (Felipo and Butterworth 2002, Reddy, Rao, and Norenberg 2008, Lores-
Arnaiz et al. 2005). It is also well known that hyperammonemia triggers neurotoxicity in
patients, reaching a state of hepatic encephalopathy (HE) due to its effects on brain energy
metabolism associated with altered mitochondrial function (Albrecht and Norenberg 2006).
Because all of these, mitochondria are considered the main target of ammonia, therefore, the
study of how ammonia affects mitochondrial function could improve the understanding of
many pathology where ammonia is involved and its mechanism of cell damage.
How ammonia goes into mitochondria is not completely clear. The theory of “The
Troyan Horse” by Albrecht and Norenberg propose that Glutamine is a “stealth” carrier of
ammonia into the mitochondria (Rama Rao and Norenberg 2014, Murthy et al. 2001).
Glutamine enters mitochondria via a histidine-sensitive glutamine carrier, which is
potentiated by ammonia. Glutamine is then hydrolyzed by phosphate-activated glutaminase,
located in the inner mitochondrial membrane, yielding glutamate and ammonia. Thus, the
generation of mitochondrial ammonia may reach high levels, reducing the pH into the
mitochondria, inducing reactive oxygen species (ROS) and mitochondrial membrane
permeability transition pore (mPT) with the consequence of morpho-functional oxidative
damage of the mitochondria and its respiratory chain. Then, the mPT-ROS induced,
characterized by the increase in the permeability of the inner mitochondrial membrane, results
in a collapse of mitochondrial membrane potential. This culminates in mitochondrial
swelling, defective oxidative phosphorylation, diminish of ATP synthesis and an increased
ROS production. These effects were observed in cultured astrocytes (Sinke et al. 2008) as
well as in vivo model (Lores-Arnaiz et al. 2005). These evidences would indicate that the
damage originated from the mitochondria, may initiate one or more mechanism of cell death,
regulated as apoptosis o mitophagy or unregulated like necrosis.
Other factors could be involved in ammonia toxicity affecting cellular homeostasis,
leading to cellular damage and cell death. Several studies demonstrate the important role of

ROS as a trigger in ammonium cytotoxicity. Also, it is well established that ammonia induced
oxidative/nitrosative stress through N-methyl-d-aspartate (NMDA)-receptor and Ca2+-
dependent mechanisms (E Kosenko et al. 2000, Hermenegildo, Monfort, and Felipo 2000).
This oxidative stage affects cellular energy metabolism, associated with alterations of
mitochondrial function, such as in reductions of brain ATP concentrations (E Kosenko et al.
1994). An excessive activation of NMDA receptors increases intracellular calcium which
binds to calmodulin and increase the formation of nitric oxide by the activation of neuronal
nitric oxide syntethase, leading to activate neurotoxic pathways, neuronal degeneration and
cell death. It has been demonstrated that large doses of ammonium in animals lead to
activation of NMDA receptors in brain, whose effect is prevented when the receptors were
previously blocked (E Kosenko et al. 1999). The activation of NMDA receptors lead to one
possible mechanism by which ammonia could induce to an excessive activation of NMDA
receptor, by increasing the concentration of glutamate, an agonist of this receptor in brain.
Also the excessive activation of NMDA receptor leads to ATP depletion, decreasing the
uptake of extracellular glutamate.
Several works have demonstrated a linkage between ammonia and different mechanisms
of cell death, such as intrinsic apoptosis, autophagy, but also necrosis in the case of metabolic
catastrophe. One in vitro study, using developing brain cell culture, has demonstrated that
ammonia induces neuronal and oligodendroglial death, with caspases and calpain activation
(unpublished data). Probably due to calpain activation, ammonia caused the cleavage of the
cyclin-dependent kinase 5 activator, p35, to p25, the cdk5/p25 complex known to lead to
neurodegeneration (Cagnon and Braissant 2008). A recent study in an experimental model of
minimal HE (mHE) from our laboratory has shown that astrocyte death is associated with
mitochondrial dysfunction (Bustamante et al. 2011). A higher ratio of the Bcl-2 family
members Bax/Bcl-xL in the outer mitochondrial membrane and cytochrome c release
revealed the activation of apoptosis. Moreover, an increase of 2-3 times in the number of
TUNEL-positive astrocytes, simultaneously marked with GFAP was also observed in the
hippocampal area. So, all of these data indicate that ammonia-neuronal damage ends in
intrinsic apoptosis.
We have seen in Organotypic Hippocampal Slice Culture (OHCS) treated with 5 -10mM
of ammonium chloride, neural but not astrocyte cell death (data not published), suggesting
that ammonia induces neuronal cell death. The treatment of OHCS with ammonium chloride
showed a drastic increase in the number of Propidium Iodide (PI) positive nuclei prior to
fixation, indicating a loss of membrane integrity and reduction of cell viability. While cell
death was most extensive with 10 mM. NH4Cl, 5 mM already induced a clear toxic effect in
the hippocampus. The slices co-stained with PI and antibody markers for neurons (NeuN) or
astrocyte marker (GFAP) shown that neurons were mostly affected (Figure 2). This result was
surprising as it is known that astrocytes, rather than neurons are the primary target of
ammonium toxicity.
On the other side, in an in vivo model of acute ammonia intoxication, Kosenko et al.
(Elena Kosenko et al. 2007) did not observe caspase-9 or caspase-3 activation, cytochrome C
release to cytosol and the mitochondrial membrane potential remain unaltered in non-synaptic
brain cell, indicating that ammonia did not induce apoptosis or permeability transition pore
(PTP). Authors did not find apoptotic nuclei in the brain, but they reported disturbances in the
mitochondrial electron transport chain in brain, that probably contribute to the reduced state 3
respiration.

While autophagy is mostly a survival mechanism, recycling vital components to maintain

cellular viability, under certain conditions, autophagy can also promote so called type II cell
death, for example by stimuli such as hypoxia, reactive oxygen species, DNA damage, ER
stress, protein aggregates, damaged organelles, or intracellular pathogens. In this case
autophagy is often induced when mitochondria fail to maintain ATP production, a process
that not only occurs during starvation (Levine and Yuan 2005) but also when mitochondria
are damaged (Elmore et al. 2001) (Gozuacik and Kimchi 2004) and the outer mitochondrial
membrane is permeabilized (MOMP). These processes constitute one of the hallmarks of
imminent apoptotic or necrotic cell death (Kroemer, Galluzzi, and Brenner 2007).
Thus, important common regulators of apoptosis and autophagy are mitochondrial
dysfunction and ROS, which have been both suspected to be involved in the pathophysiology
of HE (Albrecht and Norenberg 2006). For that reason, autophagy could play and important
role in hyperammonemic conditions, such as a survival process trying to rescue the cell from
death or to lead it to die.
Ammonia not only affects the CNS, but also, in the case of liver disease ammonia
detoxification will take in kidney, muscle and brain, so these organs could also be a target for
ammonia. Unpublished data from our laboratory on skeletal muscle and central nervous
system cells in hyperammonemic conditions showed different patterns of cell death. Probably
the elapsed time for achieve the specific tissue cells and the specific characteristic of each
tissue determine different pathways, for example, a caspase dependent or independent on
intrinsic apoptosis; and/or a death receptor or a dependence receptors in extrinsic apoptosis.
Besides, preliminary data indicates that a possible necrosis route could be also related. It
could also be speculated that the increased consumption of ATP due to activation of Na+-K+-
ATPase and decreased synthesis of ATP in mitochondria due to impairment of calcium
homeostasis, could be the trigger of a state of metabolic and energetic catastrophe that would
lead to necrosis.
Ammonia and Manganese
Manganese (Mn) was considered to be neurotoxic 170 years ago when workers employed
in grinding black oxide of Mn developed an unsteady gait and muscle weakness (Atsushi
Takeda 2003). Since that time many cases of Mn neurotoxicity (manganism), a neurologic
disease characterized by psychological and neurologic abnormalities, have been reported,
particularly in miners, smelters, welders, and workers involved in the alloy industry (Keen,
Fransson, and Lönnerdal 1984).
Manganese is also an essential element that is normally excreted via the hepatobiliary
route (Davidsson et al. 1991) and has a key role in the normal functioning of several
enzymes including mitochondrial superoxide dismutase, glutamine synthetase, and
phosphoenolpyruvate carboxykinase (Aschner, Vrana, and Zheng). Manganese acts as a
cofactor for many enzymes and therefore, it plays important biological functions (Atsushi
Takeda 2003). High accumulation of Mn (Siger-Zajdel and Selmaj 2002) in the basal ganglia
produces an irreversible neurological syndrome similar to Parkinson's disease (MIM 168600)
(manganese-induced Parkinsonism). Typically, patients exhibit extrapyramidal changes that
include hypokinesia, rigidity and tremor (Spahr et al. 1996) (Pomier-Layrargues, Spahr, and
Butterworth 1995).

Patients with CLF have been shown to exhibit increased serum and brain levels of Mn
and display many of the clinical and pathological features associated with manganese toxicity
(Spahr et al. 1996) (Hauser et al. 1996).
It has also been demonstrated in a rat model of cirrhosis an excessive deposition of Mn in
brain (A Takeda, Sotogaku, and Oku 2002). Quadri et al. (Quadri et al. 2012) have studied
two unrelated families with hepatic cirrhosis plus an extrapyramidal motor disorder and
polycythemia. A manganese primary disorder was suspected, and it was diagnosed when a
recessive mutation in SLC30A10 (MIM 611146), was demonstrated. This gene plays a
pivotal role in the transport of Mn and the mutation is the ethiology of Manganese primary
liver pathology.
Our laboratory has reported (Prestifilippo et al. 2008) increased deposits Mn, NOS
activity and the release of gamma-aminobutyric acid (GABA) in the hypothalamus in rats
with prehepatic portal hypertension. This is also a model of minimal hepatic encephalopathy
with moderate hyperammonemia. Considering these results, it could be speculated that
ammonia and Mn have a synergic action that should be explored deeply.
CONCLUSION
Much work has been done, and it is possible to elucidate a mechanism of how ammonium
induces cell death (Figure 3); likewise, the relationship between hyperammonemia and cell
death opens a broad avenue, especially to the central nervous system. Although many studies
from several laboratories have uncovered a number of factors and pathways that appear to be
critically involved in the pathogenesis of hyperammonemia, the mechanism by which
ammonia induces cell death remains unclear. Even more, the studies must be focused on
evaluating whether ammonia is toxic per se, or if its harmful effects are the result of toxic
mediators, such as ROS production and mitochondrial damage.
Viewing new and important knowledge in the field of sublethal and lethal cell injury, and
because of the relevance of hyperammonemia in congenital disorders, ALF and CLF, a new
deeper molecular vision is necessary to clarify and create opportunities for new therapeutic
strategies.
OHCS were first stained with PI, washed and then fixed and permeabilized and stained
with an antibody against NeuN (neuronal marker). Control (a-a´´), 10 mM ammonium-treated
(b-b´´) hippocampal culture. Magnification of the pyramidal cell layer: Control (a-a´´´),
ammonium-treated (b-b´´) hippocampal culture. Arrows indicate cell NeuN+/PI+
colocalization (yellow staining). Scale bar = 20 µm (data not published).

Figure 2. NH4Cl induce cell death (PI staining) preferentially in the neuronal cell population (NeuN) of
the OHCS.
Figure 3. Cell death mechanisms triggered by Hyperammonia.

Ammonio enters the cell which is used by the glutamate to produce glutamine by
glutamine synthetase. Glutamine enters the mitochondria and is metabolized to ammonio and
glutamate. High levels of ammonio into mitochondrial matrix affect its pH, increase
ROS/NOS production, induce mitochondrial membrane permeability lead to the collapse of
mitochondrial membrane potential resulting fall ATP production, release of Ca+2 into the
cytosol. These mitochondrial alterations are common signals for different cell death
pathways, such as intrinsic apoptosis (activated by the release of cytochrome C) and
mitophagy. Necrosis may also occurs by increasing water intake due to the failure of the
ATPase Na+/K+ pump leading to cell swelling (Oncosis). Also the ROS produces cytoskeleton
and plasma membrane damage by the activation of enzymes, enhanced by the releasing Ca+2
that increase cellular degradation.
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Chapter 5
CANNABINOIDS:
IMPLICATIONS IN LIVER DISEASE
Laura Caltana and Alicia Brusco
ABSTRACT
Cannabinoid receptors, their endogenous ligands and the enzymes for synthesis and
degradation are the components of the endocannabinoid system. Furthermore, there are
both natural cannabinoids, such as phytocannabinoids including tetrahydrocannabinol
(THC) or cannabidiol, and synthetic cannabinoids.
Cannabinoid receptor type 1 (CB1R) is expressed in liver including hepatocytes and
biliary epithelial cells while Cannabinoid receptor type 2 (CB2R) is expressed in
hepatocytes in patients with cirrhosis.
Endocannabinoids (eCBs) and CB1R are up-regulated in experimental model liver
disease, as hepatocarcinoma. Phytocannabinoids and eCBs have anti-proliferative effects
in cancer cells, inhibiting proliferation and stimulating apoptosis in cancer cell lines.
Experimental data remain controversial about cannabinoids and hepatic protection.
For example, while CB1R is related in the development of steatosis, CBR2 shows a
protective effect on the damaged liver. Cannabinoid treatment of liver pathologies should
also consider its implications in psychiatric disorders due to its psychotropic effects. In
this context, Cannabidiol (CBD) is a promissory compound without this psychotropic
impact.
Keywords: endocannabinoids, cannabinoid receptor, liver disease
ABBREVIATIONS
2-AG: 2-arachidonoyl glycerol
AEA: Anandamide
CB1 mRNA: cannabinoid receptor type 1 messenger RNA

98 Laura Caltana and Alicia Brusco
CB1R: cannabinoid receptor type 1

CB2 mRNA: cannabinoid receptor type 2 messenger RNA
CB2R: cannabinoid receptor type 2
CBD: Cannabidiol
CBR: cannabinoid receptors
eCBs: endocannabinoids
FAAH: fatty acid amide-hydrolase
HSCs: hepatic stellate cells
THC: tetrahydrocannabinol
Δ9-THC: Δ9-Tetrahydrocannabinol
ENDOCANNABINOID SYSTEM
Marijuana and its compounds have been used for centuries as recreational drugs, due to
their psychotropic effects, and as therapeutic drugs for the treatment of different disorders
(rheumatism and abdominal symptoms) due to their analgesic, antiemetic and appetite-
stimulating properties. In particular, they have been used in India and China with medical
purposes.
Δ9-tetrahydrocannabinol (Δ9-THC), a phytocannabinoid and the main psychoactive
constituent of the marijuana plant was isolated in 1964. The following years, research focused
on elucidating the properties of these compounds and also on generating synthetic
cannabinoids with similar effects.
The endocannabinoid system is composed of cannabinoid receptors (CBR), their
endogenous ligands (endocannabinoids - eCBs) and proteins for synthesis and degradation
(Pacher et al., 2006).
Over the last decades, experimental and clinical evidence has demonstrated that the
regulation of the endocannabinoid system may be effective in the treatment of pain (Ablin et
al., 2016), glaucoma (Pinar-Sueiro et al., 2011; Cairns et al., 2016), stroke (Caltana et al.,
2015) and neurodegenerative disorders such as Parkinson’s disease (Connolly and Lang,
2014; Carroll et al., 2012) and multiple sclerosis (Novotna et al., 2011; Zettl et al., 2016;
Russo et al., 2016) among others.
CB1R and CB2R are receptors with a seven-transmembrane domain structure and are
coupled to G protein. CB2R have 68% homology with the amino acid sequence in the
transmembrane domains of CB1R, and only 44% homology in the total protein (Munro et al.,
1993).
CB1R are expressed mostly in certain regions of mammalian brain, where they act as
synaptic modulators, and in peripheral tissues, liver cells and gastrointestinal tract. CB2R are
expressed in the immune system (spleen, tonsils and immune system cells such us B
lymphocytes, T cells, and monocytes) and hematopoietic cells (Munro et al., 1993; Galiegue
et al., 1995; Schatz et al., 1997) and have also been detected in the liver in certain
pathological conditions. In addition, CB1R has been found expressed in hepatocytes and
biliary epithelial cells, while CB2R has been detected in hepatocytes and cholangiocytes in
patients with primary biliary cirrhosis (Floreani et al., 2009). Non-CB1R and non-CB2R have
also been identified including the orphan GPR55 and GPR18 receptors, which interact with

Cannabinoids 99
the eCB system (Haugh et al., 2016; Yang et al., 2016). eCBs may also bind vanilloid
receptors (Carletti et al., 2016) and peroxisome proliferator activated receptors (Del Rio et al.,
2016).
CBRs have different ligand including eCBs, phytocannabinoids and synthetic
cannabinoids (Di Marzo 2008; Manera et al., 2016; Ranieri et al., 2016).
Anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG) are
the eCBs most widely studied eCBs. Anandamide shows a higher affinity for CB1R than
CB2R while 2-AG shows a similar affinity for CB1 and CB2 (Keereetaweep and Chapman
2016). Other eCBs described include as noladin ether, virodhamine, N -arachidonoyl
dopamine.
eCBs are synthetized on demand from membrane phospholipid precursors,
via phospholipid-dependent pathways involving phospholipase D (of N-
acylphosphatidylethanolamine –NAPE- phospholipase D) for AEA and the combined actions
of phospholipase C (PLC) and diacylglycerollipase (DGL) diacylglycerol lipase for 2-AG
(Keereetaweep and Chapman 2016).
AEA is hydrolyzed and degraded by the enzyme anandamide amidohydrolase, also called
fatty acid amide-hydrolase (FAAH) and 2-AG, by monoacyglycerol lipase (MGL) to yield
arachidonic acid and glycerol (Di Marzo and Deutsch, 1998).
THC is the most abundant phytocannabinoids of Cannabis Sativa with psychoactive
effects and binds CB1R and CB2R.
ENDOCANNABINOID SYSTEM IN LIVER DISEASE

During the last decades, research has focused on the implication of eCB system in the
pathogenesis of human diseases, such us neurological disorder, cancer, obesity,
cardiovascular diseases and the development of therapeutic strategies.
CB1R and CB2R have a low expression in adult liver and hepatocytes and liver non-
parenchymal cells synthetize eCBs.
CB1R expression is induced by chronic alcohol intake, high-fat diets and liver fibrosis
(Jeong et al., 2008).
In humans, acetaldehyde has been shown to upregulate CB1 mRNA in hepatic stellate
cells (HSCs) but to leave CB2 mRNA unaffected. In addition, CB1R are involved in the
evolution of alcoholic liver disease suggesting an additive effect of cannabis with high
alcohol consumption (Patsenker et al., 2011).
Cocaine-induced seizures occur along with severe hepatic toxicity. Cannabidiol (CBD), a
Cannabis Sativa compound, inhibits cocaine-induced seizure and reduces liver injury in mice,
decreasing hepatic inflammatory process, and reducing cocaine lethality (Vilela et al., 2015).
CB1R signaling induces neurogenesis and angiogenesis during embryo development and
also in adulthood (Saez et al., 2014; Prenderville et al., 2015). Liver regeneration shows an
increase in AEA synthesis, which acts through CB1R inducing the expression of critical
mitosis genes. Hepatic regeneration increases levels of AEA in the G1 phase of the cell cycle,
with a concomitant increase in CB1 mRNA levels and a peak in protein expression later
during the S phase. Selective CB1R antagonist/inverse agonist SR141716 inhibits pERK and
pSTAT3 pathways blocking the cell cycle progression (Pisanti et al., 2015).

CB1R and eCBs stimulate de novo lipogenesis (Osei-Hyiaman et al., 2005), during liver
regeneration. AEA synthesis is activated via NFκB and Akt activation, also activated by
CB1R. So, AEA acting via hepatic CB1R controls the expression of cell cycle regulators
driving M-phase progression (Mukhopadhyay et al., 2011).
On the other hand, eCBs and CB1R are up-regulated in an experimental model of
hepatocarcinoma, inducing the expression of tumor-promoting genes through the activation of
the FOXM1/Bin1/IDO2 (Mukhopadhyay et al., 2015). These changes could be attenuated by
the blockade or genetic ablation of CB1R, suppressing the growth of HCC.
Phytocannabinoids and eCBs show anti-proliferative effects on cancer cells, inhibiting
proliferation and stimulating apoptosis in cancer cell lines (Calvaruso et al., 2012; Giuliano et
al., 2009). WIN55,212-2, a synthetic cannabinoid, decreases proliferation and induces
apoptosis of BEL-7402 HCC cells problably acting via a mitochondrial pathway (with up-
regulation of Bax expression, down-regulation of Bcl-2 expression and a significant reduction
in mitochondrial membrane) and inducing the cleavage of caspase-3 and PARP (Hong et al.,
2013).
Steatosis is a reversible process characterized by an excessive accumulation of
triglycerides within hepatocytes. Despite being considered a benign disease, its persistence
can trigger inflammation, fibrosis and cirrhosis. Steatogenic agents such as ethanol and high-
fat diet can upregulate the activity of CB1R by increasing the synthesis of eCbs, 2-AG and
AEA (Purohit et al., 2010).
Chronic ethanol consumption produces an increase in CB1R expression, with higher
release of 2-AG in the liver. In addition, ethanol increases the release of 2-AG by HSCs, and
2-AG then binds CB1R in hepatocytes, producing an increase in the expression of lipogenic
genes as a consequence. Moreover, steatosis produced by ethanol can be abolished with the
administration of CB1R antagonist. (Jeong et al., 2008).
2-AG induces cell death in activated HSCs reducing levels of glutathione and the
expression of degradation enzymes such as FAAH and increasing COX-2 and the consequent
production of pro-apoptotic PGD2-GE (Siegmund et al., 2016).
Cirrhotic human livers exhibit a three-fold increase in CB1R expression in isolated
vascular endothelial cells. Experimental data show that rats with biliary cirrhosis have low
blood pressure, which is elevated by CB1R antagonist (Bátkai et al., 2001).
Trebicka et al. (2011) have demonstrated that CB2R has a protective role in the liver
damage produced by chronic ethanol intake while CB1R is related with the development of
steatosis.
CB2R displays beneficial effects on alcohol-induced inflammation by regulating M1/M2
balance in Kupffer cells, thereby reducing hepatocyte steatosis via paracrine interactions
between Kupffer cells and hepatocytes. These data identify CB2R agonists as potential
therapeutic agents for the management of alcoholic liver disease (Louvet et al., 2011).
Mallat et al., (2007) have proposed therapies for treatment of chronic liver diseases
combining CB2R agonist and CB1R antagonist, due to the profibrogenic effect of CB1R and
antifibrogenic effect of CB2R.
Experimental and clinical data demonstrate that the regulation of eCB system, including
the increase in CBR, is implicated in liver diseases including liver injury and cirrhosis due to
alcohol, hepatitis, primary biliary cirrhosis and cancer (Xu et al., 2006; van der Poorten et al.,
2010). However, the efficacy of CB administration in liver diseases remains controversial due
its ambiguous effects on hepatic tissue and its non-specific side effects, including increased

Cannabinoids 101
risk of developing psychiatric disorders such as schizophrenia and major depression (Rubino
et al., 2015; Schrot and Hubbard 2016).
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Chapter 6
EXTRACELLULAR MATRIX-LIVER RELATIONSHIP
Néstor R. Lago, Ariel Marcotegui, Lisandro Orbea,

Pablo A. Souto and Juan C. Perazzo
ABSTRACT
Extracellular Matrix (ECM) is a key factor in both the niche environment that
maintains stem and progenitor cells in an undifferentiated state during regeneration and in
the tissue microenvironments in which these cells differentiate. ECM deposition is
another key process in tissue repair and fibrosis. It will be presented here in some detail
as it has a central role in both repair and regeneration. To do this, a brief summary about
ECM precede the description of its close relationship with the liver cells.
ECM is the non-cellular component of intercellular tissue, consists of a complex
molecular framework richly hydrated which is located between the cells and is
synthesized by some of them. It consists of structural proteins such as collagen and
elastin; specialized proteins like laminin, fibronectin and fibrillin, all of them immersed
in a matrix of glycosaminoglycans, forming high molecular weight complexes. There are
also certain growth factors/cytokines, matrix metalloproteinases (MMPs) and processing
enzymes such as tissue transglutaminase and procollagen propeptidases. It is an
ecosystem with high dynamics, with precise control based on the needs of the
environment, extra- and intracellular. This balance is altered in pathological conditions
such as chronic fibrogenesis, continuous wound healing processes that result in scarring.
Importantly, the ECM directs cellular differentiation, migration, proliferation, and
fibrogenic activation or deactivation. It sends specific signals to the cells and confers
stress activation, with a resultant fibrogenic response, or stress relaxation, with a
fibrolytic response. Alternatively, ECM-derived peptides can modulate angiogenesis, or
growth factors and MMP availability and activity.
Here, we propose a new classification of ECM pathology and an overview of its
relation with liver pathology. The main focus is on a key cell, liver Stellates cells (HSC),
which are normal residents of the sinusoidal space of Disse, which is activated by a
chronic injury in a differentiated cell, the myofibroblast (MF-like), that acquires
proinflammatory capacity, contractility and pro-fibrogenic properties.

106 Néstor R. Lago, Ariel Marcotegui, Lisandro Orbea et al.
INTRODUCTION
Understanding liver pathology requires to know the interrelationship between the liver
and other organs and systems. In addition, the hepatocyte has a close interaction with its non-
cellular surrounding: the extracellular matrix (ECM). As in other fields, much has been
achieved in the knowledge of the ECM.
The regeneration of the human liver was the basis of the Greek myth of Prometheus,
whose liver regenerated daily. The study of wound healing process encompasses a variety of
cells, matrix proteins, growth factors and cytokines, which regulate and modulate these
mechanisms. Nearly every stage in the repair process is redundantly controlled, and there is
no one rate-limiting factor, except uncontrolled infection. ECM also provides cells positional
information and a mechanical scaffold for adhesion and migration.
ECM is a key factor in both the niche environment that maintains stem and progenitor
cells in an undifferentiated state during regeneration and in the tissue microenvironments
where these cells differentiate. ECM deposition is another key process in tissue repair and
fibrosis.
Extracellular matrix will be presented here in detail since it has a central role in both
repair and regeneration. To do this, a brief summary about it precedes the description of the
close relationship between the liver cells and ECM.
ECM is the non-cellular component of intercellular tissue, formed by a complex highly
hydrated molecular framework, located between the cells and synthesized by some of them.
Some of the ECM components are structural proteins such as collagen and elastin;
specialized proteins such as laminin, fibronectin and fibrillin, all of them immersed in a
matrix of glycosaminoglycans, forming high molecular weight complexes. There are also
certain growth factors/cytokines, matrix metalloproteinases (MMPs) and processing enzymes
such as tissue transglutaminase and procollagen propeptidases. ECM is a highly dynamic
ecosystem, with precise control based on the needs of the extra- and intracellular
environment. This balance is altered in pathological conditions such as chronic fibrogenesis, a
process of continuous wound healing, that results in scarring. Importantly, the ECM directs
cellular differentiation, migration, proliferation, and fibrogenic activation or deactivation. It
sends specific signals to cells and confers stress activation, with a resultant fibrogenic
response, or stress relaxation, with a fibrolytic response. Alternatively, ECM-derived peptides
can modulate angiogenesis, growth factors and MMP availability and activity.
Different types of tissues show variations in the amount and composition of the ECM,
from a small amount as in nerve tissue to the connective tissue, where ECM is its main
component. In addition, there are areas where the ECM acquires critical functional
importance and has a high specialization degree, e.g., glomerular basement membrane, and
other where its permanence is limited, e.g., in embryonic development.
The different compositions and proportions of the ECM present in tissues correlates with
the needs of adaptation to the functional requirements. So, ECM will determine the stiffness,
anchoring force, the compressive strength (rich in proteoglycan), the distensibility of shear
stress, tensile forces (collagens) and can modify the strictly regulated conditions of
permeability and hydration.

Extracellular Matrix-Liver Relationship 107
COMPOSITION AND FUNCTIONS

The ECM could be considered to be integrated by two basic groups: A. interstitial matrix,
and B. basement membrane.
A. Interstitial Matrix (IM)
It can be divided into 2 subgroups: A.1. - Structural or constituent proteins, and A.2.
Matricellular Proteins.
A.1. Structural or Constituent Proteins (Connective Tissue)

The Interstitial Matrix forms a continuum between different components, e.g., epithelia,
vascular structures and smooth muscle fibers. Among the elements that produce the
Interstitial Matrix can be found the cells that synthesize IM, fibroblasts, myofibroblasts,
adipocytes, osteocytes, chondroblasts and endothelial cells.
The main constituents of the IM are fibrillar and non-fibrillar collagens, fibronectin,
elastin, proteoglycans, glycosaminoglycans (GAG), hyaluronan and others.
Collagen
Collagen (Col), is one of the most abundant proteins in biology. It is an insoluble
fibrillary triple helix, with extracellular location and its function is to confer tissue tensile
capacity. The balance between synthesis and degradation of collagen is very sensitive. Its
defective synthesis, for example in Vitamin C deficiency, leads to poor healing, as observed
in scurvy. Another way of deregulated production generates a pathological collagen
deposition known as fibrosis, as seen in scleroderma and cirrhosis. Collagen is a major
fibrillar protein present in the ECM and represents approximately 30% of the total protein
content in the body; therefore, it constitutes the principal structural protein in mammalian
tissues. There are identified about 30 types of Col, organized by different arrangements of
about 38 chains. The members of the collagen superfamily, are classified based on structure
and supramolecular organization and may be listed: Fibrillar, Fibril-associated collagens with
interrupted triple helix (FACITs), Anchoring fibrils, Network-forming, Transmembrane,
Multiplexins and other collagens (Arriazu et al. 2014).
Fibril-forming or fibrillar collagens include type I, II, III, XI collagen and the more
recently discovered type XXIV and XXVII collagen (M. K. Gordon and Hahn 2010). They
are the most abundant and widely distributed collagens in the body. Their role is mainly
mechanical, as they provide tensile strength to both tissues and organs (Ricard-Blum and
Ruggiero 2005).
FACITs constitute the largest collagen subclass, including type IX, XII, XIV, XVI, XIX,
XX, XXI, and XXII collagen. They do not form fibrils themselves, but bind the surface of
preexisting collagen fibrils contributing to fibril enlargement (Sato and Takino 2010).
Transmembrane collagens include homotrimeric type XIII, XVII, XXIII, and XXV
collagen, most of which are anchored to the plasma membrane. Transmembrane collagen
contains several extracellular domains that can be easily detached from the cell surface. They
also act as cell surface receptors and soluble extracellular molecules (Sato and Takino 2010).

Multiplexins include type XV and XVIII collagens. Finally, type XXVI and XXVIII
collagen do not properly fit within any of these subgroups, although both of them contain
collagenous domains within their structure (M. K. Gordon and Hahn 2010).
As said before, Col is a heterotrimer with different polypeptide α chains, plaited into a
triple helix. The domain encoding Col repeating Glycine is essential to generate the triple
helix, which is also rich in proline, hydroxyproline and hydroxylysine.
Any alteration in the Gly-Y-X, turns the sequence more susceptible to proteinases.
Furthermore, enzymes such as the copper dependent lysyl oxidase, are of great importance in
the post-translational modifications by catalyzing covalent crosslinks. The tensile strength of
fibrillar Col derived from this crosslinking, is a process that depends of vitamin C.
Some types of Col (e.g., Type I, II, III and V) form fibrils due to lateral crosslinking
triple helix. The fibrillar Col represents a significant proportion of connective tissue in the
wound healing and, particularly, in the scars formation. Genetic defects in these Col cause
diseases such as osteogenesis imperfecta and Ehlers-Danlos syndrome. The non-fibrillar Col
integrates basement membrane (type IV) or are components of other structures such as
intervertebral discs (IX type) or dermoepidermal unions (type VII) (M. K. Gordon and Hahn
2010).
Elastin
Elastin is a stromal unglycosylated protein. Although the tensile strength derives from
fibrillar Col, the elastic capacity of the tissues to relax-retract and return to its previous status
is conferred by elastin. Elasticity is crucial in the walls of the great vessels, it responds to
recurrent pulsatile flow and situations as shear stress. Morphologically, the elastic fibers
comprise a core surrounded by an elastin mesh of glycoprotein fibrillin. Like the Col, Elastins
require glycine in every third position, but unlike Col they have less crosslinks. A mesh
serves as a scaffold for fibrillin deposition to assembly elastin and elastic fibers. Defects in
the fibrillin synthesis and skeletal abnormalities lead to a weakening of the aortic wall
(Marfan syndrome) with a possible catastrophic clinical condition as is the induction and
rupture of aneurisms (Qi and Elson 2016).
Proteoglycans
Proteoglycans form compressible highly hydrated gels, conferring the tissues elasticity
and lubrication (as in articular cartilage). They are a long chain of polysaccharides, called
glycosaminoglycans (e.g., Dermatan sulfate and heparan sulfate) responsible of the anionic
charge of the glomerular basement membrane, conferring a basic feature for the filter function
(Kesimer and Sheehan 2008).
Hyaluronate (Hyaluronic Acid)

It is a molecule composed of repeated disaccharides, without a protein core. Because of
its ability to bind water, forms a gelatinous viscous matrix. In addition to providing tissue
compressibility, like proteoglycans, it also serves as reservoir for secreted growth factors in
ECM (e.g., Fibroblast growth factor).
Proteoglycans can also be part of the membrane proteins and participate in
differentiation, migration and cell adhesion; e.g., the transmembrane proteoglycan syndecan

has chains linked to hyaluronate growth factors that can set the matrix, such as Fibroblast
Growing Factor, facilitating their interaction with receptors of the cell surface.
Glycoproteins and Adhesion Receptors

They are structurally different molecules that are involved in intercellular adhesion,
binding between cells and extracellular matrix (ECM) and establish the connection between
the components of the ECM. Adhesive glycoproteins include fibronectin (main component of
interstitial ECM) and laminin (major constituent of basement membrane); as prototypes of the
group. Adhesion receptors, also called adhesion molecules (CAM) are grouped into four
families: immunoglobulins, cadherins, selectins and integrins (Theocharis et al. 2016). This
document will focus particularly in the characteristics of integrins.
Integrins are heterodimeric receptors. These matricelular proteins have high sensitivity
and flexibility with the ECM (Hanein and Horwitz, 2012; Hohenester, 2014). Because of this,
they have specific responses in both physiological and pathological conditions. In humans, 18
α and 8 β subunits have been characterized enabling about 24 heterodimeric combinations.
Some integrins mediate leukocyte adhesion and transmigration across the endothelium during
inflammation. Integrins are present in the plasma membrane of most animal cells except
erythrocytes. The tripeptide Arg-Gly-Asp (RGD) was originally identified as the sequence
that ligates to fibronectin mediating cell attachment. Integrins bind to many components of
the ECM, initiating signaling cascades that can affect motility, cell proliferation and
differentiation. Their intracellular domains bind to actin filaments in focal adhesion
complexes, through adapter proteins such as talin, vinculin and kindlins. Binding integrins
belong to the most studied subfamily, including avb1, avb3, avb5, avb6, avb8, a5b1, and
aIIbb3. The RGD motif is present in ECM components such as fibronectin, vitronectin,
osteopontin, and fibrinogen. In the binding process to collagen or laminin, the cell integrins
involved are the ones that contain either B1 (A1B1, A2B1, a10b1, a11b1, a6b1.) or B4
(a6b4.) subunits (Wheaton et al. 2016).
The integrins intracellular signaling transduction is shared with growth factor receptors;
e.g., adhesion to fibronectin can activate integrins mediated elements using MAP kinase, PI3
kinase and protein kinase C. Thus, the mechanical forces can be coupled from the
extracellular matrix.
Fibronectin
Fibronectins are large heterodimers with disulfide bonds synthesized by a variety of cells
including fibroblasts, monocytes and endothelial cells. There are two main types, tissular and
plasmatic fibronectins. They present specific domains that bind to a broad spectrum of ECM
components (e.g., Collagen, fibronectin, heparin and proteoglycans) and may also bind to
cellular integrins through RGD. Fibrillar tissue fibronectins form aggregates at wound healing
sites; plasma fibronectin binds to fibrin to form a clot in a provisional wound, which serves as
substrate for deposition of ECM and repair.
Laminin is the most abundant glycoprotein of basement membrane. It is a cross-shaped
heterotrimer, connecting the cells to the underlying ECM components such as collagen type
IV and heparan sulfate. In addition to mediate the binding to the basement membrane, laminin
may also modulate proliferation, differentiation and cell motility (Wang and Ni 2016).

A.2. Matricellular Proteins (MP)

In contrast to the adherent ECM proteins (collagen, fibronectin, etc.), there is a set of MP
in the ECM that doesn´t promote extracellular adhesion, even more, they can antagonize it.
These proteins are present in a soluble state and can induce focal reorganization in stress
fibers such as actin, and because of that, are considered an intermediate adhesion.
Summarizing the 3 core proteins are: I. Secreted Protein Acidic Rich in Cysteine (SPARC),
also known as osteonectin or BM-40; II thrombospondin (TSP-1) and III tenascin-C. These
proteins are secreted by various cell types, i. may be associated with insoluble fragments as
fibrillin, ii. are non-stick in certain circumstances, iii, are involved in physiological processes
such as embryogenesis as well as in pathological.
The extracellular environment consists of a macromolecular matrix that is specific for
each tissue. During injury, resident inflammatory cells interact with the ECM, using this
scaffolding for migration along a chemokine gradient. Collagen, elastic fibers, basement
membrane proteins, glycoproteins and proteoglycans are among the ECM components. MP
are secreted macromolecules that can link cells to the ECM or disrupt cell–ECM interactions.
Cytokines and growth factors influence associations among cells, the ECM and matricellular
MP proteins include:
 SPARC (secreted protein acidic and rich in cysteine) is a multifunctional

glycoprotein that organizes ECM components and modulates growth factor activity.
It affects cell proliferation, migration, differentiation and is counter adhesive,
especially for endothelial cells.
 Thrombospondins are secreted glycoproteins that affect cell–matrix interactions,
influence platelet aggregation and support neutrophil chemotaxis and adhesion.
 Tenascins C, X and R are counter adhesive proteins expressed during development,
tissue injury and wound healing.
 Syndecans are heparan sulfate proteoglycans implicated in coagulation, growth factor
signaling, cell adhesion to the ECM and tumorigenesis.
 Osteopontin is a phosphorylated glycoprotein important in bone mineralization. It
also mediates cell–matrix interactions, activates cell signaling (mainly in T cells), is
chemoatractant and supports adhesion of leukocytes and has anti-inflammatory
effects.
MP have substantive participation in pathophysiological processes such as: cell shape,

protein secretion, motility, cytoskeletal organization, signaling activation, nitric oxide
signaling inhibition, growth factor receptor agonism and antagonism, cell-cell and cell-ECM
de-adhesion (intermediate adhesion), cell motility, cell cycle regulation, anoikis resistance,
senescence, synapse formation, pain perception and cell fate determination. In the
endoplasmic reticulum (ER) through TSP1, TSP4 and COMP; SPARC regulates the ECM
chaperone, ECM pre-assembly, the calcium regulation, regulating ultimately the ER function
(Figure 1) (Murphy-Ullrich and Sage 2014).

Figure 1. The Matricellular protein actions. From ECM to the Nucleus. (Modified from Murphy-Ullrich
and Sage 2014).
B. Basement Membrane
The arrangement of the interstitial matrix in connective tissues becomes very organized
around epithelial, endothelial cells and smooth muscle cells, forming a specialized structure:
the basement membrane. The basement membrane is located under the epithelial, endothelial
and mesenchymal cells that synthesize and tends to form a mesh. As a specific area of the
ECM, its main structural components are: Col type IV, laminin, heparan sulfate
proteoglycans, entactin and osteopontin. Basement membrane collagens, endostatin
precursors, endostatin-XVIII, and endostatin-XV and are secreted by proteolysis (Arriazu et
al. 2014).
Basement membrane components are well characterized in their structures and amino
acid sequences of the molecular interactions sites. These interactions include self-assembly
processes and heterotypic binding between the individual components, as well as calcium
binding (laminin, BM-40) and are used for the assembly of the basal membrane. Laminin,
entactin and col IV also possess several binding sites and interact with cells through different
cell receptors. There is evidence that such interactions are involved in the control of cell
behavior, providing a more defined understanding of the basement membrane function.
Alterations in the basement membrane give rise to a large number of skin diseases
grouped as bullous diseases of genetic origin. Alterations in the highly specialized basement

membrane of the glomerular capillaries can generate a dysfunctional state and produce kidney
diseases that clinically present with proteinuria and/or hematuria (Lopes Pinheiro et al. 2016).
SUMMARY OF ECM FUNCTIONS

ECM regulates proliferation, movement and differentiation of living cells in their
microenvironment, and participates in:
 Physical support for the anchorage and maintenance of cell polarity.

 Cell migration facilitation.
 Growth control regulation and cell differentiation, signaling pathways through
integrin receptor family.
 Repairing. Maintaining normal tissue structure requires a basement membrane or
stromal scaffold. The integrity of the basement membrane or stromal cells is critical
for organized tissue regeneration. The restoration of a normal structure only takes
place if the MB is not damaged. Disruptions in these structures lead to deposition of
collagen or scar formation.
 Tissue microenvironment generation. The basement membrane acts as a custom
between the epithelium and connective tissue. E.g., the ECM structure modulates
renal filtration.
 Storage and presentation of regulatory molecules like growth factors, e.g., FGF and
HGF are excreted and stored in the ECM. This allows a rapid utilization after local
injury or regeneration.
Given the critical functions performed by the ECM, its constant remodeling (synthesis
and degradation) and the numerous processes in which it intervenes, it is clear that any
situation that modifies some of these conditions is capable of inducing a pathological
condition.
ECM PATHOLOGY
ECM pathology can be grouped considering whether there is an alteration in the
synthesis, remodeling or degradation and if there are deposits of substances that are not
normally present in ECM. In addition, it could also be considered the origin or acquired
genetic imprinting, especially regarding ECM synthesis.
ECM pathology can be classified into 4 groups according to the origin of the disturbance,
1. Altered ECM synthesis

1.A. Acquired
1.B. Genetic
2. Increased synthesis of ECM
3. Abnormal deposits in the ECM
4. Increased degradation of ECM

1. Altered ECM Synthesis
1.A. Acquired
Scurvy is caused by deficiency of Ascorbic acid (Vitamin C) in the diet. It is a pathology
known since the fifteenth century and is developed in countries or regions with nutritional
deficit. Ascorbic acid is essential for the proline and prolyl oxidation to form lisilhidroxilase
hydroxyproline Col. Scurvy, therefore, generates a delay in growth. The development of
altered collagen rich structures such as bone and the blood vessels basement membrane,
promotes bleeding and bone disorders with skeletal deformities and scarring because of the
alteration of the osteoid matrix composition. Another example of an acquired alteration of the
ECM is the alteration in the calcium deposits related to a vitamin D deficiency, which will
generate a disease called rickets in children and osteomalacia in adults.
Rickets consists in an inadequate bone mineralization due to hypocalcemia or vitamin D
deficiency. Inadequate mineralization of osteoid matrix by calcium reluctance is perpetuated
by poor absorption and alteration in the new matrix formation. The growth plate is thickened,
so epiphysis remains open and unmineralized. There are numerous etiologies, some
associated with hypophosphatemia or liver disease, tumors and the use of anticonvulsants in
children. In osteomalacia there is also an impaired bone mineralization that leads, in advanced
stages, to fracture predisposition and disabilities.
1.B. Genetic
Collagen disorders:
Altered synthesis.
This group is characterized by defects in the synthesis and structure of collagen. If the
collagen involved is Col I - III, a fibrillar type, the disease will be called Ehlers-Danlos
Syndrome, if it is Col I it will be an osteogenesis imperfecta, if it is a Col III in basal
membrane it will develop the Nail-Patella Disease (Figueroa-Silva et al. 2016).
If the basal membrane non-fibrillar Col is involved, the kidney will suffer Alport disease.
Alport disease is an inherited, X-linked pathology, caused by genetic alterations in the
COL4A5 gene that produces a defective synthesis of a chain in Col IV in the glomerular
basement membrane (Wickman et al. 2016).
The clinical state is a progressive renal disease with renal failure. It is associated with
familial deafness and visual disturbances. Mutations in more than a dozen genes have been
found to cause the Ehlers-Danlos syndrome, disease that is clinically presented with six major
types and at least five minor types, that correspond to various genetic mutations characterized
by the production of poor Col. Hyperelasticity clinically present with skin and joints. The
classical type results most often from mutations in either the COL5A1 gene or the COL5A2
gene (Parapia and Jackson 2008).
2. Increased Synthesis of ECM
A prototype of this ECM alteration can be seen in the hepatic tissue. A persistent liver
noxa induces a tissue response characterized by excessive deposition of ECM proteins.
During fibrogenesis, the ECM undergoes continuous changes in quantity as well as in

composition, and the amount of ECM proteins in fibrotic livers is increased by up to six-folds
compared with that in healthy individuals. This deposit deranges the normal disposition of
liver cells, so not only Col deposits trigger pathological paths, but also altered
cytoarchitecture. The pro-fibrogenic cells are the hepatic stellate cells, fibroblasts and
vascular smooth muscle cells (Xu et al. 2014). Cytokines play a decisive role in the balance
between production and degradation of ECM. Therefore, the unbalance may cause abnormal
and / or fibrotic repair. One of the most studied cytokines is the transforming growth factor β
(TGF β) plus TNFα, PDGF, BFGF, MCP-1, MIP-1-α and interleukins 1, 13 and 8. The TGF
β1 is much related to fibrotic processes in different organs, inducing the expression of
proteins like Col I, III and V, fibronectin and proteoglycans and also some glycoproteins that
inhibits protease production in the ECM. Glycoproteins also induce the metabolic inhibition
or enzymatic degradation of the ECM. There are many diseases that express these pathways,
one of them is scleroderma. Even though it is a disease of a yet unknown origin, there is
consensus linking it with an inadequate immune response. It is more common in women (3:
1) in the 6th. decade of life. It can affect several organs, skin, gastrointestinal tract, kidney,
muscle, heart and lung. It is characterized by increased production of growth factors, leading
to a systemic fibrosis (J. K. Gordon and Domsic 2016).
3. Abnormal Deposits in the ECM
The characteristics of the deposits can be very different. Immune complexes can be
deposited in the renal glomeruli, generating significant renal pathology, e.g., membranous
glomerulonephritis.
The amyloid deposition in the ECM refers to a type of protein with β pleated structure
that has variations in its amino acid sequence but keeping similar physical and ultrastructural
properties. In Alzheimer’s disease (AD), amyloid β peptide is deposited in senile plaques in
the brain vessels of cortex and meninges (Kisilevsky, Raimondi, and Bellotti 2016).
Edema is another abnormal deposition, being considered as an increment in the ECM
liquid from the increased exchange in tissue microcirculation.
Infiltration of the ECM by mucinous fluid (gel) that produces a skin induration is called
myxedema. This condition is often seen in the shins area in hypothyroid patients.
Calcium deposition can occur in different tissues such as heart valves, arterial walls or
mammary glands. These forms are called dystrophic calcification. If the deposit occurs in
renal tissue due to hypercalciuria is called nephrocalcinosis or metastatic calcification.
Uric acid deposition in the form of urate crystals in the joints and in renal tissue due to
hyperuricemia generates a heterogeneous group of diseases generically called Gout. Iron is
stored as Intracellular reservoir through two proteins, hemosiderin and ferrritin. In some
anemias, iron deposition is increased, giving rise to what is called hemosiderosis. Excessive
accumulation in different tissues may result from a genetic alteration in controlling intestinal
iron absorption leading to Hereditary Hemochromatosis. Hemochromatosis is an autosomal
recessive disorder and is due to a homozygous mutation of the HFe gene in the chromosome 6
short arm. There is a toxic intracellular iron accumulation mainly in heart, liver and pancreas.
In liver, it can lead to cirrhosis and a possible complication is the hepatocellular carcinoma.
Also it can generate diabetes and heart failure (Powell, Seckington, and Deugnier 2016).

4. Increased Degradation of ECM
This group may include fibrinoid necrosis as a major feature within the rheumatoid
arthritis and rheumatic fever. The necrosis is generally avascular with fibrin deposits, which
may also be placed in artherial walls leading to hypertension and a form of vasculitis.
Fibrinoid is the description of the intense eosinophilia observed in the ECM as a result of a
stronger staining due to the increase in eosinophilic binding sites. There are two pathologies
that show this Fibrinoid injury more broadly, located rheumatoid arthritis and rheumatic
fever, both showing the disruption in the Col chains and fragmentation of the Col-ECM
fibers. (McCourt and Dutz 2013).
ECM AND LIVER

Hepatic stellate cells (HSC) are normal residents macrophages located in the sinusoidal
space of Disse. They can be activated, in chronic injury, into a differentiated Mio-Fibroblast
like (MF-like) cell, acquiring contractile, proinflammatory and pro-fibrogenic properties
(Friedman 2008a).
An outstanding feature of MF-like cells is the capability to migrate and accumulate at the
sites of tissue repair, secreting large amounts of ECM, mainly type I collagen, regulating in
ECM remodeling. Furthermore they also secrete metalloproteins generating a close
synergistic action with hepatocytes (Nieto 2007). The fibrogenic response to injury is
triggered by both, Kupffer cells (KC) and HSC, leading to a fibrogenic response (Cubero and
Nieto 2008).
The Portal connective tissue in normal liver contains only quiescent fibroblasts and no
Portal fibroblasts (PF), which only appear when a lesion occurs in the portal zone. PF are
derived from small portal vessels and express different markers than HSC (Iwaisako, Brenner,
and Kisseleva 2012). So, PF are the other main liver cells population involved in fibrogenesis
that undergo myofibroblastic differentiation in the chronically injured liver, expressing large
numbers of α-SMA-containing microfilament bundles arranged in a parallel fashion along the
major axis of the cell. Rough endoplasmic reticulum and Golgi complexes are more
prominent in myofibroblastic PF than in normal PF. PF also proliferates around biliary cells,
acquiring an MF phenotype, making them part of the early deposition of ECM in Portal
zones. The cell populations involved differ according to the pattern of human liver fibrosis:
PF in the septa and HSC at the interface between septa and parenchyma in portal diseases
such as chronic viral hepatitis and chronic bile duct obstruction and HSC and second-layer
fibroblasts in centrilobular diseases such as alcoholic liver disease (ALD) and nonalcoholic
steatohepatitis (NASH). Matrix Metalloproteases (MMPs), Matri-cellular proteins (MP),
Tissue Inhibitors of Metalloproteases (TIMPs) and cells with transmembrane receptors (e.g.,
integrins and MMPs 14-15-16-24) and surface differentiations (invadopodia) play all together
a regulatory role, both in physiology and pathology (Nakahara et al. 1997) Hepatic and
extrahepatic MF works together with the stem cells that induced extrahepatic MF. They
contribute to ECM deposition during liver fibrosis. Studies by Li et al. and Russo et al.
demonstrated that Mesenchymal Stem Cells (MSC) are a source of extra-hepatic MF in the
damaged liver (Li et al. 2009).

It is still a matter of discussion regarding hepatocytes and biliary epithelial cells if they
contribute or not to liver fibrosis by undergoing epithelial-to-mesenchymal transition (EMT).
There are three basic mechanisms that are necessary for repair, but sometimes, when
pathological pathways are triggered, it doesn´t end up in a normal repair. This is evident when
the repair mechanisms do not achieve their goal and fibrosis, like in cirrhosis, is developed.
These basic mechanisms are (i) Cellular migration, (ii) Extracellular matrix organization and
remodeling, and (iii) Cell proliferation.
The Nomenclature Committee on Cell Death (NCCD) recommends to consider the whole
picture regarding cell death (2015). Hepatocytes with necrosis and /or apoptosis are a strong
repair stimulus (Issa et al. 2004), but if this is so, it could be suggested that sublethal damage
could also be a starter of the repair machinery. It is accepted that cell injury related with acute
inflammatory reaction cause moderate cell necrosis and ECM damage. Hepatocyte response
to parenchymal cell loss is an attempt to restore the physiological liver mass by self-
replication. However, if the noxa persists, a deregulation of the normal healing occurs and
regeneration fails, resulting in liver fibrosis, massive ECM deposition, scar formation, and
progressive and persisting injury that drives to hepatic failure (Friedman 2008b).
So, going back to integrins, we shall recall that they transmit intracellular signals that also
regulate cellular survival, proliferation and differentiation. Integrin functions are affected by
additional matrix receptors, such as collagen-binding discoidin domain receptors (DDRs),
tetraspanins and other cell activators (e.g., Growth factors and chemokines). These molecules
allosterically alter the binding avidity of the extracellular portion of integrins by signaling
through activation of their cytoplasmic tails (inside-out signaling). Thus, cytokines can also
influence organization and tension in matrix and tissue.
Many different noxas may damage the liver and drive to a chronic injury, and will
determine the characteristic of liver fibrosis. Despite this fact, there are common features that
involve the first steps; the damage of the sinusoidal/endothelial barrier and the caveolae
system; the persistent hepatocyte reaction; the release of inflammatory mediators; the
recruitment of cells related to the inflammatory and fibrogenic response with ROS generation
and active ECM response with collagen deposits that alter the normal liver histology. Among
the etiologies that can be listed are: alcohol abuse, viral hepatitis, drugs, toxins, insulin
resistance, metabolic disorders, venous outflow obstruction and autoimmune disease.
It is interesting to note that fibrosis could begin in different histological areas, as in Portal
space in chronic alcoholism or centrilobular area in chronic diseases such as cholestatic or
viral hepatitis. But despite its beginning, the final common pathway is similar and known as
chronic liver failure. Before this, a special form of regeneration is established, each normal
tissue has its own histological features, but the arrangement and balance between tissue
constituents may lead to liver failure. Thus, there is a histological rearrangement in the
regenerative nodules surrounded by fibrosis (in particular Collagen I and III) with a change of
flow direction. This new arrangement of the tissue is ineffective and even worse, avoids the
basic metabolic pathways as the Krebs cycle. (Kage et al. 1997).
These effects, exacerbated by alcohol consumption, contribute to the fibrogenic response.
Lastly, Wilson’s disease is an autosomal recessive disorder in copper metabolism and is
characterized by excessive copper deposition in the liver, brain, and other tissues. It is caused
by mutations in the ATP7B gene that codes for a copper carrier involved in transferring this
metal from the transGolgi system to apo-ceruloplasmin and its transport from the hepatocyte

to the bile. Copper deposition causes mitochondrial dysfunction and oxidative stress, hence
promoting scarring in the liver (Dong and Wu 2012).
ECM AND OTHER IMPLICATIONS
ECM and Cancer
The Nobel laureate O. H. Warburg in 1924, hypothesized that cancer growth is caused by
the fact that tumor cells mainly generate energy by aerobic glycolysis rather than by the non-
oxidative breakdown of glucose, glycolysis. This is unlike the normal cells which mainly
generate energy from oxidative breakdown of pyruvate. Nunes et al. conclude that mtDNA-
driven OXPHOS dysfunction correlates with an increased motility and migration capacities,
through a mechanism that may involve the cross talk between the cancer cell mitochondria
and the ECM. (Nunes et al. 2015).
It can be concluded that all of the tumor cells specific tasks will be not possible if the
ECM does not enable it. (Pickup, Mouw, and Weaver 2014).
INTEGRINS AND CANCER

Cell survival is also sustained through ECM-integrin interactions, both in development
and in tissue homeostasis.
Anoikis, a type of cell death, is the consequence of the loss of cell adhesion. Basically it
is the result of the blocking of the pro-survival integrin-dependent signaling pathways
including PI3K/AKT, MEK/ERK, FAK, NFkB, and/or ILK (Griffiths et al. 2011).
Resistance to anoikis promotes tumor progression and favors the emergence of metastasis
(Buchheit, Weigel, and Schafer 2014). The “integrin switch” includes changes in their
expression profile and functionality during cell detachment from the ECM thus overcoming
anoikis and allowing tumor cell survival and metastasis (Janes and Watt 2004).
EVASION
The p53 gene, the most prominent tumor suppressor gene, is altered in a large majority of
tumors by alternative pathways, deletions/mutations, of endogenous activators or
amplifications of inhibitors (Brennan et al. 2013).
THERAPY RESISTANCE
As integrins support the pro-survival tumor cell capacities, they are active participants in
the resistance toward therapies including radio, chemo and targeted therapies (Naci, Vuori,
and Aoudjit 2015).

ANGIOGENESIS
The role of integrins in developmental and pathological angiogenesis has been largely
described (Avraamides, Garmy-Susini, and Varner 2008). But today, it is becoming clear that
integrins could trigger both, pro or anti-angiogenic effects (Steri et al. 2014).
Cilengitide, a specific αvβ3/β5 integrin antagonist, was the first to be used as an anti-
angiogenic compound in a clinical trial (R. Stupp et al. 2010). Cilengitide failed to improve
the overall patient survival rate in a multicentric randomized phase III clinical trial (Roger
Stupp et al. 2014). The need to understand the fine molecular events supporting integrin
biology and functions appears currently as a priority in the field (Atkinson et al. 2014).
METASTASIS
Cell adhesion to ECM is central to the migration/invasion/metastasis process and
implicates largely integrins (Naci, Vuori, and Aoudjit 2015).
EPIGENETIC REGULATION OF HSCS

Epigenetics is a process that alters gene activity without changing the DNA sequence,
leading to heritable modifications. These modifications are natural and essential for normal
development, differentiation, and tissue-specific gene expression. However, epigenetic
abnormalities could occur due to environmental factors such as toxins, drugs, or diseases such
as cancer, autoimmune diseases, age-related illness and neurological disorders. Recent studies
have highlighted the regulatory effect of epigenetic modifications on gene expression in HSC
and on the risk of developing liver fibrosis. Quiescent HSC acquire either an adipogenic or a
myogenic phenotype depending on the balance between clusters of genes during their
differentiation. Under normal conditions, adipogenic genes are dominant, and quiescent HSC
differentiate into adipogenic cells. When liver injury occurs, the prevalence of myogenic
genes such as type I collagen, TIMP-1, or a-SMA promotes the differentiation of adipogenic
HSC to myogenic HSC. Moreover, aberrant expression of different histones and chemokines
in activated HSC can aggravate inflammation and generate ROS that increase HSC
differentiation into MF, consequently enhancing liver fibrosis. The degradation of ECM is
also affected by epigenetic modulation of matrix-associated enzymes such as MMPs.
Preliminary data suggest an association between hypomethylation of the PPARc gene
promoter and mild fibrosis in NAFLD patients. Recent studies show that inhibitors of DNA
methyltransferase reduce the rate of DNA hypermethylation and that a methyl deficient diet
regulates gene methylation and the development of liver fibrosis. Lastly, a recent study
demonstrated that the necdin-Wnt pathway could be regulated to mediate anti-adipogenic
HSC trans-differentiation via epigenetic repression of PPARc, which may have a therapeutic
application.

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Chapter 7
ENDOTHELINS IN LIVER HEALTH AND DISEASE
Myrian R. Rodriguez, María Julia Guil,

Luis Cassinotti, Yanina Bota, Mercedes Scholler,
Marcelo S. Vatta and Liliana G. Bianciotti
ABSTRACT
Endothelins (ETs) are a family of structurally related peptides with greater
vasoconstrictive properties than any known factor so far. Since their discovery numerous
investigations were carried out in order to determine their role in almost every organ. The
biologic effects of the ETs are variable and depend on tissue and cell-specific expression
of ET receptors. ETs, particularly, ET-1 plays a key role in the regulation of vascular tone
in healthy individuals but it is also involved in cellular growth and proliferation as well as
tissue fibrogenesis suggesting a potential role in various diseases. Given the importance
of the ETs in vascular biology, renal, cardiovascular and liver diseases have been
emphasized. This chapter focuses primarily on the role of the ET system in the
physiology and pathophysiology of the liver. It also highlights the importance of the
endothelinergic system as a therapeutic target for various liver diseases.
1. STRUCTURE, SYNTHESIS AND RELEASE OF ENDOTHELINS

In 1985 Hickey and co-workers reported that cultured vascular endothelial cells produced
a substance with long lasting vasoconstrictor properties (Hickey et al., 1985). This substance
was then isolated and characterized from endothelial cultures and named endothelin
(Yanagisawa et al., 1988). Then, other endothelin isoforms were reported.
Endothelins (ETs) are a family of 21-amino-acid-related peptides encoded by distinct
genes that bind to specific receptors widely expressed in numerous tissues and cell types
(Yanagisawa et al., 1988; Inoue et al., 1989a; 1989b). The family, comprising ET
(endothelin)-1, ET-2 and ET-3, exerts diverse biological effects mainly in an autocrine and/or
paracrine manner (Barton and Yanagisawa 2008; Kohan et al., 2011). Therefore, the
circulating level of ETs might not directly reflect the full physiological impact of these

124 Myrian R. Rodriguez, María Julia Guil, Luis Cassinottiet al.
peptides. ET-1 is the major isoform in the cardiovascular system produced by the endothelial
cells. It is the most potent vasoconstrictor reported so far that also displays inotropic,
mitogenic and pro-inflammatory properties. In addition, it stimulates the renin-angiotensin
system and the sympathetic nervous system (Maguire et al., 2015; Davenport et al., 2016).
ETs possess two intramolecular disulphide bonds, a feature that also share a family of
peptides isolated from snake venom termed sarafotoxins (Kloog et al., 1988; Takasaki et al.,
1988). Disulphide bonds are between cysteine residues at positions 1 and 15 and, 3 and 11.
ET-1 was first isolated from porcine endothelial cells (Yanagisawa et al., 1988). The C-
terminal end is related to receptor binding whereas the N-terminal end determines the
peptide's binding affinity to the receptor. The other isoforms, ET-2 and ET-3 differ from ET-1
in two and six amino acid residues, respectively (Inoue et al., 1989b).
ET-1 biosynthesis has been more extensively studied but evidence supports that the
synthesis of the other isoforms is essentially similar. ET-1 gene encodes a pre-pro-peptide
mRNA that is proteolytically cleaved to produce pro-ET-1 which is further cleaved at dibasic
sites by furins to Big ET-1 precursor (Xu et al., 1994; Turner and Murphy, 1996). Production
of the mature 21-amino acid peptide results from the action of a family of endothelin
converting enzymes (ECE) within endothelial cells that cleave the intermediate Big ET at
Trp21-Val 22 (Takahashi et al., 1993, Xu et al., 1994; Emoto& Yanagisawa, 1995). Three
forms of ECE have been reported, namely ECE-1, ECE-2, and ECE-3 with different
specificities for the isoforms of Big ET (Xu et al., 1994; Emoto et al., 1995; Hasegawa et al.,
1998; 'Orleans-Juste et al., 2003). ECE-1 is widely expressed in different tissues and cell
types and four isoforms originated by differential splicing have been reported in humans
(Davenport et al., 1998; Jeng et al., 2002). ECE-2 is localized to the trans-Golgi network and
is expressed abundantly in neural tissues and endothelial cells. ECE-1 and ECE-2 prefer Big
ET-1 over Big ET-2 and Big ET-3. ECE-3 was shown to be specific for Big ET-3 (Hasegawa
et al., 1998).
The biosynthesis of ET-1 is controlled by mRNA transcription. In this sense several
transcriptional factors including activator protein-1 (AP-1), GATA, Smad or hypoxia-
inducible factor-1 (HIF-1), have been described to interact with specific genomic regions
within the ET-1 gene promoter (Inoue et al., 1989; Rodríguez Pascual et al., 2004). Various
studies report that particular microRNAs regulate ET-1 expression in liver sinusoidal
endothelial cells and other cells (Yeligar et al., 2009; Jacobs et al., 2013).
ET-1 is constitutively produced and released from endothelial cells. However, there is
also a regulated pathway for ET-1 release in this cell type from Weibel-Palade bodies in
response to stimuli like shear stress, hypocapnia, transforming growth factor β, angiotensin II,
cytokines, and norepinephrine that increase pre-proET-1 mRNA levels (Yohsimoto et al.,
1991, Kohno et al., 1992; Stow et al., 2011; Davenport et al., 2016). Upon activation of
endothelial cells, Weilbel-Palade bodies fuse with the plasma membrane and release their
contents by exocytosis (van Mourik et al., 2002). The constitutive pathway evokes greater
release of ET-1 than the secretory granule pathway and appears to be the primary secretory
mechanism (Russell and Davenport, 1999). ET-1 mRNA is decreased by hypoxia, atrial
natriuretic factor, nitric oxide (NO) and prostacyclin (Yoshimoto et al., 1991; Kohno et al.,
1992; Prins et al., 1994).
The release of ET-1 from endothelial cells is also accompanied by Big ET-1 molecules
that do not bind to any ET receptor but they are further processed to ET-1 by smooth muscle
ECE or through an alternative pathway mediated by chymase which converts Big ET-1 to an

Endothelins in Liver Health and Disease 125
intermediate ET-11-31 that is then processed to the mature biologically active peptide (Russell
et al., 1998; D’Orleans-Juste et al., 2008; Davenport et al., 2016).
The plasma half-life of ET-1 is less than 2 min, due to its efficient extraction in the
pulmonary and renal vascular beds. Plasma concentrations of immunoreactive endothelin
vary inversely with renal function. ET-1 is metabolized to inactive metabolites by neutral
endopeptidases (NEP) which are mainly found in the brush border vesicles of kidney
proximal tubules (Skolovsky et al., 1990). It should be noted that NEP are not selective for
ETs and also metabolize other peptides like natriuretic peptides (Charles et al., 1996). In
addition to NEP action, the endothelin receptor type B (ETB) that promotes vasodilation, also
clears ETs from the circulation by internalizing the ligand-receptor complex (Gasic et al.,
1992). The formation of the disulphide bridge in the ET peptides blocks the N-terminal amino
acid conferring resistance to enzymic degradation in plasma. Internalization by ETB
scavenging receptors is therefore particularly important for termination of the ET signal in
health and disease. The major sites of ETs clearance are the lungs, the liver, and the kidney
(Johnstrom et al., 2005).
2. ENDOTHELIN ISOFORMS
ET-1 is mainly released by endothelial cells and causes long-lasting vasoconstriction
through the activation of endothelin receptor type A (ETA). Together with vasodilators,
particularly NO, maintains the vascular tone, modulates tissue perfusion and endothelium
homeostasis. ET-1 released from endothelial cells acts initially in an autocrine manner
binding to ETB receptors to evoke NO and prostacyclin production. Then it binds to ETA
receptors present in smooth muscle cells to promote vasoconstriction. ET-1 whether centrally
or peripherally applied plays a relevant role in blood pressure elevation acting synergistically
with angiotensin II and catecholamines (Mosquera-García et al., 1992; Gulati et al., 1997;
Perfume et al., 2008). ET-1 is the most abundant isoform in the human cardiovascular system
and although the primary source is the vascular endothelial cells, the peptide is also produced
by other cell types, including epithelial cells, for example in the lungs, gastrointestinal tract,
kidney, liver, colon; macrophages and monocytes as well as neurons and reactive glial cells in
the central nervous system. It is accepted that ET-1 regulates vascular function and blood
flow in vital organs like the heart, lungs, central nervous system, kidney and liver. Plasma
levels of ET-1 are in the picomolar range (1-10 pmol/L) (Haynes and Webb, 1994).
ET-2 appears to have a more limited tissue distribution as compared to ET-1. It is
expressed in the vasculature, heart, lung, kidney, intestine, heart, lung, kidney, intestine, and
ovaries (Howard et al., 1992; de la Monte et al., 1995; Palanisamy et al., 2006). The
vasoconstrictor potency of ET-2 is similar to that of ET-1 (Guimaraes et al., 1992; Maguire
and Davenport, 1995). ET-2 shares many features with ET-1 although growing evidence
supports a distinct role. Different studies support that ET-2 is involved in ovarian physiology,
particularly in follicular rupture (Ling et al., 2013).
ET-3 is not produced by endothelial cells and it was proposed as the brain endothelin
peptide because it is predominantly expressed in the brain. The peptide is found in
neostriatum, hypothalamus, hippocampus, cerebellum and medulla oblongata (Giaid et al.,
1991). ET-3 modulates catecholamine metabolism in the hypothalamus and olfactory bulb (di

Nunzio et al., 2002; 2004; Morgazo et al., 2005; Perfume et al., 2008; Hope et al., 2008;
2010; Nabhen et al., 2009; Vatta et al., 2015). ET-3 is also expressed in the heart,
endometrium and gastrointestinal tract (Liu et al., 1998; Plumpton et al., 1993). Studies in
knock-out mice support that ET-3 is involved in the development of the enteric nervous
system (Kurihara et al., 1999).
3. RECEPTORS AND BIOLOGICAL ACTIONS OF ENDOTHELINS

Two well pharmacologically characterized G-protein coupled receptors (GPCRs), ETA
and ETB, mediate the biological effects of ETs (Arai et al., 1990; Sakurai et al., 1990; Kohan
et al., 2011). ET receptors belong to class A GPCRs, which is the target of most currently
used drugs in medicine. ETA and ETB receptors have different molecular and pharmacological
characteristics and functions depending on their location (Maguire et al., 2015).
The ETA receptor exhibits higher affinity for ET-1 and ET-2 than for ET-3, whereas the
ETB receptor binds the three isopeptides with similar affinity (Kohan et al., 2011). However,
atypical responses, where biological effects mediated by ETs fail to be mimicked or inhibited
by selective agonists or antagonists, suggest the existence of additional ET receptor subtypes.
However, up to date no additional ET receptors have been identified (Davenport et al., 2016).
ETA and ETB receptors are coupled to multiple signaling pathways and are expressed in
several tissues and cells types including hepatocytes (Barton and Yanagisawa, 2008;
Davenport et al., 2016).
Both ETA and ETB receptors are affected by posttranslational changes including
phosphorylation, glycosylation and palmitoylation although not all these changes have been
well characterized as regards receptor function (Davenport et al., 20169. Phosphorylation
induced by ligand binding desensitizes ETB but not ETA receptors whereas palmitoylation of
ET receptors is essential for ET mitogenic activity (Cramer et al., 2001). Splice variants of
ETA and ETB receptors have been reported although their physiological and/or
pathophysiological relevance still remains to be fully elucidated (Davenport et al., 2016).
ET receptors are widely distributed in almost every organ given that ETA receptors are
expressed in vascular smooth muscle cells whereas ETB in endothelial cells. However, these
receptors are also expressed in other cell types. In the brain ETA receptors are localized to the
smooth muscle of the vasculature supporting a role in the regulation of cerebral blood flow
(Adner et al., 1994). They are also expressed in the endothelial cells of the blood brain barrier
(Purkiss et al., 1994).
Both, ETA and ETB receptors are expressed in the lungs where they mediate
vasoconstriction and bronchoconstriction (Hay et al., 1993; McCulloch et al. 1996; Adner et
al., 1996). ET receptor antagonists are currently used in the treatment of pulmonary arterial
hypertension given that ET-1 is involved in its pathogenesis, and further plasma ET-1 levels
correlate with the severity of the disease (Rubbin et al., 2002).
In the heart, both receptors are expressed in the atria and ventricles (Bax et al., 1993;
Molenaar et al., 1993). ETA is predominantly expressed in myocytes whereas ETB receptors
in the atrioventricular conducting system (Molenaar et al., 1993). Activation of ETA receptors
induces positive cronotropic and inotropic effects and increases vascular resistance leading to

blood pressure elevation. In the vasculature ETB-mediated NO release counterbalances ETA-

mediated vasoconstriction.
ET-1 and ET receptors are upregulated in cardiovascular diseases including cardiac
failure and hypertension (Rodeheffer et al., 1992). Experimental studies with animal models
of hypertension support that ETA receptor blockade improves cardiac hypertrophy and lowers
blood pressure whereas ETB receptor inhibition worsens the disease (Bianciotti and de Bold,
2000; 2001; 2002). However, clinical trials using ET receptor antagonists in patients with
cardiovascular diseases brought disappointing results (Kohan et al., 2012).
In the liver, both ET receptors are expressed not only in the vasculature but also in
hepatocytes and stellate cells. Activation of ETA receptors rises portal venous pressure
whereas stimulation of ETB receptors regulates bile secretion (Tanaka 1994; Rodríguez et al.,
2013). The biological effects of ETs in the liver are further detailed in other sections.
The ETA receptor primarily mediates the vasoconstrictive and proliferative responses to
ET-1 whereas the ETB receptor has broader effects, including ET-1 clearance, endothelial cell
survival, NO production, prostacyclin synthesis, and ECE-1 inhibition (Davenport et al.,
2016). Studies using knock-out mice for ET receptors have shown that ETA deficient mice
show a different phenotype to ETB knock-out mice. ETA deficient mice die at birth of
respiratory failure as a consequence of craniofacial abnormalities supporting that this receptor
subtype is essential for development (Kurihara et al., 1999). Conversely, ETB knock-out mice
survive up to two months and show the same phenotype as ET-3 deficient mice. In these
animals the enteric nervous system precursors and neural crest-derived epidermal
melanoblasts fail to colonize the intestine, causing an aganglionic megacolon and a
pigmentary disorder in their skin (Hosoda et al., 1994; Baynash et al., 1994).
ET receptors are coupled to multiple signaling pathways (Sandoval et al., 2014;
Davenport et al., 2016). In smooth muscle cells ETA activation stimulates phospholipase C
leading to the generation of diacylglycerol that in turn activates protein kinase C and inositol
triphosphate that promotes calcium release from the endoplasmic reticulum. Elevation of
intracellular calcium causes vasoconstriction. In less classical pathways in other cell types
ETA is coupled to phospholipase D activation, arachidonic acid production and prostaglandin
release, adenylyl cyclase or protein tyrosine kinases stimulation. Activation of ETB receptor is
coupled to NO generation and subsequent cGMP intracellular increase that activate protein
kinase G.
4. ENDOTHELINS IN LIVERPHYSIOLOGY
ET-1 binds with a high affinity to rat liver plasma membranes, and induces sustained
calcium mobilization and activation of glycogenolysis in rat hepatocytes (Serradeil-Le Gal et
al., 1991). Coupling of ET-1 with the ETA on the membrane of hepatocytes activates
phospholipase C leading to the release of calcium from the intracellular pool and blocking the
efflux of calcium from the cells which results in long lasting and strong activation of
glycogenolysis (Serradeil-Le Gal et al., 1991).
Expression of ET-1 in isolated liver cells was observed in sinusoidal endothelial cells,
stellate cells, portal vein endothelial cells, bile duct epithelial cells and, to a lesser extent, in
Kupffer cells (Gerbes et al., 1998). ET-1 mRNA significantly increases in stellate and
endothelial cells following liver injury (Pinzani et al., 1996). The highest expression of ET

receptors was reported predominantly in the membrane of stellate cells and hepatocytes
(Gondo et al., 1993; Housset et al., 1993; Mallat et al., 1995).
An increase in portal venous pressure occurs following ET-1 administration through the
activation of ETA receptors coupled to a rise in cytosolic free calcium (Yan et al., 1990;
Okumura et al., 1994; Tanaka et al., 1994). The three ET isoforms are potent constrictors of
the portal vein in rats (Guimaraes et al., 1992; Oshita et al., 1993). ET-1 stimulates the
reversible contraction of stellate cells in culture, mediated by elevated intracellular calcium
via ETA suggesting that ET-1 may regulate blood flow through the sinusoids during liver
injury. The location of stellate cells in the normal liver suggests that the contraction of these
cells around sinusoids regulates sinusoidal blood flow by modulating sinusoidal resistance
(Thimgan & Yee, 1999; Soon and Yee, 2008). The magnitude and rate of stellate cells
contraction and relaxation around the sinusoid determine hepatic blood flow (Thimgan &
Yee, 1999). Isolated rat livers perfused with ET-1 reveal that the peptide reduces sinusoidal
diameter colocalized with stellate cells that correlates with portal pressure elevation (Tran-Thi
et al., 1993; Zhang et al., 1994; Kaneda et al., 1998). These findings support that ET-1
regulates hepatic blood flow through stellate cell contraction. Different studies support that
sinusoidal blood flow is regulated by the net balance of factors that induce stellate cell
relaxation as NO and factors that stimulate stellate cell contraction like ET-1 (Rockey 1997;
Soon and Yee, 2008).
ETs produce a sustained pressor response of hepatic circulation behaving as cholestatic
agents (Bluhmetal., 1993; Tran-Thi et al., 1993; Elliot et al., 1997). However, it was also
reported that in isolated perfused livers ET-1, through ETA receptors, dose-dependently raises
portal venous pressure, resulting in either choleresis or cholestasis despite pressure elevation
(Gandhi et al., 1990; Tanaka et al., 1994; Okumura et al., 1994). High doses of ET-1 decrease
bile flow whereas low doses increase it even under conditions of vasoconstriction sufficient
for portal pressure elevation (Tanaka et al., 1994). Increases in portal venous pressure tend to
decrease bile secretion but the liver possesses autoregulatory mechanisms to maintain
constant blood flow along the sinusoids. Hemodynamic changes must be abrupt or
pronounced to overcome hepatic autoregulatory mechanisms.
In vivo studies performed in our laboratory show that both ET-3 and ET-1 in doses that
do not modify portal venous pressure or portal blood flow, dose-dependently induce
choleresis through ETB receptor activation coupled to NO generation (Rodriguez et al., 2013).
ETs-induced choleresis is mediated by vago-vagal reflexes stimulated at the level of vagal
afferent fibers and results from stimulation of both bile flow acid dependent and
independent flow fractions since the peptide enhances the excretion rate of bile acids, and
gluthatione derivatives. Increased canalicular solute excretion stimulated by ETs is due to a
rapid translocation of hepatic transporters to the plasma membrane from vesicular
(endosomal) compartments. ET-3 and ET-1 stimulates Bsep (bile salt export pump), Ntcp
(Na+/taurocholate co-transporting polypeptide), Mrp2 (multidrug resistance protein-2), and
AQP8 (aquaporin 8) insertion in the plasma membrane (Rodriguez et al., 2013). Both ETs
also enhance the transcription of these hepatic transporters involved in bile formation. These
findings suggest that these peptides may have a potential beneficial role in pathophysiological
conditions where bile secretion is impaired as in hepatocellular cholestasis. In this sense
preliminary studies from our laboratory show that ET-3 inhibits estrogen-induced cholestasis
in the rat and modifies bile acid excretion profile (Rodriguez et al., 2015).

ETs also participate in the brain regulation of bile secretion in the rat. Centrally applied
ET-1 (brain lateral ventricle) through ETA receptor activation produce systemic regional
circulatory changes mediated by the sympathetic nervous system (Gulati et al., 1997). The
decrease in the perfusion of peripheral organs following brain ET-1 injection is hardly
attributable to the leakage at peripheral sites given that the same doses peripherally applied
fail to evoke hemodynamic changes. ET-1 applied to the brain impacts on cerebral blood flow
that may eventually lead to impaired perfusion of the brain eventually followed by necrosis,
although it is entirely dependent upon the administered dose (Zhu et al., 1996; Gulati et al.,
1997). Studies from our laboratory show that ET-1 and ET-3 applied to the brain exerts either
choleresis or cholestasis depending on the injected dose (nM or pM range) although through
different mechanisms.ET-1 mediated the effects through the activation of ETA receptors and
modulation of the parasympathetic activity whereas ET-3 action was mediated by ETB and a
NO pathway but independent of the autonomic nervous system (Rodríguez et al., 2005;
2006).
Binding of ETs to receptors in the liver do not have only vasoconstrictive effects.
Binding of ET-1 to ETB receptors on the cell membrane of Kupffer cells activates
phospholipase A2, which leads to the release of prostaglandin E2 and NO that mediate
vasodilation (Rockey& Chung 1994).
Current literature supports that ETs not only promote hemodynamic changes in the liver
but also participate in metabolic pathways and in the regulation of bile secretion.
5. ENDOTHELINS IN LIVER DISEASE

ETs have been implicated in the pathogenesis of a variety of diseases. Given the
relevance of these peptides in vascular physiology, cardiovascular, renal and hepatic diseases
have been emphasized. Furthermore, the mitogenic effect of ETs also drew attention for
potential therapeutic intervention in proliferative disorders as cancer and fibrosis. Numerous
investigations have focused on the role of ET-1 in the pathogenesis of cirrhosis and its
contribution to portal hypertension but most attracting was the possibility that ET antagonist
could be used in the treatment of these diseases. However, disappointing results raised from
most of clinical trials.
ET-1 modulates portal pressure and is involved in the pathophysiology of portal
hypertension. Portal hypertension is a clinical syndrome caused by a pathological increase in
portal pressure usually accompanied by complications such as ascites, esophageal varices,
and hepatic encephalopathy. In the normal liver, portal pressure is maintained within a normal
range despite the dynamic change in portal flow by adjustment of intrahepatic vascular
resistance. The development of portal hypertension results from increase of portal flow
resistance caused by collagen deposition, hepatocyte enlargement, and narrowing of the
sinusoids as well as by enhanced intrahepatic vascular tone induced by increased circulating
vasoconstrictors like ET-1 and angiotensin II and decreased local vasodilators like NO
(Mallat 1998; Laleman et al., 2005; Seo and Shah, 2011). Splanchnic vasodilatation and
hyperdynamic circulation together with the development of portosystemic collateral vessels
further aggravates portal pressure by increasing portal blood flow.

ET-1 is increased in the portal vein of rats with hepatic or pre-hepatic experimental portal
hypertension (De Gottardi et al., 1998). Furthermore, ET receptors are enhanced in the
vasculature of portal hypertensive rats (Cahill et al., 1998). Different studies in animal models
of portal hypertension show that ET antagonists reduce portal pressure (Reichen et al., 1998;
Sogni et al., 1998; Kojima et al., 2000; De Gottardi et al., 2000). Bosentan (mixed ETA/ETB
receptor antagonist) administration decreases portal pressure in vivo by reducing
hepatocollateral vascular resistance in rats (Sogni et al., 1998). Also TAK-044 (mixed ETA/
ETB receptor antagonist) attenuates acute liver injury and portal hypertension in rodents
(Ghandi et al., 1998). Selective ETA antagonists were also effective in reducing portal
pressure, both when administered acutely or chronically to mice exposed to carbon
tetrachloride (Feng et al., 2009). This study proposed that acute effects of ET receptor
antagonists were directly on the hepatic and sinusoidal vasculature, whereas chronic
endothelin receptor antagonism was complex affecting fibrogenesis and the hepatic
microcirculation. Some studies show that ETA but not ETB antagonists reduces portal pressure
in portal hypertensive rats (partial portal vein ligated rats) suggesting that selective ETA
antagonist would be more beneficial than mixed antagonists (De Gottardi et al., 2000). In this
sense, it was shown that ETB activation by sarafotoxin S6c exacerbates portal hypertension in
cirrhotic rats (Kojima et al., 2001).
Increased ETs production contributes to portal hypertension by inducing stellate cell
contraction and by increasing hepatic sinusoidal tone. Furthermore, in the injured liver,
constitutive NO production is reduced not only in rodents but also in humans (Zimmermann
et al., 1996; Sarela et al., 1999). Enhanced ET-1 and decreased NO production in the
intrahepatic microcirculation impair sinusoidal tone leading to an increase in the hepatic
sinusoidal resistance. However, in the splanchnic circulation of cirrhotic patients, the release
of NO is increased (Sarela et al., 1999). The abnormal dilation of the mesenteric vascular bed
and decreased responsiveness to vasoconstrictor agents, features of portal hypertension, are
attributable to elevation of NO in the splanchnic vascular bed (Wiest & Groszmann, 2002).
Although ET antagonists were effective in reducing portal pressure and safe in animal studies,
clinical studies could not prove the benefit of ET antagonist treatments, which could not
lower portal pressure in patients with portal hypertension. Further, ET antagonists exhibit side
effects like hepatotoxicity (Humbert et al., 2007; Digemanse et al., 2009; Eriksson et al.,
2011).
Growing evidence from cellular and animal studies supports that ET-1 significantly
contributes to tissue fibrosis. Chronic liver injury despite the etiology leads to fibrosis
mediated predominantly by stellate cells. Liver damage induced by chronic alcoholism, viral
infection or bile duct ligation triggers a fibrogenic response that progresses to excessive
scarring and organ failure as in liver cirrhosis. In response to injury stellate cells acquire an
activated phenotype (myofibroblast-like cell) characterized by enhanced cellular proliferation,
increased production of smooth muscle -actin and extracellular matrix (Friedman 1993).
Fibrogenesis is a complex process that results from an interplay among extracellular matrix
components, cytokines and factors like ETs. Hepatic injury leads to ET-1 and ET receptors
overexpression (Rockey et al., 1998). In normal hepatic cells, pre-pro-ET-mRNA is expressed
in only non-parenchymal cells, predominantly in sinusoidal endothelial cells but following
liver injury (bile duct ligation), pre-pro-ET-1 mRNA and immunoreactive ET levels increase
with progressive injury more prominently in stellate cells than in endothelial cell (Rockey et
al., 1998).

Antagonism of ET in animals with established stellate cell activation and fibrogenesis

reduced stellate cell activation and extracellular matrix mRNA expression supporting that
ETs acts in a paracrine/autocrine manner inducing proliferation and activation of this cell type
(Rockey& Chung, 1996). Furthermore, stellate cells have been shown to transform into
fibrotic fibroblasts by the action of ET-1 (He et al., 2013; Zhan &Rockey, 2011). In fact,
fibrosis is a pathophysiological process common to several chronic diseases including
idiopathic pulmonary fibrosis, liver cirrhosis, systemic sclerosis and nephrosclerosis as well
as several cardiac diseases and ETs significantly contribute to its development (Rodriguez
Pascual et al., 2014). The pro-fibrotic effect of ET-1 is mediated by mitogen activated protein
kinases (MAPKs) (Shi-Wen et al., 2006). Furthermore, the activation of rac/Rho and
phosphatidylinositide 3-kinase (PI3K)/Akt pathways have also been implicated (Shi-Wen et
al., 2004; Shafiei&Rockey, 2012). Nevertheless, despite the consistent contribution of ET-1
to fibrotic disorders, most clinical trials using ET receptor antagonists have unfortunately
given poor or negative results so far not only in the liver but also in other organs (Anand et
al., 2004; Tripathi et al., 2006; King et al., 2008; Mann et al., 2010). Only the mixed ETA/ETB
antagonist bosentan has been approved for the treatment of digital ulcers associated to
scleroderma (Korn et al., 2004).
In cirrhosis, decreased sinusoidal caliber and impaired sinusoidal wall elasticity are
considered to be responsible for the increased portal pressure (Shibayama 1988).
Hyperdynamic circulation, as well as increased hepatic resistance, contributes to portal
hypertension in cirrhosis of the liver. Advanced cirrhosis is characterized by hyperdinamic
circulation with increased cardiac output, heart rate and plasma volume and decreased arterial
blood pressure and systemic vascular resistance. In patients with cirrhosis ET-1 and ET-3
levels are elevated in arterial and venous plasma (Gülberg et al., 1992; Møller et al., 1995;
Gerbes et al., 1995; Bernardi et al., 1996). This reflects increased hepatic synthesis of ET-1,
mainly by stellate and sinusoidal endothelial cells. Furthermore, ET-1 and ET receptor
expression is increased in cirrhosis (Gandhi et al., 1996). It was shown that ETA activation
contributes to portal hypertension in cirrhotic rats, whereas ETB-mediated portal depressor
effects are attenuated compared with non-cirrhotic animals (Cavasin et al., 2009). It was
shown that TAK-044 (mixed ETA/ETB antagonist) arrests and reverses the development of
carbon tetrachloride-induced cirrhosis in rats (Thirunavukkarasu et al., 2004).
Elevated levels of ET-1 in the liver of patients with cirrhosis result from both increased
synthesis and decreased clearance (Hinterhuber et al., 2004). The isolated perfused liver
extracts proportionately more ET-1 than the lungs, with 80% uptake in a single pass through
binding to ETB receptors on hepatic stellate cells and is reduced in conditions such as
cirrhosis (Rockey and Weisiger, 1996). In patients with cirrhosis hepatic tissue ET-1 levels
are increased and correlates with the severity of the disease and the magnitude of ascites,
supporting that the peptide modulates intrahepatic resistance in cirrhotic portal hypertension
(Alam et al., 2000).
ET-1 is also implicated in the pathogenesis of the hepatopulmonary syndrome that occurs
in 10-30% of patients with cirrhosis. It results from an impairment of arterial oxygenation due
to pulmonary vascular dilation and angiogenesis leading to arterial hypoxemia (Rodriguez
Roisin and Krowka, 2008; Grace and Angus, 2013). Hepatopulmonary syndrome has also
been reported in chronic viral hepatitis without portal hypertension and in non-cirrhotic portal
hypertension (Babbs et al., 1988). Most of the investigations to reveal the pathophysiological
mechanisms have been performed in bile duct ligated rats that develop cirrhosis, portal

hypertension, and hepatopulmonary syndrome at five weeks following surgery. ET-1 levels
are increased in cirrhosis but are higher in patients with intrapulmonary vasodilation (Luo et
al., 1998; Koch et al., 2012). ET-1 infusion into the peripheral circulation causes
vasoconstriction in healthy subjects, but vasodilation in patients with advanced cirrhosis
(Vaughan et al., 2003).
Evidence from studies in animal models of hepatopulmonary hypertension showed that
the vasodilation of the pulmonary vascular bed is mediated by increased production of ET-1
in response to liver injury that acting through ETB receptors in the lung vasculature promotes
upregulation of endothelial NO synthase (eNOS) (Ling et al., 2004). In addition, macrophage
accumulation in the pulmonary vascular bed further increases NO levels through the
activation of inducible NO synthase (iNOS). Selective ETB blockade decreases pulmonary
eNOS and ETB receptor expression as well as macrophage accumulation and iNOS
expression, thus attenuating hepatopulmonary hypertension (Ling et al., 2004). Furthermore,
knock out mice for ETB showed similar results (Zhang et al., 2009). Currently, no effective
medical therapies for the hepatopulmonary syndrome exist, and liver transplantation is the
only successful treatment. Clinical trials of ETB blockade in human hepatopulmonary
syndrome have not been performed so far.
Hepatorenal syndrome is a functional severe or moderate renal impairment that occurs in
patients with advanced liver cirrhosis or fulminant acute liver failure. In this situation tubular
function is preserved but renal blood flow and glomerular filtration rate are markedly reduced
due to intense renal vasoconstriction that progresses as liver aggravates. The development of
hepatorenal syndrome has a dismal prognosis in patients with cirrhosis because renal failure
is usually irreversible unless liver transplantation is performed.
Several factors have been implicated in the pathogenesis of hepatorenal syndrome,
including peripheral arterial vasodilation, enhanced renal sympathetic activity, cardiac
dysfunction and vasoactive mediators (Wadei et al., 2006). In fact, the underlying
mechanisms are not still clear although it is accepted that increased vasoconstrictor and
decreased vasodilator factors acting on the renal circulation are involved. However, the
administration of renal vasodilators like dopamine or drugs to antagonize the action of
vasoconstrictors like angiotensin-converting enzyme inhibitors, and ET antagonist did not
significantly improve renal perfusion or glomerular filtration rate. Studies with ET
antagonists have shown controversial results. The ETA antagonist BQ123 improved renal
function in patients with hepatorenal syndrome whereas the non-selective antagonist
tezosentan was not beneficial and further showed deleterious renal effects (Soper et al., 1996;
Wong et al., 2008). Systemic vasoconstrictors aimed at reducing splachnic vasodilation in
combination with albumin were more effective in patients (Wadei et al., 2006; Davenport et
al., 2012).
An imbalance between NO and ET-1 in the microcirculation significantly contributes to
hepatic ischemia-reperfusion injury. Ischemia damages the liver but restoration of blood flow
results in further insult mediated by hemodynamic disturbances associated to an inflammatory
response mediated by the activation of Kupffer cells that release cytokines and reactive
oxygen species (ROS) (Serracino-Inglott et al., 2001). Several surgical procedures of the liver
require periods of ischemia, and when the blood flow is restored the liver suffers further
injury due to vasoconstriction, cell swelling and sinusoidal platelet aggregation that leads to
microcirculation failure. Contraction of stellate cells mediates by increased ET-1 in a context
of diminished NO production promotes blood flow impairment during reperfusion and

damage of the microcirculation. ETA receptor antagonist reduces microcirculatory

disturbances and transplant dysfunction after partial liver transplantation. (Kawamura et al.,
1995; Pannen et al., 1997). Following ischemia, NO production is reduced because NADPH
and oxygen, cofactors for its synthesis are dramatically diminished (Serracino-Inglott et al.,
2001). It was shown that pretreatment with bosentan attenuates liver injury induced by
ischemia-reperfusion (Scommotau et al., 1999). In addition, augmentation of NO levels also
improved hepatic damage supporting the relevance of ET-1 and NO in the pathogenesis of
ischemia reperfusion injury.
Postoperative liver failure is attributable to damage of the microcirculation secondary to
increased portal blood flow. It is a severe and life-threatening complication following liver
surgery that makes the hepatic parenchyma extremely vulnerable to regeneration failure and
organ dysfunction progression (Kishi et al., 2009). A recent prospective study proposes serum
ET-1 as an early predictor of postoperative liver failure (Ratti et al., 2016). The study
included patients undergoing liver resection for primary or secondary liver tumors. ET-1
levels were significantly higher in patients that had major or extended liver resection than
those who underwent minor resections (Ratti et al., 2016). A positive correlation between
postoperative liver failure and serum ET-1 early in the postoperative period was found. ET-1
on the second postoperative day resulted an independent predictor of postoperative liver
failure in multivariate analysis (Ratti et al., 2016).
In acute liver failure despite the etiology activated Kupffer, stellate and endothelial cells
release a plethora of mediators involved in further hepatic injury in addition to that induced
by the precipitating cause. In acute liver failure not associated with surgery circulating ETs is
also elevated in rodents and patients (Moore et al., 1992; Anand et al., 2002; Palmes et al.,
2005; Heiden et al., 2008). In rats with severe acute hepatic failure induced by galactosamine
it was found that ETB expression is diminished, suggesting that it may contribute to enhanced
ET-1 levels due to reduced ET-1 clearance from circulation (Heiden et al., 2008).
Numerous studies support that ETs, particularly ET-1, are involved in the pathogenesis
and maintenance of various acute and chronic liver diseases. However unfortunately, most
clinical trials targeting ET receptors resulted not effective.
6. ENDOTHELINS AS THERAPEUTIC TARGETS IN LIVER DISEASES:

WHAT WE KNOW AND WHAT WE EXPECT
Based on the physiological and pathophysiological role of ETs, the components of the
endothelinergic system, mainly ET receptors, have been extensively investigated as
therapeutic targets in most diseases involving fibrogenesis and hemodynamic disturbances.
Diverse ETA selective or mixed orally active antagonists have been developed by many
pharmaceutical companies and proved to be effective in experimental research but
unfortunately a few of them were effective in clinical trials. Three nonpeptide antagonists,
bosentan, macitentan that are mixed ETA/ETB antagonists and ambrisentan that displays ETA
selectivity, have been approved by the U.S. Food and Drug Administration (FDA) for clinical
use, primarily in pulmonary arterial hypertension.
The key in the development of pharmacological agents was the first ETA-selective
peptide antagonist, BQ123 (Ihara et al., 1992) whereas the first selective peptide ETB

antagonist was BQ788 (Ishikawa et al., 1994). Different therapeutic strategies were
approached in experimental studies and clinical trials to inhibit the unwanted effects of ETs in
several diseases. The first ETA/ETB antagonist with oral bioavailability was identified by
Clozel et al., (1993). The use of selective ETA receptor antagonists like ambrisentan or the
ETA/ETB receptor antagonists like bosentan proved to be highly efficient in diverse diseases.
In this sense, bosentan, was the first in the class to be introduced into the clinic, initially for
the treatment of pulmonary arterial hypertension (Rubin et al., 2002). Later more selective
ETA antagonists like ambrisentan and sitaxentan were approved for clinical use in this disease
(Vatter& Seifert, 2006; Benza et al., 2007; Galie et al., 2009). However, in 2010 sitaxentan
was voluntarily withdrawn by the pharmaceutical company due to cases of idiosyncratic
hepatitis leading to fatal acute liver failure (Don et al., 2012).
Several cases of patients undergoing sitaxsetan therapy for pulmonary arterial
hypertension developed liver failure, some of them, fatal. Patients showed increased liver
aminotransferases and biopsies revealing hepatocellular damage and areas of necrosis
compatible with drug-induced liver injury (Barst et al., 2002; Hoeper et al., 2009; Lavelle et
al., 2009). Nevertheless, only few cases of liver cirrhosis and liver failure have been reported
in patients with bosentan therapy. Ambrisentan is the most recent ETA receptor antagonist
approved by the FDA for the treatment of pulmonary arterial hypertension. No evidence of an
association between ambrisentan therapy and hepatotoxicity emerged from a rigorous post-
marketing monitoring to assess the hepatic safety carried out in the United States.
Hepatotoxicity may be related to the chemical structure of the drugs. Bosentan and
sitaxsentan share a sulfonamide structure and are potentially hepatotoxic whereas ambrisentan
is a propanoic acid-based drug. Furthermore, it was reported that bosentan and sitaxentan, but
not ambrisentan, inhibit hepatic transporters which may be likely related to the hepatotoxicity
observed for these drugs in the clinical setting (Hartman et al., 2010). Previous studies have
shown that bosentan inhibits hepatic canalicular transport proteins Bsep and Mrp2 reducing
bile excretion (Fattinger et al., 2001; Kemp et al., 2005). In addition to the hepatic toxicity
reported for some ET antagonists, other causes that derived in failure of the clinical trials
testing ET antagonists include study design, patient selection, and drug dosing (Barton and
Kohan, 2011). It has to be considered that most of these trials were conducted when much of
the biology of ETs and ET receptors, particularly in humans, was largely unknown.
Despite of both mixed receptor and ETA-selective antagonists, other pharmacological
strategies are being investigated. Clinical trials examine the efficacy of ET-converting
enzyme and neutral endopeptidase combined therapeutics (Wengenmayer et al., 2011). In
addition, another strategy investigated is based on ETB receptor activation by agonists,
particularly in chemotherapy and neuroprotection. IRL1620 is a truncated linear analogue in
which the N-terminus has an N-succinyl modification that reduces metabolism by nonspecific
peptidases. It was developed as an ETB agonist but now is used in clinical trials as a potential
vasodilator in the delivery of anticancer agents and in neuroprotection. Another ETB agonist,
BQ3020, has been widely used but not in clinical trials (Assal et al., 1997).
Although ETs are intimately involved in the pathogenesis of diverse liver diseases the use
of therapies based on drugs targeting the endothelinergic system is still a pending issue since
most clinical trials resulted either non-effective or negative. A wide variety of selective and
mixed ET antagonists has been developed and although they proved effective and safe in
animal studies, they resulted hepatotoxic in clinical trials.

Up to date, as previously discussed, ET antagonists have only been approved to be used

in pulmonary arterial hypertension and scleroderma-related digital ulcers. Nevertheless, the
analysis of possible causes involved in the failure of most ET antagonist clinical trials as
discussed in the Twelfth International Conference on Endothelin by a panel of experts brings
new hopes in the field of ET antagonists for possible clinical use in diverse diseases given
that most clinical trials were performed when the knowledge about the biology of ETs was
limited (Kohan et al., 2012). In addition, other targets of the endothelinergic system are
presently being investigated so hopefully new drugs will emerge to be tested in liver diseases.
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Chapter 8
HEPATITIS B VIRUS
Diego Flichman
ABSTRACT
Hepatitis B virus (HBV), which may cause self-limited or persistent infections, is a
global health problem. It has been estimated that 2 billion people worldwide have been
exposed to HBV and 400 million are chronic carriers.. HBV is the prototype of the family
Hepadnaviridae. The virion is a spherical particle of 42 nm in diameter composed of a
nucleocapsid surrounded by an outer lipoprotein coat, where viral surface antigens are
embedded. The genome is a small partially double-stranded relaxed circular DNA, which
has a very compact coding organization with four partially overlapping open reading
frames that are translated into seven proteins. Although is a DNA virus, like RNA
viruses, employs the error-prone polymerase reverse transcriptase as part of its replication
process. HBV has been classified into 8 genotypes (A to H) and several subgenotypes.
An effective vaccine has been available since the 80s, producing protection in up to 95%
of immune competent recipients. The pathogenesis of chronic infection is mainly based
on two events: a variety of molecular mechanisms by which the virus interferes with the
immune system, preventing its clearance, and successive cycles of inflammation, necrosis
and regeneration consequence of the host immune system in an attempt to clarify the
infection.
VIRAL STRUCTURE
The human Hepatitis B virus (HBV) is a prototype member of the family Hepadnaviridae
(a name that derives from the organ tropism - hepa for liver - and the nature of the nucleic
acid. All viruses of this family share similar genomic organization, organ tropism and a
unique strategy of genome replication. Moreover, they have common structural features;
particularly, HBV - one of the smallest enveloped animal viruses - is a spherical particle of 42
nm in diameter surrounded by a lipoprotein coat picked up when the virus buds from an
infected cell. The membrane is embedded with three similar forms of the viral surface
antigens (HBsAg) that are involved in viral binding of and entry into susceptible cells. In

150 Diego Flichman
addition, these antigens contain a highly conformational and cysteine-rich domain, called the
‘a’ determinant, against which neutralizing anti-HBs antibodies are directed. Inside the
envelope, there is a 22-25 nm viral capsid made of Core protein (HBcAg), which contains the
viral genome and a protein with polymerase activity (Figure 1).
The HBV genome is a small partially double-stranded relaxed circular DNA (rcDNA) of
3200 base pairs in size, consisting of a full length minus strand with a terminal redundancy of
7-9 nucleotides and a shorter plus strand with a variable length between 1700-2800 base
pairs. Despite being not covalently closed, the viral genome maintains the circular shape due
to a small cohesive overlapping region between the 5 'end of the positive strand and both ends
of the negative strand (Seeger et al., 2006).
In the hepatocyte nucleus, once infection has occurred, the viral genome undergoes a
structural transformation/modification, mediated by cellular enzymes, which convert the
rcDNA in a circular covalently close molecule, termed cccDNA. This form, structured as a
mini-chromosome surrounded with histones and non-histones proteins, remains in the
hepatocyte lifetime, is the hallmark of the establishment of persistent infection and is the
major pitfall to eradicating chronic HBV infection (Belloni et al., 2007; Pollicino et al.,
2006).
The coding information of the genome is found exclusively on the minus strand, which
contains four highly overlapped open reading frames (ORFs) - C, P, S and X. This feature
causes constraints in the evolution of HBV, since a mutation at a given ORF might involve
mutations in the overlapping ORF, which would have significant implications in biology of
HBV (Seeger et al., 2015).
The cccDNA is the transcriptional template of five RNA messengers. All transcripts have
different 5ènds and a common 3’ end because they all use the same polyadenylation signal,
located at the 5’ end of the core gene. After being synthesized, viral RNA messengers are
transported to the cytoplasm where they are translated.
Two of these transcripts, the pregenomic (pg) and the preCore (pC) RNAs, are 3.5 kb in
length and are longer than the genome minus strand itself. The first one is a bicistronic
transcript which codifies for both Core (21 KDa) and Polymerase (90KDa) proteins. Its ORFs
are shifted and partially overlapped; in addition, the pg-RNA plays the role of a template for
synthesis of the viral genome after being packaged into core particles together with a viral
polymerase molecule. On the other hand, pC-RNA translation results in the pre-Core protein,
a precursor of the e antigen (HBeAg). This antigen is not required for viral assembly,
replication, or infection but acts as a decoy antigen, playing an immunoregulatory role in
natural infection, favoring the establishment of persistent infection.
The three remaining mRNAs are subgenomics. Two of them encode the envelope
proteins and have in-frame initiation codons. Depending on which translation initiation site is
used, large (L-HBs), medium (M-HBs) and small (S-HBs) surface antigens are obtained.
These antigens differ in their N-terminal domains and share the same C-terminal. Like typical
membrane proteins, the HBV envelope proteins are synthesized and integrated at the ER
(Bruss, 2007). Finally, the X mRNA encodes for protein X (17 KDa), which is not part of the
HBV structure and has multi-regulatory activity on viral and host genes (Lucifora et al.,
2011).

Hepatitis B Virus 151
Figure 1. Schematics of the hepatitis B virus (HBV) structure and replication cycle. On the top a liver
sinusoid and the hepatocyte extracellular space (space of Disse) are depicted. HBV is a spherical particle of
42 nm in diameter, composed of an envelope which contains the viral capsid formed by Core protein, the
viral genome and a molecule with polymerase activity. HBV binds to its cellular receptor NTCP, which
posses 7 or 9 transmembrane segments. Heparan sulfate proteoglycan (HSPG) is critical for virus attachment
and helps enrich virions on the cell surface, bringing them in close proximity to the receptor. Cellular entry of
HBV is likely mediated by endocytosis, presumably after the virus binds NTCP. The virion is uncoated and
directed to the cell nucleus, driven by the nuclear localization signals present in the Core protein. The relaxed
circular DNA (rcDNA) genome is modified into the nucleus, where covalently closed circular DNA
(cccDNA) is formed. The cccDNA acts as a transcriptional template generating five viral transcripts
(mRNAs). Thereafter, protein synthesis is accomplished. A molecule of pg-RNA is packaged in the capsid
alongside the viral polymerase; subsequently, a minus strand is synthesized by reverse transcription and then
the plus strand is synthesized. Once the capsid is mature, it is assembled with the envelope and ultimately the
viral particle is released. P: polymerase, C: Core protein, e: e antigen, S: envelope proteins.

152 Diego Flichman
The expression of viral transcripts is regulated by four promoters, - L promoter for the
2.4 kb RNA encoding L-HBs, - S promoter for the 2.1 kb RNA giving rise to the M-HBs and
S-HBs, - X promoter for the 0.7 kb RNA encoding HBx and - core promoter for the 3.5 kb
RNA for the precore and core proteins; while polymerase expression is controlled at the
translational level via leaky ribosomal scanning. In addition, the HBV genome has two
enhancers involved in regulating replication (Moola et al. 2002).
Another two structures are relevant to the biology of HBV, short direct repeat (DR)
sequences on the 5òf both strands, which are involved in priming the synthesis of their
respective strands, and the encapsidation signal, characterized by a highly conserved stem-
loop structure that interacts with the viral polymerase in the early stage of the genome
replication (Jeong et al., 2000).
CELL CYCLE
The lack of a suitable cell culture system has hampered to some extent the understanding
of HBV biology. Currently, there are no stable cell lines that allow full characterization of the
HBV replication cycle. However, significant advances in this field have been made in the past
decades.
The HBV replication cycle begins with the attachment of the virus to a susceptible
hepatocyte and subsequent penetration into the cell cytoplasm as a consequence of the
interaction between the anti-receptor present in the viral envelope and its specific cell
receptor (Urban et al., 2010; 10. Schädler et al., 2009) (Figure 1).
The anti-receptor has been mapped, after genetic and functional studies, in a small region
covering residues 9–16 of the preS1 domain of the large envelope protein (Chouteau et al.,
2001). Initially, this region resides in the interior of the virus particle but subsequently, during
virion maturation, the N-terminus of the preS1 domain is modified by myristic acid and
approximately half of the preS1 molecules domain undergoes a posttranslational translocation
process, resulting in its exposure on the virion surface. This dual topology allows preS1 to
participate, on one hand, in the attachment-binding to the cell-receptor and on the other hand,
to play its role in the coating of the replicating core particles (Lambert et al., 2004).
Regarding cell factors, heparan sulfate proteoglycans act as critical binding factors for
viral attachment to the surface of hepatocytes, leading viruses in close proximity to the
receptor prior to specific engagement. Subsequently, the preS1 region of the L-HBs
specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), which has
recently been discovered to act as a functional receptor of HBV. The NTCP, encoded by the
gene SLC10A1 [Solute Carrier family 10 (sodium/bile acid cotransporter), member 1], is a
multiple transmembrane transporter, predominantly expressed in the liver (Yan et al., 2012).
The specific mechanism of viral entry has not been elucidated yet. Both endocytosis and
direct fusion of the viral envelope with the plasma membrane have been proposed as potential
pathways.
After uncoating into the cytoplasm, viral capsids are directed to the cell nucleus, driven
by the nuclear localization signals present in the Core protein. The nucleocapsid interacts with
nuclear pores and dissociates, releasing the rcDNA into the cell nucleus, then the rcDNA
genome is repaired by host enzymes, yielding a covalently closed circular molecule

(cccDNA). Specifically, - 3' downstream gap on the plus strand is filled, - 5’ terminal
structures (terminal protein in the minus strand, oligoribonucleotide primer in the plus strand)
are removed, and eventually, the gaps in both strands’ ends are covalently linked (Grimm et
al., 2011).
The viral gene expression is accomplished by the cellular RNA polymerase II, using the
negative cccDNA strand as template and regulated by viral promoters and enhancers.
Thereafter, synthesized viral mRNAs are transported to the cytoplasm, where they are
translated (Datta et al., 2012).
As mentioned in the preceding section, the pg-RNA encodes both Core and viral
polymerase protein. The Core protein has two domains. The N-terminal (residues 1 to1 49)
forms the assembly domain, necessary and sufficient for capsid assembly, whereas the C-
terminal, rich in arginine residues, is involved in the pregenome packaging.
The viral capsid, as in many cases, is formed of a single type of protein. In this case, it is
composed of 120 dimers of Core protein with an icosahedral T = 4 symmetry. The first step is
the formation of homodimers linked by a disulfide bridge between the cysteine residue at
position 61. Thereafter, Core protein dimers polymerization is triggered by the binding of
viral polymerase to the encapsidation signal located in the pg-RNA (Selzer et al., 2014).
The other protein derived from the pg-RNA is the viral polymerase, which has three
domains with specific activities: -the N-terminal, called terminal protein (PT), which acts as a
primer for minus strand synthesis; - the RT domain, possessing DNA polymerase and reverse
transcriptase activity; and - the C-terminal with RNAse activity (Jones et al., 2013).
The viral genome synthesis from the encapsidated pg-RNA involves multiple steps.
Initially, pg-RNA is packaged, together with a polymerase molecule, into subviral core
particles, forming the replication complex.
The specific packaging of the pg-RNA depends on the formation of a complex that
requires the interaction of viral polymerase, via the hydroxyl group of the tyrosine residue at
position 63 of terminal protein, with the encapsidation signal present in the pg-RNA.
Thereafter, primed by a 4-5 base oligonucleotide, a minus DNA strand is synthesized using
the reverse transcriptase activity of the RT domain and the pg-RNA as a template; this event
occurs inside the HBV nucleocapsid. Simultaneously, the C-terminus domain of the
polymerase, with RNAseH activity, removes the pg-RNA. The Hepadnaviridae family is the
only DNA virus that, like RNA viruses, employs the error-prone polymerase reverse
transcriptase as part of its replication process (Nassal, 2008).
After the negative strand has been synthesized, the viral polymerase switches its position
from the direct repeat sequence 1 (DR1) in the 3’ end of the minus strand to the homologous
DR2 in the 5’ and initiates the synthesis of the plus strand, using DNA polymerase activity of
the RT domain and the minus strand as a template. Synthesis of the plus strand continues until
it reaches 50-70% of the length of the minus strand.
Different hypotheses have been raised about whether second strand synthesis may halt
when the capsid gets too crowded, stopping short of a full-length double helix. Alternatively,
the formation of double-stranded DNA appears to be the signal that causes the capsid to seek
out the membrane and start budding out of the cell. So reverse transcription may be halted
prematurely when the supply of nucleotides is shut off as the virus buds (Goodsell, 2007).
After carrying out the synthesis of both DNA strands, mature capsids may follow two
alternative paths: be driven back to the nucleus, in a feedback process that amplify the nuclear
cccDNA pool, or follow the assembly pathway, being coated in the ER with a lipid-protein

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envelope containing the HBsAg transported to the protein secretion pathway from ER to
Golgi network and released as viral progeny into the extracellular space.
The selection of mature nucleocapsids for envelopment and subsequent virion secretion is
influenced by a change in the structure of the capsid protein. This structural change is
triggered by the maturation of the viral genomic DNA or the dephosphorylation of the core
protein, which leads to the association of mature capsids with cellular membranes (Mabit et
al., 2000). Finally, viral particle release is mediated by multivesicular bodies of the late
endosomal compartment. HBV virions have been localized to membranes of the late
endosome and large intracellular compartments (Mhamdi et al., 2007).
The mean half-life of the serum HBV pool was estimated at 4 hours with a production
rate of 1011 virions/day. As a consequence of viral replication, viral loads of ≥108 -109
virions/ml of plasma are found in HBV chronically infected patients (particularly in the
HBeAg-positive stage) (Murray et al., 2006).
The hepadnaviridae family has a peculiar feature: in addition to mature viral particles, the
envelope proteins also assemble into empty subviral particles at the post-ER/pre-Golgi
membrane. Subviral particles are found as spherical structures with a diameter of about 22 nm
and filaments of similar diameter but variable length (Patient et al., 2007).
The non-infectious subviral particles are usually produced in a 1,000- to 1,000,000-fold
excess over virions. Such extensive overproduction could induce T cell response impairment
and/or deletion and acts as a decoy for the humoral immunity. Moreover, it was recently
observed that these subviral particles carry selective pools of liver-specific miRNAs which
might play a pivotal role in HBV biology. Furthermore, the large amounts of secretion of
HBsAg have allowed easy detection in the blood of infected individuals and nowadays,
quantification of this marker has been proposed in predicting response to antiviral treatment
and in identifying the individual’s infection status (Novellino et al., 2012).
Last, two non-structural proteins are synthesized during the replication cycle. The precore
polypeptide, synthesized from pC-RNA, is transported to the ER lumen, where its amino- and
carboxy-terminal are cleaved and the resultant protein is directed to the secretory pathway
and released into the bloodstream as the e antigen (HBeAg) (Millich et al., 2003) and the X
protein (HBx), which contributes to the efficiency of HBV replication by interacting with
different transcription factors and is capable of stimulating both cell proliferation and cell
death (Kew et al., 2011).
CLASSIFICATION AND GENETIC VARIABILITY

The Hepadnaviridae family has two genera, the Orthohepadnaviruses that infect
mammals (humans, woodchucks, ground squirrels, etc.), and the Avihepadnaviruses that
infect birds (ducks, wild herons, etc.) (Schaefer, 2007).
The HBV belongs to the orthohepadnavirus genus. Because of its evolution over a long
period of time a large amount of genetic diversity, despite the constraints imposed by the
complex genetic organization of the viral genome, has occurred. Consequently, HBV has
been classified into genotypes when genetic divergence across the complete genome among
isolates is greater than 7.5%. Currently, eight genotypes designated A through H have been
described, while another two putative genotypes, 'J' and 'I,' have been proposed, but this

designation is still controversial. In addition, there is a great deal of diversity within

genotypes and this has led to the division of some genotypes into different subgenotypes
when intergroup nucleotide divergence is between 4.0 and 7.5% (Kramvis, 2014).
The genotypes and subgenotypes have a distinct ethnic-geographical distribution.
Genotypes A and D are ubiquitous, although genotype A is mainly found in northwestern
Europe, North America and Sub-Saharan and western Africa countries while genotype D is
most prevalent in southeastern Europe, the Mediterranean Basin, the Middle East, and the
Indian subcontinent. Genotypes B and C are found essentially in Southeast Asia, genotype E
is restricted to West and Central Africa and genotype F is characteristic of South and Central
America and likely originated in Amerindian populations. Genotype G was usually identified
in isolated cases in different regions, often co-infecting with the most prevalent genotype in
the region, and genotype H is mostly found in Central America. Genotype I and genotype J
have been proposed for two recombinant strains of HBV, reported from Vietnam and Japan,
respectively (Sunbul, 2014).
Epidemiological data have increasingly associated HBV genotypes and subgenotypes
with differences in clinical and virological characteristics, such as severity of liver disease
and response to antiviral therapies (Lin, 2011; Liu et al., 2013).
The extensive magnitude of HBV replication rate, estimated at approximately 1011
virions/day, coupled with the mutational frequency of the HBV polymerase that is in the
range of 1.4 to 5.0 x 10-5 nucleotide substitutions per site per year (ten times higher than for
other DNA viruses and similar to certain RNA viruses), equals the awesome amount of at
least 1010 point mutations produced per day (Whalley et al., 2001).
Therefore, as a consequence of replication rate, the viral turnover, and the error-prone
polymerase, random mutations are introduced into the HBV genome during the replication.
Thereafter, in vivo environmental pressure selects those mutants with better fitness over the
wild type virus (Durantel, 2010). Mutants on the envelope, polymerase, and basal core
promoter/precore regions have been shown to have clinical implications (Torresi, 2002; Gao
et al., 2015).
POLYMERASE MUTANTS
Interferon-based treatment was the standard therapy for HBV in the early 80s. In the next
decade Lamivudine, the first oral nucleos(t)ide analog (NA), was approved for HBV chronic
infection treatment. Subsequently, several NAs were developed, including Adefovir in 2002,
Entecavir in 2005, Telbivudine in 2006, and Tenofovir in 2008. The viral target of these
antiviral agents is the RT domain of the HBV polymerase (Zoulim et al., 2011).
This therapeutic approach does not affect the cccDNA therefore, therapy does not
eradicate HBV from the liver, and hence the ultimate goal is to achieve sustained suppression
of viral replication, preventing progression to cirrhosis, liver failure, and terminal
hepatocellular carcinoma. Accordingly, the NAs treatment is long with the consequent risk
that resistant strains are selected.
Under selective pressure by means of the administration of antiviral agents, minor HBV
quasispecies converge on a dominant mutant that can escape selection pressure, creating a
drug-resistant HBV strain. The resistance phenomenon is usually verified by a ≥1 log increase

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in HBV viral load compared to the lowest value obtained during therapy. Therefore, NAs
therapy should be monitored closely.
Resistant variants to most of the currently available NAs have been observed and
molecular approaches evidenced that are the consequence of the selection of strains carrying
mutations in the RT catalytic domains.
The mutations M204I/V, L180M and A181T/V confer resistance to Lamivudine and
Telbivudine while A181T and N236T are associated with Adefovir resistance. Due to high a
genetic barrier, Entecavir and Tenofovir are currently considered the most potent antiviral
agents and are at low risk of developing resistance. Entecavir resistance usually involves
L180M + M204V and another among I169T, 184G/S, S202I/G and M250V, whereas no
resistance has been reported in patients treated with Adefovir for up to 6 years (Locarnini
2008).
It is important to highlight that some mutations associated with antiviral resistance are
common for different NAs; therefore, the use of one antiviral may jeopardize the
effectiveness of another. For instance, common mutations that confer resistance to
Lamivudine and Telbivudine give cross-resistance and reduced sensitivity to Entecavir.
Moreover, other than the specific point mutations associated with antiviral resistance, the
frequency of occurrence of these mutations must be considered. Clinical experience shows
that resistance rates over time vary markedly among the different NAs. After five-year
therapy rates were 70% for Lamivudine - for this reason, its use is no longer recommended as
monotherapy-, 29% for Adefovir, less than 2% for Entecavir and no resistance was observed
in patients treated with Tenofovir (Bang et al., 2014).
ENVELOPE MUTANTS
Like NAs to viral polymerase, the vaccine is the main selection force of mutants in the
envelope region (Purdy et al., 2007).
The HBV vaccine consists of a yeast-derived recombinant HBsAg protein and is effective
at producing protection in up to 95% of immunocompetent recipients. It was introduced in the
early 1980s. Currently, 184 of the 194 WHO member states (94.8%) have nationwide
vaccination programs, and global coverage with three doses is estimated at 82% (Cassidy et
al., 2011).
Despite the high efficacy of the HBV vaccine, breakthrough infections due to vaccine
escape mutations have been reported in vaccinated individuals, which accentuate the
importance of these escape mutants. As stated before, neutralizing antibodies are mainly
targeted against the “a” determinant present in the HBsAg. Mutations causing a
conformational change within this epitope could affect the HBsAg antigenicity and be
responsible for escaping vaccine-induced immunity. In addition, these variants may also
provide false negative results in serological tests, which are known as false occult hepatitis
(Shepard et al., 2006; Romanò et al., 2015).
The prevalence of chronic hepatitis B has declined dramatically worldwide
since vaccination programs’ implementation; however, as a counterpart, the massive
implementation of the vaccine has acted as a powerful selective force of variants of vaccine
escape mutants. For instance, HBV prevalence in Taiwan declined from 8.6 to 2.1% between

1984 and 1999, while vaccine escape mutants increased from 7.8 to 28.1% in the same time
period (Hsu et al., 2004).
The first of these mutations, associated with vaccine escape mutants, was G145R and
although it remains by far the predominant HBsAg mutant, a wide range of other HBV
envelope changes have been described, including amino acid substitutions, deletions or
insertions across the whole ‘a’ determinant (D144A, P142S, Q129H, I/T126N/A, M133L,
etc.) (Coppola et al., 2015). Different studies have estimated, by using mathematical models,
that in the coming decades vaccine escape mutants will become the dominant HBV
quasispecies globally (Wilson et al., 1999).
As mentioned before, the HBV genome is organized in such a way that the envelope gene
is completely overlapped by the polymerase gene (Ahn et al., 2014). As a consequence,
polymerase gene mutations selected during the course of antiviral NAs therapy may affect
neutralization epitopes within the HBsAg. For instance, the triple mutational pattern causing
Lamivudine resistance (V173L, L180M, and M204V) causes two amino acid changes in the
surface gene (E164D and I195M), which reduce antiHBs binding to levels similar to those
caused by vaccine escape variants.
BASAL CORE PROMOTER AND PRECORE MUTANTS

The HBeAg was initially considered a marker of active viral replication and still remains
an important endpoint in antiviral therapy of HBeAg-positive patients (Gerlich, 2013;
Hadziyannis 2014). However, in the early 80s, by using molecular biology tools HBV-DNA
was determined in anti-HBe positive patients, which led to the idea that the virus has active
replication without HBeAg expression (Carman et al., 1989).
As mentioned previously, the HBeAg is not essential for virus replication; it is assumed
that HBeAg acts as a decoy to buffer anti-core protein immune response and is necessary for
the establishment of persistent infection since infections caused by e minus variants rarely
follow this course.
The presence of mutations affecting HBeAg expression during the acute stage is
associated with more severe clinical courses and/or fulminant hepatic failure; furthermore, in
contrast to acute HBV infections with wild-type HBV, infections with HBeAg minus variants
rarely, if ever, go into chronicity. Thus, although virus survival during the anti-HBe stage
does not require HBeAg expression, the establishment of de novo chronic infection does
(Hadziyannis et al., 2006).
During the course of chronic infections a large proportion, if not all, seroconvert from
HBeAg to anti-hepatitis B e antibody (anti-HBe), which represents a late phase in the natural
history of chronic infection. This event, consequence of host-viral interaction, is usually
associated with mutants that affect HBeAg expression, suggesting a better fitness of these
variants at this stage of infection.
Mutations reducing or abrogating HBeAg production are the hallmark of the entity
known as antiHBe chronic hepatitis and may occur either at the transcriptional level, in
regulatory elements, or at the translational level, blocking HBeAg synthesis because of
nucleotide insertions, deletions or mutations that abolish HBeAg expression (Brunetto et al.,
1999).

158 Diego Flichman
In the Basal Core promoter region (BCP), the double mutation 1762T/1764A has been
the most widely described. The 1762T/1764A mutation reduces the level of pC-RNA
transcription, and indirectly HBeAg levels; as well as affecting the expression of HBeAg,
these mutations increase the rate of virus replication (Kramvis et al., 1999).
In addition to 1762T/1764A, mutations can be detected at nearby positions such as 1753,
1757, 1766, and 1768. Site-directed mutagenesis experiments have suggested that the
additional mutations at 1753, 1766, and 1788 further reduce HBeAg expression and enhance
genome replication, with the 1762T/1764A/1766T triple mutant having greater than 10-fold
higher replication capacity than the wild-type virus (Parekh et al., 2003). Regarding the
preCore region, the most common mutation abolishing HBeAg expression is G1896A, which
converts the penultimate (28th) precore codon from TGG to TAG (Papatheodoridis et al.,
2001).
Basal core promoter and stop codon mutants appear to be frequently associated. It was
observed that BCP mutations were detectable in the late HBeAg-positive phase of infection;
therefore, the extent of this event does seem per se sufficient to justify the antiHBe
phenotype, whereas precore mutations emerged during anti-HBe seroconversion. Therefore,
the suggested order of events would be that the virus first reduces HBeAg expression through
BCP mutations and then entirely abolishes HBeAg expression with precore mutations (Kay et
al., 2007).
HBV variants carrying the double1762T/1764A mutation in the BCP region and/or
the1896A mutation in the pC region have been presumed to be strictly associated with
progressive forms of liver disease (Lin et al., 2005; Chen et al., 2011).
However, the implication of mutants affecting HBeAg expression in the progression of
the chronic infection is a controversial issue. On the one hand, numerous studies have found a
significant association between the presence of these mutations and more severe clinical
disease. On the other hand, these mutations have also been observed in inactive carriers,
suggesting that mutations per se would not be responsible for the severity of the course of
infection (Chun et al., 2000; Ledesma et al., 2011).
The selection of HBeAg minus variants is usually observed in the late stage of chronic
infection, a result of the virus and host interaction; therefore it is likely that these mutations
are present in individuals with long-standing infections, but also, the evolution to more severe
stages of infection is dependent on time. For that reason, it is possible to hypothesize that
mutations in the BCP/pC region are a surrogate marker of the duration of infection, so they
are most often in advanced stages of infection. Studies where patients have been matched by
age have shown that most of these mutations are more frequent in older HBV carriers,
regardless of their clinical status (Shinkai et al., 2007; Kreutz et al., 2002).
In summary, mutations in the BCP/pC regions are the hallmark of chronic anti-HBe-
positive individuals; nevertheless, the even distribution of mutations in active and inactive
carriers suggests that BCP/pC mutations may occur during HBV infection not strictly related
to the HBV infection activity.

PATHOGENESIS
The HBV may lead to both self-limited and persistent infections. The infection outcome
relies on a finely poised and complex interplay between the virus and the host immune system
(McMahon et al., 2009).
There are two insights of HBV infection that highlight the relevance of the host in the
evolution of the infection: first, the persistent course is observed in most neonatal/perinatal
infections but only a small minority of adult onset infections, and second, a wide spectrum of
clinical features is induced during the chronic infection, ranging from an inactive carrier state
to severe forms of liver disease, such as fulminate hepatitis, cirrhosis, or hepatocellular
carcinoma (Liaw, 2010; Ganem et al., 2004).
There are several pieces of evidence that HBV is non-cytopathic, for instance, the
immunotolerant phase of the infection usually observed in children who become infected
perinatally, in whom high viral loads without signs of liver damage are observed. Therefore,
the pathogenesis basis of chronic HBV infection mainly lies in the successive cycles of
inflammation, low-level liver cell destruction, and regeneration over long periods of time, the
consequence of an impaired immune system in an attempt to clarify the infection (Baumert et
al., 2007).
SELF-LIMITED INFECTION
In the presence of a vigorous, polyclonal and multispecific cellular immune response
during the HBV primary infection, a significant rate of inflammation is observed, because the
immune response leads to substantial destruction of infected hepatocytes, which usually
results in a greater chance of control and resolution of the infection; however, there is also the
risk of liver failure and fulminant hepatitis (Isogawa et al., 2015; Yang, 2010).
It is generally acknowledged that early virus control is accomplished by cytokines
released by liver-infiltrating HBV-specific T cells. Interferon alpha, beta, and gamma as well
as tumor necrosis factor alpha have been implicated as the major contributors to viral
clearance. Particularly, CD8 cells at the site of infection trigger two events, apoptosis of the
hepatocytes and secretion of interferon gamma, which inhibits HBV by preventing pg-RNA-
containing capsids assembly in the cytoplasm, in a proteasome and kinase-dependent process
(Bertoletti et al., 2006).
In self-limited infections, viral DNA decreases by more than ninety percent within 2–3
weeks after peak viral replication and before detection of liver damage. Most of the HBV-
DNA is eliminated from the liver by non-cytolytic processes that precede and are independent
of the immune elimination of infected hepatocytes that probably supplements these non-
cytopathic antiviral events to fully control the infection. Furthermore, it has been observed
that even the cccDNA, a form believed to have a long half-life in non-inflammatory
conditions, is also susceptible to non-cytolytic control (Guidotti et al., 1999).
On the other hand, humoral response, particularly antiHBs antibodies, plays a critical role
in viral clearance by limiting viral spread from residual productively infected hepatocytes that
are not eliminated by the CD8+ T cells, complexing with free viral particles and removing
them from circulation or by preventing their attachment and uptake by hepatocytes; however,

160 Diego Flichman
the appearance of neutralizing antibodies occurs relatively late after HBV exposure and thus
it is unlikely to contribute to the early phase of viral clearance during acute infection (Mason
et al., 2008; Oh et al.,2015).
PERSISTENT INFECTION
In contrast to what is observed in the self-limited infection, HBV persistence is a
dynamic state of interactions among HBV, hepatocytes and host immune cells, characterized
an indolent low-grade chronic necro-inflammatory liver disease in the presence of a weak or
lack of protective T-cell memory maturation and by an exhaustion of HBV-specific T-cell
responses, ranging from functional inhibition to physical T-cell deletion (Ferrari, 2015).
In order to establish a persistent infection, the virus must deal with/overcome the innate
and acquired immune response of the host. Different HBV molecular features have been
associated with this aim.
Immune cells producing IFN-alpha/beta and IFN-gamma play an important role in
mounting an innate immune response against HBV under the conditions of a chronic
infectious process. Macrophages, natural killer cells, NKT, TCRγδ+ T cells secreting IFN-
gamma, and the production of IFN alpha/beta by plasmacytoid dendritic cells and infected
hepatocytes all function to enhance a systemic antiviral response. On the other hand, there
exists an experimentally confirmed CD8+ specific T cell-induced inflammation of the hepatic
tissue that is actively supported and aggravated by the above-mentioned non-specific cellular
components of the immune system present in hepatic cellular infiltrate (Golsaz-Shiraz et al.,
2016; Balmasova et al., 2014).
A key role in CD8 cell recruitment into the liver is played by platelet activation within
the infected liver, which can facilitate platelets/cytotoxic T lymphocyte interaction and the
egress of the latter from the bloodstream, with accumulation in the infected parenchyma
(Guidotti et al., 2015).
Peripheral blood CD4 T cell responses to HBV do not directly participate in viral
clearance and tissue damage but probably contribute indirectly to controlling the infection by
facilitating the induction and maintenance of the virus-specific B cell and CD8 T cell
response (Chisari et al., 2010).
Unlike another virus, primary HBV infection is poorly sensed by the innate immune
system. In vivo studies, performed in animal models, indicate a lack of IFN inducible genes
associated with entry and expansion of the virus, reflecting a defective activation and effector
function of the innate immune response. It is believed that this feature could be a consequence
of HBV replication features such as a transcriptional template (cccDNA) hidden within the
nucleus of infected cells in its replication cycle, capped and polyadenylated viral mRNAs that
resembles the normal cellular transcripts, and protection of newly transcribed genomes within
viral capsids in the cytoplasm (Wang et al., 2015).
In addition, it is likely that HBV nonstructural proteins actively inhibit the innate
response by interfering with IFN type I signaling and thereby affecting both their production
and antiviral activity. This suggests that HBV has developed some sophisticated mechanisms
to evade or subvert key aspects of the antiviral activity of liver cells. For instance, it was
shown that X protein interacts with mitochondrial antiviral signaling protein, preventing IFN-

beta induction, and also acts as an inhibitor of virus-triggered IFN-regulatory factor-3, which
mediates the induction of type I interferons. Furthermore, HBeAg is associated with the
disruption of monocyte functions, including a significant decrease in expression of the key
component of innate antiviral immunity, Toll-like receptors type three, which brings about
HBV persistence by blocking non-specific defense processes (Woltman et al., 2011).
Because of the poor induction of innate intracellular immunity, adaptive responses are
efficiently and timely induced immediately after active virus replication begins. Although
HBV-specific T cells become detectable several weeks after infection, these responses are
only apparently delayed in relation to the time of infection because of the initial quiescence of
HBV, which probably does not provide enough stimulation to prime and expand HBV-
specific T cells.
Although control of infection and liver cell injury are strictly dependent upon protective
immune responses, because hepatocyte damage is the cost that the host must face to get rid of
the intracellular virus, a recent study showed, after analyzing the relationship between the
number of intrahepatic HBV-specific CD8 T cells, extent of liver disease, and levels of HBV
replication in chronically infected patients, that inhibition of virus replication could be
independent of liver damage, and that the functionality of HBV-specific CD8 T cells was
more important than the number of T cells to control HBV replication (Maini, 2000).
In fact, depressed cytokine production with preserved cytotoxic activity has been
suggested to be detrimental to virus control but actively involved in the maintenance of
chronic liver inflammation and cell damage.
Analogous to what was described for the innate response, some viral proteins have been
shown to regulate the adaptive immune response to HBV, suggesting that HBV may also
employ active evasion strategies targeting the adaptive immune response (Busca et al., 2014).
The degree of T-cell impairment is variable and its severity is related to the level of virus
replication and antigen load. The antiviral T-cell function is more efficient in patients who
can control infection either partially, such as inactive HBsAg carriers with low levels of virus
replication, or completely, such as patients who achieve HBsAg loss either spontaneously or
after antiviral therapy (Tjawa et al., 2011).
The effect of prolonged exposure of T cells to high quantities of viral antigens is a key
determinant of functional T-cell impairment. Indeed, HBV produces large amounts of HBeAg
protein, as well as subviral noninfectious particles containing envelope antigens (HBsAg)
(Boni et al., 2007).
The HBeAg works as a tolerogenic protein because its homology with Core protein may
facilitate, by central deletion of high-affinity HBe/HBcAg-specific CD4+ T cells, clonal
ignorance or adaptive tolerance, resulting in ineffective cytotoxic T cell lysis of infected
hepatocytes; thus, it may suppress immune elimination of infected cells by Core-specific T
cells and thereby contribute to viral persistence in chronically infected adults (Chen et al.,
2005). Due to its small size, HBeAg may cross the placenta and elicit HBe/HBcAg-specific
Th cell tolerance in utero. This would explain the high chronicity rates on newborns from
chronically infected HBeAg-positive mothers, as well as the absence of chronicity from
HBeAg-negative mothers (Li et al., 2015).
On the other hand, surface antigens might also suppress immune elimination of infected
cells by functioning as a high dose tolerogen. In fact, defective peripheral HBsAg-specific
CD8+ T cell responses in chronically infected patients correlate with serum HBsAg titers
(Chisari et al., 2010).

162 Diego Flichman
Exhausted T cells express high levels of co-inhibitory molecules, such as TIM3, CTLA4,
2B4 and PD1, that can transduce inhibitory signals to T cells. As a result of the quantity of
viral antigens and the duration of T-cell exposure to high antigen loads, T-cell exhaustion can
be more or less severe, with either a total or a partial loss of antiviral cytokine production,
cytolytic activity and capacity to expand following antigen encounter. The importance of
these mechanisms of T-cell inhibition is indicated by the partial restoration of the T-cell
function upon decline of antigen and prolonged suppression of HBV replication in nucleoside
analog treated patients.
Moreover, additional factors such as the tolerogenic effect of the liver environment can
contribute to T-cell inhibition caused by the tolerogenic features of hepatocytes and liver
resident cells and also by IL10, TGF-b, and arginase, which are enriched within the livers of
chronic patients (Pallett et al., 2015).
Epidemiological studies have proven a strong correlation between chronic hepatitis B
virus infection and the development of hepatocellular carcinoma (HCC) (El-Serag, 2012).
The underlying molecular mechanism to HCC developments is probably a multifactorial
phenomenon that includes accumulation of genetic damage due to immune-mediated hepatic
inflammation and the induction of oxidative stress, epigenetic effects through the
modification of the genomic methylation status, regulation of microRNA expression and
interference of viral proteins in cell pathways (87. Ringelhan et al., 2015; Liu et al., 2007).
In many different tissues, chronic inflammation is known to play a vital role in cancer
development. As mentioned above, the persistent HBV infection is characterized by an
indolent low-grade chronic necro-inflammatory liver disease, with continuous cycles of cell
injury, inflammation, and regeneration.
The chronic liver cell injury is a premalignant condition that initiates a cascade of events
characterized by widespread DNA damage, insertional deregulation of cellular growth control
genes, increased rates of cellular DNA synthesis and production of endogenous mutagens
coupled with compromised cellular detoxification and repair functions. If these processes are
sustained for a sufficiently long period of time, they would be expected to cause the multiple
genetic and chromosomal changes necessary to trigger the development of hepatocellular
carcinoma. Integration of HBV-DNA into the human genome is considered an early event in
the carcinogenic process (Tarocchi, 2014).
Furthermore, epigenetic effects through the modification of the genomic methylation
status may be involved in the carcinogenesis (Lin et al., 2014). Aberrant DNA methylation
has been associated with a large number of human cancers. There is a large body of evidence
that HBx up-regulates the DNA methyltransferases, enzymes responsible for the maintenance
of methylation patterns. For instance, HBx down-regulates miR-152 with the consequent up-
regulation of DNMT1, which methylates the promoters of many tumor suppressor genes (Ali
et al., 2014). Indeed, DNA hypermethylation in the promoter region of specific
oncosuppressor genes was found in HBV-related HCC. Moreover, HBx interacts with p53,
altering p53-mediated apoptosis, trans-activation properties of p53, cell cycle regulation,
DNA repair genes, and tumor suppressor genes (Chung et al., 2003).
Micro-RNAs are small non-coding RNA molecules that regulate gene expression at
transcriptional and post-transcriptional levels. In HBV-related HCC miR-143, miR-34, and
miR-19 have been found to be up-regulated and associated with a more aggressive cancer
phenotype (Braconi et al., 2011).

Finally, HBx and HBsAg have been involved in mechanisms associated with HCC.
The HBx interacts with different transcription factors and is capable of stimulating both
cell proliferation and cell death. Its expression is associated with activation of the Ras-Raf-
MAP kinase pathway, an important cellular pathway that has been implicated in
hepatocarcinogenesis. A further piece of evidence of the involvement of HBx in the
carcinogenesis is that unlike orthohepadnavirus, the avihepadnavirus lacks the X gene and
HCC is not developed chronic infection (Ali et al., 2014).
Last, several specific mutations in the preS/S gene may induce an unbalanced production
of envelope proteins that accumulate in the endoplasmic reticulum of the hepatocytes and
may activate the ER stress signaling pathways, with consequent induction of oxidative DNA
damage and genomic instability. This process happens also in the presence of pre-S2 mutants
in which the viral proteins collect in the ER. The induced ER stress up-regulates the
cytoplasmic Cyclin A, increasing infected cell proliferation (Lazar et al., 2014); at the same
time, Cyclin A up-regulation can promote, through centrosome over-duplication,
chromosome instability, which is a well-known mechanism in HBV-related
hepatocarcinogenesis (Neuvert et al., 2010).
In summary, the pathogenesis of chronic HBV infection is mainly based on two events.
On the one hand, a variety of molecular mechanisms allows the virus to interfere with the
immune system preventing its clearance. On the other hand, inflammation, necrosis, and
regeneration occur in successive cycles, as a consequence of an immune system that is
inefficient when it must clear the infection.
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Chapter 9
CHRONIC HEPATITIS B: CURRENT TREATMENT AND

NEW THERAPEUTIC OPTIONS
Maria Gimena Fernandez
ABSTRACT
Hepatitis B virus (HBV) affects approximately 250 million people worldwide.
Hepatocellular carcinoma (HCC), cirrhosis, liver failure and death are severe
consequences of chronic infection.
Hepatitis B virus (HBV) is a small DNA virus that causes liver cell infection; the
viral genome generates a highly stable intranuclear minichromosome, the covalently
closed circular DNA (HBV cccDNA). The persistence of HBV cccDNA in liver cells is
the main cause of the lack of a definitive cure with current therapy.
Hepatitis B antiviral therapy´s main goal is to suppress the replication of HBV and
ideally to eradicate HBV in order to decrease morbidity and mortality related to the
infection.
Nucleotide analogs, nucleoside analogues and immunomodulators are currently
available treatment options for HBV. Nucleos(t)ide analogues suppress viral replication
by inhibiting ADN polymerase; and Pegylated interferon is a cytokine with
immunomodulatory and antiviral effects. Nowadays the virological cure, meaning virus
eradication, including cccDNA is not a reachable goal.
New drugs with diverse mechanism of action and targets are at present under
development, with the aim of preventing HBV replication and eradication of cccDNA in
order to achieve HBV definite cure.
INTRODUCTION
Approximately 250 million people worldwide are affected by chronic hepatitis B virus
(HBV). Hepatocellular carcinoma (HCC), cirrhosis, liver failure and death are severe
consequences of chronic infection, 30% of cirrhosis and about 53% of hepatocellular
carcinoma are attributed to chronic hepatitis B (Brahmania, Feld, Arif and Janssen, 2016). In
hepatitis B e antigen (HBeAg) positive, the annual incidence of cirrhosis is 2-6% and in

170 Maria Gimena Fernandez
HbeAg negative patients is 8-10%. The estimated lifetime risk for developing cirrhosis, liver
failure or HCC reaches 15-40% in patients affected with HBV (C.-L. Lin, Yang and Kao,
2016; A. Lok, 2002).
Hepatitis B virus (HBV) is a small DNA virus that causes liver cell infection by entering
the hepatocyte through the sodium taurocholate cotransporting polypeptide surface receptor
(NTCP; Coffin and Lee, 2015). After the interaction with the receptor and entry, the viral
genome generates a highly stable intranuclear minichromosome, the covalently closed
circular DNA (HBV cccDNA), this is the transcriptional template for all viral proteins (HBV
surface or envelope (S), polymerase or reverse transcriptase (P/RT), precore (HBeAg in
serum), core or nucleocapsid (C) and X protein) (You, Lee, Jang and Yoon, 2014). The main
mechanism of HBV infection and the principal cause of the lack of definitive cure with
current therapy is the persistence of HBV cccDNA in liver cells (Coffin and Lee, 2015),
besides due to the persistence of cccDNA in the nucleus of the hepatocytes there is a risk of
HBV reactivation even in patients with serological markers of resolved infection (Terrault et
al., 2015). Since HBV is a non- cytopathic virus liver injury is mediated by HBV specific
cluster differentiation CD8 cytotoxic T cells.
Different studies have evidenced that progression to liver cirrhosis and HCC in chronic
HBV infection is directly related to circulating HBV DNA levels, therefore HBV antiviral
therapy´s main goal is to suppress HBV replication and ideally to eradicate HBV in order to
decrease morbidity and mortality related to the infection (You et al., 2014).
The natural history of HBV infection is divided into four phases: immune tolerant phase,
immune clearance phase, low replicative or inactive carrier stage, and reactivation phase. At
present HBV treatment is indicated in the immune clearance phase and in the reactivation
phase of chronic HBV infection, it is not recommended for the immune tolerant phase (You et
al., 2014).
The sustained suppression of HBV DNA replication is associated with normalization of
liver enzymes, improvement in liver histology and HbeAg loss with or without development
of anti Hbe (You et al., 2014).
Current available treatment options for HBV are divided in: nucleotide analogs,
nucleoside analogues and immunomodulators. Nucleos(t)ide analogues are able to suppress
viral replication by inhibiting ADN polymerase; and Pegylated interferon is a cytokine with
immunomodulatory and antiviral effects.
Nowadays the virological cure, meaning eradication of virus, including cccDNA is not a
reachable goal.
CURRENT TREATMENT OPTIONS
Pegilated Interferon Alfa (Peg-IFN)
Pegilated interferon´s mechanism of action against HBV is the enhancement of the host´s
cytotoxic T cell lymphocytes in order to remove infected cells (Brahmania et al., 2016; A.
Lok, 2002).
Low serum HBV DNA, ALT flares (2-5 times the upper limit of normal, meaning host
immune response to treatment), HBV genotype and IL 28 (HBsAg seroconversion rate in

Chronic Hepatitis B 171
HBeAg-negative chronic HBV patients was significantly higher in the IL28B CC patients) are
factors that predict favorable response to Peg-IFN therapy (You et al., 2014; A. S. F. Lok and
McMahon, 2009; Flink et al., 2005).
The main advantages of using peg interferon in HBV treatment are its finite duration (48
weeks) and the lack of resistance (Liu, Seto, Lai and Yuen, 2016). Frequent side effects (flu-
like symptoms, fatigue, mood disturbances, cytopenias, and autoimmune disorders) and
subcutaneous injection are the principal disadvantages of Peg-IFN treatment. Peg-IFN is
contraindicated in patients with decompensated HBV-related cirrhosis or autoimmune
disease, in patients with uncontrolled severe depression or psychosis, severe cytopenias,
severe cardiac disease, uncontrolled seizures and in female patients during pregnancy
(Terrault et al., 2015; EASL, 2012).
Peg- IFN is the treatment of choice in patients with potent immune system, elevated liver
enzymes and evidence of viral replication (Manzoor, Saalim, Imran, Resham and Ashraf,
2015), the recommended dose is 180 ug weekly during 48 weeks.
Nucleos(t)ides Analogues Nucleos (T Ide Analogues)
The approved nucleos(t)ides analogues for the treatment of chronic HBV are lamivudine,
adefovir, telbivudine, entecavir and tenofovir.
The advantages of nucleos(t)ides treatment are the oral administration, good tolerance
and potent antiviral effect. The risk of resistance, the indefinite duration and its unknown
long-term safety can be mentioned among some of the disadvantages. (EASL, 2012; Ohishi
and Chayama, 2012).
Some studies demonstrated histological improvements in necroinflammatory and fibrosis
scores after long term treatment with entecavir and tenofovir (C.-L. Lin et al., 2016).
Lamivudine (LAM)
LAM was the first approved oral antiviral agent against HBV. It is a cytidine nucleoside
analogue with anti HBV effect by inhibiting reverse transcriptase enzyme (Manzoor et al.,
2015).
The recommended dose is 100 mg administered daily.
HBeAg seroconversion rate in Chronic HBV patients treated with Lamivudine after one
year is 32% reaching up to 65% after five years of treatment (Dienstag, 1999; Liaw, 2003;
Leung, 2008).
The major disadvantage of LAM is the low genetic barrier, therefore there is high
association with drug resistance, and the emergence of tyrosine-methionine-aspartate-
aspartate (YMDD) mutants is as high as 60%-70% after five years of treatment (You et al.,
2014).
Because of the risk of resistance, LAM is not recommended as a first line treatment
option in HBV chronic infection in the latest international guidelines.
Adefovir (ADV)
ADF is a nucleotide analogue of adenosine monophosphate. It inhibits reverse
transcriptase and DNA polymerase activity and is also incorporated into HBV DNA causing

chain termination (A. S. F. Lok and McMahon, 2009). It is administered in a daily dosage of
10 mg.
ADF has shown significant histological improvement, with virological and biochemical
efficacy when administered both in HBeAg-positive and negative naïve chronic hepatitis B
patients. It has even demonstrated virological response in patients with LAM-resistant HBV,
used alone or combined with LAM therapy (You et al., 2014).
Despite the fact that ADF is accepted as HBV therapy with less development of
resistence than LAM, it is not included as first line treatment in current international
guidelines since it is less effective than entecavir and tenofovir and there is a high risk of
nephrotoxicity as side effect (You et al., 2014).
Entecavir (ETV)
Entecavir is an analogue of 2 –deoxyguanosine, the inhibition of HBV replication by
entecavir is achieved through three steps: the priming of HBV DNA polymerase, the reverse
transcription of the negative strand HBV DNA from the pregenomic RNA, and the synthesis
of the positive strand HBV DNA (A. S. F. Lok and McMahon, 2009).
Entecavir is more effective than LAM and ADF in the treatment of chronic HBV, is even
effective against lamivudine resistant mutants (A. S. F. Lok and McMahon, 2009).
The recommended dose is 0, 5 mg daily for the treatment of naïve patients and 1 mg for
those with LAM resistance (Manzoor et al., 2015).
ETV is currently considered treatment of choice for chronic hepatitis B patients,
including compensated and decompensated cirrhosis (Ohishi and Chayama, 2012).
Telbivudine (LdT)
LdT is the L-nucleoside analogue of L-deoxythymidine, it has potent antiviral activity
against HBV, and it is even more potent than LAM.
The low genetic barrier of telbivudine is a major disadvantage and there is cross
resistance with lamivudine.
It is orally taken at a daily dosage of 600 mg and is safe to use in pregnancy.
At present telbivudine is not recommended as first line treatment in chronic HBV
according to international guidelines.
Tenofovir (TDF)
Tenofovir disoproxil fumarate is a nucleotide analogue with a powerful antiviral effect
against HBV; it is also effective against HIV virus (You et al., 2014).
TDF inhibits both HBV and HIV polymerase. The recommended dose is 300 mg daily,
orally administered.
Most frequent adverse events are nephrotoxicity and decreased bone density.
There is no resistance described for TDF due to its high genetic barrier, therefore is
recommended even in case of entecavir resistance.
TDF is safe to use during pregnancy.

Drug Route Dosis Side effects Duration

Peg-IFN alfa Subcutaneous 180 ug weakly Flu-like symptoms 48 weeks
Fatigue
Mood disturbances
Cytopenias
Autoimmune disorders
Lamivudine Oral 100 mg daily Pancreatitis Indefinitely
Lactic acidosis
Adefovir Oral 10 mg daily Acute kidney failure Indefinitely
Fanconi syndrome
Nephrogenic diabetes insipidus
Lactic acidosis
Entecavir Oral 0,5 mg daily Lactic acidosis Indefinitely
Telbivudine Oral 600 mg daily Creatine kinase elevations Indefinitely
Myopathy
Peripheral neuropathy
Lactic acidosis
Tenofovir Oral 300 mg daily Nephropathy Indefinitely
Fanconi syndrome
Osteomalacia
Lactic acidosis
With the available treatments, the rate of antiHbe seroconversion in HbeAg positive
patients after 12 months is 30% with Peg IFN and 20% with nucleos(t)ides analogues
approximately, and the rates of HBsAg loss is 3– 7% with Peg-IFN, 1% with lamivudine, 0%
with adefovir, 2% with entecavir, 0.5% with telbivudine, and 3% with tenofovir. In HbeAg
negative patients virological response is 20% after Peg IFN therapy and less than 5% after
only one year of nucleos(t)ides analogues therapy, however in patients treated with entecavir
or tenofovir for more than 3 to 5 years, the virological remission reaches 95%. Rates of
HBsAg loss after treatment with Peg-IFN is 3% approximately and is extremely unusual with
nucleos(t)ides analogues (EASL, 2012).
Current guidelines consider TDF and ETV as first-line treatments for compensated and
decompensated liver disease in chronic HBV infection since they are the most effective
antiviral agents.
NEW THERAPEUTIC OPTIONS
New Polymerase Inhibitors
Several clinical trials are ongoing to develop novel nuceos(t)ides analogs that aim to
achieve new drugs without resistance
Tenofovir Alafenamide (GS-7340)

Tenofovir alafenamide (TAF) is a prodrug of tenofovir. It has shown more potent activity
against HIV infection with higher intracellular tenofovir levels as well as low-level plasma
tenofovir concentrations than TDF (Markowitz et al., 2014).

In a recent study, non-cirrhotic treatment-naive HBV patients were randomized to TAF 8,

25, 40, 120 mg and TDF 300 mg for 28 days. It showed that in patients receiving TAF
independently of the dose, the decrease in HBV DNA was similar to patients receiving TDF
(Agarwal et al., 2015), with less nephrotoxicity.
Besifovir (LB80380)
Besifovir (LB80380) is a potent acyclic nucleotide phosphonate with a similar structure
to adefovir and tenofovir. It is converted into active metabolites in liver and intestine.
Preliminary studies showed that it is effective in HBV DNA suppression for treatment of
naïve chronic HBV patients as well as lamivudine resistant patients (Coffin and Lee, 2015).
Both drugs, TAF and besifovir can suppress HBV replication in an effective and safer
way but they do not clear cccDNA (C.-L. Lin et al., 2016).
Pegylated Interferon Lambda
IFN-l is a recombinant IL-29 protein, member of the Type type III IFN family, it has a
more limited extrahepatic receptor distribution, so the risk for systemic extrahepatic adverse
events is lower than IFN alpha.
It is characterized by having antiviral, antiproliferative, and generally immuno
modulatory immunomodulatory properties.
Phase-2 studies evaluated pegylated interferon lambda (Peg-INF-l) compared with
standard Peg-IFN-alpha in chronic HBV patients. On-treatment, lambda IFN showed greater
early impact on HBV DNA and HBsAg, and similar serologic and virologic responses at end
of treatment. (Coffin and Lee, 2015; Chan, 2014).
However, post dosing Peg-IFN-alpha HBeAg seroconversion was higher and further
development of Peg IFN l was stopped by the manufacturer.
Treatments Targeting cccDNA
Site-specific nucleases that induce DNA double-strand breaks are being used at present to
inhibit HBV replication.
The main used engineered DNA-binding proteins to target cccDNA are the zinc-finger
nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the RNA-
guided clustered regulatory interspaced short palindromic repeats (CRISPR) and CRISPR-
associated (Cas) protein endonucleases (C.-L. Lin et al., 2016; Maepa, Roelofse, Ely and
Arbuthnot, 2015).
ZFNs: blocks transcription of cccDNA, it has been used to inhibit viral transcription and
replication of duck HBV, one study in cultured cells has shown effective cleavage of viral
DNA targets by HBV specific ZFN (Cradick, Keck, Bradshaw, Jamieson and McCaffrey,
2010).
TALENs: transcription activator-like effector nucleases. Enzymes These enzimes can
degrade cccDNA by cleaving sequence-specific DNA targets, they disrupt sequences and
suppress markers of viral replication, reduce the production of HBeAg, HBsAg, HBcAg and

pregenomic RNA, and decrease cccDNA levels. Combined with IFN-a they seem to enhance
the antiviral effects of interferon (Coffin and Lee, 2015; Chen et al., 2014).
CRISPR/Cas system: this is a novel genome editing tool for site-specific cleavage of
DNA targets directed by a synthetic guide RNA basepaired to the target DNA sequence (C.-
L. Lin et al., 2016; Bhaya, Davison and Barrangou, 2011; Makarova, Brouns, Horvath, Sas
and Wolf, 2012) A recent study has used the CRISPR/Cas system with eight HBV-specific
guide RNAs targeting different regions of the HBV genome, they showed a reduction of HBV
core and surface proteins (S.-R. Lin et al., 2014).
CCC-0975 and CCC-0346: inhibitors of cccDNA production targeting the conversion of
relax-circular DNA to cccDNA (Cai et al., 2012).
INHIBITION OF NUCLEOCAPSID ASSEMBLY

Inhibition of nucleocapsid assembly is another target to prevent HBV replication. The
inhibitors of nucleocapsid formation include phenylpropenamide derivatives (AT-61,
AT130), heteroaryldihydro-pyrimidines (Bay 41-4109) and sulphamoylbenzamide
derivatives.
Heteroaryldihydropyrimidines (Bay 41-4109) inhibits capside formation,
phenylpropenamide and sulphamoylbenzamide derivatives prevent the encapsidation of viral
pregenomic RNA into nucleocapsid. They are effective against wild type HBV virus and
nucleoside resistant HBV (C.-L. Lin et al., 2016).
TREATMENTS TARGETING HBV RNA

The expression of viral proteins and DNA replication can be blocked by obstructing RNA
transcription.
ARC-520 is a combination of hepatocyte-targeted, N-acetylgalactosamine conjugated
melittin-like peptide with liver-tropic cholesterol-conjugated small interfering RNAs directed
against conserved HBV RNA sequences that reduce HBV RNA, proteins and DNA levels
(C.-L. Lin et al., 2016).
HBV ENTRY RECEPTOR INHIBITOR

Sodium taurocholate cotransporting polypeptide (NTCP) is a specific binding receptor of
the pre-S1 domain of the HBV envelope protein that allows HBV entry into liver cell.
Myrcludex-B is a synthetic lipopeptide derived from the pre-S1 domain of the HBV
envelope protein that targets NTCP, it effectively inhibits HBV entry in vitro and in vivo
(Volz et al., 2013; Urban, Bartenschlager, Kubitz and Zoulim, 2014).
Myrcludex-B has shown to inhibit both amplification of intrahepatic cccDNA and
dissemination of intrahepatic infection (Volz et al., 2013).
Prevention of HBV entry constitutes the first step to avoid primary infection; this is
specially promising in the liver transplant setting to prevent reinfection of the graft
(Brahmania et al., 2016; Coffin and Lee, 2015).

TREATMENTS TARGETING HOST IMMUNE RESPONSES

Drugs that can enhance immune response are thought to play an important role in
treatment of HBV.
Lucifora et al. reported that IFN-a and lymphotoxin-b receptor agonists up-regulated
apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 3A (APOBEC3A) and
APOBEC3B cytidine deaminases in HBV-infected cells, as a result they found a non-
cytolytic clearance of cccDNA and a reduction of cccDNA, HBV DNA and HBsAg levels
(C.-L. Lin et al., 2016; Lucifora et al., 2014).
The toll-like receptor (TLR) family is an important regulator of innate and adaptive
immune responses by inducing endogenous interferon production (Coffin and Lee, 2015)
(Funk, Kottilil, Gilliam and Talwani, 2014).
GS-9620 is a selective oral TLR7 agonist; in a study with chronically infected
chimpanzees it could induce suppression of HBV DNA in serum and the liver, and reduction
of serum HBsAg and HBeAg levels (Langford, Guerra and Chavez, 2013).
A phase Ib study in chronic HBV patients showed that GS-9620 induced peripheral IFN-
stimulated gene without significant systemic adverse events (Coffin and Lee, 2015; Gane et
al., 2015). Therefore TLR7 agonist would be a potential approach to control HBV infection.
CONCLUSION
HBV is one of the main causes of liver morbidity and mortality; with the number of
treatment options that we count on nowadays, infection can be controlled but there is a low
chance of viral eradication. Hopefully in the near future, the cure of HBV will be possible by
using a combination of different drugs with diverse mechanism of action and targets.
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Chapter 10
HEPATITIS C VIRUS

ABSTRACT
Hepatitis C virus (HCV) is a small, enveloped, 9.6 kb positive strand RNA virus that
belongs to the genus Hepacivirus within the Flaviviridae family. Hepatitis related to
HCV is a progressive disease that may result in chronic active hepatitis, cirrhosis and
hepatocellular carcinoma. Since it is estimated that about 200 million individuals are
chronically infected with HCV and there is no available vaccine, the virus represents
serious global health problem. Although direct-acting antiviral agents were recently
approved and made available and that more drugs are in the pipeline, patients response to
the current standards of care therapy (pegylated interferon–α and ribavirin) is limited.
Given this fact, and since the liver is the major site of HCV replication, liver failure
arising because of HCV infection is one of the most common reasons for the organ
transplant. Its high replicative activity, together with the lack of proof-reading activity of
the viral RNA dependent RNA polymerase, is the basis of the high genetic variability of
HCV, as well as of the high degree of intra-host genetic diversity. HCV isolates are
classified into seven major genetic groups referred to as genotypes, which present distinct
geographical distribution. Moreover, HCV exists as an ensemble of closely related but
genetically divergent variants, commonly referred to as “quasispecies.” The mechanisms
leading to liver injury are under constant revision, but the fact that both immune system-
mediated reactions and viral cytopathic effects are involved in pathogenesis is widely
accepted. In this chapter, we addressed HCV structure, cell cycle, pathogenesis,
classification and genetic variability in order to understand the clinical manifestations of
HCV infection and the treatment strategies.
INTRODUCTION
Hepatitis C virus (HCV) infection is one of the main causes of chronic liver disease
worldwide (Lavanchy, 2009). The long-term hepatic impact of HCV infection is highly
variable, from minimal changes to chronic hepatitis, extensive fibrosis, and cirrhosis with or

180 Pamela Valva, Mario Alejandro Lorenzetti and María Victoria Preciado
without hepatocellular carcinoma (HCC). Globally, the number of chronically infected

persons worldwide is estimated to exceed 200 million, but most may have no knowledge of
their infection or of the ensuing hepatic condition. The prevalence of HCV infection varies
greatly from region to region, and as a result, the burden of disease exhibits important
differences worldwide. Concerning clinical care, considerable advances were introduced for
patients with HCV-related liver disease during the last two decades. As a result of the
growing knowledge on this disease’s mechanisms, remarkable progresses in prevention,
diagnostic procedures and in therapeutic approaches were made. Still, various aspects are not
completely resolved yet (European Association for the Study of the Liver, 2011).
HCV was identified in 1989 by immunoscreening an expression library with serum from
a patient with post-transfusion non-A, non-B hepatitis (Choo et al., 1989). However, the virus
was not visualized conclusively, at that moment. Given the low viral titer in serum and liver
tissue, the biochemical characterization of native viral products was precluded and, most
importantly, it was not possible to culture HCV efficiently in vitro; all of them factors that
impeded the elucidation of the virus life cycle and the development of specific antiviral
agents and preventive vaccines. Despite these obstacles, great progress has been made in the
study of HCV over the past 25 years. While the chimpanzee is the only established animal
model of HCV infection, several in vivo and in vitro models have been developed that allow
us to study various aspects of the viral life cycle – in particular, the replicon system, the
production of recombinant infectious virion pseudoparticles (engineered retroviral particles
bearing functional HCV envelope proteins) and, most recently, a complete cell-culture
systems. These new approaches revolutionized the investigation of HCV-RNA replication
and rendered all stages of the viral life cycle, including entry and release of viral particles,
amenable to systematic analysis under reproducible and conveniently measurable conditions
((Moradpour et al., 2007; Brass et al., 2007) and references herein).
VIRAL STRUCTURE
HCV is classified in the Hepacivirus genus within the Flaviviridae family. All members
of this family share a number of basic structural and virological characteristics. Namely, they
are all enclosed in a lipid bilayer in which two or more envelope proteins (E) are anchored.
This envelope surrounds the nucleocapsid, which is composed of multiple copies of a small
basic protein (core), and contains the RNA genome. Particularly, HCV particles are 40–70 nm
in diameter, with a core protein and envelope glycoproteins E1 and E2 as the principal protein
components of the virion. E1 and E2 are presumably anchored to a host cell-derived lipid
envelope that surrounds the nucleocápside, composed of multiple copies of the core protein
and the genomic RNA. The HCV genome is a positive-strand RNA molecule (about 9.6 kb in
length), with an open reading frame (ORF) encoding for a polyprotein which is flanked by a
5’- and 3’-untranslated regions (UTR). The 5’-UTR contains an internal ribosomal entry site
(IRES) which is essential for viral translation and replication. The polyprotein is processed by
viral and cellular proteases into three structural proteins (C, E1, E2) and seven nonstructural
proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B) (Moradpour et al., 2007; Houghton,
1996) (Figure 1).

Hepatitis C Virus 181
HCV core is a highly conserved protein that, as mentioned above, makes up the viral
nucleocapsid and plays a role in pathogenesis (Khaliq et al., 2011). E1 and E2 are highly
glycosylated proteins that participate in cell entry (Drummer et al., 2003). E2 contains three
hypervariable regions (HVR), known as HVR1-3 (Troesch et al., 2006), which are under
continuous selective pressure exerted by the host immune system. P7 is a 63-amino acid
polypeptide that serves as a signal sequence for the translocation of NS2 into the lumen of the
endoplasmic reticulum (ER) for further cleavage. P7 is also essential for particle assembly
and release of infectious virions (Steinmann et al., 2007; Steinmann and Pietschmann, 2010).
NS2 is a transmembrane protein that is required for viral replication while NS3 is the HCV
protease and NTPase/helicase (Gallinari et al., 1998; Stapleford and Lindenbach, 2011).
NS4A acts as a cofactor for the NS3 protease and NS4B is a small hydrophobic protein
required for recruitment of other viral proteins (Morikawa et al., 2011; Gouttenoire et al.,
2010). NS5A is a hydrophilic phosphoprotein needed for viral replication (Macdonald et al.,
2004; Reed et al., 1997). Lastly, NS5B is the HCV RNA dependent RNA polymerase (RdRp)
(Behrens et al., 1996) which lacks proofreading and error correction mechanisms, rendering
viral genome replication into a highly error prone process ((Moradpour et al., 2007; Preciado
et al., 2014) and references herein).
Figure 1. HCV genome and polyprotein. The HCV genome is composed of an open reading frame
(ORF) flanked by 5’ and 3’ untranslated regions (UTRs). IRES-mediated translation of the ORF leads
to the formation of a polyprotein that is processed into viral proteins. Picture courtesy of Mayra Cruz-
Rivera.
CELL CYCLE
The liver is the major site of HCV replication; however, extrahepatic reservoirs that
achieve very low replication levels, were proposed. Among the latter are various myeloid and

lymphoid cell types, such as dendritic cells, B lymphocytes, occasionally T lymphocytes,

monocytes/macrophages and granulocytes (Goutagny et al., 2003; Laporte et al., 2003;
Roque-Afonso et al., 2005; Bouffard et al., 1992; Caussin-Schwemling et al., 2001). HCV
presence was also documented in the lymph nodes (Forton et al., 2004; Pal et al., 2006).
Finally, the existence of viral negative-polarity RNA, which is considered the intermediary of
genomic replication, was reported within the brain (Radkowski et al., 2002; Forton et al.,
2004; Burgel et al., 2011).
HCV circulates in various forms through the infected host. It is associated with low-
density lipoproteins (LDL) and very-low-density lipoproteins (VLDL), both of which seem to
represent the infectious fraction, and it can also circulates as virion bound to
immunoglobulins as well as free virion. Viral entry into the cell involves initial binding steps,
mediated by one or more surface receptor and co-recptors, which facilitate the anchoring of
virions prior to their subsequent internalization (Bartosch and Cosset, 2006; Cocquerel et al.,
2006; Moradpour et al., 2007). Numerous receptors and co-receptors were described for
HCV, including: 1) CD81, a member of the tetraspanin family that is expressed on many cell
types; 2) the LDL receptor (LDL-R), which is expressed in all nucleated cells; 3) the
scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein
present in a variety of tissues and cell types, with a particular high expression in the liver; 4)
members of the claudin family (CLDN1, 6 and 9) and occludin, proteins present in the tight
junctions; and 5) surface glycosaminoglycans and the asialoglycoprotein receptor (Douam et
al., 2015; Lyu et al., 2015). Moreover, the existence of HCV “capture” receptors such as
lectins L-SIGN, expressed in lymph nodes and liver endothelium, and DC-SIGN on dendritic
cells was also suggested (Gardner et al., 2003; Lozach et al., 2003; Pohlmann et al., 2003). It
is plausible that the interaction of these lectins with the viral envelope glycoproteins may
facilitate the transfer of virions into contiguous permissive cells (Bartosch and Cosset, 2006).
Furthermore, the glycosaminoglycans and the LDL-R may also facilitate the initial interaction
of viral particles with the host cell which could probably be mediated by HCV-associated
lipoproteins. In addition, several entry factors including the receptor tyrosine kinases (RTK),
the epidermal growth factor receptor (EGFR) (Lupberger et al., 2011), the ephrin receptor A2
(EphA2) (Lupberger et al., 2011) and the Niemann-Pick C1-like 1 cholesterol absorption
receptor (NPC1L1) (Sainz et al., 2012) have also been identified (Figure 2A).
The initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors
is followed by coordinated interactions with the SR-B1, CD81, and the tight junction proteins
CLDN1 and occludin. This tight concert of receptor interactions ultimately leads to the uptake
and cellular internalization of HCV through a clathrin-dependent endocytosis process (Douam
et al., 2015) (Figure 2A). After internalization, the acidification of the endosome induces
HCV glycoprotein membrane fusion. Little is known about the uncoating process, which
results in the release of the viral genome from the nucleocapsid into the cytoplasm, after
which it is both translated into a polyprotein and replicated at the rough ER. The beginning of
translation is mediated by the IRES, which acts as an initiation factor that directly regulates
the assembly and function of initiation complexes that are arranged around the 40S ribosomal
subunit (Otto and Puglisi, 2004). Viral proteins, in conjunction with host cell factors, induce
the formation of a membranous compartment [designated as the membranous web (MW)]
composed of single-, double- and multi-membrane vesicles as well as lipid droplets. The
NS4B protein induces the formation of the MW that serve as a scaffold for RNA replication,
a process that proceeds via the transcription of a negative-sense copy of the viral RNA [(–)

RNA] that serves as a template for the production of excess amounts of positive-sense
progeny RNAs [(+) RNA]. After genome amplification and protein expression, progeny
virions are assembled and released. Assembly of HCV particles probably initiates in close
proximity to the ER and lipid droplets, where core protein and viral RNA accumulate. The
viral envelope is acquired by budding through the ER membrane in a process that is linked to
lipoprotein synthesis. HCV particles are thought to be released via the constitutive secretory
pathway (Figure 2B) (Chevaliez and Pawlotsky, 2006; Moradpour et al., 2007).
Figure 2. Schematic overview of Hepatitis C virus replication cycle. A) HCV interaction with the cell
receptors is shown. B) The replicative cycle of HCV is displayed. Upon entry, the HCV genome is
released into the cytoplasm. In parallel, the genome is translocated to the ER for translation and
replicated and assembled into viral particles. The membranous web, attached to the newly translated
viral proteins in the ER, is used as the scaffold for viral replication. Picture courtesy of Mayra Cruz-
Rivera.
CLASSIFICATION AND GENETIC VARIABILITY

HCV infection is a highly dynamic process with a viral particle half-life of only a few
hours and production and clearance of an estimated 1012 virions per day in a given individual.
This high replicative activity, together with the lack of a proof-reading function of the viral
RdRp, is the basis of the high genetic variability of HCV as well as the high degree of intra-
host genetic diversity (Cruz-Rivera et al., 2013). HCV´s mutation rate in vivo is estimated at
about 2.5 x 10-5 per nucleotide per genome replication (Ribeiro et al., 2012); however, higher
estimates have also been obtained (Cuevas et al., 2009). HCV exists as an ensemble of closely
related but genetically divergent variants, commonly referred to as “quasispecies” (Wilke,
2005). In HCV molecular epidemiology, the term quasispecies has become a synonymous of
intrahost genetic variation (Holmes, 2010). Analysis of the HCV intrahost genetic variation is
important for genetic relatedness and the molecular evolution dynamically evolving viral
population, drug resistance, and epidemiological studies (Gismondi et al., 2009; Gismondi et
al., 2013). This characteristic molecular plasticity of HCV is a key biological property that
enables the rearrangement of the intrahost viral population under different selective pressures,

such as the immune response and antiviral therapy, warranting viral persistence (von Hahn et
al., 2007; Ralston et al., 2011). However, despite the high degree of genetic variability, HCV
antigenic diversity is significantly convergent (Campo et al., 2012), most likely due to due to
structural or functional constrains in the antigenic proteins (Penin et al., 2001). HCV
molecular evolution plays a pivotal role in virus transmission, progression of disease and
therapy outcome. Therefore, understanding the mechanisms driving the molecular evolution
of HCV is likely to help in the implementation of measures aimed to control HCV-related
disease.
To date, seven major HCV genotypes and several subtypes have been identified (Smith et
al., 2014). While genomic diversity between HCV genotypes is about 30%, subtypes differ by
about 15% (Bukh et al., 1995; Simmonds et al., 1993), a fact that renders HCV to exhibits a
complex taxonomic structure (Smith et al., 2014). In a further level of complexity, HCV
genotypes show a characteristic distribution throughout different geographical regions
(Lavanchy, 2011). Globally distributed genotypes are referred to as epidemic, and their rapid
dissemination over the last century is primarily attributed to transmission modes, which
including the use of intravenous drugs, nosocomial transmission and blood transfusions
(Magiorkinis et al., 2009). Endemic genotypes are usually highly divergent and limited to
well defined geographic regions. Even though genotypes 1, 2 and 3 exhibit a worldwide
distribution, the first two are endemic in West Africa and the latter is endemic to the Indian
subcontinent. Moreover, genotypes 4 and 5 are primarily found in Africa, where genotype 4 is
particularly endemic in Egypt and Central Africa. Finally, genotype 6 is endemic in Asia
whereas the distribution of genotype 7 has not been fully assessed (Lavanchy, 2011; Agha et
al., 2004; McOmish et al., 1994; Kao et al., 1995; Simmonds, 2013). These characteristic
distribution patterns facilitate the identification of the origin site and enable the tracking of
the genetic history of different HCV lineages. In general, high degree of genetic variability
among HCV strains evolving in relatively small geographical regions suggests long-term
evolution. Conversely, strains exhibiting lower genetic heterogeneity have shorter genetic
histories and are often associated with recent introduction and higher transmission rates
(Jackowiak et al., 2013). In this way and regarding ancestrality, genotype 2 is the oldest
lineage, followed by genotypes 3, 5 and 6, while genotypes 1 and 4 emerged more recently
(Jackowiak et al., 2013).
HCV PATHOGENESIS
HCV frequently elicits only a mild immune response and then establishes a chronic
infection. In this way, HCV antigens persist and permanently stimulate the immune system,
which leads to profound changes in the infected host’s immune responsiveness, and
eventually contributes to the pathogenesis of chronic hepatitis. A strong anti-HCV immune
response is frequently associated with severe tissue damage. In chronic hepatitis C (CHC);
however, the effector arms of the immune system either become refractory to activation or
switches into a regulatory function. Taken together these alterations in immunity may lead to
persistent liver damage and cirrhosis. Consequently, the effector arms of the immune system
will not only be considered with regards to antiviral defense but also as the pivotal
mechanisms of inflammation, necrosis and progression to cirrhosis (Spengler et al., 2013).

During its replication HCV is sensed by pattern recognition receptors (PRRs) in the host
cell that detect pathogen associated molecular patterns (PAMP) within viral products.
Replication involves the generation of an HCV replicative-intermediate (antigenomic) [(–)
RNA], and probably an intermediate double stranded RNA (ds-RNA) product, which can
trigger intracellular PRRs. Thereafter, this process leads to a coordinated activation of the
innate and adaptive immune responses, which work together in an integrated fashion to
recognize and defend against HCV infection. Innate response to HCV comprise both the
cellular response, such as recognition of the “non-self” by various types of natural killer (NK)
cells, and also the humoral component, mainly given by interferons and a variety of other
cytokines. Thus, the development of adaptive B and T cell immunity is shaped by the initial
innate responses. However, despite these immune defenses, hepatitis C becomes chronic in
about 70%-80% of acute infections and leads to a sustained inflammatory responses, which
are the key mechanisms for tissue injury in CHC, as a consequence of failed immunity and
continued viral persistence ((Spengler et al., 2013) and reference herein).
Three types of PRRs are known to detect HCV: 1) the retinoic acid inducible gene-I
(RIG-I)-like receptors, which serves as a cytoplasmic viral sensor, since they are able to
detect viral RNA in the cytosol; 2) toll-like receptors (TLRs), such as TLR3, which detects
ds-RNA fragments in the endosomal compartment; and 3) the nontraditional pattern
recognition receptor protein kinase R (PKR), which also binds ds-RNA. RIG-I and PKR
signals are relayed via the mitochondrial antiviral signaling protein (MAVS) to trigger innate
immunity, while TLR3 signals are relayed via the TIR-domain-containing-adaptor-inducing-
interferon-β (TRIF). Downstream of these adaptor proteins, the signaling cascade is activated
to conclude in the secretion of type I and III IFNs. Binding of auto and paracrine IFNs to their
receptors then activates signaling pathways which induce the expression of hundreds of
interferon-stimulated genes (ISG) in the infected and neighboring cells, thus creating a
general antiviral state in the liver that limits HCV RNA replication and cell to cell spread
(Spengler et al., 2013). Briefly, RIG-I signaling is initiated by binding of the polyuridine
motif of the HCV genome that consists of an exposed 5’-triphosphate and the 3’-poly-U/UC-
rich untranslated region of the HCV RNA. These regions are located at opposite ends of the
viral genome but are brought together by intra-genomic interactions. RIG-I activation leads to
the formation of a multi-component complex with MAVS, which interacts with the essential
adapter protein TRAF3 (tumor necrosis factor receptor–associated factor 3, TRAF3) to
further recruit downstream kinases, IKK-ε and TANK-binding kinase 1 (TBK1). These
kinases phosphorylate the transcription factors IRF-3 and IRF-7 (interferon-regulatory factor-
3 and -7), and their binding to NF-κB leads to the induction of antiviral and
immunomodulatory genes, including types I and III IFNs, as well as chemokines and
proinflammatory cytokines that function in parallel with IFNs to mediate the response to
HCV (Loo and Gale, 2011; Rosen, 2013). On the other hand, HCV ds-RNA intermediates,
which occur late in HCV replication, activate TLR3 (Li et al., 2012) and interact with the
adaptor molecule TRIF. They also recruit downstream kinases to phosphorylate IRF-3 and -7,
and bind to NF-κB with the consequent production of interferons and pro-inflammatory
cytokines (Takeuchi and Akira, 2009). Finally, the ligand for PKR at the IRES of HCV RNA
induces phosphorylation of the α-subunit of the eukaryotic initiation factor 2 (eIF2α). RNA
binding also triggers a kinase-independent signal transduction cascade involving MAVS
which finally activates interferon-β and interferon-stimulated genes (ISGs) (Spengler et al.,
2013; Arnaud et al., 2011).

There are different types of interferon and each one of them has a specific receptor. Type
I IFNs are interferon-α and interferon-β which bind to a specific cell surface receptor complex
known as the IFN-α/β receptor (IFNAR). Type II IFN is IFN-γ, which has its own IFN-γ
receptor (IFNGR) and is released by immune cells. Type III IFNs are IFN-λ1 [also named
interleukin (IL)-29], IFN-λ2 (IL-28A), IFN-λ3 (IL-28B) which signals through a receptor
complex consisting of IL10R2 (also called CRF2-4) and IFNLR1.
In general, type I and II interferons are responsible for regulating and activating the
immune response; meanwhile the expression of type I and III IFNs can be induced in virtually
all cell types upon recognition of viral components, whereas type II interferon is induced by
cytokines such as IL-12 and its expression is restricted to a sub-set of immune cells, such as
NK and T cells (Rosen, 2013). Finally, all IFN receptors transmit signals from the cell surface
to the nucleus via the Jak-STAT pathway to activate ISGs. Specifically type I and III IFNs
induce IFN-stimulated gene factor 3 (ISGF3) consisting of phosphorylated STAT1 and 2
proteins and IRF9 which activate the IFN-stimulated response elements (ISRE) of multiple
genes contributing to antiviral activity ((Spengler et al., 2013; Rosen, 2013) and reference
herein).
Although HCV can be detected effectively by RIG-I, TLR3 and PKR, it frequently
establishes chronic persistence since it has evolved several mechanisms to counter-act innate
immunity. The multi-functional HCV NS3/NS4A protease is a key component in the innate
immunity evasion strategy. It has been demonstrated that HCV initially activates the RIG-I
pathway; however, once sufficient viral proteins have accumulated in the cytosol, NS3/NS4A
abundance increases and blocks RIG-I signaling by cleaving MAVS from intracellular
membranes (Baril et al., 2009; LiSun et al., 2005; Spengler et al., 2013). This cleavage
prevents signal transduction, abrogates interferon induction and facilitates progression to
chronic infection. The NS3/NS4A protease can also cleave TRIF, and the relative abundance
of this protein is reduced after HCV infection, probably as a result of degradation following
its cleavage by NS3/NS4A (Wang et al., 2009; LiFoy et al., 2005). In addition to actions of
the NS3/4A protease, HCV core protein interferes with STAT signaling and may contribute to
INF resistance by diminished binding of ISGF3 to nuclear ISREs. Finally, HCV proteins E2,
NS3, NS4A and NS5 provide several strategies to interfere both with PKR signaling and
PKR-regulated inhibition of translation, but these interactions are complex and mechanisms
by which they support HCV persistence are still unclear ((Rosen, 2013; Spengler et al., 2013)
and references herein).
Natural killer cells are one of the earliest lines of innate immune defense since they not
only rapidly recognize and lyse virus-infected cells and inhibit viral replication but also exert
immune regulatory functions. They exert their purpose by the secretion of cytotoxic
molecules like granzyme and perforin or through tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL)-mediated killing. They also secrete cytokines that can
regulate innate and adaptive immunity like IFNγ, TNFα, IL-10, and IL-21. NK activity is
governed by the balance between activating and inhibitory signals. The most important NK
cell receptors (and their cognate ligands) comprise the killer immunoglobulin-like receptor
(KIR) family (ligands: HLA-A, -B and -C), the CD94-NKG2A/C complex (ligand: HLA-E),
NKG2D (ligands: MIC-A and MIC-B and others) and the natural cytotoxicity receptors
NKp30, NKp44 and NKp46 (Moretta and Moretta, 2004). NK cells become activated when
there is a relative reduction of inhibitory signals, e.g., downregulated MHC class I expression
on virus-infected cells, or a relative increase in signals from activating receptors, e.g., binding

of antibody-coated antigens. NK cells are highly enriched in the liver and are expected to play
a major role in controlling hepatotropic infections. Activated NK cells with potent
degranulation and substantial cytokine production have been described in acute HCV
infection, and there is accumulating evidence to suggest that NK cells play an important role
in the antiviral immune response to HCV and later on also in the immune-mediated
pathogenesis of chronic disease. NK cells can inhibit HCV replication by IFN-γ mediated
non-cytolytic as well as by granzyme/perforin and TRAIL-mediated cytotoxic mechanisms.
However, IFNγ-mediated clearance of HCV might be more important than direct cytolysis,
because cytolytic elimination of all HCV-infected hepatocytes would lead to extensive liver
damage. Notably, during chronic HCV infection, intrahepatic NK cells are generally highly
activated and this activation correlates with the degree of liver inflammation (Oliviero et al.,
2009; Bonorino et al., 2009). NK cells expressed altered patterns of NK receptors, where
NKp46 is of particular interest, since it is considered a major activating receptor in HCV.
High expression of NKp46 defines a NK cell subset with high cytotoxic activity and IFN-
production; these subset seems to accumulate in the liver and to be associated with liver
damage (Pembroke et al., 2014). However, it was also observed that the NK cell number and
function, especially cytotoxic activity, were lower in CHC patients as compared to healthy
donors, but it should be taken into account that the various experimental designs and patient
cohorts rendered divergent results (Rosen, 2013; Spengler et al., 2013). In addition NK cells
express the tetraspanin CD81 so, as demonstrated by in vitro studies, the binding of HCV E2
to CD81 blocks antiviral functions of NK. On the other hand, it is important to note that NK
not only contributes to non cytolytic HCV clearance and tissue damage; but also NK cell-
mediated cytotoxicity against hepatic stellate cells (HSC) may contribute to the regulation of
intrahepatic fibrosis in CHC. For this reason, the effect of NK in HCV pathogenesis is
consider dual ((Spengler and Nattermann, 2007; Spengler et al., 2013) and references herein).
NK cells also interact with Dendritic cells (DCs) and this cognate interaction regulates
both innate and adaptive immunity. NK cells induce maturation of DCs by IFNγ and TNFα
production, and enhance their capacity to prime virus-specific T cells. In return, DCs produce
IL-12 and IL-15 that enhance activation of NK cells (Marcenaro et al., 2012). DCs are one of
the major antigen-presenting cells in the body, so they bridge innate and adaptive immunity
and may influence priming of HCV-specific immune responses. DCs rapidly differentiate into
mature DCs in response to various “danger” signals like the activation through PAMPs (in
particular TLR ligands), the interaction with activated innate lymphocytes (NK and NKT
cells), cytokines, and inflammatory mediators (Steinman and Banchereau, 2007). There are
two main subsets of DCs, myeloid DCs (mDCs) representing the majority of DCs and mostly
associated with antigen processing and presentation, and plasmacytoid DCs (pDCs) that can
sense viral infections and are the main producers of type I and type III IFNs. pDCs can detect
HCV RNA in a TLR-7 specific manner when presented as part of an infected cell (Takahashi
et al., 2010). Although, DCs are considered a main orchestrator of the HCV innate and
adaptive immune response, their role and function during acute and chronic HCV infection
remain highly controversial. The frequencies of mDCs and pDCs were shown to correlate
with the infection outcome, where reduced frequencies were associated with chronic
infection; however, their role and contribution in HCV pathogenesis remain under debate
((Spengler et al., 2013; Rosen, 2013) and reference herein).
A coordinated immune response involving both, antibodies and T cell responses, is
normally required for efficient adaptive immunity. The actual contribution to protection of

naturally acquired anti-HCV antibodies is controversial, since their presence cannot prevent
re-infection. Circulating antibodies against structural and non-structural components are
generated in virtually all patients irrespective from the outcome of HCV infection. A rapid
induction of neutralizing antibodies, which frequently recognize HCV envelope proteins,
early in the course of HCV has been demonstrated to contribute to HCV clearance, but broad
antibody responses usually occur at the stage of chronic infection and are not neutralizing.
Thus, due to the high genetic variability of HCV, adaptive sequence changes in the
hypervariable HCV E2 region 1 (the putative target of neutralizing antibodies) are generated
during viral replication indicating that HCV adapts to the immune selective pressure exerted
by natural anti-HCV antibodies via escape mutations. Moreover, the glycosylation of HCV
proteins and the association of viral particles with lipoproteins further prevent antibody
recognition (Spengler and Nattermann, 2007).
In parallel with the onset of acute hepatitis, HCV-specific CD4+ T and CD8+ T cells are
detected at only low frequency in peripheral blood although they are somewhat enriched in
the liver. Persistent and strong HCV-specific CD4+ and CD8+ T-cell responses were observed
in the majority of acute HCV-infected patients who eventually recovered from the infection.
Their responses coincided with a decreases in HCV quantity. By contrast, in cases that
progress to chronic hepatitis, CD4+ T cell response is weak, narrowly selected and short-
lived. Moreover, the frequency of HCV-specific CD8+ T cells is high during the acute phase
of infection but decreases after HCV persistence develops. Despite the high numbers of CD8+
T cells, some of these cells are “stunned” in the acute phase, as demonstrated by their
attenuated response to HCV antigens. Therefore, it is considered that adaptive cell-mediated
immunity is a key mechanism for resolution of primary HCV infection. While CD8+ T cells
are the primary effectors mediating protective immunity, the aid of CD4+ T cells is required
for CD8+ T cells to normally function. Unfortunately, in CHC patients HCV-specific CD4+ T
are functionally impaired (Kanto and Hayashi, 2006) and consequently, HCV-specific CD8+
T cells in possess lesser capacity to proliferate and produce less IFN-γ in response to HCV
antigens. The latter are not sufficient to eradicate HCV but are instead involved in HCV-
induced liver inflammation and damage ((Kanto and Hayashi, 2006) and reference herein).
Several plausible mechanisms have been proposed for the frequent T cell functional failure
observed in chronic HCV infection: 1) HCV escape mutation, 2) primary T cell failure or T
cell exhaustion, 3) impaired antigen presentation, 4) suppression by HCV proteins, 5)
suppression by regulatory T cells and 6) tolerogenic environment in the liver (Kanto and
Hayashi, 2006). Briefly, as previously mentioned, HCV is characterized by a quasispecies
nature, enabled by a high replication rate and the lack of proof-reading capacity of its
polymerase, which contributes to rapid virus diversification. The mechanisms adopted by
mutant virus to survive are decreased or impaired binding affinity to TCR, and impaired
antigen processing by proteasomes resulting in the attenuation of specific T cell responses.
The progressive functional exhaustion and ultimate loss of HCV specific CD4+ and CD8+ T
cells is demonstrated since they show an increased expression of inhibitory receptors, such as
programmed death-1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), and T cell
immunoglobulin and mucin domain-containing molecule 3 (Tim-3). Impaired antigen
presentation by DC might be involved in the failure of sustained HCV-specific T cell
response maintenance. It was described that mDCs have an impaired ability to stimulate
allogeneic CD4+ T cells but this functional impairment diminishes when HCV is eradicated
from the patient, evidencing a HCV-induced DC disability. Direct HCV infection of DCs

might be one of the plausible mechanisms of DC dysfunction in CHC although further study
needs to be done to clarify this point. Recently, regulatory T cells (Tregs) have become very
important due to their contribution to the pathogenesis of various diseases, including those
from the liver. These cells are functional suppressors of HCV-specific CD8+ T cells in a cell-
cell contact manner or by producing IL-10 or TGF-β1. Accumulating evidence indicates that
patients with chronic viral hepatitis display increased number of Treg cells in peripheral
blood and liver. It is considered that once the immune system failed to clear HCV infection,
Treg cells seem to exert multiple different effects: they diminish inflammatory responses
associated with reduced antiviral activity of the immune system, facilitate HCV persistence,
and also contribute to the regulation of fibrosis. Finally, the liver is a mediator of local and
systemic immunity and hence, an important immune regulation site. From experimental
observations, it is evident that the liver favors immune tolerance rather than an active
response. Their unique immunoregulatory function is mediated by local expression of co-
inhibitory receptors and immunosuppressive mediators which help to prevent inadvertent
organ damage. However, these tolerogenic properties render the liver an attractive target for
pathogens. Although most pathogens that reach the liver via the blood are eliminated or
controlled by local innate and adaptive immune responses, some pathogens (such as hepatitis
viruses) can escape immune control and persist in hepatocytes. Therefore, the liver’s
immunological environment is potentially tolerogenic to infiltrating T cells, liver sinusoidal
endothelial cells (LSEC), Kuppfer cells, stellate cells, and liver DC which may mediate this
tolerogenic effect contributing with T cell functional failure (Toubi, 2008; Alatrakchi and
Koziel, 2009; Amoroso et al., 2012; Losikoff et al., 2012; Speletas et al., 2011; Irshad et al.,
2013; Langhans et al., 2013; Valva et al., 2014; Nemeth et al., 2009; Kanto and Hayashi,
2006; Spengler and Nattermann, 2007; Spengler et al., 2013; Mengshol et al., 2007).
In summary, HCV alters the earliest lines of innate immune defense by interfering with
intracellular IFN signalling. Moreover, intact HCV particles or its fragments can interact with
cellular receptors, such as CD81, to inhibit the function of NK cells, alter chemokine release
and impair the traffic and function of DC and T cells. Insufficient innate immunity also leads
to a deficit in the generation of the adaptive immune response. In later stages of HCV
infection, there is insufficient expansion and maturation of HCV-specific T cells probably
owing to insufficient help, T-cell escape mutations, clonal exhaustion and the generation and
expansion of Tregs. Depending on the severity of CD4+ T cell dysfunction, HCV-specific
CD8+ T cells either succeed or fail to control the infection. In the case of CHC, CD8+ T cells
persist in the liver and probably contribute to sustained liver damage, since virus-specific T
cells are recruited together with non-specific inflammatory cells that also damage uninfected
bystander cells. Moreover, the interaction of Fas ligand, released from inflammatory cells,
and its specific receptor on hepatocytes, is a further candidate mechanism to enhance liver
damage by apoptosis. Finally, inflammatory cells directly activate intrahepatic fibrogenesis
by releasing inflammatory and myofibroblast-activating cytokines, such as TGFβ. Thus,
tissue damage and fibrosis in CHC are probably the result of long-term activation of non-
specific inflammatory immune responses (Spengler and Nattermann, 2007) (Figure 3).

Figure 3. Hepatitis C virus pathogenesis. Chronic hepatitis C pathogenesis is mediated by the immune response but also by the virus. However, several
mechanisms including apoptosis in addition to various other phenomena such as hepatic steatosis, oxidative stress and insulin resistance are involved
in the pathogenesis.

It is well known that CHC pathogenesis is mediated by the immune response, but also by
the virus. HCV is a non-cytopathic virus (like the virus that enter the liver cell and undergoes
replication simultaneously causing cell necrosis); however, several mechanisms including
apoptosis in addition to various other phenomena such as hepatic steatosis, oxidative stress
and insulin resistance are involve in the pathogenesis. The proteins/peptides encoded by
different sub-genomic regions of HCV genome influence the above mechanisms, and thus,
have a significant role in HCV pathogenesis.
As mentioned in chapter “Cell death issue for researchers and clinicians,” based on the
morphological characteristics of liver biopsies, it is currently accepted that hepatocyte
damage is a result, at least in part, of apoptosis induction. Many authors have demonstrated
that this process is a prominent liver feature in CHC patients since significantly higher
numbers of apoptotic cells were identified in chronically infected biopsies from pediatric and
adult patients when compared to controls (Bantel et al., 2004; Bantel et al., 2001; Bantel and
Schulze-Osthoff, 2002; Calabrese et al., 2000; McPartland et al., 2005; Rodrigues et al., 2000;
Sarfraz et al., 2008; Seidel et al., 2005; Walsh et al., 2004; Valva et al., 2014). Furthermore,
some studies indicated that hepatocyte apoptosis plays a significant role in the pathogenesis
of HCV infection, and is clinically recognized to trigger liver inflammation and fibrosis
(Albertoni et al., 2012; Bantel and Schulze-Osthoff, 2002; Calabrese et al., 2000; Fischer et
al., 2007; Kountouras et al., 2003; Rust and Gores, 2000; Valva et al., 2014). Both in vitro
studies and in vivo models have demonstrated the ability of HCV to induce apoptosis (Deng
et al., 2008; Fischer et al., 2007; Joyce et al., 2009; Mengshol et al., 2007). Many studies have
demonstrated multiple molecular interactions between different viral proteins and
components of the cellular machinery involved in the apoptotic process; however, it has been
observed in vitro that certain HCV proteins may acts as both, inducers or repressors, of
apoptosis depending on the experimental design. Particularly, HCV Core protein was
described as an inducer of apoptosis that leads to steatosis and increases oxidative stress and
mitochondrial damage (Benali-Furet et al., 2005; Berg et al., 2009; Moriya et al., 1997;
Moriya et al., 1998). HCV infection is a dynamic process with only low proportion of
infected hepatocytes during disease, mainly in adult samples. It is accepted that HCV
spreading among hepatocytes is controlled by intrahepatic immune response during chronic
infection. Thus, an excess of infected hepatocytes implies that the immune system could not
completely exert a viral control, and in these cases apoptotic hepatocytes may be most related
to virus effects. In contrast, in cases with low level of infected hepatocytes and a more
effective immune response, it may be considered that apoptosis activation is not directly
triggered by HCV, but instead could be due to the antiviral immune response. Therefore,
apoptosis induction in non-infected hepatocytes could represent a major contribution to the
overall liver injury. So, these observations indicate that the virus, apoptosis and the immune
response are all involved in CHC pathogenesis and support the "Bystander killing" theory
(Bowen et al., 2002; Gremion et al., 2004; Spengler and Nattermann, 2007), which suggests
that HCV-specific cytotoxic cells eliminate infected hepatocytes by apoptosis induction and
the release of soluble effectors. But, although these soluble effectors aid in the control of the
infection, they also attract non HCV-specific lymphocytes which in turn affect non-infected
hepatocytes. Finally, it is important to note that liver damage is not only related to the
inflammation and the effect of cytokine and mediators of immunity; that engulfment of
apoptotic bodies by hepatic stellate cells also stimulates the fibrogenic activity of these cells

and so it may be a potential mechanism by which apoptosis facilitates subsequent fibrosis in

liver tissue (Canbay et al., 2004; Canbay et al., 2003) (Figure 3).
On the other hand, HCV infection is reported to have a strong association with hepatic
steatosis. Much of the HCV life cycle is closely associated with lipid metabolism, and this
association includes entry into naïve cells, infectivity, RNA replication, viral assembly and
viral secretion (Syed et al., 2010). Adipose tissue has emerged as an important endocrine
organ due to its release of adipocytokines, which regulate lipid and glucose metabolism via
the adipoinsular axis. However, most data regarding HCV infection and adipocytokine
alterations are inconclusive (Chang, 2016). It is postulated that the mechanisms responsible
for the development of steatosis would be associated with viral genotype. Therefore, two
forms of steatosis have been defined in patients with HCV, metabolic steatosis and steatosis
induced by HCV (Gonzalez-Reimers et al., 2015). Thus, genotype 1 would be related to the
metabolic way, where obesity, diabetes, high body mass and mainly insulin resistance have a
fundamental role (Yoon and Hu, 2006; Fartoux et al., 2005). Instead, genotype 3 would be
involved in steatosis induced by HCV. The correlation between the severity of steatosis and
viral replication, together with the association between the antiviral response and the steatosis
decrease, suggests the direct action of HCV genotype 3 in this process (Castera et al., 2004;
Patton et al., 2004). Subsequent studies indicated that genotype 3 interferes with very low-
density lipoprotein (VLDL) secretion as well as that genotype 3 core protein promotes lipid
accumulation in hepatocytes with more efficiency compared to core protein from genotype 1
(Irshad et al., 2013). In turn, amino acid changes in HCV Core protein from genotype 3,
which were not found in other genotypes, were reported to be associated with increased
formation of lipid droplets in the hepatocytes. However, the molecular mechanism by which
HCV induced steatosis is still not completely defined (Hourioux et al., 2007; Jackel-Cram et
al., 2007).
All these reports concluded that HCV causes steatosis in three different ways: 1)
Impaired secretion of lipids from hepatocytes; 2) Increased de novo-synthesis of free fatty
acids (FFA); and 3) Impaired FFA degradation. The first aspect of HCV-induced steatosis
was proposed due to the impaired secretion of VLDL. In vitro studies and transgenic mouse
models expressing Core protein suggest that this would be sufficient to induce lipid
accumulation in the cytoplasm of hepatocytes. This protein is localized on the surface of lipid
droplets and its overexpression stimulates their formation (Barba et al., 1997; Moriya et al.,
1997). It is postulated that Core inhibits the Microsomal Triglyceride Transfer Protein
(MTTP), whose activity is key for the assembling of VLDL, and triacylglycerol accumulates
as a result (Perlemuter et al., 2002). Related to the novo-synthesis of FFA, different authors
have demonstrated the effects of HCV in increasing the activity of various enzymes involved
in the synthesis of fatty acids by regulating the activity of the Sterol Regulatory Element
Binding Protein -1c (SREBP-1c), a transcription factor regulator of lipid metabolism. In HCV
experimentally infected chimpanzee livers, it was observed an increased activity of the
enzymes involved in fatty acid synthesis which are under the control of SREBP-1c, such as
ATP citrate lyase (Su et al., 2002; Moriya et al., 1997). Moreover, the HCV Core protein can
also bind to DNA binding domain of the retinoid X receptor-α (RxR- α), a transcription factor
that controls many cell functions, including lipogenesis (Tsutsumi et al., 2002; Yamaguchi et
al., 2005). HCV-induced steatosis is also due to impaired FFA degradation by HCV.
Expression of HCV-core protein has been reported to reduce the expression of peroxisome
proliferation activated receptor-α (PPARα), a nuclear receptor involved in FFA degradation

and down-regulation of mitochondria β-oxidation. Genotype 3 shows significant down-

regulation of PPARα as compared to genotype 1. The core protein from genotype 3 also up-
regulates the suppressor of cytokine signaling-7 (SOCS-7) in human hepatoma cells. These
data clearly show that HCV-core protein may modulate the expression of various genes
responsible for FFA degradation via down-regulation of PPARs (Irshad et al., 2013; Cheng et
al., 2005; Dharancy et al., 2005).
Regarding the aforementioned metabolic steatosis, this is triggered by insulin resistance
since insulin is involved in both glucose and fatty acid metabolism. The mechanisms related
to insulin resistance are multiple. Deregulation of hyperglycemia/hyperinsulinemia state
stimulates the expression of various enzymes involved in FFA synthesis, while inhibiting
mitochondrial -oxidation. Moreover, insulin resistance activates peripheral lipolysis and
stimulates the mobilization of FFA from the adipocyte. Therefore, increased levels of
circulating FFA contribute to its accumulation in liver and muscle, even more, it causes
insulin resistance in these tissues by alterations in the signaling cascades of insulin (Browning
and Horton, 2004; Negro, 2010). Even though insulin resistance is a characteristic of the
metabolic syndrome regardless of HCV infection, the high prevalence of type 2 diabetes
observed among HCV patients suggests that HCV may influence, at least to some extent, the
degree of insulin resistance (Negro, 2006). Furthermore, experimental data suggest that HCV
interferes directly in the insulin signaling pathway by proteasomal degradation of Insulin
Receptor Substrate- 1 and -2 (IRS-1 and -2) (Kawaguchi et al., 2004). Moreover, HCV Core
protein prevents the translocation of the glucose transporter GLUT4 to the plasma membrane,
generating an increase in extracellular glucose level and thus an alteration in the
glucose/insulin level leading to insulin resistance (Romera et al., 2006; El-Zayadi, 2008). It
also causes insulin resistance, either directly or via increased secretion of TNF-á. HCV Core
can activate inhibitors of insulin signaling including mammalian target of rapamycin (mTOR)
and suppressor of cytokine signaling (SOCS)-3 and C-Jun N-terminal kinase (JNK). The
activation of JNK by HCV Core may follow a direct or indirect pro-inflammatory cytokine
mediated mechanism (Irshad et al., 2013).
Finally, steatosis per se does not lead to liver injury; inflammation is required as a second
event to make it plausible. It is broadly accepted that steatosis impacts on fibrosis
progression, so steatosis should not be considered a benign feature, but rather a silent killer
(El-Zayadi, 2008). Although the mechanisms by which steatosis may lead to worsening
fibrosis are not understood, the factors implicated include oxidative stress, insulin resistance
and increased apoptosis which leads to the activation of hepatic stellates cells (Zeuzem et al.,
2006; Walsh MJ, 2004; Cross et al., 2009; Negro, 2006; Murray et al., 2005) (Figure 3).
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Steatosis and liver cell apoptosis in chronic hepatitis C: a mechanism for increased liver
injury. Hepatology no. 39 (5):1230-8, 2004.
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and liver cell apoptosis in chronic hepatitis C: a mechanism for increased liver injury.
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no. 24 (8):1133-49, 2006.

Chapter 11
MECHANISM LIPOTOXICITY IN THE

PROGRESSION FROM FATTY LIVER TO
NON-ALCOHOLIC STEATOHEPATITIS
Fernando J. Barreyro and Juan José Poderoso
ABSTRACT
Non-alcoholic fatty liver disease (NAFLD) is currently the most common form of
chronic liver disease worldwide. A subset of these individuals develops hepatocyte
damage, inflammation and fibrosis, a syndrome referral as nonalcoholic steatohepatitis
(NASH). Although much progress has been made since NASH was first documented, the
pathogenesis of NASH is not fully elucidated. Recent studies suggest that free fatty acids
(FFA) induced lipotoxicity contributes to the pathogenesis of NAFLD and NASH.
Currently there are no clinically effective treatments for NASH, understanding the
pathomechanisms responsible for disease development and progression could give the
clue to a beneficial therapeutic approach. The purpose of this chapter is to discuss the
pathophysiology involving hepatocyte injury by lipotoxicity, focuses on the known
mechanisms of cellular toxicity secondary the network between lipid metabolism,
autophagy, oxidative stress, ER-stress, JNK activation, death receptors and BCL-2 family
proteins. A more comprehensive understanding of the molecular mechanism that triggers
lipotoxicity-related pathways could likely create potential therapeutic opportunities for
NASH.
ABBREVIATIONS
α-SMA Alpha-smooth muscle actin
ALT Alanine aminotransferase
AP-1 Activator protein-1
AST Aspartate transaminase
ATF Activating transcription factor
BH3 Bcl-2 homology 3

204 Fernando J. Barreyro and Juan José Poderoso
BIM Bcl-2 interacting mediator

ChREBP Carbohydrate response element binding protein
CV Cardiovascular disease
CHOP C/EBP homologous protein
CXCL2 C-X-C chemokine ligand-2
CYP2E1 Cytochrome P450 2E1
DAG Diacylglycerol
DAMPs Damage-associated molecular patterns
DNL De novo lipogenesis
DR Death receptor
ER Endoplasmic reticulum
ETC Electron transport chain
ERAD ER-assisted degradation
FAS Fatty acid synthase
FAT Fatty acid translocase
FATP Fatty acid transport protein
FC Free cholesterol
FFA Free fatty acids
GSK3 Glycogen synthase kinase 3
HCC Hepatocellular carcinoma
HH Hedgehog
HFD High fat diet
HMGB1 high mobility group box 1 protein
HMGCo-A hydroxymethylglutaryl-Co-A
HPC Hepatic progenitor cells
HSC Hepatic stellate cell
IL-1β Interleukin 1 beta
IRE1α Inositol-requiring enzyme-1α
IRS−1 Insulin receptor substrate-1
IR Insulin resistance
JNK c-Jun-N-terminal kinase
Keap1 Kelch-like ECH-associated protein
LDL Low density lipoprotein
LPC Lysophosphatidyl Choline
LPS Lipopolysaccharide
MCD Methionine choline deficient
MCP-1 Monocyte chemoattractant protein
NAFL Nonalcoholic fatty liver
NAFLD Nonalcoholic fatty liver disease
NASH Nonalcoholic steatohepatitis
NF-kB Nuclear factor-kappaB
NLR NOD like receptors
Ob Leptin
OA Oleate
PA Palmitate
PAMP Pathogen-associated molecular patterns

Mechanism Lipotoxicity in the Progression from Fatty Liver … 205
PERK PKR-like ER kinase

PLA2 Phospholipase A2
PO Palmitoleate
PPAR Peroxisome proliferator-activated receptors
PRR Pattern recognition receptors
PUMA p53 upregulated modulator of apoptosis
ROS Reactive oxygen species
SA Stearate
SCD-1 stearoyl-CoA desaturase-1
SNP Single nucleotide polymorphism
SREBP-1c Sterol regulatory element-binding protein 1c
SREBP-2 Sterol regulatory-element binding protein 2
StAR Steroidogenic acute regulatory protein
TG Triglyceride
TGF-β Transforming growth factor-β
TIMP-1 Tissue inhibitor of metalloproteinase type 1
TLR Toll like receptors
TLR4 Toll like receptor 4
TNF-α Tumor necrosis factor alpha
TRAIL Tumor necrosis factor–related apoptosis-inducing ligand
UPR Unfolded protein response
VEGF Vascular endothelial growth factor
VLDL Very low density lipoprotein
INTRODUCTION
Non-alcoholic fatty liver disease (NAFLD) is currently the most common form of chronic
liver disease worldwide, affecting 20-30% of western countries population [1], and 10–25%
in the Asian population [2]. It increased prevalence is closely associated with insulin
resistance (IR) and overweight [3]. The main feature of NAFLD is hepatic steatosis, which is
characterized by fat accumulation (in the form of triglyceride, TG) in more than 5% of
hepatocytes or hepatic TG ≥ 55 mg per g of liver [4], in the absence of excessive alcohol
intake (<20 g per day for men, <10 g per day for women) or other secondary cause of fat
deposition such as viral infections, certain drugs and genetic disorders [4, 5]. A subset of
these individuals, approximately 15 to 25%, develops hepatocyte damage, inflammation and
fibrosis, a syndrome referral as nonalcoholic steatohepatitis (NASH) [4, 6]. This hepatic
inflammatory disorder can progress to cirrhosis, liver failure, and hepatocellular carcinoma
[7]. Between 10 to 29% of individuals with NASH develop cirrhosis within 10 years [8] and
liver-related mortality is increased up to ten-fold in patients with NASH [6]. Also, about 4 to
27% of individuals with NASH-induced cirrhosis develop hepatocellular carcinoma [9].
Although much progress has been made since NASH was first documented [10], the
pathogenesis of NASH is not fully elucidated. The initial theory for the NASH pathogenesis
the “two hit hypothesis,” was proposed by Day [11]. According to this hypothesis, simple
steatosis as a result of IR and excessive hepatic TG, is the first hit of NAFLD. Then, first hit

sensitizes the liver to a second hit leading to hepatocyte injury, inflammation and fibrosis,
promoting progression to NASH. The second hit likely involves oxidative stress, lipid
peroxidation and mitochondrial dysfunction. This hypothesis implies that the accumulation of
TG is necessary for the development of NASH. However, subsequent studies indicated that
the accumulation of triglyceride in hepatocytes may have a protective role to prevent
hepatocytes from lipotoxicity [12]. Thus, “the nontriglyceride lipotoxicity hypothesis” was
proposed [13]. This hypothesis pointed out that non-triglyceride lipids play an important role
in the processes leading to hepatocyte injury and progression to inflammation and fibrosis. As
more contributing factors are identified, it is evident that NASH is a multifactorial disease.
The mechanisms underlying the progression of simple steatosis to steatohepatitis are not
known so far, however a major risk factor for NASH is insulin resistance (IR), which
occurring in the context of the metabolic syndrome [14, 15]. Certainly, IR results in excessive
lipolysis within peripheral adipose tissue [16]. This lipolysis liberates free fatty acids (FFA)
from neutral fat. The liberated intracellular free fatty acids are released into the serum, where
they are taken up by the liver [16, 17]. Indeed, the bulk of hepatic neutral fat in NASH is
derived from re-esterification of circulating FFA [17]. Given this information, it is not
surprising that elevated serum FFA correlates with liver disease severity [18, 19]. FFA appear
to cause liver injury by inducing hepatocyte derangements leading to cell death by apoptosis,
a phenomenon termed lipotoxicity [20, 21]. It is recognized that hepatocyte apoptosis
correlates with NASH [22]. Indeed, apoptosis has been implicated as cardinal feature of
NASH by liver tissue analysis, where it correlates with histological severity and fibrosis
progression [22]. Consistent with this concept, elevated serum M30 neo-epitopes
(cytokeratin-18 fragments), a marker of hepatocyte apoptosis, distinguish simple steatosis
from NASH [23].
Our chapter focuses on the mechanism of lipotoxicity, but and emerging information
arrived from the role of microbiota and gut-liver axis [24], sterile inflammation [25],
adipokines [26] and genetic predisposition [27] as contributors for the development of
NAFLD. However, these biological aspects will not be covered and the readers can find
deeply reviews on these topics elsewhere. In view of the fundamental role of lipotoxicity in
virtually all models of NAFLD, the purpose of this chapter is to discuss the pathophysiology
involving hepatocyte injury by lipotoxicity, focuses on the known mechanisms of cellular
damage secondary to obesity-related fatty liver and in particular the network between lipid
metabolism, autophagy, oxidative stress, ER-stress, JNK activation, death receptors and BCL-
2 family proteins and their connections that contribute to the progression of NASH.
ALTERED LIPIDS METABOLISM

Within hepatocytes steatosis reflects a disordered homeostasis of lipid metabolism when
lipid inputs exceed lipids utilized. The main form of lipids stored in lipid droplets is
triglycerides (TG), which are composed of three fatty acids and one glycerol backbone via
ester bonds. FFA entering the liver are mostly derived from lipolysis of adipose tissue TG in
the fasting state (constituting 60% of liver FFA in NAFLD), de novo lipogenesis (26%) and
from hydrolysis of dietary triglycerides (15%) [17] (Figure 1). Hepatic FFA taken up from
circulation is dependent on the concentration of FFA in the blood and transport proteins

including fatty acid transport proteins (FATP), fatty acid binding proteins (FABP), and fatty
acid translocase (FAT/CD36) [28, 29, 30]. As hyperlipidemia is often associated with NASH,
FFA taken up from the circulation is increased in fatty liver [18]. Moreover, the hepatic
expression of FFA transport proteins is also increased in NAFLD, which further contributes
to enhanced FFA delivery to the liver [16]. De novo lipogenesis (DNL) is a metabolic
pathway which synthesizes fatty acids from simple metabolic precursors (i.e., acetyl-CoA) in
a series of enzymatic reactions. The DNL pathway is mainly regulated by insulin and glucose
at the transcriptional level in adipose tissue and liver. Under insulin resistance (IR) condition,
the stimulatory effect of insulin on DNL is retained in the liver, whereas the inhibitory effect
on glucose production is diminished [31]. It is established that IR is the primary factor
underlying hepatic steatosis, indeed excess of FFA not only induce IR but also impair insulin
clearance [32, 33, 34]. These lead to the typical hyperinsulinemia of patients with NAFLD
[35]. In hepatocytes the disruption of normal insulin signaling and increased abundance of
fatty acids as well lead to altered lipid metabolism [16]. In IR liver, insulin is unable to inhibit
glucose production, leading to an increased glucose level [31]. Glucose stimulates DNL via
the transcription factor known as the carbohydrate response element binding protein
(ChREBP). On the other hand, insulin still stimulates DNL by increasing sterol regulatory
element-binding protein 1c (SREBP-1c), a transcription factor of lipogenesis [16]. In
addition, NAFLD patients present IR in adipose tissue and skeletal muscle as well, which
may influence hepatic steatosis [21]. Due to IR, the adipose tissue becomes resistant to the
anti-lipolytic effect of the insulin. As a result, influx of fatty acids to the liver in NAFLD
patients is increased. One major function of hepatic FFA is to provide energy for the liver
through oxidation [36]. Hepatic FFA are mainly oxidized by mitochondria through β-
oxidation (Figure 1). In NAFLD increased FFA enhance β-oxidation by mitochondria until
mitochondrial respiration becomes severely impaired [37]. Moreover, increased hepatic FFA
also stimulate β-oxidation in peroxisomes and microsomal ω-oxidation in the endoplasmic
reticulum by microsomal enzymes. Peroxisome proliferator-activated receptor α (PPAR-α) is
a key transcription factor regulating the expression of genes involved in mitochondrial,
peroxisomal and microsomal FFA oxidation [38] (see below). FFA have been shown to
induce PPAR-α in NAFLD, indicating that the enhanced FFA oxidation could be mediated by
increased PPAR-α in NAFLD [38]. Hepatic FFA can also be used to synthesize other lipids
such as phospholipids or re-esterified to form TG. Then TG can be assembled into very low
density lipoprotein (VLDL) particles for secretion or stored in lipid droplets (Figure 1).
Although defective VLDL secretion may contribute to lipid accumulation, it has been shown
that VLDL secretion is increased in NAFLD [39, 29]. Increased FFA oxidation and VLDL
secretion could be a compensative mechanism in response to the overload of hepatic FFA.
However, VLDL secretion in NASH patients is significantly lower than that in simple
steatosis patients although both NAFLD patients have elevated serum VLDL levels than
control group, suggesting that decreased VLDL secretion may contribute to disease
progression from simple steatosis to NASH [40]. Thus, various mechanisms such as increased
FFA influx to the liver, enhanced DNL, reduced FFA oxidation, and decreased VLDL
secretion can lead to hepatic steatosis.
Some of the most prominent offenders to the liver are FFA [41]. FFA with double bonds
are referred to as “unsaturated” while those without double bonds are called “saturated.”
Palmitate (PA; C16:0), the most common saturated FFA found in animals and plants, is
ingested as part of the diet or can be produced by DNL from excess of carbohydrate

consumption or IR. Oleate (OA; C18:1), an unsaturated FFA, is commonly present in the
“western diet” [42]. Several in vitro studies have demonstrated the toxic effects of unsaturated
FFA such as PA or stearate on liver cells by inducing apoptosis [43]. FFA levels are increased
in patients with NASH and correlate with disease severity [18]. There is an increased body of
evidence indicating that saturated FFAs are more hepatotoxic than unsaturated FFA [43, 44].
This difference in toxicity between saturated and unsaturated FFA is related to the ability of
unsaturated FFA to more readily be esterified into TG than saturated FFA [45, 46]. Saturated
FFA (such as palmitate and stearate) or their end products lysophosphatidyl choline (LPC)
exerts their toxicity by triggering several apoptotic processes implicating the activation of ER
stress- and JNK-dependent pathways (see below). Failure of hepatocytes to dispose of excess
FFA results in apoptosis, a cardinal feature of NASH.
Figure 1. Mechanisms of Hepatic Steatosis and free fatty acid metabolism in NAFLD. A net retention
of free fatty acids in NAFLD hepatocytes could potentially result from alterations in the (1) uptake
from visceral adipose tissue lipolysis, (2) diet, (3) de novo lipogenesis, (4) oxidation, (5) TG synthesis,
and (6) export pathways as VLDL.
AUTOPHAGY
Autophagy is the catabolic degradation of cellular components through the lysosome, and
serves as both a removal mechanism for dysfunctional cell content and an energy source [47].
Autophagy has been implicated in lipid metabolism [48]. In hepatocytes, the degradation of
lipid droplets mediated by autophagy is known as lipophagy [49]. Studies have shown that
inhibition of autophagy in hepatocytes and in mouse liver increases TG storage in lipid
droplets, suggesting lipophagy may prevent steatosis [50]. In addition, the increased TG
storage is due to impaired lipolysis and not to increased TG synthesis, suggesting that
autophagy plays a key role in the lipolysis of lipid droplet [48]. Conversely, lipid storage in
hepatocytes is decreased when autophagy is induced by pharmacological therapy [49].

Furthermore, in dietary mouse models, protein levels of autophagy-related protein 7 (ATG7),

an important component in autophagy, are reduced; and levels of autophagy are decreased as
well [50, 51]. Overexpression of ATG7 in the liver of Ob −/− mice induces autophagy and
reduces steatosis significantly. These findings support a lipolytic function of autophagy. The
lipolytic function of lipophagy in liver is thought to rapidly mobilize large amounts of lipids
in fasting state since the level of cytosolic lipases in hepatocytes is low compared to
adipocytes [52]. Insulin resistance is thought to be critical to the development of NAFLD and
a complex interrelationship exists between autophagy and both insulin resistance and lipid
accumulation [50]. Insulin downregulates autophagy in response to nutrient supplies, but
autophagy modulates insulin sensitivity as well [49]. Hyperinsulinemic high fat diet-fed mice
have decreased levels of autophagy [53]. Moreover, the direct effect of insulin occurs through
mTOR signaling, and in these studies levels of ATG5 and ATG7 were decreased, suggesting
a different mechanism for the effects of insulin on liver autophagy in obesity [53]. In both
murine model of NAFLD, diet-induced and genetically obese mice, impaired autophagy has
been associated with insulin resistance with decreased hepatic insulin signaling occurring in
concert with increased ER stress [51]. Adenoviral-mediated ATG7 overexpression decreases
ER stress and improves insulin sensitivity in these animals. Defective autophagy may lead to
insulin resistance from increased ER stress. In a immunohistochemical study on human liver
tissue has shown that the autophagy marker microtubule-associated protein 1 light chain 3
(LC3) was decreased with an increased degree of steatosis, suggesting decreased autophagy
in more severe steatosis [54]. In another study increased p62 accumulation, indicative of
defective autophagy, was observed on liver biopsies in NAFLD patients [55]. In-vitro cell
culture hepatocytes demonstrated that the saturated FFA palmitate inhibits autophagy, which
contributes to its ability to induce apoptosis [56]. In contrast, the nontoxic unsaturated FFA
oleate induces autophagy. Cell death from liver steatosis induced by oxidative stress is
increased in a rat hepatocyte in the absence of ATG5 [57]. These studies suggest autophagy is
defective in NAFLD a may be associated in lipotoxicity and NASH progression.
OXIDATIVE STRESS
One important mediator of lipotoxicity is the over production of reactive oxygen species
(ROS). Under normal physiological conditions hepatocytes process low levels of ROS that
are constantly generated as a by-product of mitochondrial respiration [58]. When ROS
production exceeds the antioxidant capacity, it leads to oxidative stress [41]. Markers of
oxidative stress are elevated in serum and livers of NAFLD subjects [59]. Accordingly,
mitochondrial dysfunction and levels of oxidative stress markers are correlated with the
disease severity from fatty liver to NASH [60, 59]. In NAFLD, the oxidation of FFA
generates ROS and reducing equivalents, such as nicotinamide adenine dinucleotide (NADH)
and nicotinamide adenine dinucleotide phosphate (NADPH) [61], promoting redox stress and
interferes with normal functioning of cellular organelles, such as the endoplasmic reticulum
and mitochondria [62]. When an excess of reducing equivalents occurs in liver mitochondria,
β-oxidation may be partially suppressed [63].
In the liver FFA are metabolized through three distinct pathways: β-oxidation, which
occurs mainly in mitochondria but also in peroxisomes, and microsomal ω-oxidation in the

endoplasmic reticulum (ER) by members of the cytochrome P450 family (Figure 1). The
extra-mitochondrial FFA oxidative pathways become increasingly important as FFA
availability increases in the liver, as is the case of NAFLD [61]. Mitochondrial β-oxidation
progressively shortens FFA into acetyl-CoA, which enter the tricarboxylic acid (TCA) cycle
or condenses into ketone bodies. Mitochondrial β-oxidation is regulated by carnitine
palmitoyltransferase 1 (CPT-I), carnitine concentration, and malonyl-CoA. CPT-I is an outer
membrane enzyme playing a key role for the entry of long-chain FFA into mitochondria,
short-chain and medium-chain FFA freely enter the mitochondria without requiring CPT-I.
FFA oxidation in mitochondria is associated with conversion of oxidized cofactors NAD+
and FAD into their reduced form NADH and FADH2. The reduced cofactors are re-oxidized
by the mitochondrial electron transport chain (ETC) in the mitochondrial inner membrane.
During oxidation of NADH and FADH2 electrons are transferred to the complexes of the
respiratory chain (Complex I-III), then migration of electrons along the ETC causes protons
to be extruded from the mitochondrial matrix into the mitochondrial intermembrane space and
a large electrochemical potential across the inner membrane to be used for adenosine
triphosphate (ATP) synthesis (oxidative phosphorylation). Most of the electrons flowing
along the ETC reach cytochrome c oxidase (Complex IV) to be coupled with oxygen and
protons to produce water despite minor electron leakages at upstream sites of the respiratory
chain [64]. Some of these electrons can directly react with oxygen (called “electrons leak”) to
form the superoxide anion radical (O2•−) that is dismuted by mitochondrial manganese
superoxide dismutase (MnSOD) into hydrogen peroxide (H2O2) and detoxified into water by
mitochondrial glutathione peroxidase and catalase, which may have a role in protection
against endogenous or exogenous H2O2 in hepatocyte mitochondria [65]. Thus, the
respiratory chain even in healthy mitochondria generates ROS. Physiologically, most
mitochondrial ROS are detoxified into water and only a small amount of ROS persists, which
play a role in several aspects of intracellular signaling and regulation of cell fate [66]. Larger
amounts of ROS leak out from impaired mitochondria and can damage other cellular
components [65]. In NASH hepatic mitochondria show ultra-structural abnormalities,
decreased mitochondrial DNA levels, decreased protein expression of several mitochondrial
DNA-encoded polypeptides, and a lower activity of complexes I, II, III, IV, and V (ATP
synthase) [67, 63]. NASH mitochondrial dysfunction lead to cytochrome C released, thereby
preventing electron flow from complex III to cytochrome c and subsequently cytochrome c
oxidase. The accumulation of electrons along the respiratory chain is further promoted by a
simultaneously enhanced mitochondrial β-oxidation rate, which increases both the formation
of NADH and FADH2, and the flow of electrons to the respiratory chain [67]. Electrons can
freely react with oxygen to form superoxide anion radical (O2•−), in turn the concentration of
H2O2 is not controlled and diffuses to the cytoplasm where participates in a series of
reactions generating other reactive compounds, such as hydroxyl radical (•HO). The
indiscriminate nature of •HO, which reacts with lipids, nucleic acids, and amino acids,
renders it short lived but highly dangerous in biological systems [68]. Thus, mitochondrial
ROS formation in NASH is enhanced [67]. Furthermore, inducible nitric oxide synthase
(iNOS) transforms O2•− into peroxynitrite, which can alter protein function via the nitration
of tyrosine residues and s-nitrosylation of cysteine moieties, indeed the components of the
ETC have different sensitivity to inhibition by peroxynitrite as is observed in NASH
mitochondria [60, 64]. Consequently, altered respiratory chain complex and further block of
the electron flow through the ETC and eventually leading to an enhanced ROS production

[64]. Increased ROS generation in fatty liver can injure hepatocytes through other mediators
as well, such as 4-hidroxy-2-noneal (4-HNE, marker of lipid peroxidation), 8-hydroxy-
deoxyguanosine (8-OHdG, a marker of oxidative DNA damage) and trigger the synthesis of
several cytokines, including TNF-α, transforming growth factor-β (TGF-β), interleukin-8 (IL-
8), and Fas ligand [41, 65, 67].
For instance, NASH is associated with mitochondrial dysfunction and lower activity of
ATP-synthase, hence, there is a shift toward peroxisomal and microsomal FFA oxidation
[69]. In the initial step of peroxisomal β-oxidation, hydrogen peroxide is formed through the
action of acyl-CoA oxidase, which donates electrons directly to oxygen [70]. The microsomal
ω-oxidation of FFA, catalyzed primarily by cytochrome P450 enzymes 2E1, 4A10, and 4A14,
forms ROS through flavoprotein-mediated donation of electrons to oxygen [65]. Additionally,
dicarboxylic acids, other products of microsomal FFA ω-oxidation, impair mitochondrial
function by uncoupling oxidative phosphorylation [65]. Consistent with this concept, activity
of the microsomal fatty acid oxidizing enzyme, cytochrome P450 2E1 (CYP2E1) is greater in
patients with NASH than in those with simple steatosis, and increased CYP2E1 activity has
been associated with oxidative stress, insulin resistance, and hepatic lipid peroxidation [71].
The net result of extra-mitochondrial FFA oxidation is thus a further increase in oxidative
stress and mitochondrial impairment in NASH. Reduction of oxidative stress is a potential
therapeutic strategy for patients with NASH. Indeed, two randomized controlled trials the
PIVENS (in adults) and TONIC (in childrens) reported that treatment of the anti-oxidant
vitamin E, improved liver injury in NASH with no significant effect on fibrosis, making
vitamin E the only treatment approved for the treatment of NASH [4, 72, 73].
ER-STRESS
The ER supports many vital cellular processes, including synthesis, maturation, folding
and transport of protein, lipid synthesis and packaging and regulation of calcium homeostasis.
Disturbances of any of these processes will initiate an ER stress [74]. The response to an ER
stress is initially mediated by the combination of several signaling pathways termed the
unfolded protein response (UPR), which serves at first to re-establish ER homeostasis and
promote survival by directing unfolded or misfolded proteins toward degradation [75].
However, a prolonged activation of the UPR will trigger apoptotic pathways causing cell
death. One of the consequences of lipotoxicity in steatosis is the activation of UPR [76].
Biosensors of ER stress include: protein kinase RNA-like ER kinase (PERK), inositol-
requiring protein-1a (IRE-1a), and activating transcription factor 6 (ATF6) [74]. PERK
dimerization drives the expression of the pro-apoptotic transcription factor C/EBP-
homologous protein (CHOP) [77]. IRE-1a activation leads to JNK activation and creates a
spliced form of XBP-1 mRNA which promotes degradation of misfolded ER glycoproteins
[74]. ATF6 optimizes protein folding during ER stress and ultimately facilitates recovery
from acute stress [78], also heterodimerizes with XBP1 and induces the ER-associated
degradation components [79]. ATF6 can also transcriptionally induce CHOP [80]. It has been
shown that saturated FFA directly induces ER stress response in hepatocytes [81]. Moreover,
increased levels of ER stress have been reported in obesity [82] and NAFLD patients [76]. ER
stress-induced JNK activation mediates insulin resistance through phosphorylation of insulin

receptor [82]. In a nutritional murine model of NASH, ER stress-induced XBP-1 mRNA

splicing and CHOP expression, correlates with the severity of apoptosis [83]. Individual fatty
acids may be important in their ability to induce the UPR as mice compromised in their
ability to synthesize mono-unsaturated fatty acids due to a deletion of stearoyl-CoA
desaturase-1 (Scd1) exhibit activation of the UPR by enhanced splicing of XBP1, and
increased expression of the stress-induced transcription factors CHOP in their livers due to a
deficiency of mono-unsaturated fatty acids [84]. Upregulation of the transcription factor
CHOP, which has low levels of expression under non-stress conditions, plays a critical role in
FFA-mediated ER stress-related lipotoxicity [81]. Unsaturated FFA do not induce ER stress
or apoptosis, and co-incubation with monounsaturated fatty acid rescues hepatocyte from
saturated FFA-induced ER stress and apoptosis [44, 85]. Palmitate-induced hepatocyte
apoptosis is mediated, in part, by ER stress-induced activation of CHOP [86]. Moreover,
CHOP upregulates the pro-apoptotic protein PUMA and the cell surface death receptor
TRAIL-R2 [86, 81]. Genetic knockdown of CHOP or the upstream protein PERK in cultured
hepatocytes attenuates palmitic acid-induced apoptosis [87]. Upregulation of CHOP is also
observed in both murine and human NASH [88, 89]. Surprisingly, CHOP knockout mice
were sensitized to the development of high fat diet-induced steatohepatitis, and methionine-
choline-deficient (MCD) diet-induced steatohepatitis [90]. This was due to a dominant in vivo
effect of CHOP deletion in macrophages, which allowed the accumulation of activated
macrophages in the liver, thus increasing the hepatic pro-inflammatory milieu [90].
Therefore, while hepatocyte CHOP might positively contribute to NAFLD pathogenesis,
macrophage CHOP might conversely protect the liver from steatohepatitis.
JNK ACTIVATION
Jun N-terminal kinases (JNKs), are a members of the mitogen-activated protein kinase
(MAPK) superfamily, of three known JNK genes, JNK1 and JNK2 are expressed in the liver
[91]. JNK1 pathway is one of the key mediators of insulin resistance and fatty acid-induced
hepatotoxicity [92, 93, 94]. The JNK pathway is known to be stimulated by both oxidative
stress and ER stress [91]. JNK1 activation in NASH has been presumed to be activated
through ER stress activation by IRE-1α/ASK pathway [95] or ER-stress independent pathway
by direct interaction between the small GTP-binding proteins CdC42/Rac1 (cell division
cycle protein 42, Ras-related C3 botulinum toxin substrate 1) and MLK-3 (mixed lineage
kinase 3) [96, 97]. In a murine model of diet induced obesity, absence of JNK1 results in
decreased adiposity, and significant improvement of insulin sensitivity [92]. Liver specific
knockdown of JNK1 in obese mice lowers blood glucose and insulin levels, but increases TG
level [98]. Furthermore, saturated FFA induce JNK-dependent hepatocyte lipotoxicity by
activating the pro-apoptotic Bcl-2 family Bim and Bax-mediated apoptosis [43]. JNK1
phosphorylates c-Jun, a member of the AP-1transcription factor complex, which induces
expression of the BH3-only protein PUMA [99] and the genetic deletion of JNK1 prevents
FFA-mediated c-Jun activation and PUMA induction by saturated FFA [99]. PUMA works
cooperatively with Bim to mediate FFA-induced hepatocyte injury leading to mitochondrial
dysfunction and culminating in cell death [86, 85]. Unsaturated fatty acids including oleate
and palmitoleate can attenuate both saturated free fatty acid-initiated JNK activation and

apoptosis [85]. However, oleate can also induce modest JNK induction and increase TRAIL-
R2-induced apoptosis susceptibility [100]. A positive correlation was found between the
expression intensity of JNK1 and the progression of liver fibrosis by inducing chronic
inflammation as ascertained in a mouse NASH model [101]. Methionine-choline-deficient
diet causes NASH coincident with the activation of JNK and caspase-12 in a murine model
[102]. In contrast, murine dietary model of NAFLD have identified an important JNK2
protective function in the liver is to down regulate the pro-apoptotic mediator Bim [93]. JNK
activation is observed in liver from patient with NASH but subjects with simple steatosis
demonstrated JNK phosphorylation levels that were similar to control subjects [76]. Taken
together, JNK activation appears to play a major role in lipotoxicity and obesity related liver
disease.
FREE CHOLESTEROL
Recently, free cholesterol (FC) has emerged as a key role in the development of
lipotoxicity in NASH. FC accumulation sensitizes hepatocytes to TNF and Fas-induced
apoptosis [103]. In a mice model of NASH was observed that TG or FC loaded hepatocytes in
response to TNF caused apoptosis, increased ROS formation and liver injury only in livers
with increased cholesterol content [103]. This observed sensitization to TNF was secondary
to a reduction of mitochondrial glutathione content. Furthermore, treatment with an HMG-
CoA reductase inhibitor decreased mitochondrial free cholesterol and increased glutathione
levels reducing liver cell death [103]. Likewise, abundancy of FC stimulates Kupffer cells
and hepatic stellate cells (HSC), which mediate inflammation and fibrosis as well as
mitochondrial dysfunction, generation of ROS, activation of UPR and downstream effects
that culminate in hepatocyte apoptosis [104]. In a murine model of NASH, where animals fed
high levels of FC results in accumulation of toxic hepatic oxysterols which contributes to
mitochondrial dysfunction and liver injury [105]. In addition, mitochondrial FC accumulation
leads to apoptosis through a toll like receptor 4 (TLR4)-regulated JNK1 pathway that
activates the inflammatory mediator high mobility group box 1 protein (HMGB1) [106].
Indeed, the expression of enzymes that regulate cholesterol homeostasis are increased in
NASH, both SREBP-2 (a transcriptional factor that plays an important role in cholesterol
synthesis) and StAR (a transporter of FC from the external to the internal mitochondrial
membranes) were overexpressed in patients with NASH compared to those with simple
steatosis [107]. Furthermore, treatment with HMG-CoA reductase inhibitor in murine model
of NAFLD reduces hepatic steatosis, inflammation, liver fibrosis. Accodingly, in-vitro
steatotic hepatocytes under HMG-CoA reductase inhibitor showed a significant reduction of
oxidative stress toxicity [108, 109, 110]. These findings suggest a role of mitochondrial FC in
lipotoxicity and disease progression from steatosis to steatohepatitis.

APOPTOSIS IN NAFLD
The Extrinsic Pathway: Death Receptors
The plasma membrane receptors termed death receptors belong to tumor necrosis factor
(TNF) receptor superfamily [111], and their activation on the cell surface initiates the
extrinsic pathway of apoptosis [112]. The death receptors expressed in the liver are Fas, TNF
receptor 1 (TNFR1), and TNF-related apoptosis-inducing ligand receptor (TRAIL-R) 1 and 2
[112]. Upon activation by its cognate ligands, leads to receptor oligomerization followed by
the formation of a death-inducing signaling complex (DISC), which consists of caspase 8 and
several adaptor proteins, such as Fas-associated protein with death domain (FADD) [111]
(Figure 2). In liver cells, caspase 8 cleaves Bid generating truncated Bid (tBid) which then
induces egress of pro-apoptotic factors from the intermitochondrial space to the cytosol that
activates apoptosome and effector caspases 3, 6 and 7 which execute cellular demise [113]
(Figure 2). Death receptors expressed in the liver have been considered as potential mediators
of hepatic lipotoxicity [41].
Figure 2. Death receptor-mediated apoptosis.
Upon binding of the cognate ligand to the extracellular portion of the death receptor, the receptor
intracellular domain recruits adaptor proteins, such as FADD (Fas-associated protein with death
domain), and pro-caspase 8 to form a signaling platform termed the death-inducing signaling complex
(DISC). Caspase 8 then undergoes proteolytic activation to truncated (tBid), resulting in Bax/Bak
activation in mitochondria, leading to activation of caspases 3, 6 and 7 that execute the final steps of
cell death.

Liver expression of Fas is significantly increased in patients with NASH compared to

simple steatosis and healthy subjects [22]. In mice models of NAFLD and in-vitro steatotic
hepatocytes were greatly susceptible to Fas-mediated apoptosis [114, 100]. In normal livers,
hepatocyte growth factor receptor (c-Met) binds directly to Fas, precluding its
oligomerization. However, this inhibitory effect is absent in NASH, facilitating ligand-
dependent activation of Fas and apoptosis [115]. Moreover, in hepatocytes under basal
condition, insulin induces proliferation upon activation of EGFR (epidermal growth factor
receptor), but in steatotic hepatocytes JNK activation by saturated FFA not only ameliorates
insulin-induced EGFR activation, but also shifts insulin-induced EGFR activation and
proliferation toward Fas activation and apoptosis [116]. In liver, Fas signaling leads to
production of IL-8, monocyte chemotactic protein-1 (MCP-1), chemokine (C-C motif) ligand
3 and chemokine (C-X-C motif) ligand 1 which are chemokines for neutrophils and
macrophages, further aggravate liver inflammation in NAFLD [117, 118].
Beside the classical outcome of a death receptor (like Fas activation) is hepatocyte death;
TNF receptor activation also affects multiple cellular responses that include survival,
inflammation and proliferation [119]. Transcription factor NF-κB (nuclear factor κB)
represents a key cytoprotective pathway that upregulates anti-apoptotic genes such as Bcl-xl
and c-FLIP and blocks prolonged activation of JNK, a key pathway over TNF induces cell
death [120]. Accordingly, TNF-mediated cell death requires inhibition of NF-κB or NF-κB
target genes [121, 122].
TNFR1 and its cognate ligand TNFα play important role in pro-inflammatory signaling,
development of insulin resistance and obesity related metabolic disorders [123]. TNFR1 is
also upregulated in liver of NASH patients [124]. In hepatocytes, FFA cause lysosomal
destabilization that result in release of cathepsin B into the cytosol and enhanced TNF-α
expression and release, further promote lysosomal permeabilization, thus exacerbating liver
injury in a feed-forward loop [125]. Likewise, TNF-α appears to be involved in development
of steatosis, in TNFR1−/− mice are protected against diet-induced steatosis and liver injury
[125]. Also, in genetically obese mice Ob−/− anti-TNF-α therapy improved steatosis [126]. It
has been observed that adipose tissue expansion in patients with NASH is associated with the
release of pro-inflammatory cytokines such as TNF [26], where in a murine NASH model,
TNFRDKO −/− mice (TNFR1 and TNFR2 KO) were protected from hepatic steatosis and
fibrosis, by decreasing Kupffer cells activation [127].
Among the death receptors, the tumor necrosis factor related apoptosis-inducing ligand
receptor 2 (TRAIL-R2), has been implicated as a key component of death receptor signaling
pathways in lipotoxicity [100, 128.] Liver expression of TRAIL-R2 is significantly increased
in patients with NASH as well as in a murine nutrient-excess or methionine and choline
deficient (MCD) diet model of NASH [100, 129, 130]. TRAIL receptor −/− mice, which have
only one TRAIL receptor gene for both human TRAIL-R1 and TRAIL-R2, under diet
induced NASH are protected against hepatocyte apoptosis and other associated pathogenic
features of NASH, such as liver steatosis, inflammation and fibrosis [131]. In addition,
exposure to saturated FFA in hepatocytes induced TRAIL-R2 expression through CHOP and
JNK1 activation, leading to TRAIL-R2 clustering into lipid rafts, recruitment of caspase-8 by
a ligand-independent activation, and cell death [132]. Conversely, unsaturated FFA sensitized
hepatocytes to TRAIL-induced apoptosis, via JNK mediated upregulation of TRAIL-R2
[100]. In cells with persistent ER stress, increased expression of TRAIL-R2 via the UPR
mediator CHOP, and ligand-independent activation of the TRAIL-R2 causing caspase 8-

dependent apoptosis have been described [133]. Because saturated FFA also cause ER stress,
thus TRAIL-R2 pro-apoptotic signaling may also contribute to FFA-induced lipotoxicity. It is
important to note that saturated FFA also upregulates pro-apoptotic proteins such as PUMA
and Bim, which contribute to lipotoxicity in the context of TRAIL-R2 activation.
Upregulation of these pro-apoptotic proteins may be critical for lipotoxicity as TRAIL
receptor ligand-dependent activation is harmless to healthy hepatocytes but is cytotoxic to
steatotic hepatocytes both in vitro and in vivo [100, 132]. Consequently, hepatocyte-specific
caspase-8 −/− attenuates murine NASH under MCD diet [134]. Additionally, the degradation
of cellular inhibitor of apoptosis 1 (cIAP-1), one of the members of the anti-apoptotic IAP
family that inhibits death receptor-mediated death signaling by caspase-8 cleavage,
contributes to lipotoxicity during the TRAIL-R2-mediated saturated FFA induced apoptosis
[135]. Accordingly, cIAP is decreased in human NASH [135]. Moreover, TRAIL signaling
may drive pro-inflammatory response, in the context of NASH, TRAIL-R is required for NF-
κB activation in macrophages and pro-inflammatory cytokine expression, TRAIL-R−/− mice
have reduced macrophage accumulation in liver and adipose tissue, and suppression of pro-
inflammatory milieu [136, 131]. Taken together, these studies suggest that TRAIL receptor
signaling plays a key role in hepatocyte lipotoxicity and NASH.
The Intrinsic Pathway: Bcl-2 Family Proteins
The mitochondrial cell death pathway in hepatocytes is regulated by interactions between

Bcl-2 family proteins [137]. The Bcl-2 family proteins regulate the permeabilization of the
outer mitochondrial membrane, triggering mitochondrial outer membrane permeabilization
(MOMP) allowing leakage of the pro-apoptotic mediators such as cytochrome c into the
cytosol where binds to monomeric Apaf-1 leading to its conformational change and
oligomerization, then pro-caspase-9 is recruited to heptameric Apaf-1 complexes forming the
apoptosome, this in turn leads to activation of caspase-9 and, through caspase-9-mediated
cleavage, activation of the executioner caspases-3 and -7 resulting in the typical morphologic
changes of apoptosis [138]. Anti-apoptotic members include Bcl-2, Mcl-1 and Bcl-xL, these
proteins act to prevent MOMP. The multidomain pro-apoptotic proteins are Bax and Bak
which oligomerize to induce the MOMP. The pro-apoptotic BH3-only proteins can induce
MOMP either by deactivating the anti-apoptotic proteins (Bmf, Bad, Hrk, Bik) or directly
activating Bax or Bak (Bim, Bid, Noxa, PUMA) [139] (Figure 3).
Both Bim and PUMA have been shown to be important in saturated FFA-induced
hepatocyte toxicity, where knockdown of Bim or PUMA protects against FFA-induced Bax
activation and apoptosis [43, 99]. Upregulation of PUMA during a lipotoxicity involves both
JNK1 and CHOP-dependent transcriptional regulation of the protein [86]. Further, under
basal condition miR-296-5p directly targets PUMA mRNA suppressing its expression, during
FFA-induced hepatocyte apoptosis a decrease miR-296-5p contribute to upregulation of
PUMA expression by enhancing its translation [140]. Accordingly, miR-296-5p is reduced in
livers from patients with NASH and varies inversely with PUMA expression [140].
Moreover, cellular level of Kelch-like ECH-associated protein 1 (Keap1) also regulate
hepatocyte expression of PUMA and Bim [141]. Thus, Keap1 which is an adaptor protein for
E3-ligases and initiates the proteasomal degradation of several proteins, under saturated FFA
lipotoxicity induces degradation of cellular Keap1 through autophagy in a p62-dependent

manner, leading to increase protein expression Bim and PUMA by a JNK1-dependent

mechanism leading to cell death, whereas maintaining Keap1 protein levels upon lipotoxic
insult protects hepatocytes from saturated FFA-induced apoptosis [141].
Figure 3. Bcl-2 proteins in NAFLD
FFAs activate the pro-apoptotic BH3-only proteins (Bim and PUMA), also Bid is activated by death
receptor pathway, causing Bax/Bak activation. Also Saturated FFAs can alter the anti-apoptotic Bcl-2
family members (Mcl-1 and Bcl-xL), releasing Bax and Bak, and causing their activation by BH3-only
proteins. Once activated, Bax and Bak promote mitochondrial dysfunction, leading to the activation of
the caspase cascade and cell death.
In the context of saturated FFA mediated lipotoxicity, Bim upregulation is dependent on

the transcription factor Forkhead box O3a (FoxO3a), activated upon dephosphorylation by the
protein phosphatase 2 A (PP2A) [142]. Unsaturated FFA, led to only modest expression of
Bim and FoxO3a activation, consistent with its lower toxicity. Also, JNK can phosphorylate
and activate Bim, Bad and Bax, which triggers the mitochondrial pathway of apoptosis [143
144]. More, another unsaturated FFA palmitoleate (PO) attenuates saturated FFA-induced cell
death. PO reduces expression of both Bim and PUMA; this protective effect of PO may be
due to more efficient sequestration of palmitate as TG in hepatocytes, diminishing palmitate-
induced ER stress [85].
Bid, is a BH3-only protein that is cleaved by caspase-8 into its active form, truncated Bid
(tBid), which links the extrinsic and intrinsic apoptosis pathways also contributes to liver
injury in NASH [145]. In-vivo Bid knockdown specific to hepatocytes in mice under NASH
induced diet, despite the degree of liver steatosis was not changed; indeed was associated

with a marked reduction in hepatocyte apoptosis, macrophage infiltration and HSC activation,
that were associated with a significant anti-fibrotic effect [145].
Both anti-apoptotic proteins Mcl-1 and Bcl-xL, by their ability to maintain mitochondrial
integrity, have a critical role for liver cell survival [146]. Cellular levels of Bcl-xL and Mcl-1
are also decreased during FFA-induced lipotoxicity [147, 148]. Hepatocytes under FFA
mediated toxicity decreased Bcl-xL protein expression engaging lysosomal permeabilization
and hepatocyte apoptosis; and overexpression of Bcl-xL prevents FFA-induced cell death
[147]. Also, the saturated FFA palmitate activates PKCθ-dependent proteasomal degradation
of Mcl-1, contributing to cell death by lipotoxicity [148].
Both extrinsic and intrinsic apoptotic pathways converge on caspase (cysteinyl aspartate
specific proteases) activation to induce cell death [149]. The caspases are constitutively
expressed as inactive proenzymes and generally require proteolytic cleavage on the aspartic
acid residue for their activation. Caspases are capable of self-activation, as well as of
activating each other in a cascade-like process [150]. Among the mammalian caspases
identified to date, some are primarily involved in apoptosis (caspases-2, -3, -6, -7, -8, -9, -10,
and -12), and other caspases, such as caspases-1, -4, -5, and -11, are involved in inflammation
[151]. Caspase activation has been well documented in both human and preclinical NASH
[114, 131]. Mice lacking caspase-1, -3 and -8 are protected against diet-induced NASH [152,
153, 134]. In addition, use of pharmacological inhibition of caspase is a useful tool to reduce
hepatocyte apoptosis, and has gained significant attention for development of novel
therapeutic for NASH. Three independent pre-clinical studies using pan-caspase inhibitors in
different mice models of dietary-induced NASH showed that reduction of hepatocyte
apoptosis by blocking pro-apoptotic caspases decrease in cell damage and the inhibition of
pro-inflammatory caspases may then interrupt the inflammatory milieu and prevent a pro-
fibrogenic process and activation of HSC [154, 155, 156]. In NASH clinical trial where used
the caspase inhibitor GS-9450, decreased M-30 neo-epitopes, a serum marker of hepatocytes
apoptosis in a dose dependent manner [157].
Recently, data suggest that injured hepatocytes, can communicate with other cells,
potentially contributing to further liver injury. Dying or injured hepatocytes release small
extracellular particles known as extracellular vesicles (EV). EV release does not seem to
require cell death and occurs before apoptosis onset, but is attenuated by JNK inhibition [158]
and caspase-3 ablation [159]. Elegant studies have shown that hepatocytes exposed to the
saturated FFA or mice model of NASH, produce and release large quantities of EV that may
act on various target cells and contribute to key processes involved in NASH pathogenesis,
including macrophage activation, angiogenesis and fibrosis [160]. More, increased serum EV
are found in patients with NASH compared to individuals with simple steatosis [160]. These
new paradigm reveal the intersection of lipotoxicity pathways can signal and influence the
surrounding liver cells in an extremely organized matter by delivering signals through EV.
CONCLUSION
NAFLD is the most common liver disease worldwide, been NASH the more aggressive
player of this entity, which can have consequences that significantly alter morbidity and
mortality. Lipotoxicity is a key mechanism of progression from simple fatty liver to NASH

(Figure 4). Hepatocyte injury by lipotoxicity occurred in steatotic liver cell overwhelmed of
FFA, where cannot be physiologically removed by lipid metabolism. This FFA can activate a
myriad of intracellular response that trigger altered autophagy, oxidative stress and ER-stress,
which in turn sustained JNK activation and enhanced susceptibility to apoptosis by Bcl-2
family proteins or death receptors. Also, lipotoxic hepatocytes release pro-inflammatory and
pro-fibrogenic mediators that contribute to the progression of NASH.
Figure 4. Schematic representation of the pathomechanism of lipotoxicity in NASH.
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Chapter 12
PORTAL HYPERTENSION
Francisco Eizayaga, Leandro Steinberg,

Carlos Brodersen and Salvador Romay
ABSTRACT
Portal hypertension is defined as the increase of portal pressure beyond 10 mmHg.
As most of the cases of portal hypertension are caused by Hepatic Cirrhosis, the
prevailing pathophysiologic theory explaining increased portal pressure in the twentieth
century was the increased resistance to blood flow due to hepatic fibrosis and
architectural distortion. Most of the research was done in dogs with complex research
models. It was in the early seventies that inconsistencies of this theory came out,
suggesting other possible explanations. In 1983 R. Groszmann and collaborators started
to work with a model of portal hypertension with rats, described in 1951 by P.C. Reynell.
These studies shed new light on pathophysiology of portal hypertension, adding new
interpretations to old findings called the forward flow theory opposed to the former
backward flow theory. These new findings provided new explanations for the managing
of hemorrhage and therapeutics of the portal pressure increase.
ANATOMY. HISTORICAL BACKGROUND AND PATHOPHYSIOLOGY
Anatomy of Hepatic-Portal System
There are two portal venous systems in the human body and are located in hypophysis
and in the liver. Portal systems are characterized by a venous capillary bed that pools in other
capillary bed before passing through the heart. The hepatic-portal system carries blood from
the digestive tract, pancreas, spleen and gallbladder to the liver. Once inside the liver, the
portal vein feeds the hepatic sinusoids and then through the supra-hepatic veins, drains into
the inferior cava vein. The hepatic artery increases the amount of oxygen in the hepatic
circulation, but still most of the oxygen comes through the portal circulation.

232 Francisco Eizayaga, Leandro Steinberg, Carlos Brodersen et al.
The portal vein is formed by the Lienal vein (which receives blood from the inferior
mesenteric vein) and the superior mesenteric vein. Before entering the liver the portal vein
receives blood form the pyloric vein, the right stomach vein, the coronary stomach vein, the
para-umbilical vein and the cystic vein.
The portal vein has an approximate length of 8 cm, enters the liver accompanied by the
hepatic artery, the common biliary duct, the hepatic plexus and numerous lymphatic vessels.
Posteriorly the vein splits in the right and the left branches. The left branch receives the cystic
vein (see Figure 1).
Figure 1. Anatomy of the hepatic-portal system (Henry Gray, 1825-1861).
Pathophysiology of the Hepatic-Portal System
In normal conditions, the portal vein has a pressure of 10 mmHg. This pressure is
determined by arterial pressure, the mesenteric system resistance and the intra-hepatic
resistance.
Portal hypertension is defined as an increase in portal pressure above 10 mmHg. This
increase in pressure is a usual complication in different kinds of chronic liver disease.
During the major part of the 20th century and before, the increase in portal pressure was
attributed to the increased resistance in intra-hepatic circulation. As most of the cases of
portal hypertension are caused by hepatic cirrhosis and intra-hepatic circulation is severely
disturbed in cirrhosis by regeneration nodules and fibrosis, this was the only pathophysiologic

Portal Hypertension 233
feature studied. The backward flow generated by an increased intra-hepatic resistance was
thought to be the origin of porto-systemic collateral vessels that tried to relieve the increased
portal pressure. In the first half of the XXth century, the diagnosis of portal hypertension was
made when there was evidence of collateral circulation and the usual treatment was to make a
spleno-renal or a porto-caval shunt. This surgery was indicated when repeated massive
hemorrhage was present. At that time, most of the research in portal hypertension was done in
dogs with complicated surgery and poor results (Wiles 1952).
I In the late 60s’ other models used included the infusion of Schistosoma in the portal
vein and the use of liver toxics like Carbon tetrachloride (alone or with Phenobarbitone) or
Dimethyl nitrosamine (McLean 1969, McLean 1969 (1)).
The prevailing evidence suggested that the hepatic blood flow was decreased by an
increased intra-hepatic resistance and this originated a high portal vein pressure (Bradley
1952).
While the most accepted theory to explain portal hypertension was the intra-hepatic
obstruction to portal blood flow, in the early 70s’ several inconsistencies of this theory begun
to come out. Supporting the “backward flow theory” was that the portal flow, as in congestive
heart failure, seemed to be passively congested and the spleen enlarged as a consequence of
this disturbance. What troubled some authors was that disturbance to the portal blood flow in
experimental animal models or the decreased portal blood flow observed in congestive heart
failure seemed neither to increase the portal pressure nor to produce esophageal varices.
While the portal blood flow was considered to be slowed down, portal oxygen content was
high, cardiac output was increased and peripheral resistance decreased. When researchers
realized that the mesenteric blood flow was increased, some experimental studies begun to
raise the possibility of an active congestion rather than a passive congestion (Witte 1974).
In 1951 an experimental procedure was described by P.C. Reynell from the University of
Oxford, to produce in a simple and inexpensive way pre-hepatic portal hypertension in the rat.
This procedure was a modification of the experimental model described by Whitaker in 1946
(Reynell 1952). The model produced portal hypertension by the partial ligation of the portal
vein. The abdomen was opened, a piece of wire was placed alongside the portal vein, a
ligature was snugly tied around the wire and the portal vein and the wire was then removed.
This very simple and inexpensive procedure was deeply studied in the subsequent years. In
1983 R. Groszmann and collaborators studied the portal vein ligation model of portal
hypertension with the infusion of two different kinds of radiolabeled microspheres. Their
hemodynamic studies clearly established that portal hypertension was influenced by
mesenteric vasodilation and a systemic hyperdynamic circulation. With a very elegant
experimental model they concluded that the hemodynamic observations of the splanchnic
circulation strongly supported the forward flow theory of portal hypertension (Vorobioff
1983).
Further studies with a simulated mathematical model determined that in the portal
ligation model of portal hypertension, backward flow accounted for 40% of the increase in
portal pressure and forward flow for the remaining 60% (Benoit 1985).
Resistance in portal vein may be modified in three different places (see Figure 2). The
increased post-hepatic, Intra-hepatic or pre-hepatic resistance (R2) ads to the increased portal
collateral circulation (R3). Conversely, the arterial mesenteric artery resistance is decreased
(R1) as described by Vorobioff et al. in 1983. This last decrease in mesenteric resistance
induces what is called hyperdynamic circulation and includes an increased heart rate and an

effective hypovolemia. This pathophysiologic feature behaves hemodynamically like an

arterial-venous fistula into which a big percentage of splanchnic blood escapes, towards cava
vein circulation, by-passing the liver. The increased portal resistance is exerted backwards
and accounts for some of the pressure increase. Portal hypertension is classified, according to
the site in which the increased resistance is placed, like pre-hepatic, hepatic or post-hepatic or
pre-sinusoidal, sinusoidal or post-sinusoidal. Portal hypertension produces, besides changes
in the local mesenteric and hepatic circulation, big changes in the organism like alterations in
cardiovascular and baro-reflex regulation (Arranz 1995), central nervous system changes in
catecholamines synthesis (Lemberg 1993, Lemberg 1998, Fernandez 1999), and
modifications in the blood-brain-barrier (Scorticati 2004).
PORTAL HYPERTENSION, HEMORRHAGE AND HEMOSTASIS

The increase in Portal pressure is accompanied by alterations in coagulation and digestive
hemorrhage. Hemorrhage produced by esophageal varices or induced by alterations in the
gastric mucosa is one of the first causes of death in patients with portal hypertension. In the
case of hepatic cirrhosis (the major cause of portal hypertension), hemorrhage is induced by
three factors.
The architecture of the gastro-esophagic junction favors the creation of esophageal
varices. These varices are formed from sub-mucous veins that progressively dilate and are
prone to rupture and massive hemorrhage. The normal gastro-esophagic junction has 4 zones
(Vianna 1987):
 Gastric zone, in which veins adopt a mostly a longitudinal distribution.

 The palisade zone, that is composed of parallel vessels laying mainly within the
lamina propia.
 The perforating zone characterized by veins adopting a treble clef shape.
 The truncal zone that is composed by few deep descending veins.
The palisade zone is characterized by a high resistance and a low pressure making the
next zone, due to a decreased resistance and a lower velocity, prone to pressure increase and
progressive dilatation. This situation is worsened by the fact that this zone is placed inside the
thorax, where there is a negative pressure surrounding, different to the positive pressure found
inside the abdomen. The Bernoulli principle that explains pressure and velocity variations of
an ideal fluid (a fluid that circulates with no resistance), according to the variations observed
in the diameter of the tube containing the fluid when this fluid circulates with a laminar flow,
helps to explain the importance of pressure variations and tendency to hemorrhages. When an
incompressible fluid of these characteristics circulates at a constant flow, pressure and
velocity are inversely related. This means that when the tube has a bigger diameter, the
pressure increases and the velocity decreases. When the diameter decreases, the velocity
increases but the pressure decreases.

Figure 2. Portal circulation. PSC: portal-systemic colateral vessels. R1: splanchnic arterial resistance.
R2: Pre-hepatic, hepatic and post hepatic resistance. R3: colateral vessels resistance (modified from
Benoit et al., 1985).
Figure 3 shows a schematic of pressures according to the total section of circulation of

the above described zone. In zone a, of circulation in the stomach, pressure is increased by the
effect of portal hypertension. In zone b, the gastroesophageal junction or palisade zone, there
is a relative decrease in pressure. In zone c, the veins gather forming big sub-mucous veins
increasing the total diameter, decreasing velocity and increasing pressure. Moreover the sub-
mucous surroundings are of low density favoring vein dilatation, the thorax negative pressure
and the increased resistance offered by the small diameter of the perforating zone when
piercing the muscular layer of the esophagus.
Other important factor for hemorrhage is the wall tension, described in Laplace’s
equation:
T= p x r
Where T is tension, p is pressure and r is radius of the vessel. Increased pressure

increases radius and as a consequence increased wall tension ends with hemorrhage.
The second factor in cirrhosis is alteration of coagulation factors due to liver failure.
These factors are dependent or independent of K Vitamin. Recombinant Factor VII is

frequently used to decrease bleeding risk in patients with liver failure that need a liver biopsy
(Kamphuisen 2002). In addition an increased fibrinolysis exists that is facilitated in many
cases by the presence of a sepsis, the increased endotoxin (Yun 2004, Minami 1997) and a
higher amount of platelet activating factor (PAF) secreted by stellar cells in the liver that is
also responsible of the increased pressure at that level (Yang 2004).
The least studied of these factors is the altered primary hemostasis. The increased liver
resistance produces a rearrangement of mesenteric microcirculation. The decreased resistance
of splanchnic arteriole has as a consequence a greater increase in portal pressure and an
increased mesenteric blood flow. This flow increase, induces stretch and shear stress in the
mesenteric endothelial cells. The response is an augmented synthesis of nitric oxide (NO) and
Prostacyclin (PGI2). Both substances decrease platelet aggregation and at the same time are
vasodilators (Gupta 1997). Albornoz et al. demonstrated that possibly NO is the most
important mediator in producing alterations in primary hemostasis. Inducing biliary cirrhosis
by chronic bile duct ligation, they measured alterations in platelet adhesion by the
Borgchevrik method and bleeding time inhibiting NO synthesis with Nitro Arginin Methyl
Esther (NAME) (Albornoz 1999). Thromboxane A2 secreted in the liver by the Kupfer cell is
also a possible factor that increases Liver resistance, helps to increase portal pressure and has
properties to increase platelet functions. Rats with portal hypertension, produced by bile duct
ligation, have an increased liver Thromboxane A2 (Yokohama 2003).
Figure 3. Venous circulation of the esophageal junction. Small vertical tubes represent the pressure
measured in each zone. Letters a: gastric zone, b: palisade zone, c: perforating zone and d: truncal zone.
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Lemberg, A. Systemic baroreflex alterations in prehepatic portal hypertensive conscious
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mechanisms of portal hypertension. Relative contributions in the rat model of portal vein
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Perazzo, J; Lemberg, A; Norepinephrine uptake modifications in circumventricular
organs, pons and myelencephalic areas and nuclei in prehepatic portal hypertensive rats,
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Minami T. Bleeding in portal hypertensive gastropathy evaluated in terms of gastric mucosal
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Chapter 13
PATHOPHYSIOLOGY
OF PORTAL HYPERTENSION
Salvador Romay, Leandro Steinberg and Francisco Eizayaga
ABSTRACT
Portal hypertension (PH) can be defined as a syndrome whose features are an
elevated blood pressure in portal vein and the development of an extra amount of porto-
caval shunts, called collateral circulation. These changes result from chronic liver disease
in most cases, nevertheless; other pathologies are able to trigger this state too.
Many questions have been addressed as, why is there a peripheral vasodilation in this
particular state in which it should be found an opposite characteristic? and, why the
hyperdynamic state triggers a vascular response to vasoactive substances, inducing post-
translational changes in receptors and/or second messengers. Could this be regarded as a
consequence due to a combination of factors, or there is an specific and unique pathway?
Many of these intriguing mechanisms and others are discussed and in some way
answered in this chapter. Although, there are a huge issues with no comprehensive
answer, this chapter focuses upon how PH is developed and how in some cases becomes
the leader symptom to be treated.
INTRODUCTION
Portal hypertension could be defined as a syndrome whose features are an elevated blood
pressure in portal vein and the development of and extra amount of porto-caval shunts called
collateral circulation. These changes are produced by chronic liver disease in most cases,
nevertheless; other pathologies are able to trigger this state too.
Understanding portal hypertension means taking into account all factors involved in such
process, that is:
1. blood and blood flow, (not only portal but also systemic blood flow,

240 Salvador Romay, Leandro Steinberg and Francisco Eizayaga
2. Heart: pathological and adaptive changes in cardiac function play a major role in
development of Portal hypertension. Cardiac function/-Portal system’s cross-talking
includes autonomic nervous system, renin - angiotensin-aldosterone system, atrial
natiruretic peptides and many others,
3. Vascular tone, Vascular tone allows an adequate control and distribution of tisular
blood flow which, in turn, provides a correct affluence to nutrients and oxygen to
tisular environment. The mechanisms involved are by one hand, the
contraction/dilation state balance of vascular smooth muscle and on the other hand
the degree of stiffness/elasticity or compliance of liver. It is helpful to recall the
number of liver vessels. That number decreases because of the oclusive process that
happens in chronic liver disease that is not counteracted by the new vessels formation
induced by vascular factors.
Groszman’s works supporting “Forward flow theory” settled down a milestone that
triggered many questions that will have been answered in the following thirty years.
Some questions like:
 what are the mechanisms and signals that lead the heart to the increase cardiac
output?,
 Is this a simple response to peripheral vasodilation mediated by autonomous nervous
system?, Why does exist peripheral vasodilation in this particular state in which it
should find the opposite feature?, Why gastrointestinal venous blood flow must be
pushed forward through the liver at a cost of developing collateral circulation such as
gastroesophageal varices?,
 Is then, the so called hyperdynamic state, a result of changes in vascular response to
vasoactive substances, changes in their receptors or second messengers, changes at
posttranslational level, or a mixture of these factors?,
 How are the relationships between all vasoactive substances that run through the
vascular system as a whole?
We will try to describe how these challenges have been solved.
CLINICAL TRIALS AND EXPERIMENTAL MODELS AS A BASIS FOR

THE DEVELOPMENT OF KNOWLEDGE IN PORTAL
HYPERTENSION’S PATHOPHYSIOLOGY
Since the early 80’s was well known the effect of vasopressin on splanchnic vascular bed,
and because of its side effects was limited, in part at least, its administration.
Groszmann et al. (Groszmann 1982) demonstrated a sinergistic effect of nitroglycerine in
cirrhotic patients receiving intravenous infusion of vasopressin. So, the co-administration was
useful not only because the sinergic effect but also because decreased the side effects of
vasopressin.
Shortly after, Vorobioff et al. (Vorobioff 1983) demonstrated in a rat model of calibrated
stenosis of Portal vein that there was no difference in portal vein resistance in experimental

Pathophysiology of Portal Hypertension 241
animals compared to sham-operated animals despite a different portal pressure. Thus, it was
most likely that the increased splanchnic blood flow was responsible of the increment in
portal pressure, having given a strong support to “the forward flow theory.”
Bosch et al. (Bosch 1983) found that bile duct ligated rat model mimics almost all the
features of cirrhotic patients including hyperdynamic circulation plus two characteristics that
the portal vein ligated model lacks: liver damage and intrahepatic portal hypertension.
The most unapproachable region to evaluate beyond the portal system is the azygos vein.
These veins drain blood flow from the portal-collateral gastroesophageal shunts. This is a
very important point because this shunting system actually exerts a role in controlling portal
pressure by diverting part of the flow and subsequently diminishing portal pressure. Trying to
evaluate this subject, Bosch et al. (Bosch 1984) [4] observed, in patients with history of
repeated gastroesophageal bleeding episodes, that they exhibited a higher azygos blood flow
than patients who received decompressive surgery as treatment.
Additionally, Vorobioff et al. (Vorobioff 1984) also demonstrated (this time in carbon-
tetrachloride intoxication experimental rat model) no changes in portal venous resistance in
spite of an increased portal pressure This increased portal pressure was promoted by an
increase in splanchnic blood flow, in turn, allowed by splanchnic vasodilation.
At this moment we have arrived to a point where some paradoxical statements need to be
unveiled. If no differences in portal venous resistance were found, how can we explain that
pathological changes in chronic liver disease are able to trigger portal hypertension?
Orrego et al. (Orrego 1981) found in a chronic ethanol oral administration rat model an
increase in intrahepatic pressure compared to controls, and this in turn, significantly correlates
to the amount of hepatocytes surface area. Similar results were found in chronic liver disease
patient´s biopsies. Besides, Orrego et al. (Colman 1983) studied what kind of relationship
exists between heptatocyte volume and portal hypertension by using an in situ animal model.
They perfusated the liver with a solution at different low osmolalities and they found that:
1. Hepatocytes enlargement produces compression of the extracellular compartment

and sinusoidal width
2. Portal perfusion pressure was increased (to maintain a constant flow rate)
3. The increment in portal pressure was additive to infusion of norepinephrine.
These two interesting and elegant experiments gave us some important clues.
a) If narrowing the space of Disse by means of increasing hepatocyte volume could be

sufficient to increase intrahepatic pressure, then, intrahepatic portal pressure and
resistance would be increased too.
b) It seems to be possible that portal hypertension lilkely appears in reversible damage
as occurs in rats’ chronic ethanol intake. Thus, portal hypertension by itself could be
a reversible state too.
Kiel et al. (Kiel 1985) found, by using a blood-perfused in situ rat small intestine
preparation, that, a significantly greater molar concentration of NE was required to achieve
the same proportional increase in intestinal vascular resistance in portal-hypertensive animals.
This new insight, called hyposensitivity or hyporesponsiveness to vaconstrictors (such as
norepinephrine) occurs in splanchnic vascular bed and needs to be explained.

Is this splanchnic bed’s behavior a result of changes in splanchinc vascular

adrenoceptors? Which receptor is involved? Is it also possible that an excess of vasodilating
substances could interfere on the contractile response to norepinephrine?
Jenkins et al. (Jenkins 1985) demonstrated that an experimental beta 2 blocker effectively
reduces portal pressure by a combination of increased splanchnic vascular resistance and
hepatic arterial resistance
Amer et al. (Arner 1985) identified smooth muscle hypertrophy in the portal vein of rats
with portal hypertension induced by partial ligature of portal vein’s hepatic branches. They
also found that the hypertrophic veins had similar Ca2+ sensitivity (in the presence of
calmodulin) but a lower force per cross-sectional area. In their study they concluded that the
low isometric force of hypertrophic veins was associated to a lower rate of cross-bridge
turnover. Moreover, this could be an effect of alterations in the activation mechanisms or in
the intrinsic properties of the contractile system itself.
Fernandez Muñoz et al. (Fernandez 1985) using a Cl4 cirrhotic rats found a
hyperdynamic circulatory state, which seems to be caused by a peripheral vasodilation of
unknown mechanism; speculating that portal-systemic shunting did not seem to be a
necessary condition for the hyperdynamic status at this early stage of the hemodynamic
disturbances.
Korthuis et al. (Korthuis 1985) observed, in portal hypertensive animals, a significant
increase in hind-quarter blood flow, while vascular resistance was significantly reduced.
Indeed, cross perfusion of control hindquarters with portal hypertensive blood resulted in a
38% reduction in hindquarter vascular resistance. This result showed a clue about that
circulating substances (or the lack of such other substances) can reproduce in control animals
the peripheral vasodilatory effect.
Benoit et al. (Benoit1985) defined indirectly the increment in portal pressure, by means
of a mathematical and theoretical method. The participation of both augmented components:
splanchnic blood flow and intra-hepatic resistance corresponds to a 40% and 60% of portal
pressure repectively.
Lee et al. (Lee 1986) demonstrated an excesive chronotropic response to isoprpterenol in
portal hypertensive animals after propranolol withdrawal. In addition, their results also
showed the existence of a transient beta-adrenergic hypersensitivity state (typical rebound
effect of beta blockers) following propranolol withdrawal in normal and portal hypertensive
rats.
Benoit et al. (Benoit 1986) studied the distribution of the increased splanchnic blood flow
in different organs and the regional distribution within them. They observed by histological
evaluation of carbonized nonradioactive 15-microns microspheres allowed for fractionation
of blood flow within the wall (mucosa, submucosa, and muscularis externa) of each organ.
They concluded the microsphere distribution pattern suggested that intramural blood flow
distribution in all organs was not dramatically affected by chronic portal hypertension.
However, Michael et al. (Davis 1985) had previously found, in an experiment with normal
rats, different responses in capillary pressure when venous pressure increases. They had also
found submucosal arterioles constriction but vasodilation in muscularis arterioles.This
simultaneously opposite behavior of both vascular regions in the same organ triggered a
single change (in this case, increased venous pressure) suggesting a highly regulated cross-
talk between both territories.

Merkel et al. (Merkel 1986) demonstrated the utility of nadolol’s administration, in

patients with cirrhosis, to decrease hepatic venous gradient and hepatic blood inflow after one
month of treatment.
Braillon et al. (Braillon 1986) warned about differences in treatment with propranolol to
cirrhotic patients with different degrees of severity of chronic liver failure. By using Child-
Pough’s classification, they observed that in patients of group C but not of group B, the mean
value of azygos blood flow after propranolol remained significantly higher than in group A.
Moreover, the fraction of azygos blood flow to cardiac output decreased in groups A and B
while slightly increased in group C.
Willet et al. (Willet 1986) observed that the high spillover of norepinephrine is reduced
by the administration of clonidine, an alfa 2 blocker, that exerts its action inhibiting
presynaptic norepinephrine release. This suggests that in alcoholic cirrhosis there is a labile
component of hepatic vascular resistance which is partly under sympathetic nervous control
and is thus potentially sensitive to pharmacological control.
Zoli et al. (Zoli 1986) identified in a small group of patients a decreased portal caliber in
patients when they received atenolol (a vasocontracting selective beta 1 blocker) but not when
they received propranolol (a vasocontracting non selective beta blocker) nor isosorbide
dinitrate (a vasodilator), in spite of the blood flow velocity reduction produced by the three
treatments. They speculated that these results could be explained because the changes in
portal blood flow are mainly due to the block of beta 1-receptors. Later it has been
demonstrated that atenolol can reduce hepatic venous gradient but does not improve the
survival in portal hypertensive patients as propranolol does.
In order to explore further mechanisms involved and try to find new possible treatments
to improve the quality of life in patients with portal hypertension, Cummings at (Cummings
1986) deigned a two steps experiment:
a) An in vitro isolated mesenteric veins experiment to prove the high affinity of

ketanserine for 5-HT2 receptors finding no changes in affinity in normal, sham-
operated and portal hypertensive rats
b) An in vivo experiment, finding a reduction in cardiac output, observed only in the
portal hypertensive rats but not in sham-operated rats. This is consistent with venous
dilatation and pooling of blood in the portal venous system. The venous pooling
could be secondary to the blockade of 5-HT2-receptors in the portal venous system.
Furthermore, they proposed that ketanserin should be explored for the treatment of
patients with portal hypertension.
Hadengue et al. (Hadengue 1987) studied the hemodynamic effects in cirrhotic patients
by ketanserin administration (another 5-HT2 blocker) where no changes in cardiac index and
systemic vascular resistance were found. The hepatic venous pressure gradient decreased a
23% after ketanserin administration. Besides, azygos blood flow was also decreased. There is
no doubt about the role of serotoninergic mediators in gastrointestinal hemodynamics, but its
usefulness, even today, still remains to be solved.
Braillon et al. (Braillon 1986), compared two types of liver shunting. They found that the
hyperkinetic circulatory state in rats with portal hypertension and a normal liver might be
related to the presence of portal-systemic shunts but not to portal hypertension per se. This

inference could be compared to an arterio-venous fistula in which deviating the blood flow
straight from arterial to venous circulation, increases both blood flow and cardiac index.
Mastai et al. (Mastai 1987) demonstrated a decreased hepatic blood flow after the
administration of methoxamine (a potent adrenergic agonist) in cirrhotic patients.
Nonetheless, they did not find further decreasing in azygos blood flow following
methoxamine administration to cirrhotic patients pretreated with propranolol.
Almost at the same time, in a smaller and slightly different study on cirrhotic patients,
Valla et al. (Valla 1987) observed an increase of azygos blood flow after the intravenous
adrenaline´s administration. Interestingly, adrenaline exacerbated the decreased azygos blood
flow following propranolol’s administration.
In tetrachloride induced cirrhosis in rats, McLaren et al. (McLaren 1987) concluded:
“there was a variable response to propranolol depending on the histological severity of
disease, the height of portal pressure and degree of shunting. There is a possibility therefore
that a potential may exist for lowering portal pressure by manipulating intrahepatic shunting.”
Intracellular calcium concentration plays a crucial role in many cell functions as
intracellular vesicles transport, and intracellular signaling, as well as the regulation of
vascular tone. Opposed to the idea suggesting that blocking calcium channels could be useful
in the treatment of portal hypertension, Koshy et al. (Koshy 1987) alerted a possible
deleterious effect of nifedipine in portal hypertensive cirrhotic patients. Macmalthuna et al.
(Macmathuna 1987) found similar results.
In a study performed in 12 patients, Westaby et al. (Westaby 1988) concluded that the
combination of vasopressin and nitroglycerin corrected all systemic haemodynamic
disturbances produced by either agent alone (arterial hypotension was produced by nitro
glicerine and higher cardiac output by vasopressin). The combination of both substances also
induced further decrease in portal pressure than either substance alone.
Benoit et al. (Benoit 1986) using a highly specific glucagon antiserum observed
reductions in blood flow in different sections of gastrointestinal tract ranging from 22% up to
27% and a reduction of portal blood flow by 30%. These changes only happened in portal
vein ligated rats, suggesting in such animals that circulating glucagon may exert its action
into that modified vascular state. The results of this study supported the hypothesis that
glucagon mediates a portion of the splanchnic hyperemia associated with chronic portal
hypertension. Indeed, Kleber et al. (Kleber 1988) observed that variceal pressure was not
influenced three to six minutes after somatostatin bolus administration and was slightly
increased during somatostatin infusion. Thus, potential haemostatic benefits of somatostatin
cannot be explained by pressure reductions in the varices. However, Kravetz et al. (Kravetz
1988) found in portal hypertensive rats that the infusion of somatostatin produced significant
reductions in the increased portal venous inflow, reductions in portal pressure, and significant
increases in portal venous resistance. The reduction of portal venous inflow was caused by
splanchnic vasoconstriction.
Hayes et al. (Hayes 1988) mentioned that the high percentage of responders with respect
to a reduction in portal pressure suggests that isosorbide 5 mononitrate may deserve further
assessment as a possible therapeutic agent alone or in combination with other ‘active agents.’
Moreover, they said “it is important to stress that there is continued controversy as to whether
a fall in portal pressure results in a decreased risk of variceal bleeding. Furthermore, any
proposed long term use of nitrates for the management of portal hypertension should be
tempered by the well documented development of tolerance found in cardiological practice.”

At this moment, the knowledge about portal hypertension can be summarized as follows:
a) Two main opposite driving conditions seem to be imprescindible to generate portal

hypertension: passive congestion and splanchnich vasodilation
b) Portal system’s congestion (unless in normal rats) can trigger two opposite actions on
splanchnic bed: submucosal arterioles constriction while muscularis arterioles dilated
with an overall result leaning to vasodilation
c) Increased cardiac output and peripheral vaodilation,
d) An apparently inefficient autonomic nervous system incapable to return to normal (if
normality exists) an excessive vasodilatory state in both peripheral and splanchnic
vascular beds.
e) The opening of preexistent portocaval shunts and the formation of new ones.
In other words there is little blood volume to distribute and too much vascular volume to
contain it. It seems to be logical that the sympathetic tone were activated to try to compensate
such misbalance. To evaluate regional sympathetic activity, and hemodynamics Gaudin et al.
(Gaudin 1991) designed a trial in patients with cirrhosis. They concluded that their results
indicate that both overall and splanchnic sympathetic activities are dependent on altered
hepatic function. Indeed, they also suggested that splanchnic sympathetic nervous activity
could either play a role in the systemic hyperkinetic syndrome or be a consequence of this
hyperkinetic syndrome. Moreover, Henriksen et al. (Henriksen 1992) measured the arterial
norepinephrine level, as an index of overall sympathetic nervous activity. They found that the
concentration of norepinephrine was doubled in cirrhotic patients (3.08 nmol/L vs. 1.36
nmol/L in controls; p < 0.01). Indeed, there was negatively correlated (r = -0.54, p < 0.01)
with estimated central blood volume (mean = 23 ml/kg in patients vs. 27 ml/kg in controls; p
< 0.05). Arranz et al. (Arranz 1995) studied the baroreflex system behavior in partially portal
vein ligated-portal hypertensive conscious rats by means of adrenergic stimulation by
Phenylephrine. Their conclusions were that baroreceptor reflex sensitivity was significantly
decreased. No differences appeared in the mean arterial pressure and in the reflex threshold.
They suggested that portal hypertension induces alterations in baroreflex regulation of arterial
blood pressure.
Battarbee et al. (Battarbee 1989) had previously found similar result using the opposite
mechanism: nitroprusside induced hypotension. Later the same group (Battarbee 1995)
concluded in an experiment to assess the vagal and sympathetic branches of baroreflex that an
increased gain of the parasympathetic (vagal) limb of the cardiac baroreflex was responsible
for attenuated pressor responses to phenylephrine in portal vein- and beta-adrenoceptors
contributed to skeletal muscle vascular hyporesponsiveness to phenylephrine in portal
hypertensive animals. Thus, the altered beta-adrenoceptor function also appears to contribute
to impaired chronotropic and skeletal muscle conductance responses to hypotension.
In conclusion: autonomous nervous system fails to compensate the vasodilatory
state, at least in part, due to a failure in baroreflex arch and the hyporesponsiveness of
vasoconstrictors. How can be explained that behavior? There is some hidden or undiscovered
vasodilator? Is the vasodilatory state triggered by only passive congestion or by an unknown
vasocontracting endogenous substance?
Lautt et al. (Lautt 1985) based on the concept that a reduction of portal flow by occlusion
of the superior mesenteric artery results in rapid increase in hepatic arterial flow, as an intent

to maintain the hepatic flow, they investigated the role of adenosine in the control of hepatic
arterial flow and they suggested that the hepatic arterial buffer response is mediated by local
concentrations of adenosine that are controlled by the rate of washout into portal blood.
PGI2 is produced by mesenteric vascular endothelium and normally has a short half-life
due to rapid hepatic degradation (Tsunoda 1982). Based on that information, Stitzmann et al.
(Stitzmann 1989) performed an experiment using calibrated portal vein stenosis in white
rabbits. and they concluded “Our findings of elevated systemic arterial levels of PGI2 in
portal hypertensive rabbits would be difficult to explain unless a significant amount of PGI2
produced by the splanchnic vascular bed avoids hepatic metabolism by virtue of
portosystemic shunting. In this way, the PGI2 produced in the splanchnic vasculature of
portal hypertensive rabbits could gain access to the systemic circulation. Thus, PGI2 may act
here as a systemic hormone, and thereby mediate much of the mesenteric hyperemia of portal
hypertension.” In other experiment Stitzmann et al. (Stitzmann 1989) observed a higher dose
at 50% (ED50) of maximal superior mesenteric artery resistance, in response to
norepinephrine in portal vein ligated rabbits. They also observed that the blockade of
cyclooxygenase by indomethacin reversed the high ED50 in experimental animals but also in
normal ones, ablating the difference between the groups. They inferred that prostacyclin may
be responsible of those results.
Soriano et al. (Guarner 1992) suggested that the increased urinary excretion of 2,3-dinor-
6-keto-PGF1 alpha observed in cirrhotic patients is not directly related to portal hypertension
itself but to portal blood factors that bypass the liver. Some such factors may be of intestinal
bacterial origin.
Stitzmann et al. (Stitzmann 1993) provided evidence in humans supporting the hypothesis
that PGI2 is elevated in portal hypertension and is related to both the degree of portal venous
obstruction and portal pressure.
Oberti et al. (Oberti 1993) compared normal, portal vein stenosis and secondary biliary
cirrhosis. In normal rats, prostacyclin infusion increased cardiac output by 21% and portal
pressure by 41%. Similar increases were observed in rats with portal vein stenosis,
nevertheless, prostacyclin did not affect cardiac output nor portal pressure in cirrhotic rats.
Indomethacin induced a more marked vasoconstrictive effect in normal rats than in cirrhotic
rats. They stated that prostacyclin plays a role in the hemodynamic alterations in portal
hypertension. Moreover, the hyporeactivity observed in cirrhotic rats suggests that
prostacyclin may play a major role in the circulatory changes of portal hypertension due to
chronic liver disease.
Many endogenous substances were evaluated to determine their role in the genesis of
portal hypertension, but, from the works of Furchgott et al. [46] to the identification of the
synthesis of nitric oxide (NO) by nitric oxide synthase (NOS) made by Vallance, Coller and
Moncada (Vallance 1989) only a few substances have raised such interest on discovering
their role in portal hypertension’s pathophysiology as NO did. Pizcueta et al. (Pizcueta 1992)
observed that NG-monomethyl-L-arginine (L-NMMA) reduced the augmented cardiac output
and PVI, and increased the diminished peripheral and splanchnic vascular resistance in portal
vein ligated rats. Whereas portal pressure was unchanged. These findings indicate that the
chronic hyperdynamic circulatory characteristics following portal vein stenosis can be
attenuated by L-NMMA. Thus, the excessive formation of endogenous NO may be implicated
in the pathogenesis of the haemodynamic disturbances and splanchnic vasodilatation
associated with chronic portal hypertension. Sieber et al. (Sieber 1992) in order to evaluate if

NO is a causative factor in the hyporesponsiveness to endogenous vasopressors in portal

hypertension performed an in vitro experiment and informed that portal hypertension is
accompanied by a significant in vitro hyporeactivity of splanchnic vessels to norepinephrine,
arginine-vasopressin, and potassium chloride, and secretion of nitric oxide in this preparation
seems responsible for this blunted response. Pizcueta et al. (Pizcueta 1992) suggested the
same, but in carbon tetrachloride-cirrhotic induced animals. Besides, Lee et al. (Lee 1993) by
inhibiting NOS by means of N omega-nitro-L-arginine (NNA) reduced portal-systemic
shunting without reducing portal pressure in portal hypertensive rats. They suggested that NO
plays a role in the collateralization of the portal system. Wu et al. (Wu 1993) identified that
cyclooxygenase blockade induces dose-dependent vasoconstriction in normal and portal-
hypertensive animals. Indeed, indomethacin induced further vasoconstriction after LG-nitro-
L-arginine methylester and reduced portal venous pressure in portal-hypertensive animals.
Karatapanis et al. (Karatapanis 1994) observed a significant impairment in vascular function
in aortic rings in this model of portal hypertension and a nitric oxide synthase inhibitor partly
corrected these changes, suggesting that although nitric oxide is likely an important mediator.
Moreover, other factors may also be involved in the pathogenesis of these alterations in
vascular function. Michelsen et al. (Michelsen 1995) observed that the inhibition of NOS by
NG-nitro-L-arginine induced an additional significant increase in the maximal response to
noradrenaline in sham operated as compared to portal hypertensive rats in aortic rings.
Despite the role of NO in portal hypertension was identified, being easily related to both
the hyperdynamic state and the hyposensitivity to vasoconstrictors, the absence of an
adequate explanation about why intrahepatic resistance and portal pressure are hardly
modifiable required additional ways to be explained.
One important step was done by Lopez Talavera et al. (Lopez-Talavera 1995) when
identified that an anti-serum treatment, (the anti-tumor necrosis factor alpha or anti-TNF-α)
induced a significant increase in mean arterial pressure, heart rate, and systemic vascular
resistance. Moreover, a significant decrease in cardiac index, portal pressure and TNF-alpha
levels were also found in comparison with placebo animals. No significant effects were
observed in sham rats.
Fernandez et al. (Fernandez 1995) found that constitutive (NOS) and inducible NO
synthase (iNOS) activities in portal-hypertensive or cirrhotic rats were similar to those
observed in sham-operated rats. Pilette et al. (Pilette 1996) demonstraded that a specific
biosynthesis inhibitor of NO, like, N-omega-nitro-L-arginine (L-NNA) has a dose-dependent
effect in conscious portal hypertensive rats. Gadano et al. (Gadano 1997) observed the
involvement of the endothelial constitutive Ca2+-calmodulin dependent nitric oxide synthase
isoform in the overproduction of nitric oxide in portal hypertension. Lopez Talavera et al.
(Lopez-Talavera 1997) stated that the hyperdinamic state seems to be mediated, at least in
part, by TNF and NO by the activation of protein kinases (PTKs and their signaling pathways.
PTK activity inhibition ameliorates the hyperdynamic abnormalities that characterize animals
with cirrhosis and PHT. Li et al. (Li 1997) found that L-NNA could partially restore
contractile responses to KCl and phenylephrine in portal hypertensive rings. Hori et al.
(Muñoz 1999) found direct evidences of an increased NO synthesis by the superior
mesenteric arterial vascular endothelium of portal vein ligated animals in response to shear
stress. The increased NO output in response to shear stress suggests an adaptive mechanism
developed by the vascular endothelial cells in response to a chronic increase in flow-mediated
shear stress. Muñoz et al. (Muñoz 1999) concluded that the specific role played by TNF-alpha

in the development of the hyperdynamic state of portal hypertension appears to be mainly

mediated through an increased release of nitric oxide and prostacyclin. Maintenance of the
splanchnic hyperemia after long-term TNF-alpha inhibition could be due to a compensatory
release of glucagon. Wiest et al. (Wiest 1999). In summary, in acute portal hypertensive rats
without hyperdynamic splanchnic circulation superior mesentery arteriy (SMA) beds already
express a significant hyporeactivity to methoxamine and increasing flow rates. This result is
at least partly mediated by NO. eNOS-derived NO overproduction in the SMA vascular
endothelium. Moreover, precedes the development of a hyperdynamic splanchnic circulation,
supporting the hypothesis that eNOS upregulation and subsequently increased NO release in
response to the physiological stimulus shear stress are playing a primary role in the
pathogenesis of the hyperdynamic circulation in portal hypertension. Finally, these results
emphasize the etiopathogenic character of an increased vascular NO synthesis initiating
arteriolar vasodilatation in portal hypertension.
Heme oxygenase (HO) catalyzes the conversion of heme into biliverdin, iron, and carbon
monoxide (CO). Two isoforms of HO have been identified: the inducible HO-1 and the
constitutive HO-2. CO, like nitric oxide, is an endogenous vasodilator that could contribute to
modulation of systemic and local vascular tone. Fernandez et al. (Fernandez 1999) identified
hem-oxygenase 1 gene expression in liver cells and splanchnic organs from portal
hypertensive rats, suggesting a possible pathophysiological role of HO-1 in chronic portal
hypertension.
Kamath et al. (Kamath 1999) observed, in isolated perfused liver studies, that portal vein
rings in non-cirrhotic bile duct ligated (BDL) rats showed increased maximal tension in
response to endothelin 1 (ET-1), as well as a shift of the dose-response curve to the left as
compared with sham-operated animals. Removal of the endothelium further increased
contractility and ET-1 increased portal resistance in both sham operated and BDL rats. The
endothelin Type A receptor antagonist BQ 123 lowered the high portal resistance in BDL rats
to levels comparable with sham operated animals. Shah et al. (Shah 1999) demonstrated that
heat-shock protein 90 (Hsp90) exerts aq role in regulation of endothelial nitric oxide synthase
contributing to vascular control in portal hypertension. Yu et al. (Yu 2000) transferred the
neuronal nitric oxide synthase (nNOS) gene to bile duct ligated and carbon tetrachloride
intoxicated animals. The expression of nNOS in each liver cell type, whether from normal or
injured liver, caused increased NO production and inhibited endothelin-1-induced
contractility of perisinusoidal stellate cells. Besides, in 2 different in vivo models of cirrhosis
and portal hypertension, transduction of livers with recombinant adenovirus (Ad.nNOS)
significantly reduced intrahepatic resistance and portal pressure. The data highlight the
feasibility of gene transfer to diseased liver and hepatic cells and demonstrate the potential of
a novel therapy for portal hypertension caused by cirrhosis.
Garcia et al. (Garcia 2001) found that anandamide elicited an increase in both portal
venous flow and pressure, along with a decline in mesenteric vascular resistance.
The reduced responsiveness in PVL was not due to changes in eNOS expression but was
due to an increase in enzyme-specific activity was detected by Iwakiri et al. (Iwakiri 2002).
They suggested a post-translational modification of eNOS. Moreover, the phosphorylation of
eNOS at Ser(1176) was significantly increased by two fold in the PVL group.
Van de Casteele et al. (Van de Casteele 2002) transferred in vivo eNOS gene, a lower
portal pressure was detected in transfected cirrhotic animals. To confirm the hypothesis that
NOS and its product NO are the main responsible of splachnic vasodilation and portal

hypertension they evaluate eNOS and eNOS/iNOS knockout mice. They fail to porve the
hypothesis because lacking eNOs animals when portal hypertension was produced, caused a
significant reduction in peripheral resistance. They concluded that, despite gene deletion of
eNOS, the knockout mice developed hyperdynamic circulation. Compensatory vasodilator
molecule(s) are upregulated in place of NO in the systemic and splanchnic circulation in
portal hypertensive animals.
The later data demand further explanation. NO production plays a major role in portal
hypertension, but why in sipte the lack of NO production still remains triggering peripheral
vasodilation? What does happen into the liver where intrahepatic resistances are hard to be
modified?
Two different types of vascular resistances coexist within the liver. On one hand the
natural progression of chronic liver disease produces changes in architectural histologic
organization in the liver that produce a non-variable resistance. On the other hand, adaptive
changes in intrahepatic vasculature makes less prone to respond to vasoactive agents mainly
NO. This point was solved by Shah et al. (Shah 1999) documenting deficient eNOS activity
that was associated with a several fold increase in binding of eNOS with caveolin. Protein
levels of caveolin-1 were markedly increased in the cirrhotic liver. Shah et al. (Shah 2001)
also demonstrated the regulation of hepatic eNOS by caveolin and calmodulin after bile duct
ligation in rats. They concluded that, in cholestatic portal hypertension, caveolin may
negatively regulate NOS activity in a manner that is reversible by excess.
Van de Casteele et al. (Van de Casteele 2003) aported additional information observing
that in contrast to prehepatic portal hypertension, cirrhotic livers had a decreased endothelial
nitric oxide synthase protein and enhanced endothelin-1 messenger RNA amount. They
hypothesized that a vasodilator/vasoconstrictor imbalance may be further aggravated by the
reduced activity of superoxide dismutase. Decreased activity allows enhanced superoxide
action, which may lead to breakdown of nitric oxide in liver sinusoids. Hendrikson et al.
(Hendrikson 2003) in order to examine the mechanism of the process by which a diminished
endothelial nitric oxide (NO) synthase (eNOS)-derived NO production from the hepatic
vascular endothelium contributes to hepatic vasoconstriction in portal hypertension, tested the
influence of a constitutively active form of eNOS (S1179DeNOS) in both primary and
propagated liver cells in vitro and in the sham and bile duct ligated (BDL) rat liver in vivo.
They indicated that recombinant S1179DeNOS protein functions appropriately in normal
liver cells but evidences dysfunction in the cirrhotic rat liver. Moreover, that caveolin
expression and inhibition in BDL nonparenchymal cells, including hepatic stellate cells, may
account for this dysfunction. Goh et al. (Goh 2006) observed no significant change in either
inducible NOS (iNOS) or neuronal NOS (nNOS) expressions while endothelial NOS (eNOS)
was up-regulated in cirrhotic livers. Concomitantly, caveolin-1, an established down-regulator
of eNOS, was up-regulated. Inducible HO-1 and constitutive HO-2 were found to show
increased expression in cirrhotic livers albeit in different localizations. They concluded “The
elevated expression of HO-1 and HO-2 suggest that CO may compensate in its role as a
vasodilator albeit weakly. It is possible that CO and NO have parallel or coordinated
functions within the liver and may work antagonistically in the pathophysiology of portal
hypertension.” Sing et al. (Singh 2016) tried to localize where caveoline 1 is up-regulated
because a tight relation between hepatic stellate cells and sinusoidal endothelial cells. They
stated that the normal liver caveolin-1 is expressed predominantly in HSCs and SECs but

after liver injury there is upregulation of caveolin-1 in HSCs, but not in SECs. These data
have functional implications for the cells in which caveolin-1 is regulated.
In summary: It has been postulated that portal hypertension is not only produced by
increased liver resistance but by an active increased portal venous inflow in the genesis and
perpetuation of portal hypertension. Thus, important information came out about how that
original passive congestion triggers an opposite active force that strengthens portal blood flow
to pass through the liver, even with the cost of increasing the portal pressure. This active
splanchnic vasodilation ultimately becomes in an allied of passive congestion that helps to
maintain the process. All the studies performed and commented, as many others, in this
chapter have been successful to improve our knowledge about this pathology, in spite that
they have partially failed to find the ultimate mechanism involved, perhaps because such
mechanisms are chronic liver diseases themselves.
It cannot be denied the role of many factors like the relationship between portal
hypertension pathophysiological steps and chronic liver disease/cirrhosis development, whose
mechanisms are tightly related. Besides, the role of endothelins and fibrogenesis have been
avoided in order to let this topics to be exposed in other chapters.
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Chapter 14
HEPATIC ENCEPHALOPATHY
Silvina Tallis
ABSTRACT
Hepatic Encephalopathy (HE) refers to a wide spectrum of neurological and
psychiatric alterations that may occur in patients with liver disease. Liver failure leads to
the accumulation in brain of neurotoxic substances that are normally metabolized and
eliminated by hepatic or biliary routes. Two neurotoxic substances have been identified
in liver failure, ammonia and manganese. While it is generally held that ammonia plays a
key role, the pathogenic mechanisms involved in HE still remain to be defined. Minimal
HE (MHE) is an early and subclinical state of HE. MHE induced by the portal vein
stricture was associated with moderated hyperammonemia and increased manganese
levels related with a marked increase in portal blood pressure, as well as
histopathological changes in astrocytes, cortical neurons and capillary vessels.
Neurotoxins present in MHE, probably are involved in the cytoskeletal alterations
observed in this MHE model, and they may affect the structural and functional neuronal-
astrocytic relationship. These could be mediated by the Hypoxia Induced Factor 1
stabilization resulting in a focal hypoxic tissue state. There is now a substantial body of
research that strongly suggests whether ammonia and/or manganese induce these
alterations showing a new pathway in MHE pathogenesis.
Consequences of liver failure have the capacity to compromise central nervous system
(CNS) function. The pathogenic mechanisms involved still remain to be defined. While is
generally held that ammonia plays a key role the mechanisms of its neurotoxicity remain
largely unknown.

258 Silvina Tallis
DEFINITION
Hepatic Encephalopathy (HE) is defined as the spectrum of neuropsychiatric
abnormalities seen in patients with liver dysfunctions and/or portal systemic shunting after
exclusion of other known brain diseases (Butterworth and Vaquero, 2009). It includes the
totality of the cerebral alterations which can occur during the course of acute or chronic liver
failure. The neurological and mental symptoms, which as a rule are potentially reversible, can
be develop in different degrees of intensity and in different combinations, so that it is possible
to subdivide HE into several well defined grades of severity or distinct stages (Kuntz and
Kuntz, 2006). Clinical symptomatology ranges from moderate neuropsychiatric disorders to
coma.
CLASSIFICATION OF HEPATIC ENCEPHALOPATHY

A multiaxial classification of HE was adopted to reflect both the types of underlying liver
disease and the characteristics of neurological manifestations, while still recognizing a
common pathogenetic basis (Ferenci et al., 2002). Three major types of HE were
distinguished, Type A, Type B and Type C (Table 1). HE in patients can occur in any of these
ways, which have been recently reclassified by the committee of the International Society for
Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN) (Butterworth et al., 2009).
Table 1. Classification of Hepatic Encephalopathy. Table modified from ISHEN

guidelines (Butterworth et al., 2009)
Type A HE associated with acute liver failure
HE associated with portal-systemic bypass without

Type B
intrinsic hepatocellular disease
HE associated with cirrhosis and development of PH
Type C
portal-systemic shunts
Pathogenesis of Hepatic Encephalopathy
Results of previous studies suggest that the pathogenesis of HE is complex and not yet
fully elucidated due to the multifactoriety of its genesis; while it is generally held that
ammonia plays a key role in the development of the disease (Perazzo et al., 2012). Views
have ranged from systemic factors in addition to ammonia such as manganese toxicity, with
alterations in the permeability of the blood brain barrier (BBB) that may increase the toxic
source trough the brain (Rose et al., 1999). More recently, a role for mitochondrial
dysfunction, oxidative and nitrosative stress as sources of generation of free radicals has been
considered (Norenberg et al., 2004). Impaired bioenergetics with redistribution of cerebral
blood flow (CBF) and disruption in the brain metabolism is present in patient with liver
cirrhosis (James and Garassini, 1971; Kosenko et al., 1996; Rama Rao and Noreberg, 2001).

Hepatic Encephalopathy 259
Liver failure leads to the alteration of several neurotransmitter systems contributing to

determine the neuropsychiatric symptoms of the syndrome (Butterworth and Vaquero, 2009).
There are a large number of possible sites or molecular targets for interference of
neurotransmission by hyperammonemia or other factors associated to liver disease.
Alterations in neurosteroids and in all classical neurotransmitter systems such as
glutamatergic, with overexpression of N-methyl D-aspartate (NMDA) receptors,
serotoninergic, gamma-aminobutyric acid acidergic (GABAergic) and cholinergic have been
reported (Kosenko et al., 2000; Loveza-Thomas, 2004; Felipo, 2008; Butterworth, 2015).
Neuropathology of Hepatic Encephalopathy
Morphological changes to neuroglia, both astrocytes and microglia, occur in HE

consisting of cytotoxic brain edema, astrocyte swelling in acute liver failure and Alzheimer
Type II astrocytosis in cirrhosis (Butterworth, 2015). Examination of material from patients
with end stage acute liver failure reveals signs of severe cytotoxic brain edema and the
primarily cell involve is the astrocyte showing massive swelling of the astrocytic end foot
(Kato et al., 1992). In contrast, the chronic form is an astrocytic alteration known as
“Alzheimer Type II astrocytosis”; a phenotype characterized by swelling of both, the cell and
nucleus, pale nucleus, prominent nucleolus and margination of chromatin (Norenberg, 1977).
These changes suggest that normal key astrocytic functions, such as ammonia metabolism,
uptake and metabolism of amino acid neurotransmitters, are impaired (Norenberg, 1981).
Since brain is devoid of an effective urea cycle, ammonia removal depends almost entirely on
glutamine formation via glutamine synthetase, an enzyme with a predominantly astrocytic
localization. Glutamine concentrations are increased in cerebrospinal fluid (CSF) and brain of
experimental humans and animals with liver failure. While glutamine is not neurotoxic, its
accumulation in astrocytes has been linked to astrocyte swelling and related with the
hypothesis that HE is a primary gliopathy (Norenberg, 1992).
Clinical Stages of Hepatic Encephalopathy
The main neurologic alterations in HE may affect cognitive and motor function and
circadian rhytms. In the worse cases, HE may lead to loss of consciousness, coma and death.
Cognitive and intellectual impairment in HE may affect attention, memory, learning and
related processes. Patients with HE may include impairment of motor coordination,
hypokinesia and bradykinesia. Alterations in sleep-waking are also often present (Felipo,
2008). Chronic HE evolves slowly; early symptoms include altered sleep patterns and
personality changes, followed by shortened attention span and asterixis progressing through
stupor to coma as the severity of the liver disease progresses (Kumar et al., 2010). The early
and subclinical states of HE are known as Minimal HE (Figure 1). Patients with liver
cirrhosis and Minimal HE show mild cognitive impairment and spatial learning dysfunction.
Hyperammonemia acts synergistically with inflammation to induce cognitive impairment in
MHE. Hyperammonemia induced neuroinflammation in hippocampus could contribute to
spatial learning impairment in MHE (Hernández-Rabaza et al., 2016).

260 Silvina Tallis
Figure 1. Stages of Hepatic Encephalopathy.
Minimal Hepatic Encephalopathy
Minimal HE (MHE) is an entity which was first described in the early 1970s in patients
with portal systemic shunts. The term MHE was endorsed to identify patients with brain
dysfunction that do not have recognizable signs on clinical examination; the subtle
abnormalities present in MHE are detected only by using specific neuropsychometric and/or

neurophysiological tools (Quero and Schalm, 1996; Ferenci et al., 2002). There is no
consensus, however, on which specific tests should be used to make the diagnosis. The
current recommendation is to apply short diagnostic batteries of neuropsychological tests, that
measure concentration, motor speed, and visuospatial control, adapted to the cultural
characteristics of the population being evaluated as age, education and repetitive testing
(Weissenborn et al., 2001). One of these tests, which is being increasingly used in different
countries, is the psychometric HE score (PHES) derived from subtests of the Wechsler Adult
Intelligence Scale (WAIS) (Figure 2). There are a total of 5 parts. 1. Number connection test
A (NCT-A): The patient is instructed to join numbered circles in order on a piece of paper.
The time required to complete the task is scored. 2. Number connection test B (NCT-B): The
patient is instructed to join numbered circles and alphabets, e.g., 1, A, 2, B and so on. 3. Digit
symbol test (DST): The patient is asked to learn a code in which a digit is represented by a
symbol. He has to reproduce the symbol corresponding to the digit. 4. Serial dotting test: The
patient is asked to dot the centre of circles on a piece of paper. 5. Trail tracing test (line
drawing): The patient is asked to trace a path of 5 mm wide as fast as possible without
touching the borders.
Figure 2. Psychometric Hepatic Encephalopathy Score. Figure from “The Neuropsychology Center”.
The prevalence of MHE has been reported to vary between 30% to 84% in patients with
liver cirrhosis. This large variation is due to differences in diagnostic methods, patients

262 Silvina Tallis
studied, and definitions of MHE used in various studies (Quero and Schalm, 1996). The
diagnosis of MHE have a clinical importance, as its presence has a negative effect on the
quality of life, capacity to perform manual labor or exhibit cognitive complaints, including
impaired psychomotor performance, decreased attention and poor memory (Quero et al.,
1995; Groeneweg et al., 1998; Prasad et al., 2007). A decline cognitive function increases the
risk of automobile accidents. For this reason, based on neuropsychological tests, it was
estimated that about half the patients with cirrhosis are unfit to drive a car (Schomerus et al.,
1981). Cirrhotic patients with MHE develop episodes of overt HE more frequently than those
without MHE (Yen and Liaw, 1990; Hartmann et al., 2000; Romero-Gomez et al., 2007).
Significantly, 72% of patients with abnormal psychometric test examinations develop overt
HE while only 21% of patients without abnormalities do. In general, HE, even in its minimal
expression, represents an index of bad prognosis and liver transplantation should be
considered (Bustamante et al., 1999). MHE could be a marker of advanced liver failure
because it is associated with a shorter survival time, especially among patients with high
concentration of plasma ammonia. However, is important to note that the decision to proceed
to liver transplantation should not be based only on the presence of MHE. This is an area in
which a correlation between new experimental results and neuropsychological evaluation
need further investigation. Knowledge in MHE is needed in order to find an early
biochemical marker of the subclinical stage.
Role of Ammonia and Manganese in Hepatic Encephalopathy
Liver failure, whether acute or chronic, leads to the accumulation in brain of neurotoxic
substances that are normally metabolized and eliminated by hepatic or biliary routes. Two
such substances have been identified, ammonia and manganese.
Ammonia Neurotoxicity
Depending upon pH, ammonia is present either in its gaseous form (NH3) or in its ionized
form (NH4+), with an ionic radius and properties similar to that of potassium (K+) or a gas,
with free access across cellular and subcellular membranes. Ammonia is an important
substrate for several enzymatic reactions in the brain and is a product of others. Ammonia is
an acid, a base and, at elevated concentrations, is a toxic to both neuronal and astrocyte cells
in the CNS. In aqueous solution, ammonia (NH3) is in equilibrium with the ammonium ion
(NH4+). The ratio NH3/NH4+ is a function of pH as defined by the Henderson Hasselbach
equation:
log10[NH3/NH4+] = pH-pKa
At 37ºC, the pKa of ammonia is 9,15 (Bromberg et al., 1960). Consequently, under
normal physiological conditions, more than 98% of ammonia is present as NH4+. An alkaline
environment favors the non-dissociated NH3 with diffuses or is transported more efficiently
than NH4+ into the tissues. Since diffusion of ammonia into brain is pH dependent, the pH
gradient between blood and brain may affect brain ammonia concentrations. Large quantities

of ammonia normally enter the portal vein circulation from protein digestion in the
gastrointestinal system. However, arterial ammonia levels are maintained at low
concentrations, a finding which illustrates the efficiency with which the liver normally
removes gut derived ammonia. Reduced hepatic capacity for ammonia associated with liver
disease results in increased blood and brain ammonia concentrations. Ammonia enters the
brain from blood by diffusion rather that via saturable transport system (Felipo and
Butterworth, 2002). Metabolic trapping of ammonia is the principal mechanism responsible
for maintaining brain ammonia levels. Since brain is unable to remove ammonia in the form
of urea, brain ammonia is metabolized almost exclusively to glutamine via the glutamine
synthetase (GS) reaction, predominantly in astrocytes because of the enzyme localization.
Glutamine synthesis remains the major route for ammonia removal in brain under both
normal and hyperammonemic conditions (Cooper and Plum, 1987). Growing evidence
suggests that glutamine may mediate some of the deleterious effects of ammonia; Albrecht
and Norenberg (2006) proposed that glutamine is a “stealth” carrier of ammonia. Once
glutamine is synthesized, it is transported into mitochondria via a glutamine carrier and, in the
mitochondrial matrix it is hydrolyzed by glutaminase resulting in glutamate and ammonia
with the concomitant generation of ROS. On account of this proposed mechanism is that
glutamine is known as a Trojan horse (Rama Rao and Norenberg, 2013). It is important to
note that, chronic liver failure results in the accumulation of other neurotoxins such as
manganese (Pomier Layrargues et al., 1995), which may have deleterious effects on brain
function.
Manganese Neurotoxicity
Manganese (Mn2+) is an essential trace metal that is involved in the metabolism of

carbohydrates, lipids and proteins. Moreover, it has an important function as a cofactor of
enzymes and is an integral component of key enzymes such as GS and mitochondrial
superoxide dismutase. However, manganese in excess becomes neurotoxic and causes a CNS
disorder that resembles Parkinson`s disease (manganism). Nevertheless, during the last years
authors have suggested that in addition to ammonia, manganese is also involved in the
mechanism of HE (Krieger et al., 1995; Spahr et al., 1996; Butterworth, 2000). Manganese in
brain is mainly deposited in astrocytes because of the presence of high capacity transporters
in these cells. Manganese brain deposits have been demonstrated in a rat model of cirrhosis
(Rose et al., 1999) and recently high plasma levels of Mn 2+ have been demonstrated in a rat
model of MHE (unpublished results). Brain manganese accumulation is believed to take place
due to elevated blood levels of manganese resulting from impaired elimination of manganese
via biliary excretion, and to increased systemic availability due to portal-systemic shunting
(Spahr et al., 1996; Rose et al., 1999). While mechanisms of manganese neurotoxicity are not
completely understood, oxidative and nitrosative stress have been implicated in its toxicity
(Butterworth, 2010). Manganese is also known to cause mitochondrial dysfunction, which
might be the mechanism for ROS production (Rama Rao et al., 2005). Jayakumar et al.
(2004) have studied the combined effects of ammonia and manganese on astrocytes in culture
and proposed that ammonia plus manganese exert additive/synergetic effects on the induction
free radicals and mitochondrial inner membrane depolarization, which may contribute to the
tissue injury associated with chronic forms of HE.

264 Silvina Tallis
Mitochondrial Dysfunction and Brain Energy Metabolism
Ammonia causes significant alterations of mitochondrial function and consequently,

changes in cerebral energy metabolism (Felipo and Butterworth, 2002). In addition,
hyperammonemia is associated with profound effects on the CBF. These effects are
dependent upon the exposure of hyperammonemia and show region selectivity (Posner and
Plum, 1960). Clinical and histopathological findings hint at regional differences in the brain's
sensitivity to metabolic changes in cirrhosis (Ahl et al., 2004). Another study that assessed
brain function imaging in patients with MHE, showed a reduction in CBF in the hippocampus
and furthermore, these patients often showed abnormalities in the neurological tests which
assessed memory and concentration (Watanabe, 1998). It is known that HE is a condition
where the energy metabolism and CBF are reduced and/or altered but it is still not clear
whether the energy metabolism or the CBF are the limiting factors that induced the cerebral
dysfunction observed in these patients (Gjedde et al., 2010).
Experimental Model of Minimal Hepatic Encephalopathy
As for all pathologic conditions, the use of animal models is of enormous importance.
Thus, studies of the pathogenesis of a wide range of human neuropsychiatric disorders
including neurodegenerative diseases (e.g., Alzheimer disease, Parkinson disease), stroke,
epilepsy and psychiatric disease (e.g., psychosis, depression) have been significantly
enhanced by the use of animal models. However, HE has proven difficult to model since liver
diseases in humans have many varied etiologies (alcoholic, viral, toxic, autoimmune,
ischemic or genetic), which involve varying degrees of portal systemic shunting and damage
to other organs (Butterworth et al., 2009). Surgical models in the rat, as partial Portal Vein
Ligation (PVL) model have been widely used in the study of the pathophysiology of PH
(Vorobioff et al., 1983). This model has been extensively used because the procedure is easy
to perform, inexpensive, reproducible and PH develops very fast. One week after surgery, the
complete portal hypertensive syndrome with splanchnic hyperdynamic circulation and portal
systemic shunting is established with almost normal hepatic features. PVL also affords a
model of MHE (Abraldes et al., 2006). The most relevant characteristics in this model are
PVL leading to an increase in portal pressure, increase in ammonia levels in blood and
cerebral tissue, and increase proteins in CSF. Regarding to neurological alterations, rats with
PVL develop impairments in day-night rhythms and alterations in the electroencephalography
(EEG) (Scorticati et al., 2001). In a recent work it was demonstrated alterations in brain
mitochondrial function at day ten after surgery (Bustamante et al., 2011). Hyperammonemia
without the concomitant liver failure resembles most of the mechanisms present in the brain
of patients with HE (Abraldes et al., 2006).
Changes in CNS Cells in Hyperammonemic Portal Hypertensive Rats
Rats with pre-hepatic portal hypertension because of partial PVL develop MHE with
hyperammonemia, impaired BBB, mild brain edema, and severe mitochondrial changes in the
hippocampus. Structural and functional alterations of neural cells were described in

astrocytes, neurons, and capillary endothelial cells in the brain cortex and hippocampal CA1
area. One important finding in this experimental model was the presence of Alzheimer type II
astrocytes, the hallmark of HE, Alzheimer type II astrocytes (Figure 3). It was specially
searched in the Cx and hippocampal areas and they were present neighboring the pyramidal
layer of the hippocampus.
Figure 3. Pyramidal layer of the hippocampus of MHE animals showing the presence of Alzheimer type
II astrocytes. (Hematoxilin and Eosin original magnification x1000)
Sham operated rats underwent identical surgical procedure of MHE, except for the
portal vein which was exposed and narrowed but not stenosed. In sham rats, GFAP-ir
(immunoreactivity) astrocytes showed a classical appearance, large and thin cellular
processes in both areas. In PVL rats, in hippocampal CA1 and Cx areas, GFAP-ir astrocytes
presented an enlarged cell body with more tortuous and thicker process, a hypertrophy that is
typical of astroglial reaction.
Intracellular S100b-ir was observed only in astroglial cells of both analyzed brain
regions. S100b protein immunostaining labeled the cell body and some of the primary
cytoplasmic projections of astrocytes. In astrocytes of sham rats, S100b protein had a
cytosolic localization limited to the cellular body and the primary or main cytoplasmic
processes. In the PVL rats, S100b-ir had the same distribution pattern, but the intensity of the
intracellular immunostaining was significantly increased in both areas.
Neuronal cell number, counted as NeuN positive cells, was not significantly different in
PVL rats in the hippocampal CA1 area or in Cx area as compared with the sham group.
MAP-2-ir rendered a filament, or fiber like, immunolabeling corresponding to the
neuronal dendritic process. The immunolabeled processes were observed as round stains or
longitudinal tracts, according to whether dendrites were transversally or tangentially
sectioned. As in case of MAP-2-ir, Nf-200-ir also rendered a filament or fiber like
immunolabeling, but in this case corresponding to the neuronal body, axon and dendritic
process. The immunolabeled processes were observed as round stains or longitudinal tracts,
according to whether the fibers were transversally or tangentially sectioned. From a
morphological point of view, some apical dendrites of the pyramidal neurons of the PVL rats
showed a clear waving shape. In the sections immunostained for MAP-2 and Nf-200, the PVL
animals showed fibers (corresponding both to axons and dendrites) with a clear waving and

266 Silvina Tallis
zigzagging shape in the Cx area. This morphological change was observed in particular in the
cortical layer V, but not in the other layers of the cortex. The pyramidal neurons of the PVL
animals, compared with the sham group, showed waving shape dendrites that, taking into
account that the section thickness was of 50 µm and it probably included the whole thickness
of the main dendrites, might be described as a corkscrew-like structure. The apical dendrites
seemed to be the most affected type of dendrites in the pyramidal neurons of the MHE group.
However, this structural alteration could be also observed in the basal dendrites.
Capillaries were labeled with specific antibodies against Nestin. In PVL rats, Nestin-ir
presented modifications of the diameter of blood vessels showing a thicker process in both
areas studied.
Figure 4. GFAP-ir astrocytes of the hippocampal CA1 (a and b) and brain Cx (c and d) areas of Sham (a
and c) and MHE (b and d) animals. (Scale bar = 50 µm).

HIF-1a staining was expressed in the Cx of the PVL animals. To assess whether this
increase corresponds to astrocytes or neurons, a double immunostaining was performed using
S-100b-HIF-1a and NeuN-HIF-1a showing that the expression of the transcription factor HIF-
1a occurred in cortical neurons. As a control, to confirm that the HIF-1a expressed under
these conditions was functional, we studied the expression of two HIF-1a target proteins, P-
gp and Epo-R, in the same area. Both showed an increased label in MHE animals.
Figure 5. S100b-ir astrocytes of the hippocampal CA1 (a and b) and brain Cx (c and d) areas of Sham (a
and c) and MHE (b and d) animals. (Scale bar = 25 µm)

268 Silvina Tallis
It could be speculated that a new pathway could involve NH4+, mimics hypoxia by
occupying the von Hippel Lindau binding domain of the HIF 1 a and thereby preventing its
degradation and causing the stabilization of HIF-1α with the concomitant multidrug
resistance-1 P-gp and the Epo-R over-expression. Because of the presence of Alzheimer type
II astrocytes in MHE animals, it is think that hyperammonemia, three fold higher in PVL rats,
is the main key factor in the model; although other factors may play a secondary role. The
neurotoxics involved in PVL, probably are closely related to the cytoskeletal alterations, and
they may affect the structural and functional neuronal-astrocytic relationship.
Figure 6. Nuclear neuronal marker (NeuN)-ir neurons of the hippocampal CA1 (a and b) and brain Cx
(c and d) areas of Sham (a and c) and MHE (b and d) animals. (Scale bar = 50 µm)

These pathogenic mechanisms could be mediated by the HIF 1 a stabilization resulting in a

hypoxia-like state. The focal hypoxic state is not accompanied with hypoxemia. There is
enough evidence that suggests whether NH4+ induce alterations showing a new pathway in
experimental PVL pathogenesis (Tallis et al., 2014).
Figure 7. Microtubule associated protein-2 (MAP-2)-ir fibers of the cortical layer V of Sham (a) and
MHE (b) animals. The photomicrographs show the neuronal filaments 200 kDa (Nf-200)-ir fibers of the
cortical layer V of Sham (c) and MHE (d) animals. (Scale bar = 25 µm)

270 Silvina Tallis
Figure 8. Nestin-ir vessels of the hippocampal CA1 (a and b) and brain Cx (c and d) areas of Sham (a
and c) and MHE (b and d) animals. (Scale bar = 50 µm)

Figure 9. Hypoxia-inducible factor 1 a (HIF 1 a) immunostaining in brain Cx showing the increased

staining of this transcription factor in MHE (b) animals compared with the Sham group (a). Double
immunostaining showing HIF-1 a and nuclear neuronal marker (NeuN) co-expression in the brain Cx
(e) indicating that HIF-1 a abundance was increased in NeuN positive neurons in MHE group. NeuN is
labeled with green (c) while HIF-1 a is labeled with red (d). P-gp immunostaining in brain Cx showing
the increased label in MHE (g) animals compared with the Sham group (f). Erythropoietin receptor
(Epo-R) immunostaining in brain Cx showing the increased label in MHE (i) animals compared with
the Sham group (h). (Scale bar = 50 µm)
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334.

Chapter 15
HEPATIC ENCEPHALOPATHY THERAPY
Sara Beatriz Chao
ABSTRACT
Hepatic encephalopathy is a metabolic neuropsychiatric syndrome that most
commonly occurs in decompensated cirrhosis and that has the potential for full
reversibility with liver transplantation. Within the context of the evolution of acute and
severe liver failure, there are clinical characteristics which are shared with the HE due to
cirrhosis decompensation.
At the moment of making decisions for the treatment of patients suffering from HE,
general practitioners and specialists should be fully aware, in their daily practices, of the
not so complex use and easy application of the grading scales as well as of the context in
which this liver condition is being developed.
In a context of severe liver failure, the HE must be treated at a level of intensive care
so as to define and maintain the feasibility of the liver transplant. On the other hand, in
the context of a chronic liver condition, the beginning of the HE, together with an
inappropriate treatment, leads to the deterioration of the patient quality of life, which
contributes to malnutrition and worsens the performance status at the moment of the
transplant.
In other cases of decompensated liver cirrhosis, serious situations such as variceal
bleeding or infections may trigger a condition of HE. The subsequent neurological
deterioration can complicate the patient’s situation to such an extent that if the diagnoses
and the treatment are not implemented in due time, this could cause the patient’s death.
This chapter intends to develop the subject of HE in order to simplify the daily
practice of the general practitioner and, at the same time, to inform about the different
situations that we must face so as to arrive at the right treatment considering the context
of the liver disease in which it can be developed and, finally, to make use of all the health
systems resources in the best possible way.

276 Sara Beatriz Chao
Definition
Hepatic encephalopathy is a metabolic neuropsychiatric syndrome that most commonly

occurs in decompensated cirrhosis and that has the potential for full reversibility with liver
transplantation [1]. Within the context of the evolution of acute and severe liver failure, there
are clinical characteristics which are shared with the HE due to cirrhosis decompensation.
However, there are also important differences in the evolution and prognosis of the patient
because of the efforts we must make to reverse the condition in both cases.
Hepatic Encephalopathy Grades
Clinical features range from clinically imperceptible symptoms in minimal hepatic

encephalopathy which require neuropsychometric testing to identify, to a comatose state in
the worst cases. The Working Party for Hepatic Encephalopathy established nomenclature for
HE in 1998. Type A hepatic encephalopathy refers to HE secondary to Acute Liver Failure,
Type B refers to enteric hyperammonemia (without liver disease), and Type C is associated
with chronic liver disease. The severity of HE is graded using the West Haven Criteria (Grade
I-IV), but alternative terminology has been suggested and gained some traction. In the new
lexicon called SONIC (Spectrum of Neuro-cognitive Impairment in Cirrhosis) covert hepatic
encephalopathy (CHE) includes minimal and Grade I HE, and overt hepatic encephalopathy
(OHE) encompasses Grade II-IV HE (Table 1) [2].
Table 1. Hepatic Encephalopathy Grades
Grade Impairment SONIC

0 normal normal normal
MHE Normal Examination findings, Minor abnormalities of COVERT(CHE)
Subtle changes in work or visual perception or on
driving psychometric or number
tests.
1 Personality changes, attention Tremor and incoordination
deficits, irritability, depressed
state.
2 Changes in sleep-wake cycle, Asterixis, ataxic gait
lethargy, mood and behavioral speech abnormalities OVERT(OHE)
changes, cognitive dysfunction
3 Altered level of consciousness Muscular rigidity,
(somnolence) confusion, nystagmus, clonus,
disorientation, and amnesia Babinski sign,
hyporeflexia
4 Stupor and coma Oculocephalic reflex,
unresponsiveness to
noxious stimuli

Hepatic Encephalopathy Therapy 277
Mechanisms of Development of the HE
Among the many factors involved in the development of the HE, there are
neurotransmission disorders in the central nervous system and in the neuromuscular system,
which could lead to impairment of fine motor coordination and to alterations in sleep patterns
and cognition. Besides, in most cases, only minor morphological changes were found in the
brain; these are astrocyte swelling and Alzheimer type II astrocyte.
There are many neurotoxic substances and among them related to HE, ammonia and
manganese. It is well known that hyperammonemia, astrocyte changes and impairment in
neurotransmission could lead to HE. Patients with liver injury concomitantly decrease the
capability of detoxification of ammonia to urea, shifting this metabolic pathway to the
muscular system and to the astrocyte in the central nervous system. In ALF, the progression
of HE is associated with an increased risk of brain edema that could lead to brain herniation, a
major cause of death. Therefore it is clear that when the ALF is complicated by HE, the risk
to life is increased. The severity of HE is also associated with difference in survival.
Manganese (Mn) is neurotoxic, particularly affecting the actions of certain proteins (i.e.,
receptors) that interact with the neurotransmitter dopamine, probably via striatal dopamine
depletion, N-methyl-D-aspartate (NMDA) excitotoxicity or oxidative/nitrosative stress.
Moreover, three basic Mn cellular neurotoxicity mechanisms can be described; 1-mito-
chondrial dysfunction and disruption of energy metabolism; 2- the inflammatory activation of
the glia; and 3- disruption of the synaptic transmission and neuronal-glial communication.
Furthermore, manganese has pro oxidant activity and direct toxic effects have been observed
in dopaminergic neurons. Manganese induces a decrease in the content of peroxidase and
catalase in the substantia nigra. This metal produces active oxygen species, i.e., superoxide
hydrogen peroxide and hydroxy radical, and also produces 6-hydroxydopamine or other toxic
catecholamines.
Cerebral edema is a response to injuries such as stroke and HE and it is well known that
the initial step involves the swelling of astrocytes. The main determinant molecule involved
in astrocyte swelling, at least triggering this pathological condition, is ammonia. Astrocyte’s
glutamine synthetase (GS) plays a detoxifying role of ammonia, by amidation of glutamate
(GLU) to glutamine (GLN). In hyperammonemic conditions, GLN is increased in the
astrocyte and astrocyte swelling occurs. Glutamime could have a relevant role in oxidative
stress/nitrosative as a critical factor in ammonia-induced cell injury. Astrocytes are important
components of the BBB. Any change in astrocytes is also a potential change in the integrity
of the BBB. Besides this, three major causes of astrocyte swelling are considered: 1-cellular
edema 2-vasogenic edema; and 3-aquaporins (AQP) [3].
Treatment
For the treatment of hepatic encephalopathy (HE), it is important to consider the clinical
situation where the symptoms occur fore, the treatment of HE can be addressed from three
different places of the liver disease: 1) treatment of encephalopathy in acute liver failure
(prevention and management of cerebral edema), 2) treatment of acute encephalopathy in
cirrhosis and 3) treatment of chronic encephalopathy in cirrhosis.

Case Report
A 28 year old woman with a background of epilepsies and under a treatment with
phenobarbital during the last two years. When she came for a consultation to the ER she
presented a condition of nausea, vomits and jaundice. In the lab test, she showed a high level
of hepatic enzymes, hyperbilirubinemia, and coagulopathy. Two days later, she had confusion
and gross disorientation.
Which could be your procedure? Would you decide to observe the patient’s evolution
during the shift or define her hospitalization for her study and treatment? Do you consider this
is an episode of worsening of her illness or it is another problem? Do you think the treatment
she received was the reason of this situation?
1. TREATMENT OF ENCEPHALOPATHY IN ACUTE LIVER FAILURE

With an annual incidence of 2000 cases in the United States, the ALF is a rare condition;
anyway, it determines a mortality rate of 40% in three weeks. Liver transplantation is the only
treatment which has substantially improved the survival of patients with ALF [4]. In a
prospective study of the year 2002, the Acute Liver Failure Study Group of the US showed
that the survival of the patients included in a waiting list was 84%; if the transplant was
possible within the right time (with an average of 3.5 days after list entry). But only 35%
could survive if they had no access to transplant [5]. Conceptually, the ALF forecast depends,
as a whole, on the process that leads to hepatocellular damage and its capacity to recover. In
addition, the development of complications, such as the increase of intracranial pressure (ICP)
and multi-organ failure, overshadows the poor results. Brain herniation and sepsis are the
causes of death in the ALF [4].
The formal recommendations in patients with ALF are hospitalization and monitoring in
an intensive care unit, as well as contact with a transplant center and plan for their referral to
have an early assessment. This determines the urgency for list entry which is essential for the
effectiveness and success of the transplant. If this action is delayed during the time of
progression of encephalopathy to III-IV degrees, the transfer may not be feasible.
The AASLD guides published in 2011 recommended different items to treat patients in
this situation [6].
Prevention and Management of Cerebral Edema in Patients
Despite continuous progress in the management of critical care, the ALF represents a
threat to life with a multisystem failure and an unfavorable prognosis. Unlike cirrhotic
patients, the development of hepatic encephalopathy in patients with acute deterioration in
liver function is often associated with important brain edema and increased intracranial
pressure (ICP). The ICP is still a serious complication and requires a continuous
implementation of studies in critical care due to the high mortality associated with it [7].

1.1. Prevention of Cerebral Edema
Reduction of Cerebral Ammonia

Experimental and clinical evidence of the association of hyperamonemia with the
pathogenesis of cerebral edema is well known; therefore, it is reasonable to look for
treatments that generate a reduction in the availability of ammonium in order to prevent
cerebral edema.
The current pharmacological agents used for the treatment of encephalopathy due to
hepatic cirrhosis have not been formally tested in ALF. From a theoretical perspective, the
colonic debugging from ammonium with non absorbable disaccharides (lactulose and lactitol)
is much lower than the rate at which ammonium is generated in the splanchnic bed and it is
distributed to the systemic circulation, all of which gives low effectiveness in this case. These
agents may also result in colonic distention and complicate the implementation of the
emergency transplant (class III) [8].
Ammonium can also be removed by dialysis. Continuous hemofiltration dialysis is the
therapy for renal replacement in patients with ALF. A large flow of dialysis is the most
effective method to remove ammonium. It is difficult to be performed in patients with
vasodilatation and ALF, which may result in a change of direction of the sharp flow to the
brain [8].
Correction of Hyponatremia
Hyponatremia is frequently observed in patients with ALF, approximately in 1/3 of
patients with ALF at entry. If it is severe, it can cause brain edema by itself and it has been
demonstrated that it enhances the ammonium-induced edema in animal models [8].
Inflammation Control and Treatment of Infections

Inflammation and infection are associated with the presence as well as the progression of
the HE in ALF.
In the absence of positive microbiological results, a state of inflammatory activity may be
suspected due to the presence of systemic inflammatory response (SIRS). Patients in whom
SIRS is detected, have a high prevalence of HE [9].
Positive bacteriological cultures precede the progression of moderate to severe
encephalopathy in approximately 70% of patients with ALF.
As cerebral edema is seen in patients with severe mental status changes, it is logical to
think that in patients with HE grades III-IV should be treated empirically with broad spectrum
antibiotics. However, the capacity to revert the effects of the inflammation/ infections may be
limited in the context of ALF.
The administration of antibiotics as a prophylaxis for infections in early stages of ALF
did not result in an improvement of survival. Comparatively speaking, HE progressed to
similar degrees in patients treated with antibiotic prophylaxis as those treated because it was
necessary.
Prevention of Seizures
Sub-clinical seizures were detected with the use of a continuous electro-encephalogram
in 45% of patients with severe HE. While the administration of phenytoin was allegedly

associated with a low degree of cerebral edema in autopsies, this was ineffective to
considering clinical parameters [8].
The reasons for the development of brain excitability and seizures can occur due to the
increase of cerebral extracellular glutamate in ALF. Cerebral hypoxia generated by the crisis
can also cause cerebral edema. However, the incidence of overt seizures in ALF is relatively
low and it could also be because of the role of other elements such as hydration, renal failure
or medication rather than a primary-expression of HE. Before the suspicion or presence of
seizures, these must be treated quickly with phenytoin. Benzodiazepines can also be used for
brief periods in the case of refractoriness to phenytoin therapy (Class III).
Currently, the prophylaxis with phenytoin cannot be recommended.
1.2. Management of Cerebral Edema
Monitoring of Intracranial Pressure

In patients with encephalopathy III-IV-degree progression, intubation and mechanical
ventilation are mandatory. The drug of choice for sedation is propofol because it can reduce
cerebral blood flow (CBF). Lower to the usual of propofol doses are appropriate because its
half-life is prolonged in patients with liver failure (Class III).
These patients require an intense monitoring and handling of hemodynamic and the
parameters of renal function, as well as of glucose, electrolyte and acid-base status.
The neurological examination to detect signs of elevation of ICP should be frequent. The
size and the pupillary reactivity, decerebration signs and changes in peripheral reflexes are
signs of advanced brain edema (PIC > 30 mm Hg); therefore, those patients require an
invasive method to measure and maintain an ICP that will allow the viability of neurological
functions.
There are basic steps as the position of the head of the bed; this should rise to 30 degrees
to facilitate venous return. Stimuli such as pain and noise must be reduced to the maximum.
Valsalva maneuvers should be avoided because of the intolerance to the endotracheal tube or
cough reflex since they increase the intrathoracic pressure and the ICP.
Images of the brain cannot be used as a tool to monitor the evolution of the situation
owing to the risk involved in the transfer and the effects that can be determined over the ICP.
The role of the ICP monitoring is still controversial as it has been noted in recent
publications. A noninvasive system is not yet available; this could provide information
equivalent to the ICP monitoring (class III).
The main disadvantage of the IPC monitoring is the intracranial hemorrhage associated
with the placement of the measurement system. The estimated frequency of bleeding was
22% in a survey conducted at the beginning of the 1990s. More recently and, according to
data collected by the Acute Liver Failure Group of USA, this complication occurs in 10% of
the cases [10]. This difference is probably due to the replacement of epidural catheters by
subdural or intraparenchymatous ones. On the other hand, the use of ICP monitoring was
associated with an increase in the use of medication to control ICP. This suggested that an
increase of ICP could be sub-diagnosed and sub-treated in the absence of this invasive
monitoring. The correction of the coagulopathy might reduce the bleeding complications [8].

Monitoring of Central Venous Oxygen Saturation

The monitoring of the venous oxygen saturation (SvjO2) complements the monitoring of
ICP. A fall of less than 55% of the SvjO2 may result in an increase of the rate of lactate
/pyruvate, cerebral ischemia and edema aggravation. One of the reasons for the decline of the
SvjO2 may be the decrease in the cerebral perfusion pressure (CPP) due to arterial
hypotension. It is recommended to keep a CPP above 55 mmHg in patients with ALF.
Theoretically, during the infusion of norepinephrine to increase blood pressure, ICP should
decrease by self-regulatory vasoconstriction. However, in patients with ALF, self-regulation
of the CPP is absent and how ICP will be affected cannot be guaranteed [7].
In patients with deep encephalopathy, the management with vasopressors to correct
arterial hypotension or to maintain CPP is rationally performed with the ICP monitoring.
Terlipressin produces vasoconstriction mediated by V1 receptors and has proved to be
effective in the reversal of hypotension resistant to catecholamines and imminent cerebral
hypoperfusion in patients with ALF [11].
Strategies for the Management of Cerebral Edema
Use of Hypertonic Saline and Mannitol

The infusion of hypertonic saline and mannitol are the treatments of choice for the
reduction of ICP. The usefulness of mannitol to correct the increased ICP in patients with
ALF and to improve their survival has been presented in very small series. Its effect is
transitional. The administration of intravenous mannitol in bolus at a dose of 0.5 - 1 g/kg is
recommended as a first-line therapy. This dose can be repeated once or twice as needed if the
plasma osmolarity is less than 320 mOsm/L. Volume overload is a risk in patients with
impaired renal function and the use of dialysis may be needed to remove the excess of fluid
(class IIa)
In patients with ALF and serious HE, a controlled study showed that the induction of
hypernatremia as a prophylaxis for the development of brain edema with hypertonic saline
solution (to an a 145-155 mEq/L plasma sodium) suggested a low incidence of ICP, as
compared with the management under normonatremia conditions. The induction of
hypernatremia is recommended in patients with high risk of cerebral edema and ALF (blood
amoniemia > 150 μmol/L, hepatic encephalopathy degrees III - IV, acute renal failure,
requirement of vasopressors to maintain mean arterial pressure) (class I) [12].
Hyperventilation
Some patients with FHA develop cerebral hyperemia (defined as a SvjO2 >75%); such
condition is associated with cerebral edema. Patients with cerebral edema and cerebral luxury
perfusion have a very poor prognosis. In this context, an attempt to reduce ICP with the
lowering of the CBF and the induction to cerebral vasoconstriction, is recommended.
Hyperventilation induces pre capillary hypocapnic vasoconstriction, which decreases the CBF
and the ICP. However, the decline of the CBF maintained through the reduction of the partial
pressure of CO2 in arterial blood (PCO2), represents a risk due to the onset of hypoxia and
brain swelling. Consequently, hyperventilation with a PCO2 of 25-30 mmHg could be made,
if the rise of the ICP is a threat to the life of the patient and cannot be controlled by other
means. So far, this has not been recommended [7].

Indomethacin
Intravenous indomethacin is a cerebral vasoconstrictor. It decreases IPC and increases the
CBF in patients with ALF. Indomethacin does not compromise cerebral oxygenation or the
concentrations of lactate in the brain and it is currently used as a rescue treatment in the cases
of increased ICP in humans with ALF. Its effects on the gastric mucosa and the renal
circulation preventing its role in prophylaxis [7].
Hypothermia
Hypothermia is a therapy of great interest. It can reduce ICP in patients with ALF. The
effects are rapid and the body temperature of 32-34°C can easily be reached with cold
blankets or with the use of renal replacement therapy. In this way, hypothermic patients
require treatment to cause neuromuscular paralysis so as to avoid tremors due to shivers. The
reduction of ICP can be maintained in the operating room during the emergency transplant.
As mentioned above, due to the loss of brain self-regulation, the use of vasopressors on
ALF leads to an increase in CBF and it may worsen the ICP. Several studies have shown that
hypothermia improves the hyperdynamic circulation, a characteristic hypotension of the ALF;
this is reflected by a reduction in requirements of vasopressor medication. Since it restates the
self-regulation of brain hypothermia, it could facilitate the hemodynamic attention of these
patients.
The adverse effects of the hypothermia, such as coagulopathy, arrhythmias and metabolic
disorders, are observed at temperatures below 32°C. In the ALF, a moderate hypothermia of
33-34°C could be effective. It can be considered in patients with ICP refractory to osmotic
agents as a bridge to liver transplantation (class II3) [10].
Corticoids
Unlike other cerebral pathologies, corticoids have not shown any benefit in the cerebral
edema by ALF and should not be used to control ICP in these cases (class I) [5].
Barbiturates
Barbiturates agents may be considered when the ICP levels do not respond to other
measures. Their administration has proved to be effective to reduce ICP (class II 3) [5].
2. TREATMENT OF ACUTE ENCEPHALOPATHY

IN HEPATIC CIRRHOSIS
Case Report
A 49 year-old patient who came to ER for consultation. He was suffering from a

deterioration of conscience condition. His family informed us that this episode had started
two hours before consultation. Previous to this, he has shown some inclination to sleep. That
was the reason why he had not received the ordinary treatment. At the moment of the
questionare, they declared he had a background of cirrhosis which had been diagnosed 6
months before. And he was going through a treatment with spironolactone, furosemide
lactulose and propranolol.

Which could be your procedure? Would you decide to observe the patient’s evolution
during the shift or define his hospitalization for his study and treatment? Do you consider this
is an episode of worsening of his illness or it is another problem? Do you think the treatment
he received was the reason of this situation?
Decompensated cirrhosis is commonly determined with the appearance of ascites, HE,
bleeding due to esophageal variceal and jaundice, and it is associated with reduced survival.
The average survival of the patients with cirrhosis decreases from > 8 years to about 2 years.
Also, the development of HE has been associated with survival time reduction among patients
with terminal liver disease [13].
The MELD score (Model for End - Stage Liver Disease) is an objective indicator of liver
disease severity. Only laboratory variables are used for this logarithmic calculation (total
bilirubin, plasma creatinine and the international normatized reason (INR). This has been
adopted in most of the countries that perform liver transplant for the procurement and
distribution of organs; it has avoided subjective variables such as ascites and encephalopathy,
and made this system as fair as possible. Since then there have been questions and studies on
whether complications such as refractory ascites, spontaneous bacterial peritonitis could
provide additional information to the forecast. HE is an important issue in the natural history
of cirrhosis which subsequently affects the quality and survival of patients. Different studies
strongly suggest that it could provide additional information to the prognosis regardless of the
MELD [13].
Following this line of thought, the appearance of acute encephalopathy in a patient with
liver disease in a condition of known decompensated cirrhosis or who is suspected of having
the disease in progression, it is important in the first instance to identify, remove and treat the
precipitating factor. If there exists the suspicion that the patient could be having a
gastrointestinal bleeding episode, it is essential to detect bleeding, remove the blood from the
digestive tract and correct uremia as the initial treatment. If the patient also has ascites, the
indication is to perform a diagnostic paracentesis for the physical chemical study, cell count
and ascites fluid cultures in order to detect spontaneous bacterial peritonitis and start
antibiotic therapy. It is also important to rule out other infectious focus as precipitating causes
of HE.
The hydroelectrolyte imbalances such as the hyponatremia (defined as natremia < 125
mEq/L) must be carefully corrected. An increase in the prevalence of HE has been observed
in cirrhotic patients with hyponatremia, an independent finding of portal hypertension. These
patients may have an increased risk of neurological complications in pre and post-transplant
associated with a rapid correction of the serum sodium in the peri-transplant period [15].
Also, it is important to rule out the presence of medication such as psychotropic drugs or
medication with sedative effect such as benzodiazepines, antihistamines or metoclopramide
that could contribute to a situation of HE.
In this type of liver disease, intestinal cleansing still is the key for the treatment to reduce
the production and absorption of ammonium in the colon.
Neomycin is an antibiotic of poor absorption; it has been used to eliminate the bacteria
from the colon. However, several adverse effects have been associated with neomycin; they
involve nervous deafness, renal toxicity, malabsorption and severe alteration of intestinal
flora. Those were the reasons why they were no longer indicated often unless the patient has
intolerance or no response to nonabsorbable disaccharides [16].

Lactulose (1-4 galactoside fructose) is a synthetic nonabsorbable disaccharide that

reduces production and the absorption of ammonium. Lactulose as the treatment of choice for
hepatic encephalopathy has been considered since the 1980s [17].
If the patient can receive the medication orally, the treatment consists of doses of
lactulose or lactitol 30 ml every 1-2 hours until intestinal evacuation. An alternative way are
the lactulon enemas. Then, the treatment must continue with 15 to 45 ml every 6 hours until
2-3 soft stools a day [16].
Although there have been no serious adverse effects, this may not be very well tolerated
due to its sweet taste and gastrointestinal reactions that may not respond to the reduction of
the dose. Lactitol (b-galactoside sorbitol) emerged as a more tolerable alternative.
Two meta-analyses compared lactulose to lactitol for chronic hepatic encephalopathy and
concluded that they were equivalent [18, 19].
In a systematic review, in which the beneficial and harmful effects of disaccharides in HE
patients were evaluated and nonabsorbable disaccharides were compared with antibiotics, the
effectiveness of disaccharides was questioned since not enough high quality evidence was
found to support or refute this treatment. On the other hand, it was found that antibiotics were
apparently superior to nonabsorbable disaccharides to improve hepatic encephalopathy, but it
is not clear whether this difference is clinically important. More research studies are needed
to show that nonabsorbable disaccharides actually have beneficial effects on HE [19].
As regards the dietary treatment, proteins should be removed from the diet for a few days
and manage carbohydrates such as intravenous glucose on the first day. When proteins are
introduced again, it must be in doses of 20 grams per day at the beginning up to the maximum
dose tolerated by the patient [20].
3. TREATMENT OF CHRONIC ENCEPHALOPATHY IN CIRRHOSIS

In patients with chronic encephalopathy without response to dietary treatment and
cathartic such as nonabsorbable disaccharides, the possibility of food transgressions (for
example, a diet excessive in proteins) must be investigated. The possibility of constipation
should also be evaluated.
If nonabsorbable disaccharides are not tolerated or if they are not effective, antibiotics are
the alternative treatment.
Currently, the rifaximin, an antibiotic of wide spectrum with a minimal orally absorption,
seems to be an effective option for the treatment of the HE. In a randomized study at phase
III, rifaximin (550 mg twice per day) reduces the risk in the progression of HE in 57.9%
compared to placebo (p < 0.0001) in patients with cirrhosis and a history of recurrent
episodes (2 or more episodes). Rifaximin also reduced the number of hospitalizations by HE.
The result of this analysis indicates that rifaximin significantly improves the quality of life for
patients with liver cirrhosis and recurrence of HE episodes. [21].
Another study showed that rifaximin significantly decreases venous ammonium
concentrations and this was correlated with the decline of the progression of HE episodes.
Anyway, further studies are still necessary so that rifaximin can be recommended [21].

As regards the diet of these patients, the increase in protein derived from vegetables and
dairy products should be indicated. In patients who do not tolerate the necessary proteins, the
administration of a branched chain amino acids should be considered.
A special situation is that one in which patients that require the placement of TIPS
(intrahepatic portosystemic shunt imaging) for the management of refractory ascites or
variceal bleeding. These could have progression of the encephalopathy as a complication and
if this does not respond to the above mentioned measures, the treatment is the reduction of
light or occlusion of the anastomosis portosystemic [20].
New Treatments
Currently, the HE therapy involves the use of nonabsorbable disaccharides or antibiotics

with poor intestinal absorption. This standard therapy is ineffective in patients with severe
degrees of hepatic failure. Under these circumstances, the concept of holding the liver failure
for some time while correcting the events that precipitated into that situation could help
patients recover from HE to be stabilized until they receive a liver transplant.
Dialysis with extracorporeal albumin (ECAD) that uses the adsorbents molecule
recirculation system (MARS) is a new method of haemodiafiltration where blood is dialyzed
in contact with a solution that contains albumin and through a high flux membrane. The
technique allows the combined removal of toxins linked to albumin and which are soluble in
water. A randomized study for the standard therapy (nonabsorbable disaccharides and/or
antibiotics: neomycin or Metronidazole) with or without ECAD which uses MARS (it was
done once a day during 6 hours until the patient responded with improvement of HE, that is,
with a reduction of at least 2 degrees of encephalopathy since randomization, or withdrawal
from study after receiving a liver transplant, death or suspension of the consent). The purpose
was to assess the efficacy, safety and tolerance to ECAD with the use of MARS. They could,
in this group, observe an early response of the severe HE in terms of improvement with
respect to standard treatment. The role of this treatment has not yet been defined [22].
The variety of symptoms, the different ways it shows itself and the evolutions of HE in
the different stages of the liver disease, make this clinical entity a permanent object of study.
The degrees of HE have been defined by general consensus in different studies. In the
future, this will allow for a better assessment of the efficacy of treatments which will open a
door to the knowledge about how to improve them and to the investigation of new therapies
in this field.
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[5] Ostapowicz G Fontana R, Schiodt PV Larson ADavern TJ, Han SHB, et al. and the US
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[9] Rolando B. Wade J, M Davalos, Wendon J, Philpott-Howard J, Williams R. The
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(2000).vol. 32 No.34 734-739.
[10] Vaquero J, Fontana RJ, Larson AM, Bass NM, DarvenTJ, Shakil AO, et al. and
Complications of intracranial pressure monitoring in patients with acute liver failure
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Comparison of terlipressin and noradrenalin on cerebral perfusion, intracranial pressure
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Update Software Ltd. available in: http://www.update-software.com. Translated from
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[17] Conn HO, Leevy CM, Maddrey WC, JB, Seeff L Rodgers, Vlachevic ZR. Lactulose in
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[19] Camma C, Fiorello F, Tine F, Marchesini G, Fabbri, Pagliaro l. Lactitol in treatment of
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Encephalopathy in Advanced Cirrhosis. Hepatology (2007) 46, N°6: 1853-1862.

Chapter 16
THE ROLE OF SYSTEMIC INFLAMMATION

IN ENCEPHALOPATHY CONSEQUENCE
OF CHRONIC LIVER FAILURE
Ariel R. Marcotegui, Soraya Wilke Saliba,

Nizar Yousif and Bernd L. Fiebich
ABSTRACT
Hepatic Encephalopathy (HE) is one of the most feared complications of liver
failure. In this context, elevated ammonia levels in the central nervous system (CNS) is
known to be a major ethiological factor.
There is a rapidly growing evidence supporting the role of inflammation in
exacerbating the neurological manifestations of both acute and chronic liver failure.
Systemic inflammation is developed after liver injury as a result of the
hyperammonemic condition, triggering many pathways and, lately reported the release of
pro-inflammatory cytokines into the circulation. The consequence is neutrophil
degranulation and release of reactive oxygen species that together with the ammonia
cross the blood-brain barrier.
This chapter is an overview of the inflammatory conditions in CNS as a result of
chronic liver disease. Considering that there are not satisfactory results in HE treatments,
this particular inflammatory condition could add a new and major player in the
developing and treatment of HE.
LIVER FAILURE
The most feared and severe course of liver disease is liver failure which is generally
induced by drugs, toxins or infections (Bernuau, Rueff, and Benhamou 1986). Liver failure
can be classified according to the time of evolution in acute liver failure (ALF), showing a
fast hepatic deterioration or chronic liver failure (CLF) which follows years of liver injury. It
can be the case that some patients with chronic liver disease develop acute on chronic liver
failure (ACLF), where there is an acute deterioration of liver function, either secondary to

290 A.R. Marcotegui, S. Wilke Saliba, N. Yousif, B. L. Fiebich
superimposed liver injury or due to an extra hepatic precipitating factor like an infection
(Jalan et al. 2012). Whichever the cause is, there has to be over an 80% of hepatic functional
capacity lost, before there is a hepatic failure (van de Poll et al. 2007).
ACUTE LIVER FAILURE

The most common cause of ALF is either an accidental or intentional overdose of drugs
such as acetaminophen (paracetamol). This induces liver failure in the first week of the onset
of the symptoms by centrolobulillar necrosis. Other causes such as autoimmune hepatitis and
hepatitis A and B, take longer to develop and have a different organ morphology (Kumar,
Abbar, and Aster 2014)
ALF was formerly known as “fulminant liver failure”. It is defined as a rapid liver illness
associated with alterations in coagulation (normally an INR ≥1.5) and some grade of
encephalopathy. It is caused by massive hepatic necrosis that occurs before 26 weeks of the
initial liver injury in the absence of cirrhosis or pre-existing liver disease (Polson and Lee
2005). It is useful to know the interval between the liver failure and the onset of the
symptoms, since it may provide information about the etiology. Therefore, ALF has been
divided in hyperacute (<7 days), acute (7-21 days) and subacute (>21 days and <26 weeks)
according to the time passed from the liver failure to the onset of the symptoms (Kumar,
Abbar, and Aster 2014).
CHRONIC LIVER FAILURE

Chronic liver disease can be a consequence of many kinds of noxas (drugs, toxins, virus,
etc.) that act in an insidious sub-lethal way and overpass the capacity of regeneration and
defense of the liver. Its morphological changes are heterogeneous cell regeneration,
inflammation and fibrosis.
The perpetuation of the damage and liver reaction produce morphological and functional
abnormalities. The final result is cirrhosis from the morphological point of view and chronic
hepatic failure from a functional one.
There are two basic types of chronic liver damage:
1. Chronic hepatitis, a limited but persistent hepatocellular damage with a progressive

inflammatory reaction that slowly develops into cirrhosis.
2. Cirrhosis: Defined as the presence of regenerating nodules of hepatocytes and
fibrosis of connective tissue depositions between the nodules.
The leading causes of CLF are chronic hepatitis B and C and alcoholic and non-alcoholic
liver disease. It is important to emphasize that patients with well treated hepatitis B or C
infections or autoimmune disease, may not progress to liver failure even though they have an
established liver cirrhosis (Kumar, Abbar, and Aster 2014).

The Role of Systemic Inflammation in Encephalopathy Consequence … 291
ACUTE ON-CHRONIC LIVER DISEASE

ACLF is defined as an acute deterioration in a diagnosed or not chronic liver disease
produced by an exacerbation in a chronic liver condition or a systemic disease that disrupts
the compensated condition of the patient. This is the case of patients with stable advanced
chronic liver disease that develop signs of acute liver failure. It is clinically important since
the mortality of these patients is around 50%. In these patients, the underlying condition is an
established cirrhosis with vascular shunting and collaterals that alter the blood supply and
leave the liver parenchyma exposed to any minor change that alters this fragile state of
“homeostasis,” precipitating the acute phase of the disease (Kumar, Abbar, and Aster 2014).
Hepatic encephalopathy (HE) is an impairment on the central nervous system (CNS) as a
result of complication of ALF and CLF and in many cases leads to death of the patient. The
pathogenesis of HE is multifactorial and one of the fundamental characteristic is the increase
of circulating ammonia by the non-functional liver to eliminate neurotoxins (Parekh and
Balart 2015; Suraweera, Sundaram, and Saab 2016). The hyperammonemia in CNS leads to a
subsequently damage like astrocytes swelling, mitochondrial dysfunction, increasing the
reactive oxygen species (ROS) or production of pro-inflammatory cytokines (Suraweera,
Sundaram, and Saab 2016).
Furthermore, patients with HE have neuropsychiatric manifestations, such as personality
changes, motor-sensory abnormalities or cognition impairment. The stages of HE are
proposed by Harold Conn and collaborators in West-Heaven Criteria, a popular method to
classify the degree of HE (stages 1 to 4). It´s based on changes of neurological findings (e.g.,
asterixis), consciousness, intellectual functions and behavioral changes (Conn 1977; Conn et
al. 1977).
SYSTEMIC INFLAMMATION
Inflammation is a response of the immune system against pathological conditions,
characterized by local vasodilatation, increased capillary permeability and infiltration of
immune cells (Medzhitov 2008; Bettcher and Kramer 2013).
In 1992 the American College of Chest Physicians (ACCP) defined the Systemic
Inflammation Response Syndrome (SIRS) as the presence of two or more of the following
symptoms: 1. Change of the body temperature: more than 38°C (100.4°F) or less than 36°C
(96.8°F); 2. Heart rate of more than 90 beats per minute; 3. Respiratory rate of more than 20
breaths per minute or arterial carbon dioxide tension (PaCO2) of less than 32 mmHg and 4.
Abnormal white blood cell count (>12,000/µL or <4,000/µL or >10% immature forms) (Bone
et al. 1992). This systemic inflammation response is produced by the activation of immune
cells and the consequent release of pro-inflammatory cytokines such as Interleukin-1 (IL-1),
Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-α), as well as vasoactive

substances and production of Reactive Oxygen and Nitrogen Species (RONS) (Medzhitov
2008).
It has been described the injury in hepatocytes like acetaminophen-induced toxicity or
cirrhosis can induce SIRS and moreover, the inflammation aggravates HE (Aldridge, Tranah,
and Shawcross 2015). During hyperammonemia, the gut flora can induce inflammatory
responses in the liver and in the spleen. (More details in the chapter “Molecular insight into
cell damage induced by hyperammonemia”)
In cirrhotic patients, the proinflammatory response against a bacterial infection is
enhanced. It has been shown that circulating levels of proinflammatory cytokines are
significantly higher in septic patients with cirrhosis that in those without it (Byl et al. 1993).
In rats, the Toll Like Receptor 4 (TLR-4) activation by lipopolysaccharide (LPS) induces
higher circulating TNF-α and IL-6 levels (Lechner et al. 1998). This was also seen in isolated
peripheral blood monocytes and mononuclear cells of cirrhotic patients (Devière et al. 1990).
It was also demonstrated that in cirrhotic rats, the LPS product of the bacterial infection,
induces the release of TNF-α and nitrates and the induction of the liver Nitric Oxide Synthase
2 (NOS2) (Heller et al. 2000). This suggests that circulating endotoxin in cirrhosis is
responsible for excessive synthesis and release of nitric oxide by the vasculature and this
increases the nitrate and nitrite plasma levels (Guarner et al. 1993). The increased circulating
Nitric Oxide (NO) binds to superoxides to form Reactive Oxygen Species (ROS), which
mediate damaging effects in the respiratory chain in the mitochondria, precipitating cell
necrosis (Szabo, Romics, and Frendl 2002). In ALF models, the levels of TNF-α (Odeh
2007), Interleukin 1b (IL-1 β) and IL-6 are increase and correlate with the severity of HE.
Furthermore, LPS also induces the increase of endothelin-1, potent vasoconstrictor, in
liver injury and it has been shown
The absence of the inhibitor IL-1 receptor-associated kinase M in the LPS-stimulated
monocytes from cirrhotic patients and the defective production of IL-10 might explain, at
least in part, the increase in the TNF-α production (Tazi et al. 2006; Le Moine et al. 1995).
Another possibility is that the increased cytokine production is mediated by the up regulation
of endothelin-1 production, as seen in a study where the administration of tezosentan, a non-
specific endothelin receptor, prevented the liver injury by decreasing the intrahepatic
neutrophil infiltration (Urbanowicz et al. 2004).
Some of the coagulation factors are synthetized in the liver. Cirrhotic patients with sepsis
present greater abnormalities than the non-cirrhotic ones, since the consumption of the
coagulation factors by sepsis worsens the pathology. Protein C shows the same pattern of
behavior. In terminal cirrhotic patients, this protein is decreased, making this a possible
mechanism underlying the susceptibility of these patients to sepsis.
SYSTEMIC INFLAMMATION IN CLF’S ENCEPHALOPATHY

Different models to induce chronic hyperammonemia have demonstrated to increase the
sensitivity to inflammation resulting in intensification of the HE. Marini and Broussard
(2006) have shown in chronic hyperammonemic mice stronger neurocognitive deficits and
more pronounced and prolonged inflammatory response to LPS (Marini and Broussard 2006).
In the Bile Duct Ligated (BDL) rat model with hyperammonemic diet, a model to study the

acute HE as result of CLF, revealed a strong impairment in motor coordination and activity,
an increase of brain water content and glutamine, following higher values in plasmatic
nitrites/nitrates, TNF-α and IL-6 (Jover et al. 2006). It was also proved that LPS exacerbates
the brain edema present in BDL rats, induces pre-coma and inflammatory response (Wright et
al. 2007) and also nitrosylation of the frontal cortex proteins (Häussinger and Schliess 2008),
supporting the idea that inflammation and ammonia have a synergistic mechanism in the
context of a cirrhotic HE animal.
Moreover, the correlation between ammonium levels, inflammation and HE was shown
to up regulate microglial activation by the increase of the marker ionized calcium-binding
adaptor molecule-1 (Iba-1) in the cerebral cortex from patients with cirrhosis and HE but not
in cirrhotic patients without HE. (Zemtsova et al. 2011). The activation of microglia was also
shown in BDL rats, resulting in an impairment of cognitive and motor function and increase
of the levels of inducible NOS, IL-1β, and prostaglandin E2 (Rodrigo et al. 2010).
In a model of chronic hyperammonemia, the presence of increased TNF-α, C-X-C
chemokine receptor 4 (CXCR4) and Stem cell Derived Factor 1 alpha (SDF-1α) levels in the
hippocampus was described (Merino et al. 2006). This increase could be related to the
remodeling process, since SDF-1α regulates neurodevelopmental processes in the CNS and
neuronal migration besides being a chemokine (Paredes et al. 2006). Other alterations such as
a decreased uptake and increased release of norepinephrine, astrogliosis and an altered Blood
Brain Barrier (BBB) permeability are also present in CLF models
To emphasize the important role of inflammation in CLF’s encephalopathy, studies have
proven the treatment with anti-inflammatory drug can improve the cognitive deficits observed
in HE. Ibuprofen, a nonsteroidal anti-inflammatory drug (NSAID), ameliorates learning
abilities (Cauli et al. 2007), restored cognitive and motor functions (Rodrigo et al. 2010),
normalized cyclooxygenase (COX) and inducible NOS activities but not IL-6 levels (Cauli et
al. 2007) and also decreased microglial activation (Rodrigo et al. 2010).
NEUTROPHILS
Neutrophils are important cells with major innate immune functions. Upon recruitment to
the inflammation site, neutrophils undergo what is called “respiratory burst,” a mechanism
they try to kill pathogens by generating ROS. On the other hand, the produced ROS might
damage the surrounding tissue, thus a prolonged or exaggerated immune response can be
defective rather than effective.
Hyperosmotic and hypoosmotic conditions, such as hyponatremia have been shown to
activate essential intracellular signaling such as the p38 MAPK pathway and induce cell
swelling. The p38 Mitogen Activated Protein Kinase (p38-MAPK) pathway is a well
preserved intracellular signaling pathway, critical osmosensor, and cell volume regulator
(Han et al. 1994).
p38-MAPK is also a potent pathway in neutrophils driving these cells to apoptosis in
response of hypoxia, LPS and stress (Alvarado-Kristensson et al. 2004). In ammonia treated
neutrophils, an increase in phosphorylated p38-MAPK has been found (Shawcross et al.
2008). Interestingly, isoproterenol, which is a p38-MAPK agonist, prevented both, ammonia
and hyponatremia cell swelling, indicating that the activation of this pathway is not the cause
but a consequence of the cell swelling in neutrophils. By these results, Shawcross et al,

showed that ammonia leads to cell swelling that leads to p38-MAPK phosphorylation. In the
same study, they showed that hyperammonemia and hyponatremia act synergistically and
significantly impair neutrophil phagocytosis, possibly by affecting cell volume (Shawcross et
al. 2008). This condition maybe involved in the predisposition to infection and inflammation
in the presence of liver disease.
AMMONIA
Ammonia is delivered to the liver from the large and small intestine through the portal
vein. In the periportal hepatocytes, it is metabolized through the urea cycle and the small
quantities left are transformed into glutamine in the hepatocytes near the central vein.
Besides the liver, ammonia can be metabolized in other tissues such as the kidney and
skeletal muscle. Around 30% of the ammonia generated in the kidney is excreted in the urine.
In the case of liver failure, the kidney has the ability to increase the ammonia excretion up to
70% (Olde Damink, Jalan, and Dejong 2009). The plasmatic ammonia and creatinine
concentrations and the glomerular filtration rate have been shown to be correlated, proving
why renal dysfunction seems to increase the cognitive impairment in patients with CLF and
HE.
In the presence of liver injury, there is an impaired capacity in ammonia detoxification by
this organ. When this occurs, the kidneys, skeletal muscle tissue and also the astrocytes in the
CNS gain importance in the ammonia metabolism. This situation produces changes in these
cells and in the neurotransmitter release. Notably, all these changes are aggravated by the
presence of oxidative stress, inflammation and secondary infections.
It is well known that hyperammonemia induces the elevation of glutamine levels, which
acts not only as an osmolyte but also as a “Trojan horse”, that allows ammonia to enter the
mitochondria. The mitochondrial high levels of ammonia may result in the Mitochondrial
Outer Membrane Permeability (MOMP) mediated by a calcium dependent process. This can
lead to free radical formation and ROS damage (Murthy et al. 2001).
Taken together, the direct brain toxicity involving neurotransmission, the changes in
astrocytes, the BBB integrity alterations and the induction in the synthesis of pro-
inflammatory cytokines; stablished that hyperammonia has a key role in HE and
neuroinflammation..
LACTATE AND MANGANESE

When hyperammonemia is developed in the CNS, the activity of the enzyme a-
ketoglutarate dehydrogenase from the tricarboxylic acid cycle is reduced (Lai and Cooper
1986), producing a reduction in the pyruvate oxidation and accumulation of lactate. This
process increases the lactate synthesis that is correlated with the degree of microglial
activation, pro-inflammatory cytokine production (Jiang, Desjardins, and Butterworth 2009),
and presence of brain edema (Zwingmann et al. 2003) in experimental models of ALF.
Furthermore, studies in cultured microglia showed that the addition of lactate to the media
promoted the release of TNF-α and IL-6 (Andersson, Rönnbäck, and Hansson 2005).

The importance of manganese (Mn) in inflammation and neurotoxicity might be

explained through its interaction with specific proteins (i.e., receptors) that bind with the
neurotransmitter dopamine. The intracellular mechanisms by which it is neurotoxic are: 1.
activation of the glia; 2. mitochondrial dysfunction producing an impairment in the energy
metabolism and 3. disruption of the synaptic transmission and neuronal-glial communication
(Takeda 2003).
Because of the impaired hepatobiliary elimination of manganese increasing its systemic
availability due to portal-systemic shunting, cirrhotic patients show an increase in this metal
deposit in the basal ganglia (Rose et al. 1999), producing a decrease in tissue catalase and
peroxidase. As a consequence, ROS accumulation induces the autoxidation of dopamine and
its ulterior depletion (Takeda 2003). Moreover, apoptosis may play an important role in the
dopaminergic neurotoxicity associated with manganese since this metal can induce the
phosphorylation of the c-JUN and SEK1/MKK4 pathways (Hirata, Adachi, and Kiuchi 1998).
This deposit is evident in most of CLF patients but not in ALF ones and it has consequences
on microglial cytokine production and neuronal cell loss due to the ROS production (Zhang et
al. 2010). It might be speculated that hyperammonemia and the increase in concentration of
manganese, exert a synergistic action in releasing inflammatory mediators in CNS.
ENCEPHALOPATHY AND NEUROINFLAMMATION

Astrocytes are one of the components of the BBB and the most commonly affected cell in
the pathogenesis of HE. Furthermore, together with the microglia they release pro-
inflammatory cytokines such as IL-1, which is known to compromise the integrity of the
BBB through the cyclooxygenase pathway (de Vries et al. 1996). Moreover, this interleukin
mediates through TNF-α and endotelin-1 the increase in the BBB permeability and, as a
consequence, the impairment of its function (Didier et al. 2003). Additionally, it was shown
in cultured astrocytes that IFN-ɤ upregulates the expression of iNOS (Falsig, Latta, and Leist
2004).
It is important to note that not only astrocytes and microglia produce inflammatory
cytokines but also immune cells in the periphery can trigger immune reactions and release IL-
1, TNF-α and IL-6 as a result of CNS inflammation or infection. These cytokines can exert
their effect on the brain by three different routes. A. peripheral tissues, which are innervated
by the peripheral and autonomic nervous system and can send direct signals to the brain via
peripheral nerves; B. brain vasculature that can convey signals through secondary
messengers, such as NO or prostanoids (produced in response to cytokines); and C. cytokines
can directly act at the level of the brain parenchyma after crossing the BBB or after entering
brain areas that lack BBB (Licinio and Wong 1997). Inflammatory cytokines can also inhibit
astrocyte glutamate uptake by a mechanism involving NO (Hu et al. 2000) and may modulate
the ammonia diffusion (Duchini et al. 1996).
Ammonia is also an essential promotor of neuroinflammation. For instance, the addition
of glutamine (Murthy et al. 2001) and ammonia to cultured astrocytes stimulates the
production of ROS. Furthermore, ROS induce an increase in the permeability of the inner
mitochondrial membrane that subsequently lead to the impairment of the respiratory chain
function (Rama Rao, Jayakumar, and Norenberg 2003).

There are contradictions in the definition of neuroinflammation. However, a common

event occurs during neuroinflammation and many authors agree about the activation of
microglial cells. Authors have some differences about it, but there is a general consensus that
due to this process to exist, there is microglial activation. Häussinger et al. demonstrated that
ammonia directly activates rat microglia, however, there has been no increase in the induction
of iNOS, COX-2 and the release of prostaglandins, glutamate, proinflammatory cytokines and
Monocyte Chemoattractant Protein 1 (MCP-1). Furthermore, they also found microglial
activation in the cerebral cortex of ammonia-challenged rats and post mortem brain tissue
from patients with liver cirrhosis and HE, but not from patients who had cirrhosis without HE
(Zemtsova et al. 2011).
BLOOD BRAIN BARRIER INTEGRITY

BBB is an anatomical and functional barrier essential for the normal function of the CNS.
It is formed by the endothelial capillary cells and its basal membrane is surrounded by the
astrocyte end feet and pericytes interposed in some areas. Pericytes are involved in the
maturation and maintenance of the BBB and the integration of the endothelial and astrocyte
function at the neurovascular unit (Armulik et al. 2010).
The most distinctive feature in the endothelial cells forming the BBB are the tight
junctions. This structure is formed by the junctional adhesion molecule (JAM)-1, occluding,
claudins, and three intracellular proteins zonulin-1, -2 and -3 (ZO-1, ZO-2 and ZO-3) that
links the transmembrane proteins to the actin from the cytoskeleton (Skowronska and
Albrecht 2012). Under normal circumstances, these tight junctions restrict the molecular
trafficking through the endothelial cell instead of the paracellular pathway. There are a few
exceptions to this, such as O2, CO2, and some small lipophilic agents. In both ALF and CLF,
pro-inflammatory cytokines such as TNF-α, IL-1 and IL-6 are increased in plasma. These
mediators are increased in HE and induce the formation of NO and prostanoids by the BBB
endothelium. Through this mechanism, the inflammatory effect in the CNS takes place
without the histological damage of the BBB. The tight junction disruption in the BBB
hippocampal capillaries has been proven by electron microscopy in a Minimal Hepatic
Encephalopathy (MHE) model, displaying also extracellular and astrocytic edema (Perazzo et
al. 2012). Additionally, the endothelial cells show pathological changes in their mitochondria
together with decreased NOS activity (Lores-Arnaiz et al. 2005). Furthermore, it has been
suggested that this impairment in the BBB induces microglial reaction such as cytokine
release or modulation of immunomolecules (Jensen, Finsen, and Zimmer 1997). Another
study showed that chronic hyperammonemia is sufficient to induce microglial activation and
the resulting neuroinflammation in HE (Rodrigo et al. 2010).
Astrocytes are located in a sensitive position surrounding the endothelial capillaries thus
any changes emerged from them can be a potential threat to the BBB integrity. Because of
their singular placement, they are involved in vasogenic and cytotoxic edema, as seen in both
ALF and CLF. The control of ion channels is important in the cell volume regulation and this
has consequences in the pathogenesis of the vasogenic edema. Changes in the ion channel
control affect the cellular homeostasis and can eventually promote the accumulation of
intracellular water. One of the most important ion exchangers is the Na-K-Cl cotransporter-1

(NKCC1) that has been shown to have an important role in astrocyte swelling by ammonia
and resultant brain edema (Arumugam R Jayakumar et al. 2008). Importantly, astrocyte
swelling is classified into two categories: cellular edema and vasogenic edema. Additionally,
there are cellular channels such as aquaporins (AQP) that also play an important role in
maintaining the BBB integrity.
AQP are a family of water channels expressed in many organs, including the CNS. Many
isoforms have been identified in different regions of the CNS, but isoforms that seem to be
more associated with brain edema are AQP-1, AQP-4 and AQP-9. In CNS, AQP-4 and AQP-
9 are mainly found in astrocytes, while AQP-1 is in the choroid plexus. AQP-4 has two
different isoforms and both are expressed in the CNS (Amiry-Moghaddam and Ottersen
2003). They are highly expressed in the plasma membrane of astrocytes near to the
subarachnoidal space, ventricles and blood vessels. The repeated expression pattern of AQP
all over the brain suggest its main role in the water transport across the BBB and the
cerebrospinal fluid.
AQP-4 seems to be one of the most important isoforms in the development of brain
edema observed in both ALF and CLF. Furthermore, this aquaporin has been related with the
apoptotic pathway through the activation of caspase 3 and 8 and the stimulation of TNF-α and
IL-1 (Chu et al. 2014). The later cytokine has been proven to impair the BBB integrity
through the cyclooxygenase pathway in the endothelial cells (de Vries et al. 1996).
Alterations in the aminoacid transport have also been proven to be altered in HE (James,
Escourrou, and Fischer 1978).
In animal models of chronic hyperammonemia, once the portal pressure returns to
normal, the integrity of the BBB is reestablished (Eizayaga et al. 2006). Furthermore, in
ACLF animal models, BBB impairment and behavioral changes were observed (Scorticati et
al. 2004). All together, we conclude that BBB is a key structure essential for understanding
the pathophysiology of HE and its relationship with microglial and astrocytic activation and
neuroinflammation.
ASTROCYTES
Astrocytes are the most abundant cells of the CNS and together with the microglia,
oligodendrocytes and ependymal cells, form the glial compartment. They are also the most
affected cells in HE because of having almost exclusively the glutamine synthetase (GS)
enzyme, responsible for ammonia detoxification (Martinez-Hernandez, Bell, and Norenberg
1977). As glial cells, astrocytes are involved in many functions, being some of the more
relevant ones for this matter, the mechanical support to neurons and the relationship with the
capillary endothelium, pericytes and extracellular matrix to constitute the BBB. This
relationship is essential in their ammonium detoxifying activity.
Ammonium crosses the BBB through the astrocyte end-feet. There are many studies
showing the swelling of the astrocyte feet in animal models, cultured astrocytes (M. D.
Norenberg 1988) from ALF patients, and in patients with deficiencies in enzymes of the urea
cycle such as ornithine carbamoyl-transferase, where big amounts of ammonium are produced
(Kendall et al. 1983). The chapter of HE includes a figure, where an astrocyte type II is
present in an experimental model of subclinic HE.

In CLF, the astrocytes adopt a characteristic morphology called Alzheimer type II, where
they show a large swollen nucleus with a prominent nucleolus, chromatin margination and a
significant enlargement of the cytoplasm (M. D. Norenberg 1977). Even though the swelling
is not as significant as in ALF, it may have functional consequences in the cross-talk between
astrocytes and neurons that ultimately impair the cerebral function.
To explain the development of astrocyte swelling, Norenberg and collaborators proposed
the “Trojan horse” hypothesis, by which they explain how the elevated amounts of glutamine
synthesized in the astrocyte due to the hyperammonemia is transported inside the
mitochondria where it is metabolized by the glutaminase to ammonia and glutamate. By this
process, the ammonium accumulated inside the mitochondria may drive to oxidative stress
and astrocyte swelling (Albrecht and Norenberg 2006). Furthermore, in ammonia-induced
astrocyte swelling model, it has shown ammonia increased the phosphorylation of MAP-
kinases: ERK1/2, p38 MAPK (Schliess et al. 2002; A. R. Jayakumar 2006), and JNK (A. R.
Jayakumar 2006). Inhibitors of MAP-Kinases have shown to diminish astrocyte swelling (A.
R. Jayakumar 2006). Furthermore, astrocytes release glutamate and prostanoids due to an up-
regulation of iNOS, providing a complimentary mechanism by which brain edema can be
produced in HE.
MICROGLIA
Microglia are the resident macrophages of the CNS and as part of the innate immunity
are a key path against pathogens that managed to cross the BBB. In their quiescent state they
are ramified accurate sensors of their environment throughout their filopodia (Marty, Dusart,
and Peschanski 1991), being crucial for the homeostatic state maintenance. When activated,
they increase the expression of the calcium-binding adaptor molecule-1 (Iba-1). This protein
produces a reorganization of the cytoskeleton allowing the cell migration and acting as an
actin cross linking adaptor necessaries also for phagocytosis (Sasaki et al. 2001).
Microglial (and astrocyte) activation can be triggered by the rise in intracranial NO levels
and increase of prostaniod release (Schiltz and Sawchenko 2003). This also can be possible,
after the systemic release of pro-inflammatory cytokines such as IL-1 and TNF-α that
activated the endothelial cells of the BBB and increased its permeability. The microglia
activation is considered a parameter of neuroinflammation and many studies are being
conducted to observe their involvement in the development of multiple neurological
disorders.
In HE, microglia came recently into focus since some studies have shown the activation
of microglia in response to ammonia in different models (Rodrigo et al. 2010; Zemtsova et al.
2011). In late stage of HE (stage IV), microglial motility was slight reduced (Rangroo Thrane
et al. 2012) and in longer exposure of ammonia (Zemtsova et al. 2011), microglial
phagocitosis was inhibited (Zemtsova et al. 2011), suggesting the response of microglia was
compromised.

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Chapter 17
HYPERAMMONIA AND SKELETAL MUSCLE
Lisandro Orbea, Ariel R. Marcotegui, Pablo A. Souto,

Juan Skerl and Juan C. Perazzo
ABSTRACT
Sarcopenia is a common feature of cirrhosis. The loss of muscle mass and function
contributes to its morbidity and mortality. Its prevalence in patients with cirrhosis is
estimated to be 40–70%. Hepatic encephalopathy (HE) has similar prevalence, and
moreover there is correlation between both (Schuppan and Afdhal 2008).
Patients with chronic liver injury decrease the capability of ammonia detoxification
to urea; instead, the muscular cells and the astrocytes in the central nervous system
(CNS) become the main ammonia metabolism pathway. Skeletal muscle tissue, due to its
large size, emerges as the main ammonia detoxifying organ (Desjardins, 1999).
Experimental portal vein ligation (PVL) induces minimal hepatic encephalopathy (MHE),
a subclinical state with nearly normal liver. Therefore, it could be considered a starting
point or very close to the early events in skeletal muscle damage.
When skeletal muscle damage starts?
The MHE, PVL induced, could give a clue to early damage of skeletal muscle. The
high-resolution optical microscopy showed that the skeletal muscle triad was dilated with
a convergent structure resembling the streets on a map. This membrane system was
increased in surface, in detriment of the fibrillar structure. The diad is a way for ions and
also for membrane trafficking. The balance of Ca2+ is basically regulated by SERCA,
closely related to the Triad. Therefore, sarcolemma showed an increase in surface,
probably due to edema, with detachment of the fibrillar structures. The edema was
accompanied by an increase in vacuoles and membranes in the subsarcolemal area.
Moreover, focal cell death was documented, and this may be regarded as
myofibrillolysis. Rupture of the nuclear membrane was also documented and it may
suggest that cell death observation could be imminent. The triad (sarcolemma, T tubes
and sarcoplasmic reticulum) was also altered and the dilation of this system and the focal
disruption was evident.
These results disaggregates the fact that it may be no necessary a damaged liver,
perhaps hyperammonemia is the one that triggers skeletal muscle pathology, with or
without liver damage.

308 Lisandro Orbea, Ariel R. Marcotegui, Pablo A. Souto et al.
INTRODUCTION
Sarcopenia is a common feature of cirrhosis, and the loss of muscle mass and function
contributes to its morbidity and mortality. Its prevalence in patients with cirrhosis is estimated
to be 40–70%. Hepatic encephalopathy (HE) has similar prevalence, and moreover there is
correlation between both (Schuppan and Afdhal 2008).
The skeletal muscle damage in cirrhosis is underestimated by physicians although its
prevalence is higher than other gastrointestinal complications. Perhaps the not so well
established criteria for the diagnosis, its poorly understood pathogenesis and, that none of the
proposed treatment options have been well explored in randomized clinical trials, help to this
clinical conduct.
Patients with chronic liver injury concomitantly decrease the capability of ammonia
detoxification to urea; instead, the skeletal muscular cells (SMC) and the astrocytes in the
central nervous system (CNS) become more relevant in the metabolic pathway of ammonia.
In the case of chronic liver failure, SMC and astrocytes turn into very important effectors in
detoxifying ammonia. Skeletal muscle tissue, due to its large size, emerges as the main
ammonia detoxifying organ (Desjardins et al. 1999). Muscular glutamine-synthase becomes
important because hypoproteic diet is a very common procedure in treating patients with liver
failure, although a normal proteic diet may be metabolically more adequate and can be safely
administered to the cirrhotic patient (Córdoba et al. 2004). A hypoproteic diet may decrease
the muscular mass and therefore its ammonia detoxifying ability. On the other hand, recently,
diet supplementation with branched chain amino acids has been shown to decrease minimal
hepatic encephalopathy and to increase muscular mass (Les et al. 2011).
The kidney is an organ capable of synthesizing and degrading ammonia. In normal
conditions, kidneys and liver interact closely to maintain the ammonia homeostasis. They are
ammonia producers and only 30% of the ammonia produced is excreted in urine. In liver
failure and metabolic acidosis, kidneys have the ability to increase ammonia elimination to
70% of its production (Olde Damink, Jalan, and Dejong 2009; Wright et al. 2011). Aquaporin
2 plays an important role in the water regulation. Its expression is increased in the urine of
cirrhotic patients, with a significant increase in patients with ascites, and even higher in
compensated cirrhotic patients (Eggers et al. 2009). The Rh B and C glycoprotein groups
participate in the elimination of ammonia through the kidney (Bishop et al. 2010; Lee et al.
2010). Plasma ammonia concentration has been shown to be related to serum creatinine and
the glomerular filtration rate. Renal dysfunction seems to increase cognitive impairment in
patients with liver cirrhosis and might be implicated in the pathogenesis of hepatic
encephalopathy (Kalaitzakis and Björnsson 2007). The extra-hepatic ammonia metabolism
appears to be the target of novel ways of treatment in chronic liver failure.
In the Model for End-Stage Liver Disease (MELD) sarcopenia becomes a very useful
tool and should be included as a way to assess the nutritional and functional status of cirrhotic
patients. AJ. Montano-Loza et al. evaluated skeletal muscle by computed tomography and
they conclude that sarcopenia is associated with higher prediction of mortality in patients with
cirrhosis, primarily in patients with low MELD scores (Montano-Loza et al. 2015).
In hospitalized cirrhotic patients there is a correlation between protein malnutrition and
sepsis (Merli et al. 2010).

Hyperammonia and Skeletal Muscle 309
From the Montano-Loza point of view, as the muscle mass and malnutrition are not taken
into account in MELD and Child-Pugh scores, there is no possibility to reflect these important
features as parameters in the prognosis of mortality risk in association with the significant
decrease in muscle mass (Montano-Loza 2014). So it could be regarded that sarcopenia and
cirrhosis have a closely related pathogenesis so that simple dietary interventions are
insufficient. Efforts should focus on improving the understanding of the multiple mechanisms
triggered in chronic liver disease and the overlap of the different pathogenesis involved in
order to arrive to the development of more effective therapies. (Sinclair et al. 2016).
When all paths have been activated it is very difficult to assess which of them began the
cascade of events. In this regard, the experimental model of Portal vein ligation (PVL) in rats
(Vorobioff, Bredfeldt, and Groszmann 1983) was used to evaluate the skeletal muscle relating
two basic characteristics of chronic liver disease in its early stages, mild hyperammonemia
and minimal hepatic encephalopathy (MHE).
The structural, ultrastructural and functional alterations observed in skeletal muscle in the
experimental model of induced MHE-PVL, were evaluated 14 days after Portal vein stenosis
(unpublished results). This MHE model, besides pre-hepatic portal hypertension, is associated
with moderated hyperammonemia, increased plasmatic manganese levels and disruption of
the blood-brain barrier, as well as histological changes in astrocytes, cortical neurons and
capillary vessels with tissue damage, similar to a hypoxic state. Although experimental MHE
showed similar changes as those described in hypoxia, Tallis et al. demonstrated that there
were no hypoxemia and/or a hypoxic state (Tallis et al. 2014). These experimental data are of
great importance when skeletal muscle is evaluated.
The MHE is a subclinical condition that can be clearly differentiated from chronic liver
disease, because PVL induces no liver damage, but shares the by-pass of the Urea cycle. So,
the skeletal muscle damage could be regarded as very closely to the starting point. The
classification in type B of HE, was addressed by the International Society for the Study of
Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN) in 2009.
Glutamine synthase (GS) is of great importance in the hyperammonemia condition. When
liver falls in failure and/or it is by-passed because an occlusion or stricture of the Portal vein
GS becomes a key metabolic path. It has been suggested that muscle ammonia uptake is
increased in chronic liver failure and that the subsequent increase in glutamine synthesis
capacity is a major alternative pathway for ammonia detoxification (Lockwood et al. 1979;
Girard and Butterworth 1992). Desjardins et al. in 1999 demonstrated in a porto-cava
anastomosis model that GS was significantly increased and also suggested that increased GS
activities observed could be the result of a post-translational modification of the enzyme
(Desjardins et al. 1999).
Our results showed that the basic biochemical parameters and the optical microscopy of
skeletal muscle had no differences when the MHE group was compared with the sham group.
These results were disappointing, but the fact that the loss of skeletal muscle is nearly
universal in cirrhosis and that adversely affects survival, inducing the development of other
complications, and that negatively affects outcome after liver transplantation decreasing
quality of life, encouraged us to go deeper. There are no effective therapeutic modalities for
sarcopenia associated with cirrhosis, due to its poorly understood pathogenesis. One of the
potential mediators of sarcopenia in cirrhosis is hyperammonemia. Since the liver is the major
responsible for ammonia detoxification, the hepatocellular dysfunction and portosystemic
shunting in cirrhosis induce hyperammonemia. However, a reduction in skeletal muscle

protein synthesis alone is not sufficient to account for continued reduction in muscle mass in
cirrhosis, and an increase in proteolysis is necessary. On the other hand, cirrhosis is a state of
accelerated starvation and the increased muscle catabolism may serve as a source of essential
amino acids for critical cellular functions. When does skeletal muscle damage starts? There is
not yet a comprehensive answer, but to date the loss of skeletal muscle is associated with
advanced stages cirrhotic. The results presented herein may be useful to broaden the
perspective. Thus it is important to say that, for the first time, skeletal muscle is evaluated in a
model with normal liver histology and MHE. These results disaggregates the fact that a
damaged liver may not be necessary to trigger SMC associated damage. Hyperammonemia
“per se” can cause skeletal muscle pathology, with or without liver damage.The high-
resolution optical microscopy (HROM) showed surprising results. The triad of skeletal
muscle was indirectly observed in dark cells as a long and convergent structure resembling
the streets on a map (Figure 1). These membrane systems increased at the expense of the
fibrillar structure. This observation was confirmed by transmission electron microscopy
(ETM).
The diad is a path of ions and membrane trafficking. The primary involved is Ca2+ ions,
and the ratio of the Triad with SERCA is very close. Sarcolemma showed an increase of the
surface, which can be mainly due to edema, and also involved the detachment of fibrillar
structures. Furthermore, an increase of vacuoles and membranes, accompanied by edema with
subsarcolemmal localization was observed. Moreover, the focal cell death was documented,
and this can be considered as myofibrillolysis. The breakdown of the nuclear membrane was
also documented, suggesting that lethal cell damage was initiated. Triad (sarcolema, tubes T
and sarcoplasmic reticulum) also was altered and the expansion of this system with focal
disruption was evident.
Observations with HROM (Figure 1) were corroborated with the ultrastructure (Figure 2).
The ETM showed severe morphological changes in the myofibrils. Consistently with the
HROM findings, the wide range of changes varied from myofibrillar lysis to changes in
sarcolemma along with initial repair stages. The immunohistochemistry (Figure 4) showed an
increased number of striated muscle cells and satellite death cells (TUNEL assay).
The ultrastructural pathological findings could be described as follows:
a) damaged fibers, with derangement of all the muscle bands. Loss of the structural
order in all the bands, bands like Z was reinforced, shortened and disrupted in the
damaged areas;
b) Increased, enlargement and fusion of vacuoles;
c) Triad with pathological changes;
d) Mitochondrial damage, including loss of the matrix density, loss of cristae, swollen,
fusion and membrane rupture into the cytosol;
e) Myofibrillogenesis;
f) Nuclear damage;
g) Early repair stage.

Figure 1. HROM, skeletal muscle Triad was observed in the dark cells as a dilated and convergent
structure that resembles streets in a map. 1000 X.
Figure 2. ETM of SM, The triad (sarcolemma, T tubes and sarcoplasmic reticulum) was altered with
dilation and the focal disruption.
The T tubes (transverse tubules) are deep invaginations of the sarcolemma constituting a
specialized region of the cell membrane of mammalian myocytes, which play a key role in
excitation–contraction coupling, associated with ion fluxes (Orchard, Pásek, and Brette 2009).
However, although cells share many ion flux pathways with normal excitation–contraction
coupling, the role of the T tubes in myocytes pathology has not been comprehensive
understood. The T tubes are the major site of transmembrane Ca2+ flux. It could be expected a
major role in normal Ca2+ balance. Conversely, in the development of the Ca2+ overload the
T tubes induce a release of sarcoplasmic reticulum Ca2+. In hyperammonemia, Ca2+ flux is
altered due to the role played by the NMDA receptors. Felipo et al. demonstrated that
blocking NMDA receptors in astrocyte cell culture, the balance of Ca2+ flux is restituted
avoiding cell damage. So, it could be speculated that the Triad is playing a role, unknown up
to now, in this hyperammonemic experimental condition and in sarcopenia.

It is of interest to relate mitochondrial damage with some pathologies and/or special

conditions as aging. It was demonstrated that advanced age is associated with mitochondrial
skeletal muscle dysfunction (Figueiredo et al. 2008). Moreover, from the correlation between
morphological and biochemical data they suggested that citrate synthase activity measured in
mitochondrial suspensions is a more accurate marker of mitochondrial mass than the total
protein content.
Figure 3. ETM, showing bands like Z reinforced, shortened and disrupted. Increased, enlargement and
fusion of vacuoles. Triad with pathological changes; Mitochondrial damage, including loss of the
matrix density, loss of cristae, swollen, fusion and membrane rupture into the cytosol and
myofibrillogenesis.
Figure 4. IHC, TUNEL assay showed an increased number of skeletal muscle and satellite (brown)
dead cells.
In an experimental model in mice fed with a high fat and sucrose diet, oxidative stress
induced mitochondrial changes in skeletal muscle (Bonnard et al. 2008). Although these
authors find a decreased mitochondrial number in subsarcolemmal and intermyofibrillar, their
results differ from our observations, although it the proposed mechanism is similar.

In the PVL model we find oxidative stress in the MHE group within alterations in the
respiratory chain. These data are very similar to that found in hippocampal area in MHE rats
(Lores-Arnaiz et al. 2005). Zhou et al. suggested that NFκB and AP-1 are important
mediators of redox-responsive gene expression in skeletal muscle, and that at least NFκB is
actively involved in the upregulation of the GPx and CAT in response to oxidative stress that
induce mitochondrial damage (Zhou, Johnson, and Rando 2001). Like these authors, we also
found changes in GPx and CAT. Zhou et al. also describe that GPx and CAT genes revealed
putative binding motifs for NFκB and AP-1, transcriptional regulators that are activated in
response to oxidative stress in different tissues, including skeletal muscle. Moreover
mitochondrial damage was oxidative stress-induced in their model. Thus, despite the diverse
mitochondrial damage, in agreement with many authors we suggest that the oxidative stress
condition developed by hyperammonemia could also induce mitochondrial changes in
skeletal muscle. Further data corroborated, at least partially, that mitochondrial oxidative
stress could trigger cell death. It was observed by HROM and ETM focal fibrillar disruption
and cell death. TUNEL assay and cleaved caspase3 could suggest an apoptotic mitochondrial
pathway, moreover if mitochondrial oxidative stress and respiratory chain alterations were
registered. It is also of interest that, a cell cycle marker as the Proliferating Cell Nuclear
Antigen (PCNA), showed positive activity in the critical Satellite cell area, cells which
activity drive to the muscle repair and cell reposition. Although the isolated presence of
collagen fibers in an area with previous myofibrilillar destruction can be considered as a
beginning of cellular repair, this cannot be fully validated when there is not sight of peri
fibrillar cell mobilization. Anyway this could have been generated by increased activity
registered by satellite cells.
It was decided to go even deeper and study the mitochondrial DNA. The aim to study
DNA was that approximately one fifth of the proteins involved in oxidative phosphorylation
are encoded by the mitochondrial genome (mtDNA). Additionally, this circular genome
encodes 22 mitochondrial-specific transfer RNAs and 2 ribosomal RNA species. The
remainings of the mitochondrial enzyme complexes are encoded in the nuclear genome.
Mutations in both nuclear and mitochondrial genes cause the so-called mitochondrial
myopathies. Diseases that involve the mtDNA show maternal inheritance, since only the
oocyte contributes mitochondria to the embryo. There is a high mutation rate for mtDNA
compared with nuclear DNA. The mitochondrial diseases may be present in young adulthood
and manifest with proximal muscle weakness. The weakness may be accompanied by other
neurologic symptoms, lactic acidosis and cardiomyopathy, so this group of disorders is
sometimes classified as mitochondrial encephalomyopathies. At this point is of great
relevance to recall that hyperammonemia develops with encephalopathy and maybe there is a
myopathy added.
Conflicts may arise between our results and those of other diseases such as mitochondrial
myopathy, encephalopathy, lactic acidosis, and stroke (MELAS).
Besides, the most consistent histopathological finding in the overall pathology of skeletal
muscle are aggregates of abnormal mitochondria, provable only by special techniques. This
occurs in the subsarcolemma in the early stages, but can severely evolve along the fiber.
Since they are also associated with distortion of the myofibrils, the muscle fiber contour
becomes irregular on cross-section, and the descriptive term ragged red fibers has been
applied to them (Suzuki et al. 2016). Electron microscopy shows a greater number of

mitochondria with irregular shapes and contain paracrystalline parking lot inclusions or
alterations in the structure of cristae (Bisceglia et al. 2014).
The study of metabolic close relationship between skeletal muscle and chronic liver
disease needs much more research. The new views of this document may show new
possibilities for understanding the pathophysiology of skeletal muscle in hyperammonemic
conditions.
CONCLUSION
It can be concluded that hyperammonemia plays a major role in the pathophysiology of
skeletal muscle. In summary,
1) It could be speculated that hyperammonemia triggers skeletal muscle pathology, with

or without liver damage.
2) Like in other tissues with moderate experimental hyperammonemia, skeletal muscle
mitochondrias appears to be the target, morpho-functional alterations here presented
supported this point.
3) New mitochondrial changes: loss of the matrix density, loss of cristae, swelling,
fusion and membrane rupture into the cytosol.
4) Oxidative stress is one of the most important ways triggered by hyperammonemia to
induce skeletal muscle pathology.
5) Severe changes of the Triad are described, enlargement especially in the
subsarcolemmal area, displacement of the fibrils and detachment from the
sarcolemma.
6) Satellite cells are conspicuously marked. Are they inducing some kind of tissue
repair?
7) Cell death, myofibrillolysis, nuclear membrane disruption, are described in the
myofibrils.
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pubmed/11728812.

Chapter 18
CELLULAR THERAPIES TO APPROACH SEVERUS

LIVER PATHOLOGIES
Gustavo A. Moviglia
ABSTRACT
The liver is one of the most essential organs for the animal survival. Probably for this
reason, even in superior vertebrates, it is an organ that has an enormous regenerative
capacity that allows it to regenerate itself from a tiny portion of remaining parenchyma
after acute or chronic injuries. Probably, in humans, it has a unique condition that, after
severe injuries sometimes is able to recover all of its functions (regulation of metabolism,
synthesis, storage and redistribution of nutrients, detoxification, as well as immune
modulation).
Because of the above described characteristics, the study of the liver response to
different to external insults as well as its capacity to regenerate after severe parenchyma
reduction provides a unique opportunity to understand the cellular process associated
with the tissue repair process.
In 1931 Higgins and Anderson, in a mouse model of hepatectomy, showed that the
70% ablation of the liver parenchyma was recovered completely after a 2-week period
post resection. So, cellular theraphy emerges as a notable possibility and some significant
results have been achieved using different cellular therapies: traumatic diminishing of its
mass, toxic parenchyma destruction, viral infections, autoimmune diseases, liver fibrosis
etc. Each one demand different cellular strategies that attend not only the actual condition
but also based on its ethiopathogenesis.
Unfortunately, despite the great advances regarding on early diagnosis and new
therapeutic agents, there are a group of diseases (acute intoxications, alcoholic liver
disease, chronic viral hepatitis, non-alcoholic liver disease, cirrhosis and hepatocellular
carcinoma) that remain unresolved and generate important morbidity conditions, with
high mortality rate and an important economic burden.
Since the last decade of the last century, different kind of cellular therapies have
been studied to approach these above mentioned unmet medical needs.
The Liver has a unique condition, after severe injury is able to recover its full
functions (regulation of metabolism, synthesis, storage and redistribution of nutrients,
detoxification, as well as immune modulation).

320 Gustavo A. Moviglia
Because the above described characteristics, the study of the liver response to
different external insults as well as its capacity to regenerate after severe parenchyma
damage provides a unique opportunity to understand the cellular process associated with
the tissue repair process.
Higgins and Anderson in 1931, in a mice model of hepatectomy showed that 70%
ablation of liver parenchyma, was completely restored after 2 weeks post resection. So,
cellular theraphy emerge as a notable possibility, and some significant results has been
achieved using different cellular therapies in: traumatic diminishing of its mass, toxic
parenchyma damage, viral infections, autoimmune diseases, liver fibrosis, etc. Each one
demand different cellular strategy that attend not only the actual condition but also based
on its ethiopathogenesis.
Unfortunately, despite the great advances done regarding on early diagnosis and new
high mortality rate and economic burden.
The liver is one of the most essential organs for the animal survival. Probably for this
reason, even in superior vertebrates, it has an enormous regenerative capacity, allowing a
regeneration from a tiny portion of a remain parenchyma after acute or chronic injuries.
Probably, in humans, it has a unique condition that, after severe injury is able to recover
its full functions (regulation of metabolism, synthesis, storage and redistribution of
nutrients, detoxification, as well as immune modulation) (Saxema 2003).
Because the above described characteristics, the study of the liver response to
different to external insults as well as its capacity to regenerate after severe parenchyma
reduction provides a unique opportunity to understand the cellular process associated
with the tissue repair process (Si-Tayeb 2010, Higgins 1931).
In 1931 Higgins and Anderson, in a mouse model of hepatectomy, showed that the
70% ablation of the liver parenchyma was recovery completely after a 2-week period
after resection (Higgins 1931).
Unfortunately, despite the great advances done regarding on early diagnosis and new
high mortality rate and economic burden.
CELLULAR THERAPY
In 1998 the FDA has defined the concept of cellular therapy: “Somatic cell therapy is the
administration to humans of autologous, allogeneic, or xenogeneic living cells which have
been manipulated or processed ex vivo.” (FDA 1991). In the present this concept has been
adopted for most, if not all, the national regulatory agencies worldwide. From the beginning
the use of cells where considered as medicine but not pharmaceutical products. The main
difference strives in the concept that a pharmaceutical product is characterized for a defined
chemical structure and its mechanism of action depend on it. Opposite, a cellular medicine is
an alive organism that should be identified by biological markers and dynamic physiologic
characteristics in relation with the humoral and physical environment. Also its relationship

Cellular Therapies to Approach Severus Liver Pathologies 321
with other cells as well as the function that is supposed to exert has to be proven. Regardless
this clear definition given by the regulatory authorities has been ignored for the majority of
researchers who has consider that cells are only spare parts to replace or supplement damaged
ones. Probably this false concept started with the first academic recognized cellular therapy:
the bone marrow transplant. This procedure was developed in the decade of the fifties by E.
Donnall Thomas (Appelbaum 2007). He decided to use donor Bone Marrow cells as an
antidote to overcome the bone marrow aplasia occurred after a success ablative radio and
chemotherapy hematologic malignancy treatment. With time what was considered a side
effect, the graft versus the leukemic effect, and its complication, the graft versus host
reaction, became important treatment success predictors in the most aggressive leukemia
cases where complete malignancy ablation could not be achieved (Gürman 2001).
Later on, several researchers found a second side effect, the healthy donor cells may
spontaneously repair damaged cells of the host. In effect, as cited by Darwin Prockop, “In
female patients who received bone marrow transplants from male donors, male cells were
found in the liver as hepatocytes and cholangiocytes, in kidney as tubular epithelial cells, in
lung as epithelial and endothelial cells, in heart as cardiomyocytes, and in brain as neurons
and Purkinje cells” (Prockop 2003).
At the light of the above mentioned concepts we may conclude that the cells, used as
biological medicines, interact with the cells of the target organ as well as the rest of the cell of
the organism, becoming not only actors but also directors of the therapeutic process. During
this interaction process, they produce tissue, appropriate, functional and stable modifications.
CELLULAR THERAPIES OF THE LIVER

As it was previously mentioned, diverse liver pathology as, traumatic diminishing of its
mass, toxic parenchymal injuries, viral infections, autoimmune diseases, liver fibrosis etc.,
could be approached using different cellular therapies. Each one demand different cellular
strategy that attend not only the actual condition but also based on its ethiopathogenesis.
In 1993 Guo et al. (Guo 1993) developed, in animal model of hepatocellular carcinoma
(HCC), an interesting and original tumor vaccine. This approach was based on the HCC
characteristic of elicit poor immune response. This lack of immune recognition was based on
the absence of MHC and co stimulatory molecules in the cell membrane of the tumor cells.
Therefore, they hybridized the hepatoma cells with activated, but not committed, B cells.
After the fusion, the hybrid kept the original tumor antigens as well as the antigen presenting
molecules provided for the B cell part. It was a successful experimental model, never
translated into clinical setting for this pathology.
However, in a recent review, Noveiry et al. (Noveriry 2016) found 12144 references in 7
databases of which 21 controlled studies with 1885 HCC patients in different stages were
included in this systematic review after the primary and secondary screenings. Overall,
patients undergoing specific immunotherapy had significantly higher overall survival than
those in control group (HR = 0.59; 95% CI = 0.47-0.76, P < 0.0001). There was a significant
difference in recurrence-free survival between patients undergoing specific immunotherapy
and patients in control groups. Patients in immunotherapy groups overall had less recurrence

than control group (HR = 0.54; 95% CI = 0.46-0.63, P < 0.00001). Based on these facts they
conclude that “the immunotherapeutic approaches could be beneficiary for the treatment of
patients with HCC.”
More than 100 of these immunotherapy approaches are related with the use of antitumor
Dendritic Cell (DC) Vaccines. In a recent study Sun et al. (Sun 2015) using an autologous DC
vaccine, in a randomized double blinded clinical trial that implicate the treatment of 160
patients (80 patients in each group) were able to reduce the relapsing of the HCC after the
partial hepatectomy from 48,75% in the control group to 17,50% in the treated group after 18
months of follow up. The DC vaccine approach is based on the fact that in the HCC setting,
the liver DC resident cells elicit a T regulatory cell population that induce tumor tolerance.
The Dendritic cell vaccine to elicit specific and powerful immune response have been
used for several authors to induce also a specific treatment for chronic B and C Hepatitis,
associated or not to HCC (Wang 2015, Chen M 2005, Chen W 2009, Luo 2010, Zhou 2013,
Hong 2012, Song 2010).
In 1993, Lohse et al. have used a cellular treatment named T Cell Vaccination to induce
tolerance in a mouse model of Autoimmune Hepatitis (Lohse 1993, Lohse 1998). T-cell
vaccination is an experimental immunotherapy that has been successfully applied in several
animal models of autoimmune disease. The principle of T-cell vaccination takes its cues from
microbial vaccinations: immunization with attenuated pathogens can induce protective
immunity to the pathogenic agent. In this case auto-aggressive organ specific T cells are
isolated from the patient, activated and expanded in vitro. Then they are irradiated and given
back to the patient. Using this methodology Loshe et al. specifically reestablished the Liver
Immune Tolerance without affecting the rest of the patient immune-response (Lohse 1992,
Lohse 1993, Lohse 1998, Cohen 1983). Regardless the experimental preclinical results the
treatment has not yet been translated to clinic. On other hand, a similar approach but using
specific T regulatory cells has been successfully tested in a pilot study of 19 patients (Oo
2013).
The above rich immune pathology associated with the concept that this organ may also
serve as a powerful tolerance inducer organ have built up the concept of Liver as an Immune
Organ (Crispe 2009). This concept associated to the fact that autoimmune reactions are
essential in the repair of organs (Moalem 1999), help to understand the concept of how
chronic inflammation may induce a fibrotic tissue disease (Wick 2010), as is the cirrhosis in a
failure attempt of tissue repair (Robinson 2016, Eckert 2015).
In effect, Moalen et al. in a model of spinal cord and optic nerve damage, has
demonstrated that the acute specific autoimmune response that happens during the first 17
days post injury is essential to promote the repair of the damaged organ, in between many
possible causes, the direct action on the arrived extra tissue Mesenchymal Stroma Cells helps
to induce the specific tissue differentiation of the progenitor cell (Moviglia 2006, Moviglia
2012). After a short lapse, the Th1 immune reaction switch to a Th2 reaction. Th2 cytokines
have been proved to be responsible of the myofibroblast differentiation (Wick 2010,
Robinson 2016, Eckert 2015).
To support this last fact, in a mouse model of hepatic regeneration has been observed that
if the liver ablation is accompanied by the bile duct ligation, after 15 days, the animals
developed liver fibrosis (Zhang 2016). In a second experiment, the addition of an immune
suppressor molecule may significantly reduce the liver fibrosis burden (Chuang 2016).

The modern biology has showed that the liver has two different sources of stem cells to
regenerate its structure: intra hepatics and extra hepatics Stem cells (Kordes 2013, DeLeve
2013, Wang 2012, Harb 2009).
There are two intrahepatic liver stem cell population: hepatic stellate cells and the intra
Hering channel stem cells. The first are localized in Disse space, in between the hepatic
endothelium sinusoidal cells (LESC) and the hepatocytes. The seconds are located in between
the last ramifications of the bile ducts and the first cells of the Hepatocytes walls in the so
called Canals of Hering (Kordes 2013).
The extrahepatic source of stem cells are the Bone Marrow Mesenchymal Stem Cells
(BM MSC). These cells are attracted for cytokines produced for the altered LESC. They
attached on the endothelium surface and penetrate into the endothelium wall and them into
the hepatocyte wall regenerating both structures in parallel.
Based in the above mentioned concepts different centers have used stem cells from
different origins to treat the severe liver failure, associated with drug intoxication, Hepatitis C
and B in terminal stages, cirrhosis and end stage liver failure as an alternative to the liver
transplant. These human trials have been resumed in the review of Tsolaki E and Yannaki E.
(Tsolaki 2015).
The different kind of Stem cells have been administered mainly using the hepatic artery
or the portal vein system (intra splenic or intra portal vein) and less often intra peripheral vein
with a dose range of 80,000,000 to 40,000,000 cells per patient (Khan 2010, Cardinale 2014,
Mohamadnejad 2007, Kharaziha 2009, Amer 2011, Amim 2013, Jang 2014, Peng 2011, El-
Ansary 2012, Zhang 2012, Wang 2013, Terai 2006, Shi 2012, Mohamadnejad 2013,
Baershiger 2009, di Bonzo 2008, Yannaki 2006, Gasbarini 2007, Levicar 2008, Pai 2008,
Han 2008, Piscaglia 2015). The Source of Stem Cells have been Fetal Liver Stem Cells (Khan
2010, Cardinale 2014); BM-MSC (Mohamadnejad 2007, Kharaziha 2009, Amer 2011, Amim
2013, Jang 2014, Peng 2011, El-Ansary 2012, Zhang 2012, Wang 2013, Terai 2006, Shi
2012, Mohamadnejad 2013), peripheral G-CSF mobilized CD34/CD133 stem cells, collected
by apheresis and re infused through the hepatic artery or the Portal Vein System (Yannaki
2006, Gasbarini 2007, Levicar 2008, Pai 2008, Han 2008, Piscaglia 2015). The main
indication was the treatment of the cirrhosis conditions that were in the waiting list for
transplant originated from all kind of causes (alcoholic abuse, associated with Hepatitis B or
C virus), or acute-on-chronic liver failure patients.
Most of the patients shown improve in the overall Liver condition that was also reflected
in the MELD index. Only one randomized, double blind, clinical trial, done in Iran, that
involved 12 control and 15 BM-MSC treated patients shown not benefit but not worsening on
any of the patient from the treated group regarding to the control group (Mohamadnejad
2013).
However, because more of the reports were successful, to avoid all the invasive
maneuvers that imply the stem cell infusion, treatment with only BM SC mobilization from
the bone marrow, using subcutaneous injections of G-CSF was also tried (Gaia 2006, Gordon
2006, Spahr 2008, Garg 2012, Singh 2014, Di Campli 2007, Lorenzini 2008). Despite some
isolated encouraging results obtained, the method did not show major benefits, therefore, after
G-CSF mobilization the cells were collected by apheresis and infused into the patient using
the intra hepatic artery or intra porta system route. Later, a comparative study done using G-
CSF mobilization only and G-CSF mobilization followed by apheresis collection and infusion
into the hepatic artery was done demonstrating the superiority of the second therapeutic

schema respect the first one (Han 2008). The results of this last trial has the opposite result
regarding to the above mentioned Iranian Trial Mohamadnejad 2013) and is in accordance
with the majority of case reports found in the literature.
On other hand, Baertschiger et al. (Baertschiger 2009) and di Bonzo et al. (di Bonzo
2008) analyzing animal and clinical data pointed the dangerous that the only SC
administration may have short term benefits regarding to hepatocyte function but may also
increase the fibrosis burden. To avoid this danger, a combination of G-CSF SC mobilization,
followed by apheresis collection, associated with Total Plasma Exchange (TPX) done in the
same procedure and ending with the intra-artery infusion have rendered outstanding results.
The purpose of TPX was to modify the micro environment of the liver, removing from the
organ and the general circulation all the Th2 Cytokines associated with the chronic
inflammatory condition of the cirrhotic livers (Piscaglia 2015).
The last clinical experience opens new further avenues to treat, in a more effective
fashion, the cirrhosis and other liver conditions. At Maimonides University, in Argentina, has
been treated chronic and complete spinal cord injury patients. The scar tissue that block the
union in between the two ends of the sectioned spinal cord have been modified for the
treatment of anti-nerve tissue Th1 activated effector cells. After this immune modulation the
scare changed into a permissive and pro-regenerative scar allowing the success of the stem
cell treatment that was applied afterward (Moviglia 2006, Moviglia 2009).
In a further clinical trial, using similar approach not only the paracrine effect of the stem
cells could be granted but also the plastic remodeling of the fibrotic tissue and more effective
liver regeneration.
Finally, different groups have been working in the tissue engineering of a liver
construction with autologous cells (Lin 2010, Kazemnejad 2009, Piryaei 2011, Li 2010, Li
2015, Soto-Gutierrez 2006). The in vitro hepatocyte maturation of MSC have been achieved.
With the use of the 3D bioprinting, scaffolds with different materials, to reproduce the spatial
liver structure, have also been successfully.. In a pre-clinical setting, the use of this small
biologic device have supported the life of complete liver failure of a mouse (Soto-Gutierrez
2006).
CONCLUSION
Cellular therapy is a modern academic therapeutic approach that in its shorts 60 years of
age has proved promising results, not only in the resolution of the previous and urgent unmet
medical needs, but also helped to change the therapeutic paradigm that characterized the 20
century. The mechanic conception of the chemical reactions is given pass to the modern
concepts of small organism interaction, where the genome does not determine the fate of the
metabolic process but is dynamically modified for the epigenetic signals of the environment
and other cell populations.
The liver cellular therapy seems to be an example of this new paradigm.

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INDEX
adenosine triphosphate, 210

# adenovirus, 248
adhesion, 18, 27, 47, 49, 74, 106, 108, 109, 110, 117,
5-HT2 receptors, 243 118, 236, 296
adhesion receptors, 109
A adipocyte, 193
adipose, 64, 206, 208, 215, 216, 220, 222, 328
Abraham, 93, 119, 144, 145, 300, 302, 303, 304, 316 adipose tissue, 64, 206, 208, 215, 216, 220
abuse, 28, 323 adiposity, 212
access, 61, 246, 262, 278 adjustment, 129
acetaldehyde, 28, 99 adolescents, 223
acetaminophen, 18, 21, 33, 35, 36, 37, 38, 40, 41, 82, ADP, 7
290, 292 adrenaline, 244, 254
acetylation, 166 adrenoceptors, 242
acetylcholine, 251 adult stem cells, 329
acid, 23, 28, 46, 67, 68, 69, 73, 74, 76, 80, 83, 88, adulthood, 99, 313
98, 99, 103, 113, 114, 123, 127, 128, 134, 145, adults, 26, 113, 161, 178, 199, 211, 219
152, 185, 192, 193, 197, 202, 204, 207, 208, 211, adverse effects, 282, 283, 284
212, 220, 221, 224, 225, 228, 259, 262, 280 adverse event, 172, 174, 176
acidic, 44, 68, 110, 137 affluence, 240
acidosis, 85, 173 Africa, 155, 184
activation complex, 6 after abrupt cessation, 253
activation state, 46, 47, 49, 51 age, 59, 70, 82, 118, 158, 261, 312, 324
active oxygen, 277 aggregation, 9
active site, 12 agonist, 86, 99, 100, 101, 103, 134, 135, 138, 176,
acute infection, 160, 185 177, 244, 293, 326
Acute liver failure, 21, 33, 38, 82, 132, 133, 134, Akan, 326
139, 142, 259, 277, 286, 289, 291 alanine, 2, 41, 199, 202
acute renal failure, 281 alanine aminotransferase, 2, 41, 199, 202
acute stress, 211 albumin, 68, 132, 285
adaptation, 106 alcohol abuse, 28, 116
adaptive immune response, 50, 161, 176, 185, 187, alcohol consumption, 28, 99, 116
189 alcoholic cirrhosis, 243, 255, 326, 330
adaptive immune responses, 50, 176, 185, 189 alcoholic liver disease, 18, 22, 28, 51, 69, 75, 99,
adaptive immunity, 37, 167, 186, 187, 197 100, 102, 115, 290, 319, 320
adefovir (ADV), 155, 156, 171, 173, 174 alcoholism, 116, 130
adenine, 9, 20, 209 aldosterone, 240
adenosine, 29, 171, 210, 246 alkalosis, 81
alpha-1-antitrypsin, 22, 29

332 Index
alport disease, 113 apoptotic pathways, 25, 211, 218

ALT, 2, 170, 203 appetite, 98
alzheimer’s disease (AD), 32, 37, 56, 74, 101, 114, aquaporin 8, 128
201, 202, 224 ARC, 175
amino, 11, 22, 27, 65, 79, 80, 82, 83, 98, 111, 114, ARC-520, 175
123, 124, 125, 154, 157, 181, 192, 210, 259, 285, Argentina, 275, 324
308, 310 arg-gly-asp (RGD), 109, 122
amino acid, 11, 22, 27, 65, 79, 80, 82, 83, 98, 111, arginine, 153, 247
114, 124, 125, 157, 181, 192, 210, 259, 285, 308, argininosuccinate lyase, 81
310 argininosuccinate synthetase, 81
ammonia, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 257, arrest, 11
258, 259, 262, 263, 264, 271, 272, 273, 277, 289, arrests, 131, 145
291, 293, 294, 295, 296, 297, 298, 307, 308, 309 arrhythmias, 282
ammonium, 81, 82, 85, 86, 88, 262, 279, 283, 284, arterial hypertension, 126, 133, 134, 135, 136, 137,
293, 297, 298 141
amnesia, 276 arteries, 135, 141
analgesic, 98 arterioles, 242, 245
anandamide, 97, 99, 102, 103, 248, 251 arterio-venous fistula, 244
anastomosis, 285, 309 artery, 59, 135, 231, 232, 233, 245, 246, 323, 324
anatomy, 81 arthritis, 115
anatomy of hepatic-portal system, 231 articular cartilage, 108
anchorage, 112 ascites, 129, 131, 135, 237, 251, 253, 283, 285, 308,
anchoring, 106, 182 330
angiogenesis, 48, 72, 99, 105, 106, 118, 131, 218 Asia, 177, 184, 219
angiotensin II, 49, 124, 125, 129, 140 aspartate, 2, 5, 86, 171, 218, 259, 277
anoxia, 16, 21 aspartic acid, 218
antagonism, 110, 130, 143, 146, 226 assessment, 42, 200, 220, 244, 278, 285
antibiotic, 279, 283, 284 asterixis, 259, 291
antibody, 86, 88, 157, 186, 188, 198, 202 astrocytes, 67, 79, 82, 84, 85, 86, 257, 259, 263, 265,
anti-cancer, 34 266, 267, 268, 272, 273, 277, 291, 294, 295, 297,
anticancer drug, 30, 36 298, 307, 308, 309
antigen, 36, 50, 67, 150, 151, 154, 161, 162, 164, astrogliosis, 293
165, 166, 169, 187, 188, 193, 321, 327 asymptomatic, 25, 82
antigenicity, 156 ATF, 203
antigen-presenting cells, 187 atherosclerosis, 70, 73, 74
antihistamines, 283 atomic force, 72
antioxidant, 28, 209 ATP, 9, 11, 13, 20, 21, 22, 23, 36, 85, 86, 87, 90, 92,
antitumor, 322 192, 210, 211
antiviral agents, 155, 156, 173, 179, 180 atria, 126
antiviral drugs, 167 atrium, 135
antiviral therapy, 157, 161, 169, 170, 183, 198, 200, atrophy, 3, 84
201 attachment, 109, 151, 152, 159, 199
aplasia, 321 authorities, 321
apoptosis, 1, 2, 3, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, autoimmune disease, 116, 118, 171, 201, 290, 319,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 30, 31, 32, 320, 321, 322
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 50, 51, autoimmune hepatitis, 2, 290, 327
53, 54, 56, 57, 71, 86, 87, 90, 92, 97, 100, 102, autonomic nervous system, 129, 240, 245, 295
116, 122, 159, 162, 186, 189, 190, 191, 193, 194, autonomous nervous system, 240, 245
195, 196, 197, 198, 200, 202, 205, 206, 208, 209, autophagy, 1, 2, 3, 15, 16, 17, 18, 19, 21, 22, 25, 26,
212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 27, 28, 31, 32, 34, 35, 38, 39, 40, 41, 42, 86, 87,
222, 223, 224, 226, 227, 228, 293, 295, 299, 300, 91, 93, 203, 206, 208, 216, 219, 222
301 autosomal recessive, 81, 114, 116
apoptosis pathways, 217 axons, 265

Index 333
blood, 20, 29, 30, 48, 60, 61, 62, 63, 64, 65, 66, 69,
B 70, 75, 80, 81, 82, 83, 100, 113, 125, 126, 127,
128, 129, 131, 132, 133, 140, 141, 142, 154, 160,
bacteria, 50, 72, 79, 81, 82, 83, 283
184, 189, 193, 195, 196, 198, 206, 212, 231, 232,
bacterial infection, 292
233, 234, 236, 237, 238, 239, 240, 241, 242, 243,
bacteriostatic, 83
244, 245, 246, 250, 252, 253, 254, 257, 258, 262,
baroreceptor, 245
263, 264, 266, 272, 274, 281, 283, 285, 289, 291,
baroreflex system, 245
297, 309, 326
basal core promoter and preCore mutants, 157
blood circulation, 66
basal ganglia, 88, 274, 295
blood flow, 20, 48, 62, 80, 125, 126, 128, 129, 132,
basal lamina, 47, 70
133, 142, 231, 233, 236, 238, 239, 240, 241, 242,
base, 5, 150, 153, 262, 280
243, 244, 250, 252, 253, 254, 258, 271, 280
base pair, 5, 150
blood pressure, 63, 100, 125, 127, 131, 239, 245,
basement membrane, 60, 61, 68, 106, 107, 108, 109,
257, 281
110, 111, 112, 113
blood supply, 20, 291
batteries, 261
blood transfusions, 184
BBB, 85, 258, 264, 277, 293, 294, 295, 296, 297,
blood vessels, 113, 141, 266, 297
298
blood-brain barrier, 82, 237, 289, 309
Bcl-2 family, 7, 8, 9, 12, 19, 40, 42, 86, 212, 216,
bloodstream, 29, 154, 160
217, 219
BM-40, 68, 110, 111
Bcl-2 proteins, 19, 217
bonds, 109, 124, 207
behavioral change, 276, 291, 297
bone, 66, 68, 76, 110, 113, 172, 195, 321, 323, 325,
beneficial effect, 100, 284
326, 327, 328, 329
benefits, 244, 323, 324
bone marrow, 76, 195, 321, 323, 325, 326, 327, 328,
benign, 26, 100, 193, 196
329
benzodiazepines, 80, 83, 280, 283
bone marrow transplant, 321
besifovir, 174
brain, 34, 80, 81, 82, 85, 86, 87, 88, 114, 116, 125,
beta 2 blocker, 242
126, 129, 140, 143, 182, 196, 234, 237, 257, 258,
beta blocker, 242, 243
259, 260, 262, 263, 264, 265, 266, 267, 268, 270,
beta-adrenoceptors, 245
271, 272, 273, 277, 278, 279, 280, 281, 282, 286,
Big ET, 124
293, 294, 295, 296, 297, 298, 321
bile, 23, 34, 35, 39, 60, 83, 115, 117, 127, 128, 129,
brain herniation, 82, 277
130, 131, 134, 137, 141, 143, 145, 152, 236, 241,
brainstem, 142, 147
248, 249, 250, 254, 322, 323
breakdown, 5, 51, 117, 249, 254, 310
bile acids, 23, 83, 128
bronchoconstriction, 126
bile duct, 23, 34, 39, 115, 127, 130, 131, 141, 236,
budding, 153, 166, 183
241, 248, 249, 250, 254, 322, 323
building blocks, 65
bile duct ligated, 34, 131, 241, 248, 249
bile flow, 23, 128
bile salt export pump, 128, 137 C
bile secretion, 127, 128, 129, 143
biliary cirrhosis, 77, 100, 236, 246 Ca2+, 9, 53, 86, 242, 247, 250, 307, 310, 311
bilirubin, 283 Ca2+ flux, 311
biliverdin, 248 calcification, 114
bioavailability, 49, 59, 62, 69, 134, 254 calcium, 13, 14, 21, 27, 33, 47, 49, 80, 87, 110, 111,
biological behavior, 31 113, 127, 128, 194, 211, 244, 251, 253, 273, 293,
biological markers, 320 294, 298
biological systems, 210 calcium deposition, 114
biopsy, 3, 42, 236, 237 caliber, 131, 243
biosynthesis, 124, 247, 253 CAM, 109
birds, 154 cancer, 3, 8, 18, 30, 31, 33, 34, 38, 40, 41, 42, 61, 97,
bleeding, 113, 236, 238, 241, 244, 275, 280, 283, 99, 100, 117, 118, 129, 141, 162, 165, 328
285 cancer cells, 8, 31, 38, 97, 100
bleeding time, 236 cancer progression, 34

334 Index
cancer therapy, 41 cell differentiation, 77, 112

cannabidiol, 97, 98, 99, 104 cell division, 30, 212
cannabinoid receptor type 1 (CB1R), 97, 98, 99, 100, cell fate, 110, 210
101 cell killing, 21, 23, 41
cannabinoid receptor type 2 (CB2R), 97, 98, 99, 100 cell line, 2, 25, 26, 31, 34, 37, 97, 100, 152, 195
cannabinoids, 97, 98, 99, 102, 103 cell signaling, 61, 110
cannabis, 99 cell surface, 41, 107, 109, 151, 185, 186, 212, 214
capillary, 59, 60, 69, 72, 231, 242, 257, 265, 281, cellular energy, 86
291, 296, 297, 309 cellular homeostasis, 86, 296
capillary pressure, 242, 251 central nervous system (CNS), 79, 80, 83, 84, 87, 88,
capsule, 47 103, 125, 196, 200, 234, 257, 274, 277, 289, 291,
carbohydrate, 207 307, 308, 327
carbohydrates, 263, 284 centrosome, 163
carbon, 75, 80, 130, 131, 138, 145, 146, 237, 238, ceramide, 23, 221
241, 247, 248, 254, 273, 291, 328 cerebellum, 125
carbon dioxide, 80, 146, 273, 291 cerebral blood flow, 80, 126, 129, 258, 271, 280
carbon monoxide (CO), 101, 202, 248, 249 cerebral cortex, 103, 293, 296
carbon tetrachloride, 75, 130, 131, 138, 145, 237, cerebral edema, 82, 272, 277, 279, 280, 281, 282
238, 247, 248, 254, 328 cerebral function, 274, 298
carbon-tetrachloride intoxication, 241 cerebral hyperemia, 281
carcinogenesis, 38, 162, 163 cerebral perfusion pressure, 281
carcinoma, 22, 30, 169, 204, 205 cerebrospinal fluid, 259, 297
cardiac output, 131, 233, 240, 243, 244, 245, 246 ceruloplasmin, 116
cardiomyopathy, 313 challenges, 240
cardiovascular disease, 99, 127 chaperones, 17, 29, 224
cardiovascular system, 124, 125, 141 chemical, 65, 134, 224, 283, 320, 324
cascades, 32, 109, 193 chemical reactions, 324
Caspase-8, 33, 299 chemokine receptor, 293
caspases, 1, 5, 6, 7, 8, 11, 12, 13, 19, 23, 24, 25, 30, chemokines, 27, 45, 49, 62, 116, 118, 185, 215, 226,
35, 38, 39, 86, 90, 214, 216, 218, 228 330
CAT, 313 chemotaxis, 43, 44, 45, 110
catabolism, 28, 76, 310 chemotherapy, 31, 35, 134, 321
catabolized, 18 children, 26, 113, 159, 165, 196, 199, 223
catalyst, 28 chimpanzee, 180, 192
catecholamines, 125, 234, 277, 281 cholestasis, 21, 23, 34, 128, 129, 143
CBD, 97, 98, 99, 104 cholestatic liver injury, 22, 23, 302
C-C, 215 cholesterol, 27, 73, 175, 182, 200, 204, 213, 225
cccDNA, 150, 151, 153, 155, 159, 160, 166, 169, choline, 204, 208, 212, 213, 215, 222, 225
170, 174, 175, 176, 178 chondroitin sulfate, 74
CD8+, 37, 159, 160, 161, 188, 189 choroid, 297
CD95, 7, 13, 36, 38, 41, 226, 300 chromosome, 114, 150, 163
cDNA, 144, 196 chronic active hepatitis, 37, 179
cell biology, 5, 75 chronic diseases, 116, 131
cell body, 265 chronic liver failure, 81, 82, 84, 116, 243, 258, 263,
cell culture, 23, 86, 101, 152, 209, 311 289, 308, 309, 323, 325, 326, 328, 329
cell cycle, 7, 11, 30, 99, 100, 103, 110, 162, 179, chronotropic response, 242
200, 313 cilengitide, 118, 121
cell death, 1, 2, 3, 4, 6, 8, 10, 11, 12, 13, 14, 15, 18, circulating waste, 65
19, 20, 21, 22, 23, 24, 27, 29, 30, 31, 33, 34, 35, circulation, 22, 46, 48, 59, 64, 65, 66, 67, 69, 70, 72,
37, 38, 39, 40, 41, 42, 51, 79, 84, 85, 86, 87, 88, 76, 81, 82, 125, 128, 129, 130, 131, 132, 133,
89, 90, 100, 101, 103, 116, 117, 154, 163, 191, 159, 206, 231, 232, 233, 235, 236, 237, 239, 240,
194, 200, 206, 209, 211, 212, 213, 214, 215, 216, 241, 244, 246, 248, 249, 251, 253, 254, 255, 262,
217, 218, 224, 227, 228, 307, 310, 313, 314 264, 274, 279, 282, 289, 324

Index 335
cirrhosis, 2, 25, 26, 35, 43, 44, 51, 55, 59, 68, 69, 71, communication, 295
72, 73, 75, 76, 77, 82, 83, 88, 90, 92, 93, 94, 97, community, 39
98, 100, 101, 107, 114, 116, 120, 129, 130, 131, comparative analysis, 196
132, 134, 136, 137, 138, 141, 144, 145, 146, 155, competition, 12
159, 169, 170, 171, 172, 179, 184, 201, 205, 231, complement, 17
232, 234, 235, 236, 237, 238, 243, 244, 245, 246, complexity, 3, 77, 184
247, 248, 250, 251, 252, 253, 254, 255, 258, 259, compliance, 240
262, 263, 264, 271, 272, 273, 275, 276, 277, 279, complications, 26, 79, 80, 81, 84, 129, 278, 280, 283,
282, 283, 284, 287, 290, 291, 292, 293, 296, 300, 289, 308, 309
301, 302, 304, 307, 308, 309, 315, 316, 319, 320, composition, 45, 47, 106, 113, 114
322, 323, 324, 325, 326, 327, 328, 329, 330 compounds, 83, 98, 176, 210
citrulline, 80 compressibility, 108
classes, 1 compression, 241
classification, 6, 76, 105, 178, 179, 201, 243, 258, computed tomography, 308
309 conception, 324
cleavage, 5, 6, 7, 8, 9, 25, 47, 86, 100, 174, 175, 181, condensation, 5, 10, 19
186, 198, 216, 218 conditioning, 326
clinical application, 326 conductance, 245
clinical examination, 260 conference, 219
clinical oncology, 37 congestive heart failure, 143, 233
clinical presentation, 81 conjugation, 18
clinical syndrome, 129 connective tissue, 66, 106, 108, 111, 112, 115, 290
clinical trials, 127, 129, 131, 133, 134, 135, 173, 308 consciousness, 276, 291
clone, 25, 196 consensus, 114, 261, 285, 296, 299
clonus, 276 consent, 285
closure, 18, 19 constipation, 284
clustering, 63, 215 constituents, 13, 14, 107, 116
clusters, 118 construction, 324
CNS, 79, 84, 87, 90, 95, 102, 103, 257, 262, 263, consumption, 28, 80, 82, 87, 100, 208, 292
264, 274, 289, 291, 293, 294, 295, 296, 297, 298, contour, 313
300, 302, 307, 308, 316 control group, 207, 321, 322, 323
CO2, 281, 296 controlled studies, 321
coagulopathy, 278, 280, 282 controlled trials, 211
cocaine, 99, 104 controversial, 22, 48, 73, 79, 97, 100, 132, 155, 158,
coding, 81, 149, 150, 162 187, 280
codon, 158 controversies, 25
cognition, 79, 80, 85, 277 convergence, 42, 195
cognitive deficits, 293 cooperation, 2, 32
cognitive dysfunction, 276 coordination, 259, 277, 293
cognitive function, 262, 273 copper, 108, 116
cognitive impairment, 259, 294, 308 coronary artery disease, 224
COL4A5, 113 correlation, 141, 162, 164, 192, 262, 293, 307, 308,
collagen, 28, 47, 48, 51, 53, 55, 66, 68, 105, 106, 312
107, 108, 109, 110, 112, 113, 115, 116, 118, 121, cortex, 114, 265, 266
129, 253, 313 cortical neurons, 257, 267, 309
collagen-binding discoidin domain receptors cost, 161, 240, 250
(DDRs), 116 cough, 280
collateral, 82, 129, 233, 239, 240, 241 cough reflex, 280
collateralization, 247 covering, 152
colon, 125, 283 CPP, 281
coma, 81, 82, 258, 259, 276, 293 CPT, 210
combination therapy, 137 creatinine, 283, 294, 308
combined effect, 263 crystallization, 27

336 Index
crystals, 114 derivatives, 128, 175

CSF, 259, 264, 323, 324, 325, 326 destruction, 3, 159, 165, 313, 319
cues, 322 detachment, 117, 307, 310, 314
culture, 37, 44, 45, 70, 71, 88, 128, 144, 180, 263, detectable, 158, 161
272 detection, 36, 154, 159, 193, 274
cure, 169, 170, 176, 177 detoxification, 21, 80, 84, 87, 162, 277, 294, 297,
CXC, 37, 49 307, 308, 309, 319, 320
cycles, 149, 159, 162, 163 developed countries, 21
cycling, 60 developing brain, 82, 86
cyclooxygenase, 246, 247, 255, 293, 295, 297 diabetes, 30, 32, 114, 173, 192, 221, 229
cyclosporine, 136 diabetes insipidus, 173
cysteine, 5, 68, 110, 124, 150, 153, 210 diabetic nephropathy, 141
cytoarchitecture, 114 diacylglycerol, 99, 127
cytochrome, 8, 9, 10, 11, 21, 23, 25, 35, 41, 86, 87, diad, 307, 310
90, 210, 211, 216 dialysis, 136, 279, 281, 285, 287
cytokines, 10, 21, 27, 28, 33, 35, 44, 45, 47, 49, 51, dialysis with extracorporeal albumin, 285
62, 84, 105, 106, 114, 116, 124, 130, 132, 159, diamonds, 177
185, 186, 187, 189, 211, 215, 289, 291, 292, 294, dichotomy, 199
295, 296, 298, 322, 323 diet, 18, 100, 103, 113, 118, 204, 207, 208, 209, 212,
cytomegalovirus, 24 215, 217, 218, 222, 223, 224, 225, 228, 284, 285,
cytoplasm, 5, 9, 11, 12, 16, 19, 30, 61, 66, 67, 150, 292, 308, 312
152, 153, 159, 160, 182, 183, 192, 210, 298 diffusion, 262, 295
cytoplasmic tail, 116 digestion, 5, 12, 16, 40, 262
cytoskeleton, 5, 7, 20, 44, 63, 72, 85, 90, 296, 298 dilation, 130, 131, 240, 307, 311
cytotoxicity, 86, 186, 225 dimerization, 7, 211
direct action, 192, 322
directors, 321
D disability, 188
discs, 108
danger, 187, 324
disease progression, 1, 2, 3, 26, 199, 200, 207, 213,
deaths, 39, 82
223
decerebration, 280
diseases, 1, 2, 3, 12, 14, 15, 16, 30, 31, 42, 70, 80,
decontamination, 251
99, 100, 108, 111, 114, 115, 118, 123, 127, 129,
defects, 30, 108, 113, 274
131, 133, 134, 135, 143, 189, 228, 258, 313, 319,
defense mechanisms, 23
320
deficiencies, 81, 297
disorder, 81, 85, 88, 99, 114, 116, 127, 144, 205,
deficiency, 22, 29, 81, 107, 113, 212
263, 273
deficit, 113, 189
dispersion, 77
degradation, 5, 11, 12, 13, 15, 16, 18, 19, 20, 26, 27,
displacement, 314
29, 31, 43, 47, 65, 66, 67, 73, 76, 90, 97, 98, 100,
disposition, 114
107, 112, 114, 118, 125, 137, 178, 186, 192, 193,
dissociation, 15, 18
204, 208, 211, 216, 218, 223, 228, 246, 268
distribution, 21, 33, 125, 155, 158, 167, 174, 179,
degradation process, 5
184, 198, 234, 240, 242, 250, 265, 283
degradation rate, 28
divergence, 154, 195
dendrites, 265
diversification, 188, 200
dendritic cell, 50, 160, 167, 182, 197, 201, 325, 326,
diversity, 49, 155, 164, 184
327, 329, 330
DNA, 5, 7, 8, 10, 11, 13, 19, 20, 25, 26, 30, 41, 50,
dephosphorylation, 154, 217
87, 118, 149, 150, 151, 153, 154, 155, 157, 159,
depolarization, 13, 20, 263
162, 163, 165, 169, 170, 171, 172, 174, 175, 176,
deposition, 47, 49, 70, 88, 105, 106, 107, 108, 109,
192, 210, 313
112, 113, 114, 115, 116, 129, 205, 274
DNA damage, 8, 11, 30, 87, 162, 163, 211
deposits, 88, 112, 113, 114, 115, 116, 263
DNA polymerase, 25, 153, 171, 172
depression, 171, 264
DNA repair, 7, 162
deregulation, 24, 30, 116, 162

Index 337
DNAs, 177 emission, 140

dogs, 231, 233 emphysema, 29
domain structure, 98 encephalopathy, 80, 82, 85, 88, 90, 91, 92, 93, 94,
donors, 187, 193, 198, 321 95, 96, 129, 257, 258, 259, 260, 261, 262, 264,
dopamine, 99, 132, 277, 295 272, 273, 274, 275, 276, 277, 278, 279, 280, 281,
dopaminergic, 277, 295 282, 283, 284, 285, 286, 287, 289, 290, 291, 292,
dosage, 172 293, 295, 296, 299, 300, 301, 302, 303, 304, 307,
dosing, 134, 174 308, 309, 313, 315
double bonds, 207 encoding, 11, 12, 19, 108, 144, 152, 180, 330
double helix, 153 endocannabinoid system, 97, 98, 101, 103
down-regulation, 22, 27, 51, 75, 100, 193 endocannabinoids (eCBs), 97, 98, 99, 100, 101, 102,
drawing, 261 103
drug metabolism, 70, 77 endocrine, 147, 192
drug resistance, 30, 163, 171, 183 endonuclease, 5, 8, 9
drug targets, 164 endothelial cells, 21, 43, 44, 45, 47, 48, 49, 59, 60,
drug-induced hepatitis, 326 61, 65, 66, 72, 73, 74, 75, 76, 100, 107, 109, 110,
drug-induced liver injury (DILI), 1, 2, 21, 22, 37, 111, 123, 124, 125, 126, 127, 130, 131, 133, 139,
134 143, 144, 146, 189, 229, 236, 247, 249, 251, 254,
drugs, 3, 11, 21, 63, 70, 98, 103, 116, 118, 126, 132, 265, 296, 297, 298, 321, 325
134, 135, 169, 173, 174, 176, 179, 184, 205, 289, endothelial NO synthase, 132
290 endothelin 1, 248
endothelin converting enzyme, 124, 145, 146
endothelin receptor antagonists, 136, 137, 138, 140
E endothelin receptors, 135, 136, 139, 141, 145
endothelins, 123, 126, 127, 129, 133, 136, 142, 145,
ECE-1, 124, 127, 146
146, 147, 250
ECE-2, 124
endothelium, 21, 48, 60, 63, 64, 70, 73, 74, 109, 125,
ECE-3, 124
182, 246, 247, 248, 249, 254, 296, 297, 323
ECM, 43, 44, 45, 47, 48, 49, 51, 105, 106, 107, 108,
endotoxins, 28, 68, 70
109, 110, 111, 112, 113, 114, 115, 116, 117, 118,
enemas, 284
119
energy, 13, 16, 18, 22, 74, 76, 79, 80, 85, 117, 207,
ECM degradation, 51
208, 264, 273, 277, 295
ECM pathology, 112
England, 91, 94
ECM-integrin interactions, 117
enlargement, 107, 129, 241, 251, 298, 310, 312, 314
ecosystem, 105, 106
entecavir, 155, 156, 171, 172, 173
ECs, 65
envelope mutants, 156
edema, 82, 237, 259, 264, 277, 278, 279, 280, 281,
environment, 105, 106, 110, 162, 188, 240, 262, 298,
286, 293, 294, 296, 297, 298, 307, 310
324
editors, 73, 77, 166, 329
environmental factors, 118
education, 261
enzyme, 6, 16, 39, 80, 81, 82, 99, 132, 134, 136, 137,
EEG, 264
138, 139, 145, 146, 171, 176, 201, 204, 210, 211,
Egypt, 184, 193
248, 259, 263, 294, 297, 309, 313
Ehlers-Danlos syndrome, 108, 113
enzyme inhibitors, 132, 139
elastin, 47, 105, 106, 107, 108
eosinophilia, 115
electroencephalography, 264
ependymal cell, 297
electrolyte, 280
epidemic, 184
electron, 9, 61, 63, 74, 75, 77, 87, 140, 210, 211,
epidemiology, 165, 167, 183, 198, 219
273, 296
epigenetic modification, 118
electron microscopy, 61, 140, 296
epigenetic regulation of HSCs, 118
electrophoresis, 5
epilepsy, 264
elongation, 18, 19
epiphysis, 113
elucidation, 180
epithelia, 84, 107
embryogenesis, 110
epithelial cells, 66, 80, 97, 98, 116, 125, 127, 321
emergency, 279, 282

338 Index
epithelial-to-mesenchymal transition (EMT), 116

epithelium, 64, 112
F
epitopes, 157, 206, 218, 330
FAD, 210
equilibrium, 2, 262
false negative, 156
erythrocytes, 109
families, 88, 109
esophageal varices, 129, 233, 234
family history, 221
esophagus, 235
family members, 8, 12, 19, 30, 44, 86, 217
ester, 74, 206
FAS, 13, 15, 204
estrogen, 128
fasting, 206, 209
ET isoforms, 128
fat, 26, 27, 70, 77, 99, 100, 204, 205, 206, 221, 224
ET-1, 46, 123, 124, 125, 126, 127, 128, 129, 130,
fatty acids, 27, 36, 64, 65, 80, 83, 192, 203, 204, 206,
131, 132, 133, 146, 248
208, 212, 220, 221, 225, 226, 227
ET-2, 123, 124, 125, 126
FDA, 133, 134, 320
ET-3, 123, 124, 125, 126, 127, 128, 129, 131
fetal development, 30
ETA, 125, 126, 127, 128, 129, 130, 131, 132, 133,
fever, 115
134, 139, 141, 143, 144
fibers, 107, 108, 110, 115, 128, 265, 269, 310, 313
ETB, 125, 126, 127, 128, 129, 130, 131, 132, 133,
fibril-associated collagens with interrupted triple
134, 135, 143
helix (FACITs), 107
ethanol, 28, 35, 70, 73, 100, 142, 241
fibrillar, 43, 107, 108, 109, 113, 307, 310, 313
etiology, 30, 46, 80, 130, 133, 220, 290
fibrillin, 105, 106, 108, 110
ETM, 310, 311, 312, 313
fibrin, 109, 115
eukaryotic, 16, 185
fibrinogen, 109
eukaryotic cell, 16
fibrinoid necrosis, 115
evacuation, 284
fibroblasts, 44, 46, 67, 107, 109, 114, 115, 131, 144
evidence, 19, 23, 24, 27, 46, 48, 51, 63, 69, 98, 111,
fibrogenesis, 2, 18, 27, 28, 36, 46, 103, 105, 106,
124, 125, 130, 134, 144, 145, 159, 162, 163, 171,
113, 115, 123, 130, 131, 133, 144, 189, 197, 226,
187, 189, 195, 200, 208, 220, 233, 246, 263, 269,
228, 250
279, 284, 289
fibronectin, 47, 53, 57, 105, 106, 107, 109, 110, 114,
evoked potential, 274
121
evolution, 80, 99, 150, 154, 158, 159, 165, 183, 184,
fibrosis, 2, 18, 23, 24, 27, 32, 34, 37, 43, 44, 46, 49,
195, 197, 200, 275, 276, 278, 280, 283, 289
50, 51, 68, 69, 70, 71, 99, 100, 103, 105, 106,
examinations, 262
107, 114, 115, 116, 118, 129, 130, 131, 140, 145,
excitability, 280
147, 171, 179, 187, 189, 191, 193, 195, 196, 200,
excitation, 311
203, 205, 206, 211, 213, 215, 218, 220, 225, 227,
excitotoxicity, 277
228, 229,232, 290, 319, 320, 321, 322, 324, 328,
exclusion, 258
330
excretion, 128, 134, 140, 246, 263, 294
fibrotic livers, 114
execution, 2, 5, 7, 14, 17
fights, 40
exocytosis, 124, 146
filament, 265
experimental condition, 25, 77, 311
filtration, 63, 64, 112, 132, 294, 308
experimental design, 187, 191
fitness, 155, 157, 274
exposure, 28, 63, 103, 152, 160, 161, 162, 215, 264,
fixation, 61, 63, 86
298
flexibility, 109
extracellular matrix, 43, 44, 45, 63, 106, 109, 130,
flora, 84, 292
131, 297, 330
fluid, 64, 114, 234, 281, 283
extracellular matrix (ECM), 43, 44, 45, 47, 48, 49,
food, 80, 284
51, 52, 55, 63, 105, 106, 107, 108, 109, 110, 111,
Food and Drug Administration, 133, 329
112, 113, 114, 115, 116, 117, 118, 119, 121, 130,
force, 106, 156, 242, 250
131, 297, 330
forebrain, 147
extraction, 125
formaldehyde, 68, 72
extracts, 131
forward flow theory, 231, 233, 240, 241
extrinsic apoptosis, 7, 87
fragments, 50, 110, 185, 189, 206
extrusion, 20
France, 194, 196, 287, 316

Index 339
free radicals, 223, 258, 263 glucose, 60, 117, 192, 193, 207, 212, 225, 280, 284
frontal cortex, 293 GLUT4, 193
frontal lobe, 143 glutamate, 80, 81, 82, 84, 85, 86, 90, 263, 277, 280,
fructose, 22, 226, 284 295, 296, 298
fulminant hepatitis, 25, 159 glutamine, 80, 82, 84, 85, 87, 90, 259, 263, 277, 293,
functional changes, 85 294, 295, 297, 298, 308, 309
fusion, 152, 182, 310, 312, 314, 321 glutamine synthase, 82, 84, 309
glutathione, 11, 21, 38, 80, 100, 210, 213
glycans, 67
G glycerol, 97, 99, 103, 206
glycine, 22, 108
GABA, 83, 88
glycogen, 60
gait, 87, 276
glycogenesis, 60
gallbladder, 231
glycolysis, 60, 117
gastric mucosa, 234, 237, 282
glycoproteins, 47, 67, 109, 110, 114, 180, 182, 196,
gastroenterologist, 40
200, 211
gastrointestinal bleeding, 283
glycosaminoglycans, 105, 106, 107, 108, 182
gastrointestinal tract, 67, 98, 114, 125, 126, 137, 141,
glycosylation, 126, 188
145, 244
Gly-Y-X, 108
gel, 5, 114
GPx, 313
gelatinase A, 47
grades, 258, 279
gene expression, 51, 118, 136, 162, 224, 248, 251,
grading, 275
313
gravity, 82
gene promoter, 118, 124
growth, 1, 10, 17, 30, 44, 47, 48, 61, 100, 105, 106,
gene therapy, 178, 329
108, 109, 110, 112, 113, 114, 117, 123, 141, 144,
gene transfer, 248, 254
162, 182, 205, 215
general practitioner, 275
growth factor, 10, 17, 44, 47, 48, 61, 105, 106, 108,
genes, 11, 12, 13, 15, 17, 19, 30, 50, 51, 99, 100,
109, 110, 112, 114, 141, 144, 182, 205, 215
113, 118, 123, 139, 150, 160, 162, 167, 185, 186,
GS-9620, 176, 177
193, 207, 212, 215, 222, 226, 313
GTPases, 76
genetic alteration, 113, 114
guardian, 11
genetic disorders, 81, 205
guidelines, 171, 172, 173, 178, 258, 272
genetic diversity, 154, 179, 183
genetic mutations, 16, 113
genetic predisposition, 206 H
genetic screening, 17
genetics, 202, 220 half-life, 125, 154, 159, 166, 183, 246, 280
genome, 11, 149, 150, 151, 152, 153, 154, 155, 157, harbors, 164
158, 169, 170, 175, 177, 180, 181, 182, 183, 185, harmful effects, 88, 284
191, 196, 313, 324 HBV, 2, 3, 22, 24, 25, 26, 39, 51, 149, 150, 151, 152,
genomic instability, 163 153, 154, 155, 156, 157, 158, 159, 160, 161, 162,
genomic regions, 124, 191 163, 164, 165, 166, 169, 170, 171, 172, 173, 174,
genotypes, 149, 154, 155, 165, 166, 167, 179, 184, 175, 176, 178
192, 193, 195, 197, 198, 200, 201 HBV infection, 24, 25, 150, 157, 158, 159, 160, 162,
genus, 154, 179, 180 163, 165, 170, 173, 176
Germany, 273, 302 HBV replication, 26, 39, 152, 154, 155, 160, 161,
gland, 3 162, 169, 170, 172, 174, 175
glaucoma, 98, 101, 103 HCC, 2, 11, 16, 22, 24, 29, 30, 31, 100, 162, 163,
glia, 277, 295 166, 169, 170, 180, 204, 321, 322
glial cells, 125, 297 HCV, 2, 3, 22, 24, 25, 26, 33, 34, 37, 39, 51, 179,
Glioblastoma, 119, 121 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,
glucagon, 15, 244, 248, 250, 252, 254 191, 192, 193, 194, 195, 196, 197, 199, 200, 201,
glucocorticoid, 139 202, 326
gluconeogenesis, 60

340 Index
healing, 28, 43, 44, 56, 57, 105, 106, 107, 108, 109, hepatocarcinoma, 97, 100, 102
110, 116, 143, 144, 146 hepatocellular carcinoma, 2, 22, 25, 26, 30, 34, 35,
health, 34, 38, 39, 42, 125, 149, 179, 273, 275 37, 40, 41, 102, 104, 114, 155, 159, 162, 163,
heart, 3, 114, 125, 126, 131, 135, 136, 138, 140, 142, 164, 165, 167, 169, 179, 180, 199, 204, 205, 219,
143, 145, 231, 233, 240, 247, 250, 252, 291, 321 319, 320, 321, 330
heart disease, 3 hepatocytes, 2, 3, 7, 8, 11, 21, 22, 23, 24, 25, 27, 28,
heart failure, 114, 233 29, 30, 34, 37, 43, 44, 46, 47, 48, 52, 59, 60, 61,
heart rate, 131, 233, 247, 250 63, 64, 67, 69, 70, 72, 81, 91, 97, 98, 99, 100,
heart valves, 114 115, 116, 126, 127, 128, 138, 140, 152, 159, 160,
heat shock protein, 50 161, 162, 163, 170, 178, 187, 189, 191, 192, 198,
heat-shock protein 90 (Hsp90), 17, 248, 254 205, 206,208, 209, 211, 213, 215, 216, 217, 218,
height, 244 219, 220, 222, 224, 225, 226, 228, 229, 241, 290,
hematology, 61 292, 294, 321, 323, 327, 329
hematuria, 112 hepatocytes enlargement, 241
heme, 248, 251 hepatoma, 25, 39, 193, 202, 228, 321
heme oxygenase, 251 hepatomegaly, 253
heme oxygenase (HO), 210, 248, 249, 251, 287 hepatopulmonary syndrome, 131, 132, 138, 141,
hemochromatosis, 114 144, 146
hemorrhage, 231, 233, 234, 235, 280, 299 hepatorenal syndrome, 132, 136, 142, 145, 146
hemostasis, 121, 236 hepatotoxicity, 21, 22, 23, 32, 33, 35, 37, 40, 41,
hepatic encephalopathy, 80, 82, 85, 88, 129, 272, 130, 134, 212, 227
273, 274, 275, 276, 277, 278, 281, 284, 286, 287, heteroaryldihydro-pyrimidines, 175
291, 307, 308, 309 heterogeneity, 60, 73, 76, 184, 195
hepatic failure, 21, 69, 72, 75, 80, 116, 133, 141, high fat, 18, 209, 212, 228, 312
157, 272, 285, 290, 329 hippocampus, 86, 125, 259, 264, 265, 272, 293
hepatic fibrosis, 48, 73, 77, 137, 143, 231 histamine, 143
hepatic injury, 21, 34, 35, 103, 133 histidine, 85
hepatic necrosis, 41, 290 histology, 116, 170, 199, 310
hepatic obstruction, 233 histones, 118, 150, 166
hepatic stellate cell (HSC), 18, 27, 28, 37, 43, 44, 45, history, 5, 32, 35, 157, 164, 165, 166, 170, 184, 195,
46, 47, 48, 49, 50, 51, 59, 61, 62, 70, 71, 77, 98, 219, 241, 283, 284
99, 103, 105, 114, 115, 118, 131, 138, 141, 142, HIV, 39, 172, 173, 178
145, 146, 187, 191, 204, 213, 218, 226, 249, 254, HLA, 50, 186
323 HO-1, 248, 249
hepatic stellate cells, 18, 27, 28, 45, 59, 61, 62, 77, HO-2, 248, 249
98, 99, 103, 114, 131, 138, 142, 146, 187, 191, homeostasis, 2, 16, 18, 21, 45, 47, 48, 64, 67, 76, 79,
213, 226, 249, 254, 323 85, 87, 125, 206, 211, 213, 220, 273, 291, 308,
hepatitis, 1, 2, 16, 21, 24, 25, 30, 32, 33, 35, 36, 37, 329
38, 39, 40, 41, 42, 51, 67, 73, 100, 115, 116, 131, hormone, 28, 246
134, 139, 151, 156, 157, 159, 162, 163, 164, 165, hormones, 16, 63
166, 167, 169, 172, 176, 177, 178, 179, 180, 184, hospitalization, 278, 283
185, 188, 190, 193, 194, 195, 196, 197, 198, 199, host, 23, 67, 149, 150, 152, 157, 158, 159, 160, 161,
200, 201, 202, 290, 319, 320, 325, 326, 327, 328, 170, 177, 179, 180, 181, 182, 183, 184, 196, 198,
329, 330 201, 321
hepatitis a, 115, 139, 157, 164 HPC, 204
hepatitis B virus, 2, 32, 33, 34, 36, 39, 40, 41, 149, HROM, 310, 311, 313
151, 162, 163, 164, 165, 166, 167, 169, 170, 176, human, 1, 2, 6, 16, 23, 25, 27, 31, 33, 34, 35, 36, 37,
177, 178, 325, 326 41, 59, 60, 67, 69, 70, 72, 74, 75, 79, 80, 82, 99,
hepatitis C virus, 2, 32, 33, 35, 36, 37, 38, 39, 40, 41, 100, 101, 102, 106, 115, 125, 132, 135, 136, 138,
42, 67, 73, 104, 164, 179, 183, 190, 193, 194, 139, 141, 142, 143, 146, 149, 162, 163, 164, 165,
195, 196, 197, 198, 199, 200, 201, 202, 330 167, 193, 195, 199, 201, 209, 212, 215, 218, 220,
hepatitis d, 189, 195 221, 222, 223, 224, 225, 226, 227, 228, 231, 251,
hepatocarcinogenesis, 33, 163, 166, 167 254, 264, 323, 325, 326, 327

Index 341
human body, 60, 231

human brain, 138
I
human genome, 162
ICAM, 49
human health, 34, 222
ICP, 278, 280, 281, 282
human immunodeficiency virus, 67
ideal, 234
human subjects, 221
identification, 32, 184, 246
humoral immunity, 154
identity, 73
hyaluronate, 108, 109
idiopathic, 131, 140
hybrid, 321
idiosyncratic, 134
hydrogen, 28, 210, 211, 277
IFN, 55, 160, 170, 171, 173, 174, 175, 176, 186, 187,
hydrogen peroxide, 28, 210, 211, 277
188, 189, 295
hydrolysis, 13, 140, 206
IFNγ, 21, 186, 187
hydroxyl, 28, 153, 210
IHC, 312
hyperbilirubinemia, 278
IL-13, 21
hypercalciuria, 114
IL-8, 197, 211, 215
hyperdynamic state, 239, 240, 247, 248, 253
ileum, 84
hyperemia, 244, 246, 248, 250, 254, 281
imbalances, 283
hyperglycemia, 193
immune defense, 185, 186, 189
hyperinsulinemia, 28, 193, 207, 221, 222
immune function, 293
hyperinsulinism, 81
immune modulation, 319, 320, 324
hyperlipidemia, 207
immune reaction, 21, 295, 322
hypermethylation, 118, 162
immune regulation, 189
hypernatremia, 281
immune response, 23, 114, 157, 159, 160, 161, 163,
hypersensitivity, 242
164, 170, 176, 183, 184, 186, 187, 189, 190, 191,
hypertension, 26, 56, 57, 82, 84, 88, 90, 94, 95, 115,
194, 293, 321, 322
126, 127, 129, 130, 131, 132, 133, 134, 135, 136,
immune system, 8, 11, 20, 24, 66, 67, 83, 84, 98,
137, 138, 139, 140, 141, 143, 144, 146, 231, 232,
103, 149, 159, 160, 163, 171, 179, 181, 184, 189,
233, 234, 235, 236, 237, 238, 239, 240, 241, 242,
191, 199, 201, 228, 291
243, 244, 245, 246, 247, 248, 249, 250, 251, 252,
immunity, 39, 67, 76, 156, 161, 184, 185, 186, 187,
253, 254, 255, 264, 271, 274, 283, 300, 309, 316
188, 191, 200, 201, 322, 326
hypertonic saline, 281
immunization, 322
hypertriglyceridemia, 70
immunodeficiency, 75
hypertrophy, 127, 242, 265
immunoglobulin, 109, 182, 186, 188
hyperuricemia, 114
immunohistochemistry, 310
hyperventilation, 281
immunomodulatory, 169, 170, 174, 185
hypokinesia, 88, 259
immunoreactivity, 138, 142, 145, 265
hyponatremia, 283, 293
immunosuppression, 327
hypophosphatemia, 113
immunotherapy, 164, 321, 322, 328, 330
hyporeflexia, 276
impairments, 264
hyporesponsiveness of vasoconstrictors, 245
imprinting, 112
hypotension, 244, 245, 281, 282
improvements, 171
hypotensive, 20
in utero, 161
hypothalamus, 88, 125, 137, 142, 237
in vitro, 2, 5, 21, 23, 24, 25, 26, 27, 30, 31, 44, 46,
hypothermia, 282, 286, 301
48, 50, 61, 72, 79, 86, 141, 175, 180, 187, 191,
hypothesis, 27, 64, 205, 244, 246, 248, 259, 273, 298
195, 197, 200, 208, 216, 243, 247, 249, 251, 322,
hypovolemia, 234
324
hypoxemia, 131, 269, 309
in vivo, 3, 22, 23, 24, 26, 27, 46, 48, 50, 61, 71, 74,
hypoxia, 3, 9, 20, 39, 87, 124, 146, 268, 269, 280,
86, 87, 130, 140, 142, 155, 175, 180, 183, 191,
281, 293, 309
195, 199, 200, 202, 212, 216, 243, 248, 249, 254,
hypoxia-inducible factor, 124
272, 273
incidence, 169, 278, 280, 281
India, 98

342 Index
individuals, 70, 114, 123, 154, 156, 158, 179, 195, insulin resistance, 27, 28, 34, 116, 190, 191, 192,
203, 205, 218, 221 193, 205, 206, 207, 209, 211, 212, 215, 220, 221,
indolent, 160, 162 222, 224, 225
indomethacin, 246, 247, 282 insulin sensitivity, 209, 212
inducer, 50, 191, 322 insulin signaling, 193, 207, 209, 221, 223
induction, 5, 11, 12, 18, 23, 24, 26, 35, 37, 103, 108, integration, 296
144, 160, 161, 162, 163, 185, 186, 188, 191, 212, integrin, 112, 117, 118, 144
224, 263, 281, 292, 294, 296, 326 integrins, 109, 115, 116, 117, 118, 119
induration, 114 integrity, 5, 9, 86, 112, 218, 228, 277, 294, 295, 296,
industry, 87 297
INF, 49, 174, 186 intensive care unit, 278
infection, 12, 16, 24, 25, 33, 42, 75, 82, 84, 106, 149, interaction process, 321
150, 154, 155, 157, 158, 159, 160, 161, 163, 164, intercellular adhesion molecule, 49
165, 166, 169, 170, 171, 173, 175, 176, 179, 180, interface, 115, 167
183, 184, 185, 186, 187, 188, 189, 191, 192, 193, interference, 162, 259
194, 195, 199, 200, 201, 202, 279, 290, 292, 294, interferon, 159, 169, 170, 171, 174, 175, 176, 177,
295 179, 185, 186, 199, 201
inflammasome, 39, 224 interferon gamma, 159
inflammation, 2, 5, 6, 12, 21, 23, 24, 27, 29, 34, 49, interferons, 161, 185, 186
50, 100, 104, 109, 118, 149, 159, 160, 161, 162, interferon-β, 185
163, 184, 187, 188, 191, 193, 201, 203, 205, 206, interleukin-8, 211
213, 215, 218, 221, 223, 224, 227, 259, 279, 289, internalization, 12, 182
290, 291, 292, 293, 294, 295, 322, 326, 329 internalizing, 125
inflammatory cells, 21, 50, 110, 189 international normatized reason, 283
inflammatory mediators, 28, 49, 50, 116, 187, 295 interneurons, 103
inflammatory responses, 10, 50, 185, 189, 227, 292 intervention, 129, 165
ingestion, 29 intestinal flora, 283
inhibition, 10, 14, 17, 23, 24, 25, 30, 31, 32, 38, 45, intestinal vascular resistance, 241
62, 80, 110, 114, 127, 146, 160, 161, 162, 172, intestine, 84, 125, 127, 174
186, 208, 210, 215, 218, 222, 225, 228, 247, 249, intoxication, 87, 237, 241, 323
253, 255 intracellular calcium, 13, 49, 86, 127, 128
inhibitor, 7, 13, 14, 15, 27, 29, 34, 37, 42, 161, 178, intracellular calcium concentration, 244
205, 213, 216, 218, 229, 247, 253, 292 intracranial pressure, 278, 280, 286
initiation, 5, 7, 18, 19, 44, 102, 150, 182, 185, 199 intravenously, 65
injury, 1, 2, 3, 12, 18, 20, 21, 22, 23, 24, 26, 28, 31, intrinsic pathway, 9, 32, 194
32, 33, 34, 35, 36, 37, 39, 42, 43, 44, 45, 46, 48, invertebrates, 65
49, 50, 51, 59, 68, 69, 73, 74, 79, 84, 85, 88, 99, involution, 3
100, 105, 110, 112, 115, 116, 118, 127, 128, 130, ion channels, 296
132, 134, 138, 143, 144, 161, 162, 170, 179, 185, ion exchangers, 296
191, 193, 194, 195, 199, 202, 203, 206, 211, 212, ions, 14, 307, 310
213, 215, 217, 218, 219, 225, 226, 227, 229, 250, Iran, 323, 328
254, 263, 277, 289, 290, 292, 294, 307, 308, 319, iron, 28, 114, 248
320, 322 irritability, 81, 276
innate immune response, 28, 77, 160 IRS, 193, 204
innate immunity, 39, 185, 186, 189, 194, 198, 298 ischemia, 12, 14, 16, 18, 20, 37, 132, 140, 144, 281
inner ear, 3 ischemia-reperfusion injury, 14, 16, 18, 20, 37, 132,
inositol, 127, 211 140, 144
insertion, 128 ISHEN, 258, 272, 309
insulin, 22, 27, 28, 34, 116, 190, 191, 192, 193, 197, Islam, 252
205, 206, 207, 209, 211, 212, 215, 220, 221, 222, isolation, 16
223, 224, 225, 226 Israel, 137, 251, 253

Index 343
lipid metabolism, 27, 34, 41, 192, 201, 203, 206,

J 208, 219, 222
lipid peroxidation, 28, 206, 211
Japan, 155, 178, 193
lipids, 27, 28, 46, 64, 192, 206, 209, 210, 220, 263
jaundice, 278, 283
lipolysis, 28, 193, 206, 208
joints, 113, 114
lipoproteins, 65, 67, 74, 182, 188, 220
liquid interfaces, 73
K liver cancer, 39, 75, 163, 166, 329
liver endotelial cell (LSEC), 43, 45, 48, 49, 59, 60,
K+, 20, 53, 87, 90, 262 61, 62, 67, 68, 70, 71, 72, 73, 75, 76, 77, 124,
kidney, 84, 87, 112, 113, 114, 125, 140, 173, 294, 146, 189, 325, 329
308, 321 liver enzymes, 170, 171
kidney failure, 173 liver transplant, 20, 132, 133, 146, 175, 262, 275,
kidneys, 294, 308 276, 282, 283, 285, 286, 309, 323
kill, 24, 50, 197, 293 liver transplantation, 20, 132, 133, 146, 262, 275,
killer cells, 186 276, 282, 286, 309
kinase activity, 14, 198 localization, 11, 30, 139, 141, 143, 151, 152, 194,
kinetic studies, 15 259, 263, 265, 310
kinetics, 37, 76, 220 longevity, 40
Krebs cycle, 116 longitudinal study, 35
kupffer cells, 20, 28, 44, 47, 49, 50, 59, 60, 61, 67, loss of consciousness, 259
70, 74, 76, 77, 100, 115, 127, 129, 132, 213, 215, low risk, 156
227 low-density lipoprotein, 74, 75, 182, 192, 221
Kupffer cells, 20, 28, 44, 47, 49, 50, 59, 60, 61, 67, lumen, 59, 63, 80, 84, 154, 181
70, 74, 76, 77, 100, 115, 127, 129, 132, 213, 215, Luo, 77, 132, 141, 146, 322, 327
227 lymph, 64, 66, 68, 72, 75, 182, 199
lymph node, 66, 68, 182, 199
lymphocytes, 8, 20, 24, 50, 59, 60, 98, 170, 182, 187,
L 191
lymphoid, 74, 182, 325
lactic acid, 85, 313 lymphoid organs, 74
lactic acidosis, 85, 173, 313 lysis, 161, 310
laminar, 234
laminin, 7, 47, 105, 106, 109, 111
lamivudine, 155, 156, 157, 171, 172, 173, 174, 177 M
L-arginine, 246, 247, 252, 254
LDL, 68, 182, 204 machinery, 19, 23, 26, 42, 116, 191
lead, 7, 12, 13, 15, 18, 20, 24, 27, 28, 30, 32, 59, 72, macromolecules, 64, 65, 66, 67, 68, 76, 110
82, 86, 87, 90, 108, 112, 114, 116, 129, 159, 184, macrophage, 27, 35, 56, 65, 66, 67, 74, 132, 194,
187, 193, 207, 209, 210, 223, 240, 249, 259, 277, 212, 216, 218, 224
294, 295 macrophages, 5, 21, 44, 50, 51, 60, 65, 74, 76, 115,
leakage, 129, 216 125, 182, 195, 212, 215, 216, 298, 302
learning, 222, 259, 293 magnetic resonance, 274
lethargy, 276 magnitude, 128, 131, 155
leukemia, 321 major depression, 101
leukocytes, 49, 110 majority, 81, 117, 187, 188, 321, 324
life cycle, 164, 166, 180, 192 malabsorption, 283
lifetime, 150, 170 malignancy, 321
ligand, 7, 8, 11, 12, 23, 44, 74, 99, 125, 126, 185, malignant cells, 3, 30
186, 189, 204, 205, 211, 214, 215, 227, 229 malnutrition, 275, 308, 309
light, 18, 51, 140, 209, 222, 231, 273, 285, 321 mammal, 61
lipases, 27, 209 mammalian brain, 98
lipid bilayer, 64, 180 mammalian tissues, 107
mammals, 16, 18, 61, 154, 223

344 Index
management, 36, 100, 104, 136, 166, 177, 202, 244, meta-analysis, 287
277, 278, 281, 285, 286, 327 metabolic, 73, 93, 166, 195, 221, 263, 303, 315, 316
manganese, 32, 84, 88, 210, 257, 258, 262, 263, 272, metabolic acidosis, 308
273, 274, 277, 295, 309 metabolic changes, 264
manipulation, 38 metabolic disorders, 116, 215, 226, 282
mannitol, 281 metabolic dysfunction, 224
marfan syndrome, 108 metabolic pathways, 116, 129
marijuana, 98 metabolic syndrome, 30, 193, 206, 220
marketing, 134 metabolism, 64, 65, 66, 68, 70, 73, 79, 80, 81, 82,
marrow, 68, 321 84, 85, 86, 116, 125, 134, 136, 192, 193, 202,
MARS, 285 207, 208, 221, 237, 246, 258, 259, 263, 264, 271,
mass, 67, 81, 116, 192, 222, 308, 309, 312, 319, 320, 272, 273, 277, 294, 295, 307, 308, 319, 320
321 metabolites, 76, 80, 125, 174, 220
mast cells, 141 metabolized, 90, 125, 209, 257, 262, 263, 294, 298
materials, 16, 324 metabolizing, 79, 84
maternal inheritance, 313 metalloproteinase, 37, 47, 205
matricellular proteins, 107, 110 metastasis, 117, 118, 329
matrix, 43, 47, 66, 90, 105, 106, 107, 108, 109, 110, metformin, 223
111, 113, 116, 118, 130, 210, 263, 310, 312, 314 methodology, 322
matrix metalloproteinase, 47, 105, 106 methylation, 28, 118, 162
matrix metalloproteinases (MMPs), 47, 51, 105, 106, MHC, 50, 186, 321
115, 118 mice, 22, 25, 29, 31, 32, 35, 36, 37, 38, 39, 40, 44,
MCP, 21, 27, 46, 57, 114, 204, 215, 296 46, 66, 77, 99, 101, 102, 103, 126, 127, 130, 132,
MCP-1, 21, 27, 46, 57, 114, 204, 215, 296 139, 142, 178, 197, 199, 209, 212, 213, 215, 217,
mean arterial pressure, 245, 247, 281 218, 222, 223, 224, 225, 226, 227, 249, 292, 312,
measurement, 280 320
mechanical ventilation, 280 microbiota, 81, 83, 206
media, 294 microcirculation, 69, 114, 130, 132, 133, 236, 237
medical, 3, 98, 132, 319, 320, 324 microenvironments, 105, 106
medication, 280, 282, 283, 284 microparticles, 229
medicine, 126, 137, 201, 222, 320 microRNA, 162
Mediterranean, 155 microscope, 77
medulla, 125, 237 microscopy, 48, 72, 313
medulla oblongata, 125, 237 microspheres, 233, 242
MEK, 117 Middle East, 155
melanoblasts, 127 migration, 43, 46, 103, 105, 106, 108, 110, 112, 116,
MELD, 283, 308, 309, 316, 323 117, 118, 210, 293, 298
mellitus, 30 mineralization, 110, 113
membrane permeability, 12, 85, 90 mio-fibroblast like (MF-like), 105, 115
membrane stability, 30, 64 MIP, 114
membranes, 14, 20, 36, 72, 154, 186, 213, 262, 307, mitochondria, 8, 9, 11, 14, 16, 21, 23, 25, 29, 35, 37,
310 42, 85, 87, 90, 117, 193, 198, 207, 209, 214, 263,
membranous glomerulonephritis, 114 273, 292, 294, 296, 298, 313
memory, 160, 259, 262, 264 mitochondrial damage, 25, 27, 29, 88, 191, 312, 313
meninges, 114 mitochondrial DNA, 210, 313
mental status change, 279 mitochondrial myopathy, 313
mental status changes, 279 mitochondrial myopathy, encephalopathy, lactic
mesenchymal stem cells, 325, 326, 327, 328, 330 acidosis, and stroke (MELAS), 85, 92, 313, 316
mesenteric vessels, 254 mitogen, 10, 131, 212
mesentery, 75, 248 mitosis, 1, 30, 99
mesothelium, 44 MMP-2, 47, 121
messenger RNA, 97, 98, 145, 249 MMP-3, 47
messengers, 21, 150, 239, 240, 295 MMPs, 47, 51, 105, 106, 115, 118, 121

Index 345
models, 2, 3, 24, 27, 68, 79, 82, 101, 127, 130, 132, nanofibers, 328
157, 160, 180, 191, 192, 195, 206, 209, 215, 218, National Academy of Sciences, 54, 56, 57, 120, 139,
228, 231, 233, 248, 253, 254, 264, 271, 272, 279, 145
292, 293, 294, 297, 298, 322 natural killer (NK) cells, 8, 50, 60, 160, 167, 185,
modifications, 26, 108, 118, 234, 237, 266, 321 199
molecular biology, 157 nausea, 278
molecular weight, 9, 105, 106 necroptosis, 1, 2, 3, 13, 14, 15, 22, 27, 31, 33, 41, 42
molecules, 6, 7, 8, 9, 12, 15, 16, 19, 27, 28, 32, 47, necrosis, 1, 2, 3, 5, 7, 12, 13, 14, 15, 21, 22, 23, 24,
49, 50, 59, 66, 67, 83, 107, 109, 112, 116, 124, 28, 31, 32, 34, 35, 36, 37, 39, 40, 41, 42, 44, 86,
152, 162, 186, 321 87, 90, 115, 116, 119, 129, 134, 146, 149, 163,
morbidity, 3, 169, 170, 176, 218, 286, 307, 308, 319, 184, 185, 186, 191, 205, 214, 215, 227, 228, 253,
320 290, 291, 292, 299, 300, 302, 303
morphogenesis, 3, 10, 164, 166 neomycin, 283, 285, 287, 300
morphology, 19, 36, 59, 70, 290, 298 nephrocalcinosis, 114
mortality, 2, 38, 39, 41, 169, 170, 176, 205, 218, nerve, 106, 324
278, 286, 291, 307, 308, 309, 319, 320 nervous system, 125, 126, 127, 240, 245
mortality rate, 278, 319, 320 network-forming, 107
mortality risk, 39, 309 neuroblastoma, 195
motif, 14, 17, 109, 185, 215, 229 neurodegeneration, 86
mRNA, 12, 30, 42, 80, 97, 98, 99, 124, 127, 130, neurodegenerative diseases, 264
131, 138, 150, 176, 211, 216 neurodegenerative disorders, 98
mRNAs, 47, 150, 151, 153, 160 neurogenesis, 99, 103
mtDNA, 117, 313 neuroinflammation, 259, 272, 294, 295, 296, 297,
mucin, 188 298
mucosa, 242 neurologic symptom, 313
multidrug resistance protein-2, 128 neurons, 67, 79, 82, 84, 86, 101, 103, 125, 136, 265,
multiple sclerosis, 98, 102, 104 267, 268, 271, 277, 297, 298, 321, 327
multiplexins, 107, 108 neuropathy, 173
multipotent, 325 neuroprotection, 134
multivariate analysis, 133 neuropsychological tests, 261, 262
muscle mass, 84, 307, 308, 309, 310 neuroscience, 303
muscles, 64 neurotoxicity, 85, 87, 257, 263, 272, 277, 295
muscular mass, 308 neurotransmission, 79, 84, 85, 259, 272, 277, 294
mutagenesis, 158 neurotransmitter, 79, 84, 85, 259, 272, 277, 294, 295
mutant, 29, 41, 155, 157, 158, 165, 166, 188 neutral, 125, 134, 145, 146, 206
mutation, 29, 81, 88, 114, 150, 158, 163, 183, 188, neutrophils, 20, 215, 293
195, 200, 313 New England, 95, 137, 142, 144, 177, 178
mutation rate, 183, 195, 200, 313 NFκB, 100, 313
mutations, 11, 29, 81, 113, 116, 117, 139, 150, 155, NH2, 75, 226
156, 157, 158, 163, 164, 167, 188, 189 nicotinamide, 209
myocardial infarction, 3 nicotine, 70
myocardium, 141 nitrates, 244, 292, 293
myofibroblasts, 44, 46, 51, 107 nitric oxide (NO), 13, 21, 45, 48, 59, 61, 62, 68, 69,
myopathy, 313 70, 71, 86, 110, 124, 125, 127, 128, 129, 130,
myrcludex-B, 175, 178 132, 137, 142, 143, 144, 147, 195, 210, 222, 223,
myxedema, 114 236, 246, 247, 248, 249, 251, 252, 253, 254, 255,
292, 295, 296, 298
nitric oxide synthase, 61, 143, 147, 210, 222, 246,
N 247, 248, 249, 251, 254
nitrite, 292
Na+, 20, 87, 90, 92, 128
nitroglycerine, 240
Na+/taurocholate co-transporting polypeptide, 128
NK cells, 11, 186, 187, 189, 194
NAD, 13, 80, 210
NMDA receptors, 86, 273, 311
NADH, 209, 210

346 Index
nodes, 66 organs, 3, 60, 61, 65, 70, 77, 87, 106, 107, 114, 125,
nodules, 116, 232, 290 129, 131, 237, 242, 248, 251, 264, 283, 297, 319,
nonalcoholic fatty liver disease, 26, 32, 42, 204, 219, 320, 322
220, 221, 222, 223, 227 ornithine, 81, 297
nonalcoholic steatohepatitis, 22, 26, 32, 35, 36, 42, osmium, 61, 63
115, 203, 204, 205, 219, 220, 221, 222, 223, 227, osteogenesis imperfecta, 108, 113
229 osteomalacia, 113
non-cellular component, 105, 106 osteonectin, 68, 110
non-structural protein, 154 ovaries, 125
norepinephrine, 124, 137, 146, 237, 241, 242, 243, overlap, 3, 309
245, 246, 247, 252, 254, 281, 293 overnutrition, 27
normal development, 118 overproduction, 154, 247, 255
North America, 155 overweight, 205
nuclear genome, 313 ovulation, 142
Nuclear Magnetic Resonance, 305 oxidation, 27, 113, 193, 207, 208, 209, 211, 221,
nuclear membrane, 5, 307, 310, 314 223, 294
nucleation, 18, 19 oxidation rate, 210
nuclei, 86, 87, 237 oxidative damage, 20, 85
nucleic acid, 149, 210 oxidative stress, 8, 9, 13, 20, 21, 23, 25, 28, 38, 117,
nucleolus, 259, 298 162, 190, 191, 193, 203, 206, 209, 211, 212, 213,
nucleos(t)ide analogues, 169, 170, 177, 178 219, 223, 273, 277, 294, 298, 312, 313
nucleotide sequence, 139 oxygen, 11, 20, 45, 60, 133, 146, 205, 210, 211, 231,
nucleotides, 20, 28, 65, 150, 153 233, 240, 272, 281
nucleus, 5, 10, 150, 151, 152, 153, 160, 170, 186,
259, 298
nutrient, 16, 28, 60, 209, 215, 227 P
nutrients, 17, 63, 69, 240, 319, 320
p53, 10, 19, 25, 36, 41, 51, 117, 162, 164, 205, 227
nutritional status, 28
pain, 98, 110, 280
nystagmus, 276
pain perception, 110
pancreas, 114, 231
O paracentesis, 283
parallel, 20, 25, 102, 115, 183, 185, 188, 200, 234,
obesity, 27, 34, 99, 103, 192, 206, 209, 211, 212, 249, 323
215, 220, 222, 224 paralysis, 282
obstacles, 180 parasympathetic activity, 129
obstruction, 115, 116, 246 parenchyma, 59, 64, 72, 84, 115, 133, 160, 291, 295,
occlusion, 245, 285, 309 319, 320
oesophageal, 252 parenchymal cell, 28, 63, 64, 75, 99, 116, 130
offenders, 207 participants, 117
old age, 70, 77 patents, 103
oligodendrocytes, 297 pathogenesis, 3, 21, 24, 27, 30, 39, 40, 42, 50, 82,
oligomerization, 7, 23, 41, 214, 215, 216 83, 85, 88, 99, 126, 129, 131, 132, 133, 134, 141,
oocyte, 313 143, 144, 149, 159, 163, 165, 179, 181, 184, 187,
opportunities, 88, 203, 223, 272, 329 189, 190, 191, 197, 198, 201, 202, 203, 205, 212,
optic nerve, 322 218, 219, 220, 221, 223, 246, 248, 257, 258, 264,
optical microscopy, 307, 309, 310 269, 273, 279,291, 295, 296, 308, 309
ores, 296 pathogenesis of chronic HBV infection, 163
organ, 2, 3, 20, 47, 75, 79, 80, 81, 82, 110, 123, 126, pathogens, 10, 66, 87, 189, 293, 298, 322
130, 133, 149, 179, 189, 192, 242, 278, 290, 294, pathologist, 63
307, 308, 319, 321, 322, 324, 325 pathology, 3, 20, 21, 30, 31, 66, 79, 85, 88, 105, 106,
organelle, 17, 18, 20 112, 113, 114, 115, 199, 219, 250, 292, 307, 310,
organelles, 5, 13, 16, 17, 22, 29, 67, 72, 84, 87, 209 311, 313, 314, 321, 322, 326
organism, 3, 234, 320, 321, 324

Index 347
pathophysiological, 16, 26, 28, 34, 110, 126, 128, phosphorylation events, 10
131, 133, 248, 250, 273, 286 photomicrographs, 269
pathophysiology, 2, 27, 82, 85, 87, 123, 129, 138, physical environment, 320
141, 144, 203, 206, 220, 231, 246, 249, 264, 297, physical interaction, 201
314 physicians, 308
pathophysiology of the hepatic-portal system, 232 physiology, 16, 65, 115, 123, 125, 129, 144, 227,
pattern recognition, 50, 184, 185 272
PCR, 42 PI3K, 16, 18, 26, 30, 117, 121, 131
PDL, 50 PI3K/AKT, 30, 117
pegylated interferon, 169, 170, 174, 179 Picasso, 328
pegylated interferon lambda, 174 pilot study, 135, 322, 326, 329
peptidase, 9 pioglitazone, 220
peptide, 6, 48, 114, 124, 125, 128, 131, 133, 136, pipeline, 179
138, 140, 146, 175 placebo, 102, 135, 220, 223, 229, 247, 284, 328
perfusion, 60, 61, 63, 129, 132, 241, 242, 281, 286 placenta, 161
pericytes, 48, 60, 296, 297 plants, 207
perinatal, 159 plasma ET, 126
peripheral blood, 30, 32, 50, 188, 194, 200, 292, 326, plasma levels, 263, 292
330 plasma membrane, 5, 8, 11, 14, 15, 23, 34, 63, 90,
peripheral blood mononuclear cell, 30, 32, 194, 200 107, 109, 124, 127, 128, 152, 193, 214, 297
peripheral vasodilation, 239, 240, 242, 249 plasmapheresis, 328
peritonitis, 283 plasticity, 183
permeability, 20, 21, 34, 50, 63, 80, 85, 87, 106, 258, platelet activating factor, 236, 238
273, 291, 293, 295, 298 platelet aggregation, 110, 132, 236
permit, 70 platelets, 160
peroxynitrite, 210 platform, 9, 18, 214
personality, 259, 291 Plato, 136
PGI2, 236, 246, 254 plexus, 232, 297
pH, 85, 90, 137, 262 PM, 35, 166, 197, 225, 228, 237, 274, 327
phagocyte, 65, 76 point mutation, 41, 85, 155, 156
phagocytic cells, 5 polarity, 112, 182
phagocytosis, 5, 65, 66, 294, 298 polarization, 102
pharmaceutical, 133, 134, 320 pollutants, 63
pharmacokinetics, 137, 176, 178 polycythemia, 88
pharmacological agents, 133, 279 polymerase, 7, 149, 150, 151, 152, 153, 155, 156,
pharmacology, 61, 145 157, 163, 167, 169, 170, 172, 179, 181, 188, 194
pharmacotherapy, 103 polymerase mutants, 155
phenobarbitone, 237 polymerization, 153
phenomenology, 38 polymorphism, 101, 205
phenotype, 31, 35, 43, 44, 45, 46, 48, 49, 50, 51, 59, polypeptide, 29, 108, 128, 152, 154, 167, 170, 175,
60, 61, 62, 68, 69, 71, 72, 73, 85, 115, 118, 127, 176, 181
130, 144, 158, 162, 259 polysaccharides, 108
phenylalanine, 197 pons, 237
phenylpropenamide derivatives, 175 pools, 154, 231
phenytoin, 279, 280 population, 2, 32, 41, 46, 59, 89, 115, 183, 196, 202,
Philadelphia, 197, 273, 329 205, 261, 322, 323
phosphate, 11, 18, 81, 85, 209 population structure, 196
phosphatidylethanolamine, 18 porosity, 61, 70
phosphatidylserine, 5 portal hypertension, 26, 82, 84, 88, 129, 130, 131,
phosphoenolpyruvate, 88 135, 138, 140, 141, 143, 144, 231, 232, 233, 234,
phospholipids, 207 235, 236, 237, 238, 239, 240, 241, 242, 243, 244,
phosphorylation, 10, 21, 27, 29, 36, 42, 85, 126, 185, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,
210, 211, 213, 228, 248, 294, 295, 298, 313 255, 264, 271, 274, 283, 309

348 Index
Portal hypertension, 82, 129, 141, 231, 232, 234, protein family, 10, 69, 227
237, 239, 240 protein folding, 211
portal hypertension, hemorrhage and hemostasis, 234 protein kinase C, 109
portal vein, 44, 48, 50, 59, 60, 67, 77, 84, 127, 128, protein kinases, 7, 10, 131, 225, 247
130, 137, 138, 231, 232, 233, 237, 239, 240, 241, protein synthesis, 151, 310
242, 244, 245, 246, 247, 248, 250, 252, 257, 262, proteins, 5, 6, 7, 8, 9, 10, 11, 13, 16, 17, 18, 19, 20,
265, 294, 307, 323 21, 22, 24, 25, 26, 28, 29, 30, 32, 34, 38, 39, 40,
portal vein ligation, 137, 233, 307, 309 42, 47, 48, 50, 51, 66, 67, 77, 84, 98, 105, 106,
portal venous flow, 248, 255 107, 108, 109, 110, 113, 114, 115, 134, 149, 150,
portal venous pressure, 127, 128, 247, 255 151, 152, 154, 160, 161, 162, 163, 164, 165, 170,
portal venous system, 231, 243 174, 175, 180, 181, 182, 183, 184, 185, 186, 188,
porto-caval shunts, 239 191, 200, 203, 206, 211, 212, 214, 216, 217, 218,
positive correlation, 133, 213 219, 224, 228, 263, 264, 267, 277, 284, 285, 293,
positive feedback, 36 295, 296, 313
post-transplant, 283 proteinuria, 112
potassium, 49, 247, 262 proteoglycans, 47, 107, 108, 109, 110, 111, 114, 152
precursor cells, 76 proteolysis, 16, 20, 111, 310
pregnancy, 171, 172 protons, 210
pressure gradient, 243 prototype, 109, 113, 149
prevalence, 80, 118, 156, 180, 193, 205, 261, 279, proximal tubules, 125
283, 307, 308 psychiatric disorder, 97, 101
prevention, 140, 146, 180, 277 psychosis, 171, 264
prevention of cerebral edema, 279 psychotropic drugs, 283
primary biliary cirrhosis, 98, 100, 101, 329 PTEN, 164
primate, 72 pulmonary hypertension, 139
priming, 152, 172, 187 PUMA, 205, 212, 216, 217, 224, 225, 228
procurement, 283 PVL, 84, 248, 264, 265, 266, 267, 268, 269, 307,
producers, 79, 187, 308 309, 313
progenitor cell, 105, 106, 204, 322, 325, 326, 328,
329
progesterone, 142 Q
prognosis, 35, 84, 104, 132, 262, 276, 278, 281, 283,
quality control, 224
309
quality of life, 243, 262, 273, 275, 284, 309
programmed cell death, 2, 23, 31, 40, 42
quantification, 154
pro-inflammatory, 5, 13, 27, 44, 49, 50, 51, 124, 185,
quasispecies, 155, 157, 179, 183, 188, 195, 196, 197,
193, 212, 215, 216, 218, 219, 289, 291, 294, 295,
198, 202
296, 298
proliferating cell nuclear antigen, 313
proliferation, 1, 10, 12, 18, 31, 37, 45, 48, 50, 97, R
100, 102, 105, 106, 109, 110, 112, 116, 123, 130,
131, 141, 154, 163, 192, 215, 222, 226, 329 radiation, 3
proline, 108, 113 radical formation, 294
promoter, 44, 152, 155, 158, 162, 164, 165, 166, radicals, 20, 223
167, 197 radio, 117, 321
propagation, 24 radius, 235, 262
prophylaxis, 279, 280, 281, 282 RANTES, 56
propofol, 280 reactions, 13, 21, 28, 137, 179, 207, 210, 262, 284,
propranolol, 242, 243, 244, 251, 253, 254, 282 322
prostaglandin, 127, 129, 141, 293, 296 reactive oxygen, 13, 20, 21, 27, 44, 85, 87, 132, 209,
proteasome, 16, 28, 29, 32, 159 289, 291
protection, 22, 75, 97, 149, 156, 160, 187, 210 reactivity, 252, 253, 280, 327
protective role, 100, 206 reading, 30, 31, 149, 150, 179, 180, 181, 183, 188
protein components, 180 recall, 116, 240, 313

Index 349
recognition, 3, 5, 6, 26, 64, 67, 185, 186, 188, 205, restoration, 72, 112, 132, 146, 162
321 reticulum, 8, 10, 16, 27, 28, 29, 41, 110, 115, 127,
reconstruction, 140 163, 181, 204, 207, 209, 210, 221, 223, 224, 307,
recovery, 20, 59, 101, 211, 320, 327 310, 311
recreational, 98 retinol, 46
recurrence, 284, 321, 329 reverse transcriptase, 149, 153, 165, 170, 171, 178
recycling, 16, 87 rheumatic fever, 115
redistribution, 80, 258, 319, 320 rheumatoid arthritis, 115
reduction of cerebral ammonia, 279 ribose, 7
redundancy, 150 ribosomal RNA, 313
reflexes, 128, 143, 280 rickets, 113
regenerate, 319, 320, 323 rifaximin, 284
regeneration, 39, 48, 99, 100, 103, 105, 106, 112, rings, 63, 247, 248, 252, 253
116, 133, 149, 159, 162, 163, 232, 290, 320, 322, risk, 38, 70, 82, 101, 118, 155, 159, 170, 171, 172,
324, 325, 329, 330 174, 206, 236, 244, 262, 277, 280, 281, 283, 284
regenerative capacity, 319, 320 RNA, 26, 33, 50, 55, 149, 150, 151, 152, 153, 154,
regenerative medicine, 325 155, 158, 159, 162, 165, 172, 174, 175, 179, 180,
regression, 3, 51, 71, 77 181, 182, 185, 187, 192, 194, 195, 198, 200, 211
regulatory agencies, 320 RNA-guided clustered regulatory interspaced short
rejection, 3 palindromic repeats (CRISPR), 174, 175, 176,
relaxation, 105, 106, 128, 251 178
relevance, 14, 39, 40, 88, 126, 129, 133, 159, 164, rodents, 130, 133, 227
313 routes, 7, 257, 262, 295
remission, 173
remodelling, 135
renal dysfunction, 294 S
renal failure, 113, 132, 135, 280
safety, 134, 137, 171, 176, 178, 285, 326
renal replacement therapy, 282
salts, 23, 35, 41
renin, 124, 240
sarcolemma, 307, 310, 311, 314
repair, 7, 11, 18, 105, 106, 109, 114, 115, 116, 162,
sarcopenia, 84, 307, 308, 309, 311, 316
310, 313, 314, 319, 320, 321, 322, 329
saturated fat, 221, 222, 225
replication, 23, 25, 26, 39, 41, 116, 149, 150, 151,
saturated fatty acids, 221, 222
152, 153, 154, 155, 157, 158, 159, 160, 161, 162,
saturation, 281
163, 164, 166, 167, 169, 170, 171, 172, 174, 175,
scanning electron microscopy, 61, 63
179, 180, 181, 182, 183, 184, 185, 186, 188, 191,
scar tissue, 324
192, 197, 199, 200
scavenger endothelial cell, 59, 65, 76
repression, 30, 118
schema, 324
RES, 65, 66
schizophrenia, 101
researchers, 32, 191, 233, 321
science, 178
resection, 20, 133, 143, 319, 320
scleroderma, 101, 107, 114, 131, 135
residue, 153, 197, 218
scurvy, 107, 113
residues, 6, 11, 80, 124, 152, 153, 210
secrete, 8, 21, 24, 45, 115, 186
resistance, 30, 31, 44, 49, 77, 110, 117, 125, 126,
Secreted Protein Acidic Rich in Cysteine (SPARC),
128, 129, 130, 131, 141, 145, 155, 156, 157, 165,
68, 74, 110
167, 171, 172, 173, 178, 186, 193, 196, 199, 200,
secretion, 23, 29, 60, 83, 110, 127, 128, 129, 143,
204, 209, 222, 231, 232, 233, 234, 235, 236, 240,
145, 154, 159, 166, 185, 186, 192, 193, 200, 207,
241, 242, 243, 244, 246, 247, 248, 249, 250, 252,
221, 247
268
sedative, 283
resolution, 2, 37, 51, 159, 188, 307, 310, 324
seizure, 99, 104
resources, 275
selectivity, 133, 264
respiration, 87, 207, 209
self-assembly, 111
respiratory failure, 127
self-regulation, 281, 282
responsiveness, 130, 184, 248
senescence, 11, 50, 51, 110

350 Index
sensing, 198 spasticity, 102, 104

sensitivity, 109, 156, 209, 210, 242, 245, 252, 264, spatial learning, 259, 272
292 specialists, 275
sensitization, 28, 213 specialization, 106
sensor, 185 species, 5, 11, 13, 20, 21, 27, 44, 80, 85, 87, 132,
sensors, 13, 298 164, 205, 209, 277, 289, 291, 313
sepsis, 236, 278, 292, 308 speech, 276
septum, 44 spinal cord, 322, 324
serine, 14 spinal cord injury, 324
serum, 2, 24, 31, 33, 38, 41, 44, 69, 72, 83, 88, 133, splanchnic blood flow, 241, 242
154, 161, 170, 176, 180, 195, 196, 200, 206, 207, spleen, 68, 98, 231, 233, 292
209, 218, 247, 283, 308 Spring, 223, 227
serum albumin, 72 SR141716, 99
sham, 241, 243, 247, 248, 249, 265, 266, 309 stability, 30, 64, 167
sham-operated animals, 241, 248 stabilization, 257, 268, 269
shape, 43, 110, 150, 234, 265 starvation, 16, 17, 18, 26, 36, 41, 87, 310
shear, 106, 108, 124, 236, 247 steatosis, 25, 26, 27, 35, 36, 38, 39, 40, 42, 97, 100,
sheep, 136 103, 190, 191, 192, 193, 195, 196, 197, 199, 200,
shock, 20, 142, 248 202, 205, 206, 208, 211, 213, 215, 217, 218, 220,
showing, 61, 81, 115, 257, 259, 265, 266, 267, 269, 221, 224, 225, 226, 228
271, 289, 297, 312 stellate cells, 43, 60, 61, 62, 77, 115, 127, 128, 130,
side effects, 100, 130, 171, 240 131, 132, 143, 144, 145, 189, 248, 249
sieve plate, 63, 76 stem cells, 30, 115, 323, 324, 326, 330
sieve-raft hypothesis, 64 stenosis, 84, 237, 240, 246, 250, 309
signal transduction, 12, 30, 39, 185, 186 sterile, 206
signaling pathway, 12, 16, 19, 22, 28, 30, 31, 34, 41, stimulation, 15, 61, 127, 128, 161, 245, 253, 297
68, 112, 117, 126, 127, 144, 163, 185, 211, 215, stimulus, 51, 71, 116, 248
224, 247, 293 stomach, 83, 232, 235
signalling, 2, 7, 12, 32, 33, 37, 77, 80, 189, 227 storage, 34, 43, 45, 46, 194, 208, 319, 320
signals, 1, 7, 9, 23, 25, 31, 53, 90, 105, 106, 116, stress, 8, 9, 10, 12, 13, 21, 25, 27, 28, 31, 33, 37, 40,
141, 151, 152, 162, 185, 186, 187, 218, 222, 226, 86, 87, 105, 106, 108, 110, 124, 163, 194, 197,
227, 240, 295, 299, 324 203, 206, 208, 209, 211, 212, 215, 217, 219, 221,
signs, 81, 159, 259, 260, 280, 291 222, 223, 224, 225, 236, 244, 247, 258, 263, 277,
sinusoidal resistance, 128, 130, 145 293, 313, 314
skeletal muscle, 84, 87, 207, 245, 294, 307, 308, 309, stress response, 40, 211
311, 312, 313, 314 stroke, 85, 98, 101, 120, 146, 264, 277, 313
skeletal muscular cells (SMC), 308, 310 stromal cells, 112, 325
skin, 111, 113, 114, 127 structural protein, 37, 105, 106, 107, 180
skin diseases, 111 structure, 5, 6, 16, 18, 32, 63, 72, 75, 77, 107, 108,
small intestine, 64, 241, 294 111, 112, 113, 114, 134, 150, 151, 152, 154, 165,
smooth muscle, 44, 48, 107, 111, 114, 124, 125, 126, 174, 179, 184, 266, 296, 297, 307, 310, 311, 314,
127, 130, 203, 240, 242, 250, 251 320, 323, 324
smooth muscle cells, 44, 111, 114, 125, 126, 127 stupor, 259
snake venom, 124 subacute, 290
SNP, 205 subarachnoidal, 297
sodium, 14, 152, 170, 281, 283, 286 sub-clinical seizures, 279
software, 286 subcutaneous injection, 171, 323
soluble guanylate cyclase (sGC), 61, 70, 71 subgroups, 107, 108
solution, 3, 241, 262, 281, 285 submucosa, 242
somnolence, 276 substitutions, 155, 157
SONIC, 276 substrate, 5, 6, 12, 16, 17, 47, 80, 109, 138, 197, 204,
Southeast Asia, 155 212, 262
Spain, 95, 272 sucrose, 312

Index 351
sulfate, 74, 108, 109, 110, 111, 151, 152 telbivudine, 155, 156, 171, 172, 173
sulfonamide, 134, 176 temperature, 8, 10, 282, 291
sulphamoylbenzamide derivatives, 175 temporal lobe, 101
Sun, 39, 42, 198, 322, 326, 329 temporal lobe epilepsy, 101
supplementation, 308 Tenascin-C, 110
suppression, 29, 39, 155, 162, 170, 174, 176, 177, tenofovir alafenamide, 173, 176, 178
188, 216, 224, 228 tenofovir disoproxil fumarate, 172
surface area, 241 tensile strength, 107, 108
surgical removal, 326 tension, 116, 235, 248, 291
surveillance, 139 terlipressin, 281, 286
survival, 10, 12, 13, 14, 15, 16, 18, 21, 24, 30, 31, territory, 59, 197
41, 45, 50, 51, 82, 87, 116, 117, 118, 127, 146, testing, 32, 134, 261, 276
157, 211, 215, 218, 243, 262, 277, 278, 279, 281, tetrahydrocannabinol (THC), 97, 98, 99, 101, 104
283, 309, 319, 320, 321, 326 textbooks, 66
survival rate, 118 TGF, 45, 48, 50, 56, 114, 162, 189, 205, 211
susceptibility, 25, 213, 219, 292 Th1 polarization, 325
suspensions, 312 therapeutic agents, 100, 176, 319, 320
swelling, 13, 14, 80, 82, 85, 90, 132, 259, 277, 281, therapeutic approaches, 2, 180
291, 293, 297, 298, 314 therapeutic process, 321
symmetry, 153 therapeutic targets, 33, 42, 133, 166
sympathetic nervous system, 124, 129, 138 therapeutics, 134, 231
symptoms, 35, 98, 171, 173, 258, 259, 274, 276, 277, therapy, 34, 71, 102, 134, 139, 146, 155, 156, 157,
285, 290, 291 169, 170, 171, 172, 173, 177, 178, 179, 184, 200,
synapse, 110 208, 215, 227, 248, 253, 279, 280, 281, 282, 283,
synaptic transmission, 277, 295 285, 320, 324, 325, 326, 329
syndrome, 81, 82, 85, 88, 131, 132, 138, 141, 144, thorax, 234, 235
146, 173, 203, 205, 239, 245, 259, 264, 275, 276, threonine, 14
286 thrombin, 48, 140, 141
synthesis, 45, 47, 49, 64, 66, 80, 85, 87, 97, 98, 99, thrombospondin (TSP-1), 110
100, 102, 103, 107, 108, 112, 113, 124, 127, 131, TIMP, 47, 118, 205
133, 144, 146, 150, 152, 153, 157, 162, 172, 183, TIMP-1, 47, 118, 205
192, 193, 208, 210, 211, 213, 221, 234, 236, 246, TIMP-2, 47
247, 254, 263, 292, 294, 309, 319, 320 TIPS, 285
systemic blood flow, 239 TIR, 185
systemic hyperkinetic syndrome, 245 tissue, 1, 3, 5, 21, 28, 30, 31, 36, 37, 43, 44, 47, 66,
systemic sclerosis, 131, 140 80, 84, 87, 100, 105, 106, 107, 108, 109, 110,
112, 113, 114, 115, 116, 117, 118, 123, 125, 130,
131, 135, 138, 139, 140, 160, 180, 184, 185, 187,
T 189, 192, 206, 207, 209, 257, 263, 264, 293, 294,
295, 296, 307, 308, 309, 314, 319, 320, 321, 322,
T cell, 37, 50, 98, 110, 154, 159, 160, 161, 162, 170,
324
185, 186, 187, 188, 189, 194, 197, 198, 322, 327,
tissue engineering, 324
329
tissue homeostasis, 1, 31, 117
T lymphocytes, 8, 24, 30, 50, 182, 197, 325
tissue perfusion, 125
T regulatory cells, 201, 322
TLR, 27, 50, 176, 187, 205, 292, 326
T tubes, 307, 311
TLR2, 50
Taiwan, 156, 165, 197
TLR3, 50, 185, 186
target, 11, 12, 16, 27, 41, 85, 86, 87, 101, 103, 123,
TLR4, 50, 56, 205, 213, 225
126, 139, 145, 146, 155, 174, 175, 188, 189, 193,
TLR9, 50
194, 201, 215, 218, 226, 267, 308, 314, 321
TNF, 7, 8, 11, 12, 14, 15, 18, 22, 32, 33, 36, 44, 49,
taxonomy, 166
50, 186, 193, 205, 211, 213, 214, 215, 225, 226,
TCR, 188
227, 247, 291, 292, 293, 294, 295, 296, 297, 298,
techniques, 313
301, 302, 304
technology, 65

352 Index
TNF-α, 22, 36, 205, 211, 215, 226, 227, 247, 291, trial, 118, 135, 140, 145, 218, 220, 223, 245, 287,
292, 293, 294, 295, 296, 297, 298 322, 323, 324, 326, 327, 328, 329, 330
toll-like receptor (TLR), 27, 50, 176, 187, 205, 292, tricarboxylic acid, 210, 294
326 tricarboxylic acid cycle, 294
tonsils, 98 triggers, 9, 11, 14, 28, 33, 37, 85, 130, 185, 194, 203,
topology, 152, 165 217, 239, 250, 307, 314
toxic effect, 86, 208, 273, 277 triglycerides, 64, 100, 206
toxicity, 21, 22, 79, 80, 81, 82, 86, 88, 99, 104, 134, tropism, 149
203, 208, 213, 216, 217, 218, 225, 229, 258, 263, tumor, 7, 15, 18, 30, 31, 32, 33, 44, 100, 101, 117,
283, 292, 294 159, 162, 185, 186, 214, 215, 227, 247, 253, 321,
toxin, 212 322, 330
TRA, 7, 14, 15 tumor cells, 117, 321
trafficking, 23, 28, 41, 50, 81, 166, 296, 307, 310 tumor metastasis, 30
traits, 66 tumor necrosis factor, 7, 15, 32, 33, 159, 185, 186,
transaminases, 80 214, 215, 227, 247, 253
transcription, 11, 26, 30, 40, 75, 102, 124, 128, 151, tumor necrosis factor alpha, 33, 146, 159, 205, 247,
153, 154, 158, 163, 166, 172, 174, 175, 177, 182, 253
185, 192, 203, 207, 211, 217, 267, 271 tumor progression, 117
transcription activator-like effector nucleases tumorigenesis, 31, 35, 48, 110
(TALENs), 174, 177 tumors, 30, 31, 113, 117, 133
transcription factors, 11, 154, 163, 185, 212 TUNEL assay, 310, 312, 313
transcripts, 31, 150, 151, 152, 160 turnover, 16, 17, 27, 43, 47, 65, 66, 155, 242
transduction, 109, 248 type 2 diabetes, 193, 221, 224
transfer RNA, 313 tyrosine, 127, 142, 153, 171, 182, 198, 210
transformation, 11, 30, 150 Tyrosine, 253, 304
transforming growth factor, 15, 47, 114, 124, 211 tyrosine hydroxylase, 142
transforming growth factor β, 114, 124
transfusion, 180, 329
translation, 26, 150, 180, 181, 182, 183, 186, 198, U
199, 216
ultrastructure, 61, 310
translocation, 14, 17, 23, 30, 33, 50, 80, 83, 84, 128,
umbilical cord, 30, 327, 329, 330
152, 181, 193, 228
unions, 108
transmembrane, 7, 11, 17, 98, 107, 108, 115, 120,
United States (USA), 38, 39, 41, 54, 56, 57, 73, 75,
151, 152, 181, 225, 296, 311
76, 702, 120, 134, 139, 145, 177, 273, 278, 280,
transmission, 63, 72, 165, 184, 200, 310
286, 329
transmission electron microscopy, 63, 310
untranslated regions, 180, 181
transplant, 133, 179, 275, 278, 279, 282, 283, 323,
urea, 79, 81, 84, 259, 263, 277, 294, 297, 307, 308
326
urea cycle, 81, 259, 294, 297
transplantation, 262, 278, 285, 286, 325, 326, 327,
uric acid, 114
328
urine, 294, 308
transport, 9, 26, 29, 46, 67, 87, 88, 116, 134, 145,
Uzbekistan, 193
204, 206, 210, 211, 244, 263, 297
transport processes, 9
treatment, 12, 22, 36, 42, 71, 81, 86, 97, 98, 100, V
101, 126, 129, 131, 132, 134, 136, 137, 138, 140,
143, 154, 155, 165, 169, 170, 171, 172, 173, 174, vaccinations, 322
176, 177, 178, 179, 195, 199, 211, 213, 223, 226, vaccine, 149, 156, 157, 164, 167, 179, 321, 322, 325,
228, 233, 241, 243, 244, 247, 255, 275, 277, 278, 326, 327, 329, 330
279, 282, 283, 284, 285, 287, 289, 293, 308, 321, vacuole, 18
322, 323, 324 variables, 283
tremor, 88 variations, 106, 114, 234
triad of skeletal muscle, 310 vascular cell adhesion molecule, 27
vascular endothelial growth factor, 45, 48

Index 353
vascular endothelial growth factor (VEGF), 45, 48 Vitamin C, 107, 108, 113
vascular system, 240 vitamin D, 113
vascular tone, 123, 125, 129, 138, 240, 244, 248 vitamin E, 211, 223
vasculature, 125, 126, 127, 130, 132, 136, 246, 249, VLDL, 182, 192, 205, 207, 208, 221
292, 295 voltage dependent anion channel, 9
vasculitis, 115
vasoactive substances, 44, 45, 48, 62, 239, 240, 292
vasoconstriction, 125, 126, 127, 128, 132, 137, 140, W
142, 244, 247, 249, 281
waking, 259
vasodilation, 125, 129, 132, 233, 239, 240, 241, 242,
Washington, 55
245, 248, 249, 250
waste, 64, 65, 66, 67, 69, 76
vasodilator, 48, 132, 134, 243, 245, 248, 249
waste clearance, 65
vasopressin, 48, 146, 240, 244, 247, 251, 254
water, 82, 90, 108, 210, 285, 293, 296, 297, 308
vasopressor, 282
weakness, 87, 313
VCAM, 49
wear, 65
vegetables, 285
web, 182, 183, 201
VEGF, 61, 71, 205
Wechsler Adult Intelligence Scale (WAIS), 261
vein, 21, 44, 47, 60, 130, 231, 232, 233, 234, 235,
West Africa, 184
240, 241, 242, 246, 294, 309, 323
west haven criteria, 276
velocity, 234, 235, 243, 250
white blood cell count, 291
venous oxygen saturation, 281
WHO, 156
ventricle, 129, 135
wild type, 155, 175
venules, 60, 140
WIN55,212-2, 100, 102
vertebrates, 65, 66, 76, 319, 320
windows, 63
very low density lipoprotein, 200, 207
withdrawal, 17, 137, 242, 285
vesicle, 18, 29
workers, 64, 87, 123
vessels, 64, 75, 108, 114, 115, 129, 232, 233, 234,
worldwide, 30, 149, 156, 169, 179, 184, 203, 205,
235, 240, 247, 257, 270, 309
218, 320
Vietnam, 155
wound healing, 105, 106, 108, 109, 110, 143, 144,
viral gene, 153
146
viral hepatitis, 1, 2, 22, 23, 30, 115, 116, 131, 164,
177, 189, 196, 198, 319, 320
viral infection, 23, 25, 130, 187, 205, 319, 320, 321 Y
virology, 61, 164
virus infection, 32, 33, 35, 36, 37, 42, 162, 163, 164, yeast, 17, 156
165, 166, 167, 177, 178, 194, 195, 196, 197, 199, yield, 99
200, 201, 202
virus replication, 157, 158, 161, 166, 183
viruses, 13, 23, 24, 25, 51, 66, 75, 149, 152, 153, Z
155, 165, 166, 189, 197
viscosity, 69 zinc, 174
vision, 88 zinc-finger nucleases (ZFNs), 174, 177
vitamin A, 43, 45, 46, 51, 77

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