Review
This paper contains Video
Endothelial barrier dysfunction in septic shock
S. M. Opal1 & T. van der Poll2
From the 1Infectious Disease Division, Alpert Medical School of Brown University, Pawtucket, RI, USA; and 2Academic Medical Center,
Division of Infectious Diseases & The Center of Experimental and Molecular Medicine, University of Amsterdam, Amsterdam, the Netherlands
Abstract. Opal SM, van der Poll T (Alpert Medical
School of Brown University, Pawtucket, RI, USA;
University of Amsterdam, Amsterdam; the
Netherlands). Endothelial barrier dysfunction in
septic shock (Review). J Intern Med 2015; 277:
277–293.
The endothelium provides an essential and selec-
tive membrane barrier that regulates the move-
ment of water, solutes, gases, macromolecules
and the cellular elements of the blood from the
tissue compartment in health and disease. Its
structure and continuous function is essential for
life for all vertebrate organisms. Recent evidence
indicates that the endothelial surface does not
have a passive role in systemic inflammatory
states such as septic shock. In fact, endothelial
cells are in dynamic equilibrium with a myriad
of inflammatory mediators and elements of the
Introduction
Sepsis, severe sepsis and septic shock comprise a
spectrum of increasingly common, potentially
lethal, yet poorly understood clinical syndromes.
The rising incidence is well documented and
thought to be attributable to the ageing of the
population, the prevalence of immunocompro-
mised patients and implantable medical devices
and the progressive increase in antimicrobial resis-
tance among common bacterial pathogens [1–5].
Developing new therapeutic agents to manage
patients with sepsis has proven difficult with a
large number of failed clinical trials [6–8].
Despite the repeated failure to demonstrate sur-
vival benefits for a number of promising novel
therapeutic agents for the treatment of sepsis in
Phase III clinical trials, the news regarding clinical
outcomes in patients with sepsis is not all bad [6,
7, 9]. The mortality rate is certainly improving in
many clinical studies worldwide, which is primarily
a reflection of improved supportive care and early
diagnosis and treatment protocols [10–13]. Addi-
tionally, substantial progress has been made in
ª 2014 The Association for the Publication of the Journal of Internal Medicine
doi: 10.1111/joim.12331
innate immune and coagulation systems to
orchestrate the host response in sepsis. The
barrier function of the endothelial surface is
almost uniformly impaired in septic shock, and
it is likely that this contributes to adverse out-
comes. In this review, we will highlight recent
advances in the understanding of the signalling
events that regulate endothelial function and
molecular events that induce endothelial dysfunc-
tion in sepsis. Endothelial barrier repair strategies
as a treatment for sepsis include modulation of
C5a, high-mobility group box 1 and VEGF recep-
tor 2; stimulation of angiopoietin-1, sphingosine 1
phosphate receptor 1 and Slit; and a number of
other innovative approaches.
Keywords: endothelial barrier, endothelial junctions,
protease-activated receptors, sepsis, sepsis-induced
immunosuppression, septic shock.
understanding the basic pathophysiology of sepsis
from the molecular level to the level of organ
communication and systems integration [14–24].
Currently, the nature of sepsis is viewed as a
fundamental disintegration of systems controls
from intercellular signalling networks to neuroen-
docrine and immune regulatory mechanisms dur-
ing the time of systemic injury and invasive
microbial stress. Failure to maintain or reconsti-
tute homeostasis after a major systemic injury
ultimately determines outcome in sepsis [7, 19,
22]. The immediate threat in sepsis is invasive
infection, and the need to activate immune
defences to clear the pathogen before irreparable
damage occurs due to the microorganism and its
toxins. However, in the process of eliminating the
pathogen, the systemic host response to general-
ized infection can lead to collateral damage to
normal tissues [4]. It is remarkable that many of
the same pathways, pattern recognition receptors
and innate host responses seen in sepsis are also
observed in other forms of severe injury and
systemic inflammation without concomitant
infection [2, 4, 22].
277
 S. M. Opal & T. van der Poll
The long-term consequences of sepsis can be
profound and disabling. These consequences are
increasingly recognized as a condition now referred
to as persistent critical illness (PCI). PCI may
persist for months or even years and is character-
ized by prolonged lengths of stay in the intensive
care unit, persistent organ dysfunction, dysfunc-
tional host response to repeat infections, slow or
even permanent cognitive decline and losses of
overall sense of well-being and function in society
[25–27]. Sepsis is frequently followed by a reduced
duration of healthy life expectancy, even if the
patient’s overall lifespan is preserved.
In this review, we will focus on recent findings
regarding the basic elements that drive the septic
process at the level of cellular communication, the
coagulation system and endothelial barrier func-
tion. Of note, other research priorities are clearly
important in understanding the basic mecha-
nisms that underlie the pathophysiology of sepsis,
including disordered immune function [28–30],
neuroendocrine [31–33], nutritional [34], immune
metabolism [18], mitochondrial sparing [23, 35,
36] and systems integration studies [14, 19].
The importance of immune dysfunction and ele-
ments of immune depression in sepsis is increas-
ingly being recognized, and they might prove to be
suitable targets for therapeutic intervention
[28–30]. Loss of adaptive immune function and
downregulation of the host innate immune
response have been well described and cause
adverse consequences. For example, opportunistic
viral infections are reactivated in the majority of
patients with severe sepsis, and these infections
can cause clinically significant conditions such as
systemic cytomegalovirus infection and herpes
simplex activation [37]. What is not clear is
whether sepsis-induced immune suppression is a
necessary compensatory mechanism to regulate
the systemic septic host response or a potentially
treatable, pathological state of immune suppres-
sion increasing the risk of superinfections in
patients [38, 39]. This question can only be
answered by carefully controlled clinical trials in
well-characterized patient populations.
We wish to highlight the loss of cellular barriers
and its consequences at the level of the endothelial
membrane to make our point about loss of function
of vascular integrity in sepsis. In a recent review,
Deutschman and Tracey emphasized that loss of
cell membrane barrier function is a general finding
278 ª 2014 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2015, 277; 277–293
Review: Endothelial dysfunction in sepsis
in septic patients [7]. The histopathological find-
ings of autopsy examinations of patients who have
died as a result of multiorgan dysfunction due to
sepsis (and in experimental animal studies of
sepsis) are often remarkably normal appearing,
characterized primarily by mild tissue oedema in
the intracellular and extravascular interstitium.
These anatomical findings seem incongruous with
the clinical findings in such patients with evidence
of acute kidney injury, cholestatic jaundice, acute
myocardial dysfunction and similar dysfunctions
in other organs. Despite the relative preservation of
tissue morphology, tissue function is often mark-
edly impaired. Cardiac myocytes stop contracting
normally, alveoli cease to maintain the air–liquid
barrier interface in lung tissue, hepatocytes no
longer secrete bilirubin, endothelial cells retract,
become permeable to macromolecules and lose
their anti-adhesive and anticoagulant surface
characteristics, and so on. The entire process
seems to be a manifestation of poor cellular and
tissue barrier function, loss of specialized tissue
actions and an acquired form of cellular hiberna-
tion. Tissues stop generating variability in the
integrated circuitry and stop generating cycles of
communication within and between tissues. The
loss of specialized cell function and barrier func-
tion has been noted by many investigators, sug-
gesting that this constitutes a common host
response to sepsis and other forms of critical
illness [7, 15, 22].
Here, we will review the evidence that loss of barrier
function in general, and endothelial barrier func-
tion in particular, is central to the pathogenesis of
sepsis and highly integrated into the host systemic
inflammatory and coagulopathic response. A num-
ber of authors have recently developed this concept
of the aberrant and dysfunctional endothelial bar-
rier as the central pathophysiological process in
septic shock [40–44]. We will also discuss how
endothelial barriers are influenced by coagulation
factors and elements of the innate immune
response, and review novel findings concerning
monocyte/macrophage–endothelial interactions
and signalling in sepsis.
The vascular endothelium and haemostasis
Endothelial cells are at the interface between
inflammation and coagulation in sepsis [43]
(Fig. 1). Dysfunction of the endothelium has an
important role in the disturbance of the haemo-
static balance in sepsis. Moreover, the endothelium
 S. M. Opal & T. van der Poll Review: Endothelial dysfunction in sepsis

Coagulation ↑ Anticoagulation ↓
MyD88-ARNO-ARF6

TFPI ↓ Endothelial cell

Tissue factor ↑ TM ↓ EPCR ↓ Tie2 Robo4
Antithrombin↓ Protein C (↓)
Microcirculation

Angiopoietin-2 ↑
Monocyte
APC ↓
Blood pressure ↓
Fibrinolysis ↓
NETs with trapped
platelets PAI-1 ↑ RBC deformability↓
APC ↓ - Thrombin ↑

Thrombosis VE-cadherin↓ Cell death

Neutrophil PAR1 Tight junctions ↓ Cell shrinkage
S1P3/S1P1 ↑

Tissue hypoperfusion Loss of barrier function

Capillary leak
Interstitial oedema
Tissue

Tissue oxygenation ↓

Organ failure

Fig. 1 Dysfunction of the vascular endothelium in severe sepsis. Sepsis is associated with microvascular thrombosis due to
concurrent activation of coagulation (mediated by tissue factor) and impairment of anticoagulant mechanisms as a
consequence of reduced activity of endogenous anticoagulant pathways mediated by activated protein C (APC),
antithrombin and tissue factor pathway inhibitor (TFPI) plus impaired fibrinolysis due to enhanced release of plasminogen
activator inhibitor type I (PAI-1). The capacity to generate APC is impaired at least in part due to reduced expression of the
endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). Thrombus formation is further
facilitated by neutrophil extracellular traps (NETs) released from dying neutrophils. Thrombus formation results in tissue
hypoperfusion, which is aggravated by hypotension and reduced red blood cell (RBC) deformability. Tissue oxygenation is
further impaired by loss of barrier function of the endothelium due to loss of vascular endothelial (VE)-cadherin function,
alterations in the endothelial cytoskeleton, high angiopoietin-2 levels (which interact with the endothelial cell receptor Tie2),
and activation of Myd88–ARNO–ARF6 signalling. ARF6 is adenosine diphosphate-ribosylation factor 6; ARNO is ARF
nucleotide-binding site opener. A disturbed balance between sphingosine 1 phosphate receptor (S1P)1 and S1P3 within the
vascular wall at least in part due to preferential induction of S1P3 via protease-activated receptor 1 (PAR1) secondary to a
reduced APC/thrombin ratio. Robo4 is an endothelial cell receptor that protects barrier function.

is involved in all three major pathogenetic path-
ways associated with coagulopathy in sepsis: tis-
sue factor (TF)-mediated thrombin generation,
dysfunctional anticoagulation and impaired fibri-
nolysis.
TF is the main initiator of coagulation activation in
sepsis. It is clear that monocytes and macrophages
are major sources of TF in severe sepsis; however,
endothelial cells also contribute to a considerable
extent [44]. Endothelial cells activated by bacterial
products or proinflammatory cytokines such as
tumour necrosis factor (TNF)-a or interleukin (IL)-
1b express TF at their surface. Expression of TF by
endothelial cells in vivo is restricted to certain
organs and vascular beds [42]. TF binds to and
activates clotting factor VII, which via factor X
results in the generation of thrombin and fibrin.
The pivotal role of TF in sepsis-induced coagulation
has been established in early clinical studies in
humans and primate models in which strategies
that prevent the activation of the TF–factor VIIa
pathway abrogated the activation of the common
pathway of coagulation elicited by administration
of lipopolysaccharide (LPS) or bacteria [45]. In
addition, in lethal sepsis models in baboons, TF
inhibition prevented multiple organ failure and
mortality [46, 47]. Similarly, in genetically deficient
mice with an almost complete lack of TF, coagula-
tion, inflammation and mortality induced by injec-
tion of high-dose LPS were reduced relative to
control mice [48]. In addition, in its cell-associated
form, TF can reside in microparticles (MPs) shed
from haematopoietic and endothelial cells. MPs

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Journal of Internal Medicine, 2015, 277; 277–293
 S. M. Opal & T. van der Poll
have been implicated in the activation of both
coagulation and inflammation in sepsis [49]. Fur-
thermore, upon stimulation with proinflammatory
cytokines, endothelial cells are able to release
alternatively spliced TF (lacking exon 5) which
circulates in a soluble form and may exert proco-
agulant activity [50].
Coagulation is regulated by three main anticoagu-
lant mechanisms: antithrombin, the protein C
system and TF pathway inhibitor (TFPI). Sepsis is
associated with impaired function of all three
pathways, primarily as a consequence of endothe-
lial dysfunction [44]. Resting endothelial cells gen-
erate activated protein C (APC) on the cell surface,
produce tissue-type plasminogen activator to stim-
ulate fibrinolysis and impede thrombin formation
and platelet adhesion. Antithrombin is the main
inhibitor of thrombin and activated (a) factor X
(Xa). Under physiological conditions, glycosamino-
glycans present on the vessel wall support anti-
thrombin-mediated inhibition of thrombin and
other clotting enzymes. During sepsis, antithrom-
bin concentrations are strongly reduced due to
impaired production, enhanced degradation (at
least in part by elastase from activated neutrophils)
and consumption caused by sustained thrombin
generation [45]. Antithrombin function is further
compromised by the decreased production of
glycosaminoglycans on the endothelial surface,
which is mediated by proinflammatory cytokines
[43].
TFPI is the main inhibitor of the TF–factor VIIa
complex, which is predominantly expressed by
endothelial cells [51]. TFPI binds with high affinity
both to TF–factor VIIa and to factor Xa present in
plasma and on endothelial cells. Under normal
conditions, TFPI is attached to the endothelium via
proteoglycans, which facilitates its TF–factor VIIa–
factor X-inhibiting properties on the endothelial
surface [52]. TFPI function is inhibited in sepsis by
the reduced synthesis of glycosaminoglycans on
the endothelial surfaces. The importance of TFPI as
an endogenous anticoagulant in sepsis is illus-
trated by the fact that rabbits became more
susceptible to LPS-induced disseminated intravas-
cular coagulation (DIC) and the generalized
Shwartzman reaction following inhibition of TFPI
[53, 54]. Administration of recombinant TFPI dose-
dependently inhibited LPS-induced thrombin gen-
eration in humans [55]. Pharmacological doses of
TFPI can prevent mortality during systemic infec-
tion and inflammation, indicating that high TFPI
280 ª 2014 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2015, 277; 277–293
Review: Endothelial dysfunction in sepsis
concentrations are able to modulate TF-mediated
coagulation [46, 47].
The vascular endothelium serves an important role
in the protein C system, which represents a crucial
endogenous anticoagulant mechanism by virtue of
the ability of APC to proteolytically inactivate
coagulation cofactors Va and VIIIa. APC is formed
from protein C when thrombin binds to the throm-
bomodulin receptor present on endothelial cells.
The activation of protein C to APC by thrombo-
modulin-bound thrombin is augmented by the
presence of the endothelial protein C receptor
(EPCR). During sepsis, the protein C system is
impaired as a result of several factors, most nota-
bly decreased synthesis and increased consump-
tion of protein C and decreased activation of
protein C due to reduced expression of thrombo-
modulin and EPCR on endothelial cells [45]. The
anticoagulant strength of the protein C system has
been demonstrated in many preclinical settings
[56].
In a landmark study conducted in the 1980s,
Taylor and colleagues demonstrated that infusion
of APC into septic baboons prevented DIC and
death and that inhibition of protein C activation
aggravated the response to Escherichia coli and
transformed a sublethal effect into a lethal DIC-
associated state [57]. In accordance with an impor-
tant regulatory function of the endogenous protein
C system in sepsis, treatment of bacteraemic
baboons with an anti-EPCR monoclonal antibody
exacerbated a sublethal E. coli infection leading to
lethal sepsis with massive activation of coagulation
[58]. The anticoagulant effects of APC in haemo-
stasis may be enhanced by its capacity to inhibit
fibrinolysis by suppressing the function of two
fibrinolysis inhibitors: thrombin activatable fibri-
nolytic inhibitor (TAFI) and plasminogen activator
inhibitor type I (PAI-1) [56].
Adhesion of cells to injured endothelium relies on
adhesive proteins, such as von Willebrand factor.
Monomers of von Willebrand factor (280 kDa) can
be linked by disulfide bonds to form ultra-large
multimers, with a molecular mass of up to 106 Da
[59]. The assembly of von Willebrand factor multi-
mers occurs in endothelial cells, where they are
stored in Weibel–Palade bodies. Large multimers of
von Willebrand factor, which are released upon
endothelial cell perturbation, can bind more effi-
ciently to platelet glycoprotein Iba. In normal hae-
mostasis, large von Willebrand factor multimers
 S. M. Opal & T. van der Poll
are cleaved by a protease termed a disintegrin and
metalloproteinase with a thrombospondin type 1
motif, member 13 (ADAMTS13); this process is
stimulated under circumstances of high shear
stress. Physiologically, von Willebrand factor acts
to stabilize the adhesion of platelets at sites of
vascular injury. Sepsis is associated with a relative
deficiency of ADAMTS13, resulting in increased
levels of ultra-large von Willebrand factor multi-
mers, which facilitate platelet adhesion to injured
endothelium [60]. It has been suggested that the
resulting thrombotic microangiopathy has an
important role in multiple organ dysfunction in
sepsis [61].
Activated platelets form a strong link between the
processes of primary and secondary haemostasis
by providing the phospholipid layer necessary for
the assembly of activated coagulation factor com-
plexes [62]. Disruption of the vascular integrity in
sepsis results in the adhesion of platelets at sites of
injury, where they can contribute to endothelial
cell activation through several mechanisms. For
example, stimulation of glycoprotein IIb/IIIa on
platelets enhances CD40 ligand expression on the
platelet membrane, which activates endothelial
cells to express adhesion molecules and TF [63,
64]. Expression of P-selectin on the platelet mem-
brane mediates the adherence of platelets to leu-
cocytes and endothelial cells and enhances the
expression of TF on monocytes [65].
Vascular inflammation and coagulation are ampli-
fied by the release of so-called neutrophil extracel-
lular traps (NETs) by neutrophils. NETs are highly
charged mixtures of DNA and nuclear proteins
together with serine proteases such as elastase,
cathepsin G and calprotectin [66]. NETs function to
entrap pathogens (an action that is facilitated by
platelets) and are designed to mediate swift elim-
ination of invasive microorganisms. However, as
for many components of innate immunity, NETs
can also contribute to collateral tissue damage [67,
68]. Indeed, NETs are procoagulant by promoting
adhesion, activation and aggregation of platelets
and by activating the contact system, leading to the
release of bradykinin and activation of the intrinsic
pathway of coagulation [68]. Moreover, by promot-
ing platelet and red blood cell adhesion, and by
engagement of clotting factors, NETs can provide a
scaffold for thrombus formation. Additionally,
NETs can induce endothelial cell death, an effect
that is likely to be mediated by NET-associated
proteases or cationic proteins such as defensins
Review: Endothelial dysfunction in sepsis
and histones [69]. Finally, NETs generated as a
consequence of platelet–neutrophil interactions
have been implicated in tissue damage in sepsis
[67].
Protease-activated receptors and endothelial barrier function
Thrombin and an array of circulating serine prote-
ases can cleave a set of ubiquitously expressed,
seven-transmembrane receptors known as prote-
ase-activated receptors (PARs) [43, 44]. Four such
receptors (PAR1–4) are found in humans and can
either disrupt or protect endothelial barrier func-
tion, depending on which G-protein-linked intra-
cellular signalling pathway is activated. PARs are
unusual receptors as the ectodomain contains the
internal, sequestered ligand that, through feed-
back, activates its own receptor, but only if the N-
terminus of the ectodomain is removed by partial
proteolysis by serine proteases such as thrombin
[43–45]. Thrombin binds to PAR1 expressed on
endothelial cells in the early phase of sepsis,
contributes to endothelial dysfunction by G12/13
Rho-dependent cytoskeletal derangements in
endothelial cells and induces endothelial cell con-
traction and rounding [70, 71]. Endothelial cell
contraction destabilizes cell-to-cell contacts, caus-
ing an increase in vascular permeability, which
facilitates the passage of large molecules (albumin
and other plasma proteins) and leucocytes from the
blood into the subendothelial compartment (see
Fig. 2). Gaps in the endothelial barrier also expose
the fluid compartment of blood to the basement
membrane and vessel adventitia with abundant TF
for clot initiation and collagen fibres for von Wille-
brand factor to polymerize and for binding plate-
lets.
It is interesting that thrombin-induced stimulation
of endothelial cells by PAR1 activation can be
deleterious to barrier function or beneficial to
endothelial function depending on the stage of
progression of sepsis and severity of the disease
state [72]. Over time, thrombin linked to PAR1 can
transactivate PAR2 into a PAR1–PAR2 heterodimer,
which has a protective role in endothelial barrier
function and survival in sepsis models. This ‘role
reversal’ for PAR1 signalling from endothelial bar-
rier disruption to barrier protection is mediated by
PAR1 to PAR2 combined signalling that switches
coupling of the GTPase RhoA intracellular barrier
disruptive pathway to an alternative PAR2-Gi-
Rac1-mediated intracellular signalling pathway
resulting in actin polymerization and improved
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 S. M. Opal & T. van der Poll
Tight endothelial barrier Dysfunctional barrier
Activated protein C C’5a, PAF, NO
Bradykinin
Atrial natriuretic peptide
VEGF receptor 2
Robo4-Silt2N
Ang1-Tie2 Thrombin, FXa, MMP
Protease inhibitors HMGB1
PAR1-2 VE-cadherin PAR1
S1P1
Rac1 RhoA
Tie2 Tie2
TF Ang-1 TF
TF TF
Collagen fibres Actin polymer
cytoskeleton
Fig. 2 Endothelial barrier function and dysfunction in
health and disease. The left panel shows the factors that
contribute to tight junctions, with well-formed and func-
tional endothelial cells covering the microvasculature. The
right panel highlights those molecular signals that con-
tribute to loss of barrier function and endothelial cell
rounding and retraction. Breakdown of tight junctions is
accompanied by loss of intravascular proteins to the
interstitial spaces, procoagulant and proinflammatory
processes, and loss of barrier integrity. Ang, angiopoietin;
Tie2, tyrosine kinase with immunoglobulin-like and epi-
thelial growth factor-like domains 2; VE, vascular endo-
thelium; PAR1–2, protease-activated receptor type 1 and 2
heterodimer; Rac1–Cell division control protein; TF, tissue
factor; S1P, sphingosine 1 phosphate receptor; C’, comple-
ment; PAF, platelet-activating factor; NO, nitric oxide; FXa,
activated factor X; MMP, matrix metalloprotease; VEGF
receptor 2, vascular endothelial growth factor receptor-2;
HMGB1, high-mobility group box 1; RhoA, GTPase of the
Ras homolog gene family.
barrier function. This dual signalling system might
be amenable to therapeutic intervention with
agents that target specific PARs at specific times
during the progression of sepsis (see Table 1 and
discussion below) [72–74].
Recently, matrix metalloprotease (MMP)1 has been
detected at high levels in the circulation in patients
with severe sepsis/septic shock (18-fold higher
than normal blood levels) [75]. This might have
significant prognostic and therapeutic implica-
tions. MMP1 efficiently cleaved PAR1 on endothe-
lial cells but, in contrast to thrombin with dual
signalling ability, MMP1–PAR1 signalling mediates
only barrier disruption [75]. Endothelial-derived
MMP1 activates noncanonical PAR1 signalling and
is associated with poor outcomes in experimental
models of septic shock [76, 77]. MMP1 cleaves and
activates PAR1 at a novel D39–P40 site, which leads
282 ª 2014 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2015, 277; 277–293
Review: Endothelial dysfunction in sepsis
to biased-barrier breakdown signalling in endothe-
lial cell membranes [76, 77]. This cleavage site is
located two amino acids proximal to the N-termi-
nus site for thrombin cleavage at position R41–S42.
The MMP1 cleavage site generates a slightly longer,
tethered peptide ligand which mediates barrier
disruption upon PAR1 activation [72, 77].
S1P3
Macrophage–endothelial cell interactions
TF
The occurrence of crosstalk between myeloid and
endothelial cells has been well known for many
Actin breakdown years as endothelial signals are critical for target-
ing neutrophils and circulating monocytes from the
vascular lumen to endothelial surfaces expressing
adhesion molecules in areas of acute inflammation
[65]. Activated endothelial cells secrete IL-8, mono-
cyte chemoattractant protein-1 and macrophage
inhibitory protein-1 to attract monocytes to inflam-
matory foci. Intravascular, adherent, activated
monocytes express large amounts of TF to propa-
gate procoagulant activity at the site of infection or
injury. However, the extent to which tissue macro-
phages are involved in angiogenesis, tissue repair
and maintenance of endothelial barrier function is
just beginning to be understood.
Macrophages are initially activated after tissue
injury by ischaemic necrosis of mesenchymal cells
in an IL-1a-dependent process. IL-1a primarily
exists as an uncleaved but biologically active
precursor which is located within the intracellular
space and nucleus where it regulates transcription
[78]. During necrotic, but not apoptotic, cell death,
the IL-1a precursor is released from intracellular
stores within dying mesenchymal cells and binds
to its type 1 IL-1 receptor expressed on adjacent
tissue macrophages. By contrast, during apopto-
sis, IL-1a precursor remains tightly bound to the
chromatin where it is then digested during the
apoptotic nuclear fragmentation process without
IL-1a release [79]. Necrotic cell release of IL-1a
precursor is rapidly followed by inflammasome
assembly within the macrophage cytosol, resulting
in caspase-1 synthesis, IL-1b processing and
extracellular secretion with marked upregulation
of IL-1b -induced chemokine, cytokine and adhe-
sion molecule expression on nearby endothelial
cells [79, 80]. Recruitment of circulating neutroph-
ils and monocytes activates inducible nitric oxide
synthase with rapid efflux of nitric oxide followed
by vasodilatation, opening of endothelial gaps and
loss of endothelial barrier function (Fig. 2) [78].
 S. M. Opal & T. van der Poll Review: Endothelial dysfunction in sepsis

Table 1 Endothelial barrier disrupters and promoters

Barrier disrupters Barrier promoters

Molecule Mechanism
C’ components Vasodilators, increase
C3a, C5a vascular permeability
Bradykinin Vasodilator, increases
permeability
PAF Vasodilator, increases
permeability
Proinflammatory Increase surface expression
cytokines of adhesins,
PMN–EC interaction
Ang-2 Inhibitor of Ang-1, pro-apoptotic
Tie1 Specific inhibitor of Tie 2
interaction with Ang-1
RhoA GTPase Breaks down actin cytoskeleton,
internalizes VE-cadherin
PAR1 signals Pro-apoptotic, activate RhoA
MMPs Activate PAR-1 barrier disruption
and RhoA
S1P3 Activates RhoA, de-polymerizes
actin cytoskeleton
Vascular endothelial Activates Src kinase to
growth factor 2 phosphorylate and internalize
VE-cadherin
b-arrestin-mediated Internalizes phosphorylated
endocytosis VE-cadherin, impairs
endothelial barrier
HMGB1 Breaks down VE-cadherin,
promotes cytokine and
chemokine generation
Molecule Mechanism
C’ regulators and Block anaphylatoxin
carboxy-peptidases actions, degrade C3a and C5a
Carboxy-peptidases Degrade bradykinin
PAF-acetyl hydrolase Degrades PAF in the
circulation
Anti-inflammatory Limit expression of
cytokines inflammatory cytokines
Ang-1 Inhibits NF-jB signalling,
inhibits apoptosis
Tie2 Tyrosine kinase receptor
for Ang-1, anti-apoptotic
Rac1 Stabilizes actin cytoskeleton
PAR1–PAR2 transactivation Anti-apoptotic, promotes Rac1
Protease inhibitors Limit activity of MMPs
S1P1 Membrane stabilizer,
promotes VE-cadherins,
actin polymerization
Atrial natriuretic peptide Activates Rac1, anti-
inflammatory, limits NF-jB
Slit–Robo4 Inhibits VE-cadherin
phosphorylation by
p120-catenin, blocks
apoptosis
HMGB1 mAb Inhibits HMGB1-dependent
alteration of endothelial
barrier function

Ang, angiopoietin; S1P, sphingosine 1 phosphate receptor; NF-jB, nuclear factor kappa light-chain enhancer of B cells;
VE, vascular endothelial; C’, complement; PMN, polymorphonuclear cell; PAF, platelet-activating factor; EC, endothelial
cell; Tie 1 and Tie 2, tyrosine kinase with immunoglobulin-like and epithelial growth factor-like domains; GTPase,
guanosine triphosphatase; RhoA, GTPase of the Ras homolog gene family; Rac1, a subfamily of GTPases; PAR, protease-
activated receptor; MMP, matrix metalloprotease; Src kinase, sarcoma-related nonreceptor protein kinase; HMGB1, high-
mobility group box 1; mAb, monoclonal antibody.

Extra-luminal macrophages support endothelial
cell growth, limit apoptosis and remodel the
extracellular matrix via expression of MMPs to
support endothelial membrane structure and
function [81–83]. Tissue macrophages signal to
endothelial cells by one of three mechanisms: (i)
synthesis and release of soluble growth factors
into the microenvironment, (ii) local release of
macrophage exosomes and (iii) direct cell-to-cell
contact with endothelial cells (Fig. 3). Vascular
endothelial growth factor A, colony-stimulating
factor 1, IL-1b and TNF-a are secreted by

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 S. M. Opal & T. van der Poll Review: Endothelial dysfunction in sepsis

Endothelial barrier

Activated TNF
Capillary lumen
monocytes HMGB1 MCP1
MIP-1 IL-1α
TF TF IL-1β
MCP1 TF RAGE
IL- 8 P-selectin TLR4
PSGL1

Soluble Ang2
Exosomes cytokines Tie2

Direct cell-to-
cell contact

Tissue macrophages
activating ECs

Fig. 3 Interactions between tissue macrophages in the perivascular space, circulating monocytes and endothelial cell
surfaces. High-mobility group box 1 (HMGB1) is a late cytokine-like mediator in sepsis that increases endothelial permeability
and induces inflammatory cytokine synthesis by endothelial cells (ECs). PSGL1, P (platelet)-selectin glycopeptide ligand 1;
MIP1, macrophage inhibitory protein 1; MCP1, monocyte chemoattractant protein 1; IL, interleukin; TNF, tumour necrosis
factor; TF, tissue factor; TLR4, Toll-like receptor 4; RAGE, receptor for advanced glycation end products.

activated tissue macrophages. These cytokines
and growth factors bind to their cognate receptors
expressed on endothelial cells to support growth
and to promote expression on vascular endothe-
lial surfaces of adhesion molecules such as
P-selectin, E-selectin, intercellular adhesion
molecule 1 (ICAM1) and vascular cell adhesion
molecule (VCAM) [81].
Recent evidence suggests that exosomes (i.e. small
microvesicles with cytosolic contents enclosed
within a lipid bilayer) are generated by perivascular
macrophages and can influence endothelial behav-
iour [84, 85]. Exosomes encase RNA species from
donor macrophages including microRNAs [83] and
can fuse with the cellular membrane of adjacent
endothelial cells and deliver their contents directly
into target cells. MicroRNAs are short sequences of
RNA (20–25 nucleotides in length) that bind to the
untranslated tails of specific complementary mes-
senger RNA (mRNA) sequences and limit further
translation of or degrade peptide sequences from
the mRNA template. These microRNAs have the
capacity to simultaneously inhibit the peptide
synthesis of multiple mRNA targets. One microRNA
known as miR-150 has been successfully delivered
from exosomes to endothelial monolayers in vitro
and found to alter endothelial cell behaviour by
increasing cell migration [81, 86]. It is tempting to
speculate that macrophage-derived exosomes are
generated in systemic inflammatory states and
disrupt endothelial barrier function during sepsis,
but this has not yet been confirmed within the
microcirculation and perivascular space in
patients with sepsis [81].
Macrophages appear to have the capacity to
directly influence endothelial cell function and
structure by direct cell-to-cell contact with endo-
thelial cells. Macrophages express the angiopoietin
receptor, which is a tyrosine kinase known as Tie2
(discussed in detail below). Interaction between
angiopoietin (Ang)-2 and Tie2 promotes endothelial
growth, cell survival and angiogenesis. This direct
cell contact between macrophages and endothelial
cells might be important in tissue repair and for re-
establishing endothelial barrier function during
the recovery phase of sepsis [81, 82]. Reciprocal
interactions between endothelial cells and macro-
phages maintain tissue macrophage viability and
promote a microenvironment for macrophage dif-
ferentiation and maturation [81, 87] Endothelial
cells promote differentiation to M2-like macro-
phages, which have an anti-inflammatory pheno-
type and rely on oxidative metabolism to support
resolution of tissue injury and repair.

284 ª 2014 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2015, 277; 277–293
 S. M. Opal & T. van der Poll
Endothelial cell MPs
MPs are vesicles that range in size from 0.1 to 2 lm
and are shed from the plasma membrane of
multiple cell types upon activation or apoptosis
[88, 89]. MPs contain many biologically active
molecules, including proteins, lipids, mRNAs and
microRNAs. The cellular source of circulating MPs
is revealed by the antigens they express, which
allows investigation of the function of cell-specific
MPs. It was shown that MPs are present at low
levels in the circulation of healthy subjects and
originate predominantly from platelets [90]. Proin-
flammatory cytokines and bacterial products can
induce shedding of MPs from endothelial cells that
contain ultra-large von Willebrand factor multi-
mers, which potently promote the formation of
platelet aggregates and increase their stability [91].
Activated monocytes release MPs expressing TF
[92]. Patients with septic shock had elevated total
MP levels regardless of the presence of DIC,
whereas levels of endothelium- and leucocyte-
derived MPs were positively correlated with DIC
status [93]. Of note, MPs can also exert anticoag-
ulant effects. Anionic phospholipids exposed by
MPs can promote the assembly of anticoagulant
proteins such as TFPI, thrombomodulin, EPCR and
protein S. APC can stimulate the release of MPs
from endothelial cells, which facilitates the efficient
inactivation of factors Va and VIIIa by MP-derived
EPCR. Several cytoprotective properties associated
with APC could be reproduced by APC-positive MPs
in vitro [94]. Additionally, recombinant human
APC, which until recently was registered for the
treatment of severe sepsis, has been found to
induce an increase in circulating APC-positive
MPs. This suggests a functional role of anticoagu-
lant and cytoprotective MPs in the in vivo effects of
APC [95].
The endothelium and the microcirculation
The endothelium plays a pivotal role in the normal
function of the microcirculation. Sepsis is associ-
ated with a decreased flow velocity in the microcir-
culation and a reduced density of perfused
capillaries [96]. Alterations in the microcirculation
in sepsis are characterized in particular by heter-
ogeneous perfusion of tissues due to a lack, or
intermittent perfusion, of capillaries adjacent to
those with normal perfusion [97]. Heterogeneous
perfusion of the microvascular circulation disturbs
tissue oxygenation and leads to hypoxic areas even
in the presence of preserved total blood flow to an
Review: Endothelial dysfunction in sepsis
organ [98]. Microvascular dysfunction has been
linked to organ failure and mortality in patients
with sepsis [96]; disturbed function of the vascular
endothelium plays a pivotal role.
The glycocalyx is a thin layer of glucosaminogly-
cans that covers the surface of the vascular endo-
thelium [43]. The glycocalyx contains several
components that are essential for normal homeo-
stasis, including antithrombin and superoxide
dismutase. One function of the glycocalyx is to
reduce adhesion of leucocytes and platelets to
endothelial cells. Sepsis results in the degradation
of the glycocalyx, leading to enhanced adhesion of
inflammatory cells and injury [44]. Other patho-
logical responses implicated in microvascular dys-
function include activation of coagulation
(resulting in thrombosis in the microvasculature),
alterations in red cell deformability and impaired
release of nitric oxide [99, 100]. Although these
responses that contribute to microvascular dys-
function are clearly detrimental in the context of
severe sepsis, they are important for host defence
against invasive infection by limiting dissemination
of the infection and recruiting cells to the infectious
site. Of note, it has been reported that microcircu-
latory function is improved by several anticoagu-
lants, including APC, antithrombin and low
molecular weight heparin [101–104]. It is interest-
ing that a modified antithrombin that is unable to
bind to the vascular endothelium but with intact
anticoagulant properties failed to improve the
microcirculation in animals with sepsis, suggesting
that the anticoagulant effect per se is not sufficient
to improve microcirculatory function [103].
Endothelial barrier function
Endothelial barrier dysfunction and microvascular
leak critically contribute to the pathogenesis of
organ failure in sepsis and of sepsis-related com-
plications such as acute lung injury [105]. The
vascular barrier consists of endothelial cells,
together with cell–cell junctions and extracellular
components such as the glycocalyx. Cell–cell junc-
tions include adherens junctions, mainly com-
posed of vascular endothelial (VE)-cadherin, and
tight junctions (the zona occludens), predomi-
nantly consisting of occludins and claudins [106].
Under normal conditions, the endothelial barrier is
semi-permeable, allowing transport of fluids and
solutes from blood to tissues. In sepsis, however,
barrier function is disturbed, resulting in
enhanced passage of proteins and solutes outside
ª 2014 The Association for the Publication of the Journal of Internal Medicine 285
Journal of Internal Medicine, 2015, 277; 277–293
 S. M. Opal & T. van der Poll
Animation 1 This animation describes in a time lag
fashion the pathophysiology behind endothelial barrier
dysfunction in septic shock. Watch the animation here.
Please note that the version of the video with narration will
be up shortly.
the circulation, and oedema (Animation 1). Several
mediators are involved in maintaining vascular
barrier function (see Table 1).
Vascular endothelial growth factor
Vascular endothelial growth factor (VEGF) is a
glycoprotein produced by endothelial and lung
epithelial cells, as well as by leucocytes and plate-
lets [106]. VEGF regulates vascular permeability
through binding to VEGF receptors 1–3, which are
endothelial-cell-specific membrane tyrosine kinase
receptors [107]. Activation of VEGF receptor 2
dissociates VE-cadherin from the adherens junc-
tion resulting in increased vascular permeability
[108]. Although the results of preclinical studies to
investigate the role of VEGF in sepsis have been
inconsistent [106], a pilot study to test an anti-
VEGF antibody (bevacizumab) in patients with
septic shock was announced in 2010 (clinicaltri-
als.gov identifier NCT01063010); the status of this
trial is currently unknown.
Sphingosine-1-phosphate
Sphingosine-1-phosphate (S1P) is an endogenous
bioactive sphingolipid produced in many types of
cells that are highly abundant in plasma and
regulate endothelial barrier function by the activa-
tion of its G-protein-coupled receptor S1P1 [109].
The interaction between S1P and S1P1 on endo-
thelial cells enhances (i) vascular barrier function
by downstream activation of small GTPases, (ii)
cytoskeletal reorganization, (iii) adherens junction
and tight junction assembly and (iv) focal adhesion
formation. Other barrier-enhancing agents may
286 ª 2014 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2015, 277; 277–293
Review: Endothelial dysfunction in sepsis
also transactivate S1P1 signalling to promote
endothelial barrier function [109]. For example,
PAR1 can influence vascular stability and function
via S1P1 signalling. APC potently inhibits throm-
bin-induced vascular hyperpermeability by a
mechanism dependent on transactivation of S1P1
[110], whereas thrombin-induced vascular hyper-
permeability is dependent on another S1P receptor,
S1P3 [111]. Notably, activation of PAR1 by low
doses of thrombin can (like APC) lead to a barrier
protective effect [112] and thrombin can transacti-
vate PAR1–PAR2 heterodimers in late sepsis, which
is also barrier protective [72]. Recently, a key role
for S1P2 in the permeability and inflammatory
responses of the vascular endothelium during
endotoxaemia was revealed [113]. Downstream sig-
nalling of S1P2 in vascular inflammation included
the activation of the stress-activated protein kinase
and nuclear factor kappa light-chain enhancer of
B-cell (NF-jB) pathways. Several S1P analogues
have been developed, with the aim of preserving
vascular integrity, for possible clinical use [109].
Fibrinopeptide Bb15–42
Fibrinopeptide Bb15–42 is a cleavage product of
fibrin that binds to VE-cadherin and stabilizes
interendothelial junctions [114]. Administration of
fibrinopeptide Bb15–42 may be of therapeutic value
in sepsis. Treatment with this peptide preserved
endothelial barrier function in two different shock
models, induced by Dengue virus and LPS, by
inhibiting stress-induced opening of endothelial
cell adherens junctions; fibrinopeptide Bb15–42-
treated animals showed improved survival rates
and reduced haemoconcentration and fibrinogen
consumption [115]. In a pig model of haemorrhagic
shock, fibrinopeptide Bb15–42 improved pulmonary
and circulatory function and reduced plasma IL-6
levels and neutrophil influx into the myocardium,
liver and small intestine [116]. Moreover, in a
murine polymicrobial sepsis model, fibrinopeptide
Bb15–42 treatment reduced proinflammatory cyto-
kine levels in the lung, liver and blood, decreased
neutrophil infiltration into the lung and attenuated
liver damage, possibly through maintaining vascu-
lar integrity and suppressing vascular leakage
[117]. In several models of acute lung injury,
produced by airway exposure to LPS or acid,
fibrinopeptide Bb15–42 reduced proinflammatory
cytokine levels, neutrophil influx and vascular
leak; moreover, fibrinopeptide Bb15–42 enhanced
bacterial clearance and survival in mice with acid
aspiration-induced lung injury subsequently
 S. M. Opal & T. van der Poll
challenged with Pseudomonas aeruginosa [118].
Because of its myocardial protective effects in
experimental ischaemia–reperfusion injury [114],
fibrinopeptide Bb15–42 (FX06) has been evaluated in
a multicentre Phase IIa clinical trial in patients with
myocardial infarction; in this study, fibrinopeptide
Bb15–42 significantly reduced the size of the necrotic
core zone of infarcts [119]. As such, fibrinopeptide
Bb15–42 is a promising therapeutic agent, although
its mechanism of action remains unclear.
Robo4 and Slit
Endothelial cells express the receptor Robo4
which, after binding to its ligand Slit, inhibits
inflammation-induced endothelial permeability by
consolidation of adherens junctions and modifica-
tion of cytoskeletal dynamics [41, 120, 121]. It is
likely that Slit stabilizes the vasculature by
enhancing VE-cadherin localization to the cell
surface. Indeed, it was shown that Slit2N, an active
fragment of Slit2, increased VE-cadherin and
p120-catenin localization to the endothelial cell
surface [41]. Accordingly, Slit2N reduced endothe-
lial cell monolayer permeability in vitro caused by
inflammatory mediators, including VEGF, LPS,
TNF-a and IL-1b [41, 121]. In vivo, Slit2N attenu-
ated the accumulation of neutrophils and protein
exudates in the alveolar space of LPS-treated mice.
This effect of Slit2N was absent in Robo4-deficient
mice, demonstrating the importance of this recep-
tor and also suggesting that the effect of Slit2N is
endothelial cell specific as Robo4 expression was
restricted to this cell type [121]. In addition, the
Slit2N-dependent reduction in endothelial cell
monolayer permeability was inhibited in the pres-
ence of a VE-cadherin blocking antibody, confirm-
ing the importance of the action of Slit2N on
VE-cadherin in vivo. Similarly, in mice with
abdominal sepsis caused by caecal ligation and
puncture, Slit2N inhibited the leakage of Evans
Blue dye in the kidney and spleen, indicating
preserved vascular barrier function and improved
survival. Slit2N also inhibited lung permeability in
a mouse model of avian H5N1 influenza [41].
Hence, the Slit–Robo4 interaction is important for
maintaining vascular integrity in multiple settings,
and Slit2N may be an attractive therapeutic agent
in conditions associated with disruption of the
endothelial barrier, including sepsis.
Recently, a novel pathway important for endothe-
lial cell integrity that functions independently from
NF-jB has been identified [122]. Specifically, IL-1b
Review: Endothelial dysfunction in sepsis
was shown to induce endothelial permeability via
activation of the adaptor MyD88 by a route that did
not rely on NF-jB activation. Rather, MyD88,
which also functions as the common adaptor of
Toll-like receptor (TLR) signalling, activated a dis-
tinct NF-jB-independent pathway, through the
small GTPase ADP-ribosylation factor 6 (ARF)6
and its activator ARF nucleotide-binding site
opener (ARNO; also known as CYTH2). ARNO binds
directly to MyD88, suggesting that MyD88–ARNO–
ARF6 functions as a proximal IL-1b and TLR
signalling pathway distinct from that mediated by
NF-jB. SecinH3, an inhibitor of ARF guanine
nucleotide-exchange factors such as ARNO,
enhanced vascular stability and improved out-
comes in animal models of sterile inflammation
[122]. The relevance of MyD88–ARNO–ARF6 sig-
nalling and the effect of SecinH3 have not yet been
investigated in human sepsis.
Ang-1 and Ang-2
Ang-1 and Ang-2 are widely studied biomarkers of
endothelial activation and dysfunction in sepsis
[123]. Ang-1 is produced constitutively, in partic-
ular by pericytes and smooth muscle cells, whereas
Ang-2 is produced by endothelial cells where it is
stored in Weibel–Palade bodies for quick release
upon exposure to inflammatory stimuli. Ang-1 and
Ang-2 bind to and inhibit the endothelial cell Tie2
receptor. Under physiological conditions, circulat-
ing Ang-1 levels exceed those of Ang-2, enabling
preferential interaction between Ang-1 and the
Tie2 receptor. This triggers pro-survival pathways
and inhibits pro-inflammatory responses, resulting
in endothelial cell quiescence.
The inflammatory response that accompanies sep-
sis causes exocytosis of Weibel–Palade bodies and
release of Ang-2, which results in a shift towards
Ang-2–Tie2 interaction. This interaction promotes
pro-inflammatory and pro-thrombotic pathways,
microvascular leak and angiogenic stimuli. In
clinical sepsis, a rise in the Ang-2:Ang-1 ratio,
normally low under nonstressed conditions, is an
indicator of acute vascular dysfunction [106]. In
accordance with this, elevated Ang-2 levels were
found to be correlated with severity of illness and
adverse outcomes in patients with sepsis [124,
125]. The functional role of Ang-2 was illustrated
by experiments with heterozygous mice missing
one Ang-2 allele; these animals were partially
protected against vascular leak, acute lung injury
and death in models of caecal ligation and
ª 2014 The Association for the Publication of the Journal of Internal Medicine 287
Journal of Internal Medicine, 2015, 277; 277–293
 S. M. Opal & T. van der Poll Review: Endothelial dysfunction in sepsis

puncture and LPS-induced toxicity [126]. Ang-1– vasculotide protected against vascular leak and
Tie2-targeted strategies have been tested in pre- mortality in polymicrobial sepsis in mice [127].
clinical sepsis models. The synthetic Tie2 agonist Similarly, vasculotide and the Ang-1-mimetic

Table 2 Potential new treatment options to prevent or treat endothelial barrier dysfunction in sepsis

Therapeutic agent Proposed mechanism of action Current developmental status

Selepressin and other Potent vasopressors that specifically Phase II clinical trials [138]
V1A receptor agonists bind to V1A receptors reducing
permeability and limiting
oedema formation
C’5a monoclonal antibody Blocks C’5a anaphylotoxin Early clinical trials planned
from binding to its receptor for sepsis [139]
and prevents excess
endothelial permeability
and C5a-induced apoptosis
Recombinant human Promote APC formation which Available in Japan, in Phase III
thrombomodulin and prevents endothelial apoptosis; clinical trials in Europe and North
APC variants induce carboxypeptidase activity and South America [140]
to degrade C’5a
Ang-1/Tie2 agents Inhibit NF-jB signalling and Preclinical studies [127, 128]
possess endothelial cell
anti-apoptotic activity
S1P1 agonists Stimulate VE-cadherins, Preclinical studies [109]
actin polymerization at
adherens junctions of the
endothelium
Fibrinopeptide Bb15–42 (FXO6) Fibrin cleavage product that Phase II clinical trials in myocardial
binds VE-cadherin and infarction, sepsis preclinical and
stabilizes interendothelial junctions early clinical studies [118, 119]
Slit2N agonists Stabilize tight junctions between Preclinical studies [41]
endothelial cells
Pepducins Lipidated peptides that are super-agonists Preclinical studies [136]
of PAR2, inducing Rac-mediated
barrier stabilization
Anti-HMGB1 mAb Blocks HMGB1-mediated loss of Preclinical studies [131–135]
endothelial barrier function and
upregulation of cytokine generation
and of adhesion molecules
Anti-VEGF receptor 2 mAb Blocks VEGF2-mediated breakdown Early clinical trials [108]
of VE-cadherin from the adherens
junction in endothelial cells

Ang, angiopoietin; S1P, sphingosine 1 phosphate receptor; V1A, vasopressin 1A receptor; C’, complement; APC, activated
protein C; NF-jB, nuclear factor kappa light-chain enhancer of B cells; VE, vascular endothelium; Tie2, tyrosine kinase
with immunoglobulin-like and epithelial growth factor-like domains; PAR, protease-activated receptor; Rac, a subfamily of
GTPases; HMGB1, high-mobility group box 1; VEGF, vascular endothelial growth factor; mAb, monoclonal antibody.

288 ª 2014 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2015, 277; 277–293
 S. M. Opal & T. van der Poll
MAT.Ang-1 attenuated vascular leak induced by
LPS in mice [128, 129]. Strategies that either enhance
the Ang-1–Tie2 interaction or inhibit the effects of
Ang-2 warrant further investigation in sepsis.
High-mobility group box 1
High-mobility group box 1 (HMGB1), first
described as a DNA-binding nuclear protein in
eukaryotic cells, regulates chromosomal replica-
tion, transcription and DNA repair [130]. It has
subsequently been found that HMGB1 has a crit-
ical role in the innate immune response to infection
and injury as a late-acting, proinflammatory cyto-
kine-like protein. HMGB1 release from either acti-
vated or necrotic cells (particularly myeloid and
endothelial cells), but not from apoptotic cells,
results in this inflammatory action [131, 132]. The
endothelial lining is both a source of HMGB1
release in sepsis and a target of HMGB1-mediated
inflammatory actions during septic shock or other
severe forms of noninfectious tissue injury.
HMGB1 functions as an ‘alarmin’, or damage-
associated molecular pattern, inducing and main-
taining the acute inflammatory response. Elevated
circulating levels of HMGB1 are detected by a
series of receptors expressed within the microcir-
culation including the receptor for advanced gly-
cated end products, TLR2 and TLR4 [130, 131].
HMGB1 disrupts endothelial barriers, alters the
actin filament cytoskeleton, impairs tight junc-
tions, promotes the release of large quantities of IL-
1a as well as an array of other cytokines and
chemokines, and stimulates enhanced expression
of cell surface adhesion components such as
ICAM1 and VCAM1 on endothelial membranes
[133–135]. Antibodies directed against HMGB1
can reverse many of these changes seen in exper-
imental animal models of sepsis, indicating that
clinical development of HMGB1 inhibitors should
be considered (see Fig. 3 and Table 2).
Conclusion
Recovery and maintenance of the endothelial bar-
rier is critical to survival in sepsis. New therapeutic
targets for the management of sepsis at the level of
the endothelium might become clinically applica-
ble as the cellular and molecular mechanisms that
cause barrier dysfunction become better under-
stood. Agents are in development that promote cell
survival by targeting mitochondria and cellular
energetics [7, 35, 36]. Recombinant variants of
APC, which have been specifically engineered to
Review: Endothelial dysfunction in sepsis
remove their anticoagulant properties and promote
their cytoprotective capacity, are possible novel
treatments for sepsis [110]. Agents are also in
development that directly protect the endothelial
barrier, including Ang-1 and Tie2 agonists
[127–129], S1P1 agonists [109], fibrinopeptide
Bb15–42 therapy [118, 119] and Slit2N agonists,
by stabilizing tight junctions between endothelial
cells [121]. Another strategy is the use of pepduc-
ins, small lipidated peptides that can readily cross
cell membranes and either block PAR1/RhoA bar-
rier disruption or function as super-agonists of
PAR2/Rac-mediated barrier stabilization, as
potential therapies for septic shock [136, 137].
Table 2 provides a summary of the novel treatment
options now in clinical development that specifi-
cally target endothelial barrier function in sepsis
[138–140].
Novel interventions directed at re-establishing
endothelial barrier function in sepsis could be a
major addition to the therapeutic armamentarium
at a time when antibiotics are failing and results
with other adjuvant therapies have been disap-
pointing. This is an exciting area of research at
present, and clinical trials should soon provide
information to determine whether targeting the
endothelium will benefit patients in septic shock.
Conflict of interest statement
Our institution receives a grant from Asahi Kasei to
run the clinical coordinating centre for soluble
thrombomodulin.
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Correspondence: Steven M. Opal MD, Chief, Infectious Disease
Division, Memorial Hospital of Rhode Island, 111 Brewster Street,
Pawtucket, RI 02860, USA.
(fax: 401-729-2795; e-mail: Steven_Opal@brown.edu).
ª 2014 The Association for the Publication of the Journal of Internal Medicine 293
Journal of Internal Medicine, 2015, 277; 277–293