Pathophysiology of Aortic Stenosis and Mitral

Regurgitation
Gianluca L Perrucci,1,2 Marco Zanobini,1 Paola Gripari,*†1 Paola Songia,1 Bayan Alshaikh,3
Elena Tremoli,1 and Paolo Poggio**1

ABSTRACT
The global impact of the spectrum of valve diseases is a crucial, fast-growing, and underrec-
ognized health problem. The most prevalent valve diseases, requiring surgical intervention, are
represented by calcific and degenerative processes occurring in heart valves, in particular, aortic
and mitral valve. Due to the increasing elderly population, these pathologies will gain weight in
the global health burden. The two most common valve diseases are aortic valve stenosis (AVS) and
mitral valve regurgitation (MR). AVS is the most commonly encountered valve disease nowadays
and affects almost 5% of elderly population. In particular, AVS poses a great challenge due to
the multiple comorbidities and frailty of this patient subset. MR is also a common valve pathology
and has an estimated prevalence of 3% in the general population, affecting more than 176 mil-
lion people worldwide. This review will focus on pathophysiological changes in both these valve
diseases, starting from the description of the anatomical aspects of normal valve, highlighting all
the main cellular and molecular features involved in the pathological progression and cardiac
consequences. This review also evaluates the main approaches in clinical management of these
valve diseases, taking into account of the main published clinical guidelines. © 2017 American
Physiological Society. Compr Physiol 7:799-818, 2017.

Didactic Synopsis 4. To date, the aortic valve stenosis management is repre-

sented by surgical intervention, since no pharmacological
Major teaching points treatments has been proved to be effective.
1. Understanding the anatomical structures of aortic and
mitral valves is necessary to apprehend the pathophysiol- 5. The major pathological stimuli involved in mitral valve
ogy of aortic valve stenosis and mitral valve regurgitation. regurgitation are as follows:
a. Mechanical, such as tension, shear stress, compression,
2. The major pathological processes involved in aortic valve
and flexure
stenosis are as follows:
b. Chemical, such as serotonin and TGF-β signaling
a. Valve interstitial cell phenotypic switching in
osteoblast-like cells
6. The main cardiac pathologies caused by mitral valve regur-
b. Endothelial-to-mesenchymal transition of valve gitation are as follows:
endothelial cells
a. Left atrial pressure overload
c. Inflammatory cell infiltration
b. Pulmonary edema
d. Fibrosis and osteogenesis
c. Pulmonary hypertension
3. The main cardiac implications caused by aortic valve
stenosis are as follows: * Correspondence to paola.gripari@ccfm.it
** Correspondence to paolo.poggio@ccfm.it
a. Left ventricular pressure overload
1 Centro Cardiologico Monzino, IRCCS, Milan, Italy
b. Cardiac hypertrophy 2 Department of Clinical Sciences and Community Health, University

c. Syncope of Milan, Milan, Italy

3 King Fahad Armed Forces Hospital, Jeddah, Saudi Arabia

d. Pulmonary hypertension † Thisauthor has contributed equally to Marco Zanobini.

e. Angina Published online, July 2017 (comprehensivephysiology.com)
DOI: 10.1002/cphy.c160020
f. Heart failure Copyright © American Physiological Society.

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Valve Pathology Consequences Comprehensive Physiology

d. Left ventricle dysfunction Each leaflet takes the name from the correspondent originat-
ing coronary: left, right, and noncoronary. The free edge of the
e. Heart failure
leaflet has a small bulge named the nodule of Arantius, which
meets together with the other nodules when the leaflets are
7. To date, mortality rates are extremely high without sur-
closed, providing the coaptation area that maintain the unidi-
gical intervention. However, patients with acute MR will
rectional blood flow. The rim of each valve leaflet, attached
also benefit from hemodynamically stabilization by after-
to the root, is slightly thicker than its body and it is known
load reduction accomplished either by pharmacological
as the lunula. The lunulae of adjacent cusps overlap during
(e.g., vasodilators) or by mechanical treatments (e.g., intra-
valve closure increasing valve support.
aortic balloon pump).

Introduction
The global impact of the spectrum of valve diseases is a cru-
cial, fast-growing, and underrecognized heath problem. The
most prevalent valve diseases, requiring surgical intervention,
are represented by calcific and degenerative processes occur-
ring in heart valves. Due to the increasing elderly population,
these pathologies will gain weight in the global health bur-
den. This review will focus on pathophysiological changes in
the two most common valve diseases, namely, aortic valve
stenosis (AVS) and mitral valve regurgitation (MR).
Aortic valve
The aortic valve (AV) is one of the two semilunar valves of the
heart. Its function is to prevent regurgitation of blood from the
aorta to the left ventricle (LV) during diastole. It is composed
of three leaflets, an annulus, and several commissures and
resides within the aortic root (Fig. 1).
Annulus
The aortic annulus cannot be simply defined as the junction
between the AV and the ventricle. It is a more complex struc-
ture due to the semilunar attachment of the leaflets, which
extend from the basal attachment in the LV to the distal one
in the sinotubular junction, making a crown shape ring rather
than a simple circular one.
Commissures
The junction between each leaflet attachment is called com-
missure. AV contains three commissures at the top of the annu-
lus, which are equally distributed. The commissure between
the left and right leaflet is located at the right posterior aspect
of the aortic root, whereas the commissures between the right
or left and noncoronary leaflet are located at the right or left
anterior aspect of the aortic root.

Leaflets Structure and cell types

The AV is made up of three semilunar leaflets attached right The leaflets are less than 1 mm thick and three different layers
below the sinus of Valsalva, where the coronary arteries arise. compose them. The fibrosa layer is rich in collagen (mostly

Figure 1 Aortic valve anatomy. Structure and nomenclature of aortic root components. The
upper side of the figure represents thoracic aortic traits, with highlighted distinctions between
ascending aorta and aortic arch. The lower part of the figure recapitulates the detailed aortic
root components: the sinotubular junction of aortic root with ascending aorta; the aortic valve
leaflets; the ventriculo-aortic junction of aortic root and left heart ventricle.

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type I and III) and faces the aorta; the spongiosa layer contains Table 1 Echocardiography Criteria for the Identification of Aortic
glycosaminoglycans and resides in the middle of the leaflets Valve Stenosis Stage
functioning as a bearing, whereas the ventricularis layer is
abundant in elastin fibers facing the cardiac ventricle. Aortic stenosis stages
A monolayer of valve endothelial cells (VECs) covers Mild Moderate Severe
the two outer layers, while a mixed population, called valve
interstitial cells (VIC), resides within all three. The majority Aortic jet velocity (m/s) 2.6–2.9 3.0–4.0 >4.0
of VICs are fibroblastic-like, up to 20% are myofibroblastic- Mean gradient (mmHg) <30a 30–50a >50a
like, and less than 5% are smooth muscle cells (37, 154, 169).
<20b 20–40b >40b
Every cell type is committed to several-specific func-
tions. VECs maintain valve homeostasis, while VICs repair AVA (cm2 ) >1.5 1.0–1.5 <1.0
leaflet micro-damages. In particular, VICs secrete new extra- Indexed AVA (cm2 /m2 ) >0.85 0.60–0.85 <0.6
cellular matrix (ECM) proteins (i.e., collagen, elastin, and Velocity ratio >0.50 0.25–0.50 <0.25
glycosaminoglycans) and eliminate damaged ones, at the
a EAE/ASE Guidelines (15).
same time, VECs control VIC activation, shutting them down b AHA/ACC Guidelines (147).
when the repair processes are completed. Together, these two
cell types provide architectural stability and valve elasticity
(37, 169).
The position of the AV, between the two highest-pressure due to the multiple comorbidities and frailty of this patient
chambers of the heart, and its anatomy lead the AV structures subset. In addition, only subjects with a congenital bicuspid
to be exposed to both bloodstream and mechanical forces. AV, 1% to 2% of the population, develop AVS at a younger
Therefore, it is not surprising that together biochemical and age, usually before 60 years of age (191).
mechanical stimuli may alter AV features causing persistent AV degeneration is defined by echocardiography follow-
damages. In fact, hypercholesterolemia, hyperglycemia along ing European Society (15) and American Heart Association
with hypertension could provide hints of stress to the valve guidelines (147) (Table 1). AVS can be viewed as a pro-
(56). All these stimuli may result in a progressive fibrosis gression from aortic valve sclerosis (AVSc), even if only a
process of the leaflets, remarked by early manifestation, such small percentage of people with AVSc will reach severe AVS.
as inflammation, neovascularization, onset of mesenchymal In AVSc, valve thickening and/or calcification do not affect
tissue, and, at final stages, calcification (18, 37, 154). normal leaflet motion (30, 200). However, as the disease pro-
gresses, leaflets become thicker, calcium nodules form, and
new blood vessels appear (33, 200), impairing leaflets motion
Aortic valve stenosis and causing obstruction of the LV outflow tract during systole
(33, 105) (Fig. 2). The classical triad of symptoms is angina,
AVS is the most commonly encountered valve disease nowa- syncope, and heart failure. Usually, patients are asymptomatic
days. It affects almost 3% of the population, its prevalence for a long period, but eventually these symptoms occur lead-
dramatically increases with age, from 0.7% in young and up ing to decompensation, which conveys a poor prognosis with
to 13% in people older than 75 year of age, thus, being con- median mortality of 5, 3, and 2 years, respectively to angina,
sidered an elderly disease (148). AVS poses a great challenge syncope and heart failure (176).

Figure 2 Echocardiography of normal and stenotic aortic valve in systole. 2D-echocardiography of a

healthy (left) and a stenotic (right) aortic valve in short-axis view. Red arrows (left) highlight the normal, thin
leaflets with an optimal orifice area. Red stars (right) highlight thick calcific leaflet edges with a restricted
aortic valve area.

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Figure 3 Cellular features of stenotic aortic valve. The cartoon depicts the main cellular mechanisms involved in the pathogenesis of aor-
tic valve stenosis. (A) The differentiation of monocytes into osteoclasts. (B) The endothelial damage leading to endothelial-to-mesenchymal
transition (EndMT) into activated myofibroblasts. (C) The differentiation of valve interstitial cells (VIC) into activated myofibroblast and/or
osteoblast-like cells, leading to fibrosis and calcification, respectively. Adapted with permission from (132).

Cellular mechanisms the expression of osteoblast transcription factor Runx2. Once

To date, it has been well established that valve stenosis is a
complex process due to the sum of cellular and molecular
events involving valve cells (VICs, VECs, and vascular peri-
cytes) modification and circulating cells (inflammatory cells
and bone-marrow derived cells) action (Fig. 3).
VIC phenotypic switching in osteoblast-like cells
Several studies have described VIC switching in osteoblast-
like cells, able to form bone tissue (6, 93, 131, 134, 165). The
abnormal activation of quiescent VICs leads to this phe-
notype transition. Once activated, VICs become more sen-
sible to osteogenic mediators. Among these pro-ostegenic
molecules, bone morphogenic proteins (BMPs) are the most
important, since studies on vascular calcification showed their
capability to stimulate osteoblasts and trigger bone forma-
tion (18). Experimental models showed transforming growth
factor-beta (TGF-β) family members, such as BMP-2 and
BMP-4, are secreted not only by activated endothelial cells,
in response to hemodynamic alterations (35, 193), but also
by aged-rodent VICs (187). BMP protein family exerts their
effect through SMAD and Wnt/β-catenin signaling pathway
activation as well as through Msx2, an osteochondrogenic
transcription factor (18). All these pathways lead to induce
Runx2 is expressed, cells are committed to an osteoblast
lineage, upregulating osteopontin, bone sialoprotein II, and
osteocalcin eventually leading calcification of the AV tissue
(91, 162, 181).
There are evidences indicating that BMP signaling is
activated in human calcified valves. Moreover, several studies
have also shown higher levels of SMAD 1/5/8 and Runx2 in
human valve leaflet undergoing calcification (6, 134). How-
ever, to date the in vitro studies and the animal model used are
not able to recapitulate all the complex pathway interactions.
Endothelial-to-mesenchymal transition
VICs are not the only cell population able to undergo
phenotypic switch, from quiescent to osteoblastic-like cells.
Indeed, VECs have the same capability; however, this
process needs further steps. Initially, VECs must undergo
endothelial-to-mesenchymal transition (EndMT). During
this process, VECs lose their ability to maintain homeostasis
and most of their endothelial cell properties. In particular,
EndMT leads to a decrease or completely lost of endothelial
markers expression (i.e., vascular endothelial cadherin,
platelet endothelial cell adhesion molecule (PECAM1), von
Willebrand factor) and a simultaneous acquisition of typical
features of mesenchymal cells. Among these properties,

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the VECs, undergoing EndMT, increase the expression of
α-smooth muscle actin (α-SMA), collagen type I, vimentin
as well as migratory properties (161). Several well-known
stimuli are involved in the EndMT of VECs, such as
TGF-β, transcription factor Msx2, and β-catenin signaling.
Interestingly, all of them have been linked to the progression
of the AV calcification. However, all the data on these
molecular processes have been obtained from experiments
on mitral VECs—and not aortic—involving human in vitro
studies and an ovine model (192, 209). The lack of EndMT
studies on AV is mainly because a very small percentage of
VECs undergo EndMT. Thus, further studies are necessary
to better investigate VEC behavior in stenotic human AVs.
Inflammatory cells
The unbalanced number of infiltrated macrophages and leuko-
cytes in healthy versus calcified human AVs supported the
hypothesis of valve calcification and subsequent valve steno-
sis as an inflammatory disease process (93,142). Inflammatory
cell infiltration is an early event in valve disease establish-
ment and it starts at the outer layers of the valve, which are
more vulnerable. Activated VECs, expressing typical mes-
enchymal adhesion molecules, facilitate the recruitment and
migration of monocytes into the leaflets. An interesting in vivo
study, involving the ApoE-deficient mouse model, showed an
increased number of macrophages along with the degree of
valve calcification (143).
In the initial phase of valve degeneration, the presence
of infiltrated inflammatory cells is often accompanied by
a higher concentration of proinflammatory cytokines (3, 4)
(i.e., tumor necrosis factor-α (TNF-α) interleukin-1β (IL-
1β) and nuclear factor kappa-B (NF-κB) ligand) and spe-
cific molecules secreted by macrophages and leukocytes (i.e.,
metalloproteinases (MMPs) and endoproteases). In particu-
lar, these latter proteins specifically break down ECM com-
ponents, such as collagen and elastin, well-known processes
involved in pathological alteration of valve architecture (93).
Among proinflammatory cytokines, TNF-α may play a
pivotal role on downstream mediator of inflammation-induced
calcification. Several experimental evidences revealed TNF-
α involvement in stenotic valve pathology. In interleukin-1
receptor antagonist deficient mice (IL-1Ra−/− ) occurs valve
thickening, calcification, and sclerosis/stenosis, however, in
the same model also lacking for TNF-α (TNF-α−/− /IL-
1Ra−/− ) this pathological valve phenotype was abrogated
(85). Moreover, TNF-α has been shown to induce vascular
calcification and MMP activation in diabetic mice (5).
Activation of receptors of advanced glycosylation end
products (RAGE) can increase vascular smooth muscle cell
calcification rate, both in vitro and in vivo (60,79,197). These
observations could suggest that RAGE activation may con-
tribute to the AVS process, since elevated plasma and tissue
AGE levels, ligands of RAGE, have been found in subjects
more prone to AVS (62, 96). Additionally, circulating soluble
RAGE is considered antiatherogenic being a decoy for AGEs
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Valve Pathology Consequences
and preventing RAGE activation. In this context, patients with
AVS have low soluble RAGE plasma levels than healthy sub-
jects (14).
In the last years, several studies investigated the role of
lymphocyte in calcified leaflets of AVS patients. Intriguingly,
it has been shown that isolated T-cells from diseased AVs may
play an active role in AVS pathogenesis rather than mediate a
secondary response (208). Moreover, a recent study revealed
the presence of B-cells within AV leaflets of AVS patients
and showed that these cells were also active (142). All these
observations open new questions and further studies, in the
field of immunology, are needed to clarify a number of still
unraveled key points related to AVS pathogenesis.
Fibrosis and osteogenic processes
As inferred by previously described overview on cell mech-
anisms, the main processes involved in AVS are fibrosis and
calcification. There are extensive fibrotic zones as well as
calcium nodule formation in diseased human (34) and mice
(133) AVs. Due to the overlapping features between fibrosis
and calcification, Miller and colleagues (132) asserted in a
comprehensive review that the term “fibrocalcific” could be
more suitable referring to AVS.
The early phase of degenerative AVS is characterized by
VIC activation, where VICs acquire a myofibroblastic-like
phenotype (133), and overproduction/secretion of ECM com-
ponents (217) cause tissue fibrosis. However, specific ECM
alterations, in AVS patients, will be further discussed in the
following paragraphs.
Concerning the calcification, many questions are still
open and further studies are needed to clarify the mech-
anisms underlying this process. To date, there are several
potential mechanisms involved in calcium nodules deposition
in AVS. It has been suggested that valve calcification may
progress by processes similar to those occurring in bones
(138), by accumulation of amorphous calcium deposits [crys-
talline ultrastructure lacking live cells within the calcified
mass (138, 201)], by cellular necrosis, or even by caspase-
dependent apoptosis (201). The mechanism most acknowl-
edged accounted for mineralization of valve tissues resembles
in part the skeletal ossification. In particular, only a subset of
cells that express osteogenic markers (4, 133, 134), resem-
bling thus osteoblast characteristics, produces the nucleation
sites where calcium nodules could grow. However, the signal-
ing cascade responsible for the trigger and the progression of
AVS, in vivo, is still unknown.
Molecular mediators
Molecular mediators that induce pro-osteogenic differenti-
ation stimuli are in the spotlight because clinicians and
researchers repute them triggers of AVS. In the next para-
graphs, we will analyze the main signaling pathways and the
specific molecules that are thought to be involved in AVS
initiation and progression.
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Valve Pathology Consequences
TGF-𝛽 signaling
The TGF-β family regulates a broad range of cellular func-
tions, including growth, development, and tissue homeostasis
(135). A peculiar feature of TGF-β is its localization, since this
molecule is stored in the ECM as a latent complex. TGF-β acti-
vation can be mediated by two different mechanisms, integrin
dependent or integrin independent. The former mechanism
is mediated by mechanical release of TGF-β produced by the
direct binding of specific integrins with TGF-β latent complex
(9). Otherwise, the integrin-independent mechanisms involve
plasmin (119), matrix MMPs (9), and thrombospondin-1
(185). These proteins cleave the latent complex and sub-
sequent conformational rearrangements allow its activation,
releasing the active form of TGF-β. In the last 20 years, the
integrin-mediated mechanisms of TGF-β activation has been
deeply characterized and it has been demonstrated that TGF-β
acts differently, depending on the ECM compliance (206).
TGF-β signaling starts with TGF-β dimerization and its
subsequent binding to TGF-β receptor type I (TGF-βR1) and
type II (TGF-βR2). These two form a receptor heterocom-
plex, able to recruit and phosphorylate suppressor of mothers
against decapentaplegic (SMAD) proteins, triggering a wide
range of signal transduction pathways (81). In vitro analy-
sis obtained on VICs showed that TGF-β1 strongly promotes
cell apoptosis, cellular aggregation, and calcium nodule for-
mation (42, 89, 203). However, administration of TGF-β1 to
VICs plated on a less stiff collagen matrix did not induced
neither osteogenic differentiation nor calcification (214). In
addition, as previously mentioned, it has been also highlighted
the ability of TGF-β in EndMT promotion. Paranya et al.
(160) demonstrated that ovine aortic VECs treated with TGF-
β exhibited higher levels of typical EndMT markers, increased
α-SMA with subsequent PECAM1 reduction.
Experimental evidences have shown that also circulating
plasma levels of TGF-β were associated with AVS. In par-
ticular, a threefold increase, in TGF-β levels, was found in
AVS patients compared to healthy subjects (202). In contrast,
at valve level, calcified valves presented similar expression
pattern of TGF-β when compared to normal AVs (213).
All the collected data on TGF-β signaling, in both ani-
mal models (133, 134) and AVS patients (89), candidate this
molecule as potential early driving mediator of fibrocalcific
processes.
Bone morphogenetic protein signaling
Bone morphogenetic proteins (BMP) are growth factors,
belonging to the TGF-β superfamily, not only localized in
bone but deeply involved during AV development and injuries
(36, 193). The BMP pathway starting mechanism is simi-
lar to the TGF-β one. Two molecule of BMP interact with
BMP receptors (BMPR1 and BMPR2) forming a heteroclom-
plex that phosphorylates SMAD1-5-8 complex and mitogen-
activate protein kinase (MAPK), triggering a myriad of down-
stream pathways.
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Comprehensive Physiology
It is well known that increased levels of BMPs and
phospho-SMAD1-5-8 are found in fibrocalcific AVs (164,
207). Although, the mechanisms contributing in BMP
increase are not completely clear to date. However, recent
studies suggest that hemodynamic alterations (i.e. shear
stress) may stimulate the BMP2-4 secretion by valve endothe-
lium on the aortic side (145, 196). In particular, this study
exploited porcine AV leaflets exposed to altered shear stress
conditions, showing a strong increase in both BMP4 and
TGF-β expression, which in turn stimulated inflammatory
responses, observed as an increase of VEC adhesion molecule
expression. Interestingly, these results were also validated in
human AVs (7).
Regarding the early step of AVS, a hypercholesterolemia
mouse model showed increased levels of active SMAD1-5-8
before any calcium nodule formation and valve impairment
(134). In addition, SMAD6-7, molecules acting as inhibitors
of SMAD1-5-8 play a significant role in preventing calcifica-
tion. Indeed, an important study by Galvin et al. (58) demon-
strated that SMAD6 null mice, starting at 2 weeks after birth,
were prone to develop both cardiovascular calcification and
aortic ossification.
Wnt/𝛽-catenin signaling
Wnt proteins are a family of secreted glycoproteins used for
short- or long-range signaling. The receptors of Wnt proteins
belong to the Frizzled family and they are able to regulate a
variety of cellular processes (e.g., cellular differentiation, tis-
sue morphogenesis, and tissue homeostasis) (120, 173). The
Wnt/β-catenin pathway is one of the major axes involved in
regulation of AV formation and disease (83). β-catenin is a
transcription factors bound to the cytosolic tail of Wnt recep-
tor. When Wnt receptor is activated, β-catenin do not get
degraded through ubiquitination but remain stable and subse-
quently is released to the cytosol. Finally, β-catenin translo-
cate to the nucleus where activates the transcription of specific
genes (120).
The expression of several modulators of the Wnt signaling
pathway, such as WIF-1, DKK-1, and sFRP-3, has been found
upregulated in patients with AVS, suggesting their potential
role as pathology biomarkers (11). Additionally, increased
levels of low-density lipoprotein receptor-related protein 5
(Lrp5) together with increased levels of nuclear β-catenin
were reported in human stenotic valves (28). High levels of
β-catenin were also showed in calcified valves of hypercholes-
terolemic animal models (134,170). All these data support the
hypothesis that alteration in Wnt/β-catenin signaling could
pave the way to AV degeneration.
Reactive oxygen species
Reactive oxygen species (ROS) appear to play a pivotal role
in atherosclerosis, stroke, and other cardiovascular diseases
(74). Both superoxide (O2 − ) and hydrogen peroxide (H2 O2 )
result to be significantly increased in stenotic AVs, as well as
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Comprehensive Physiology
the expression of nitric oxide (NO) synthase and the reduction
of antioxidant enzymes (131).
Several experimental evidences underline a close relation
between ROS activity and the progression of calcific valve
impairment. Indeed, it is well known that ROS levels increase
prior to valve dysfunction in mice, suggesting that this event
does not merely happen because of the calcification-related
modifications (133). Moreover, ROS have been found rele-
vant in the transduction of pro-osteogenic and pro-fibrotic
signaling cascades (41).
Despite the clear key-points associating ROS and AV cal-
cification initiation and progression, to date, several link are
still missing. For example, the precise role of NO synthase
isoforms and NAD(P)H oxidase isoforms, which contribute
to increase ROS generation. In addition, the real contribu-
tions of the different antioxidant mechanisms (e.g., catalase,
superoxide dismutases, and peroxidases) in their respective
subcellular compartments.
Nitric oxide bioavailability
The counterpart of ROS is NO that plays a key role in sev-
eral cardiovascular disease. Its bioavailability is frequently
inversely correlated with ROS levels. The major NO pro-
ducer is represented by the enzyme endothelial nitric oxide
synthase (eNOS), which has been found upregulated on the
aortic side of the impaired valves during the early stages of
valve pathology (65). This side-specific response could be due
to a protective mechanism activated by VECs. The protective
role of eNOS, which acts also as inhibitor of VIC proliferation,
deeply depends on the expression levels of its cofactors and
substrates. However, it has been notice that eNOS overexpres-
sion, in a hypercholesterolemic mouse model, could acceler-
ate the atherosclerotic process. This event is strongly related to
eNOS uncoupling (157), a peculiar phenomenon that leads to
ROS generation and occurs when eNOS is decoupled from its
cofactors or its substrates are not available (118). Moreover,
endogenous inhibitors of eNOS have been shown significantly
increase in patients with stenosis of AV (27). Thus, the modu-
lation of the molecular system underlying NO bioavailability
may represent a possible therapeutic strategy to decrease AV
calcification progression. Indeed, exogenous NO administra-
tion reduced calcium nodule formation in VICs (99) and the
administration of statins, in a hypercholesterolemic animal
model, increased eNOS levels with concomitant NO release
(171).
Renin—angiotensin system
Renin-angiotensin system (RAS) and the principal effector,
angiotensin II (AngII), are well-establish signaling known
to be involved in oxidative stress, inflammation, and several
processes able to trigger the early stages of AVS (125). AngII
overexpression is strongly related to hypertension, which is
an important risk factor for AVS (148).
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Valve Pathology Consequences
Moreover, it has been described a large presence of inflam-
matory cell infiltration (i.e., macrophages), in fibrocalcific
aortic leaflet, that are able to produce and release large
amount of AngII (154). In 2004, Helske et al. (75) discovered
that the expression of angiotensin converting enzyme (ACE)
and one of the AngII receptor (angiotensin type I receptors,
AT1R) were increased in stenotic valves and colocalized with
macrophages and mast cells. Animal models confirmed the
involvement of RAS in AVS progression using AT1R block-
ers, which leaded to reduce inflammatory cell infiltration and
myofibroblast activation, already at the early stages of the
disease (10).
RANK/RANKL/OPG axis
The interactions between receptor activator of NF-κB
(RANK), RANK ligand (RANKL), and osteoprotegerin
(OPG) modulates oxidative stress and inflammation with
important consequences on calcification progress in bones,
arteries, and probably causing also impaired aortic leaflet
motion (43). Increased RANK/RANKL activation may influ-
ence calcification on circulating and interstitial cells. Indeed,
the production of RANKL by vascular smooth muscle cells,
under calcification stimuli, is sufficient to promote both mono-
cyte recruitment (proinflammatory cell) and osteoclast differ-
entiation (interstitial cell) in an atherosclerotic mouse model
(26). The osteoclast differentiation mediated by RANKL may
be driven by ROS production, even if RANKL induces calci-
fication of vascular smooth muscle cells through the BMP4
pathway (108,159,183). The last mentioned actor of this axis
is OPG that is an endogenous RANKL decoy receptor able
to inhibit inflammation and the pro-osteogenic pathway acti-
vation. Based on animal models and human studies, it has
been suggested that OPG may act as inhibitor of calcification
in AV leaflets. Indeed, it has been shown that: (i) in OPG−/−
knockout mice aortic calcification was inhibited (152); (ii)
atherosclerotic lesion progression was quicker in ApoE−/−
OPG−/− double knockout mice then in ApoE−/− model (16);
(iii) OPG administration prevented calcification of the aorta
in ldlr−/− knockout mice (140); and (iv) RANKL expression
was higher in human stenotic valve than in control (92).
Peroxisome proliferator-activated receptor gamma
Peroxisome proliferator-activated receptor gamma (PPARγ)
is a ligand-dependent transcription factor, member of the
nuclear hormone receptor superfamily (71). Several findings
depict a strong interplay between PPARγ and AVS (97), (212).
In particular, increased PPARγ expression reduces differen-
tiation of progenitor cells into osteoblast-like cell, in vitro
(97). Accordingly, PPARγ inhibition increases differentia-
tion of embryonic stem cells into osteoblasts (212). Despite
these evidences of PPARγ involvement in AVS, to date no
study have neither deeply investigated nor elucidated this
association. However, it has been highlighted that the role
of PPARγ ligands are important in antioxidant defenses and
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Valve Pathology Consequences
Figure 4 Aortic valve stenosis pathophysiology. Phenotype of stenotic aortic valves and the involvement of valve stenosis in cardiac
diseases. In the left side are depicted normal and stenotic aortic valves in diastole and systole. In the stenotic picture are present several
leaflet regions of spotty calcification, which stiffen the valve structure and alter the dynamic of valve opening. In the right side of the figure
are mentioned the main cardiac pathologies caused by aortic valve stenosis.
antinflammatory gene expression induction (175). Since
PPARγ regulates osteoblast modulation and plays an impor-
tant role as antioxidant and antinflammatory agent, it may be
enrolled as therapeutic target for calcific AV disease, but we
need to be very careful because PPARγ affects a large number
of genes (98).
Notch signaling
Polymorphisms accounted for Notch1 gene have been
reported to be strongly associated with AVS early develop-
ment, in particular in bicuspid AVs (59). In Notch1 haploin-
sufficient mice, the calcification of the aortic leaflets occurs
through the repression of BMP2 expression (117, 146). How-
ever, recently has been proposed that decreased levels of
Notch1, in the endothelium, provoke a subsequent decrease
of matrix Gla protein (MGP), which allow increased BMP2/4
availability and, at the same time, promotes osteogenic dif-
ferentiation of VICs (8).
Matrix metalloproteinases and cathepsins
MMP and cathepsin degrade both matrix and non-matrix pro-
teins in the extracellular environment. Both of these prote-
olytic enzyme classes have been found upregulated in AVS tis-
sues and therefore corroborating the hypothesis regarding the
association with fibrocalcific processes occurring at AV lev-
els. In particular, the expression of MMP-1, MMP-2, MMP-3,
MMP-9, and cathepsins S, K, V, and G has been reported in
valve pathology (50,55,76,77,88,93). Although the functional
role of a single MMP or cathepsin, in valve stenosis, is still
unclear, the complex remodeling of ECM could be the main
process involved in calcified nodule formation and growth,
along with elastin and collagen deposition/brake down (166).
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Comprehensive Physiology
Pathophysiology
AVS will lead to narrowing of the aortic orifice and once aortic
valve area (AVA) reaches measurements below 2.0 cm2 will
cause a progressive increase in pressure gradient (Fig. 4). This
will cause LV pressure overload and subsequent increased
wall stress. To overcome these stress changes, LV reacts with
compensatory mechanisms. As per Laplace law, wall stress is
proportional to pressure × radius/thickness. In other words,
any increase in pressure will cause an increase in wall stress,
so to compensate, the heart has to reduce diameter and/or
increase its thickness (hypertrophy), thus normalizing the wall
stress (30, 63, 174). Usually, it begins with a slow LV remod-
eling leading eventually to concentric hypertrophy associated
with increased LV mass. Concentric hypertrophy is a mech-
anism that increases the number of sarcomeres reducing the
intraventricular radius, thus the ventricular volume results
reduced.
Hypertrophy is considered a double-edged sword, ben-
eficial in some aspects and deleterious in others (30). This
can be simply explained by supply/demand mismatch. Once
hypertrophy begin, the heart requires more oxygen to perform
its normal functions, however, the impairment of blood flow,
due to decrease LV compliance consequential to AVS, leads
to compression of coronary arteries limiting their flow (30).
AVS could also cause syncope, but the exact mechanism
is still unclear. However, it could be explained by exercise
induced vasodilatation associated with fixed cardiac output
state and inability to augment the cardiac output. Other clini-
cians have postulated that very high intraventricular pressure,
developed during exercise in people affected by AVS, causes
a reflex depressor response causing in turn syncope (i.e.,
vasoplegic syncope) (30). In addition, arrhythmias occurring
in AVS patient, induced by exercise, lead to ischemia that
impairs ventricular filling either by reducing filling time or
loss of atrial kick, causing syncope (30, 95).
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Comprehensive Physiology
These symptoms can be either due to diastolic or systolic
dysfunctions. The former is due to concentric hypertrophy
and subendocardial fibrosis that will lead to impairment of
LV relaxation. This impairment has deleterious effects on
diastolic filling pressure leading: (i) to reduction of LV com-
pliance; (ii) to increase pressure of the left atrium (LA). Con-
sequently, these modifications eventually lead to pulmonary
congestion and heart failure.
The reduced LV compliance elevates LV diastolic pres-
sure impairing the LV filling, reducing the stroke volume
and subsequently the aortic pressure. These mechanical alter-
ations lead to the activation of neurohormonal pathways to
compensate the reduction of cardiac output.
Another potential problem in AVS patients, underesti-
mated in most instances, is represented by pulmonary hyper-
tension. The process starts with LV diastolic dysfunction and
the subsequent increase in diastolic filling pressure, which
leads LA and pulmonary venous hypertension. Raise in LA
pressure will activate pulmonary vascular protective mech-
anism, by which pulmonary arterioles start to constrict. In
addition, this will cause a further reduction of cardiac output,
due to the secondary obstruction caused by the developed pul-
monary hypertension, causing right ventricle (RV) pressure
overload and eventually an overt RV failure (101).
The systolic dysfunction exact mechanism is still contro-
versial. However, it could be explained by myocyte exhaustion
that can no longer compensate for the increase in afterload,
leading the fall of the ventricular functions (30, 73, 78).
Management of AVS
From the prognostic point of view, patients with AVS and
symptomatic heart failure have the worst outcomes.
In severe symptomatic AVS only mechanical relief will
improve the prognosis. Medical treatment for sever AVS is
not effective (30, 163). Several studies linked AVS inflamma-
tory processes with atherosclerosis because they share many
risk factors (94, 144, 158). Indeed, some studies have been
trying to prove that statin therapies can retard the progres-
sion of stenosis. However, all of these studies failed since
randomized controlled trials showed no difference between
statin and placebo group, in term of inducing regression or
preventing progression of AVS (40). Thus, surgical aortic
valve replacement is the treatment of choice when it comes to
severe symptomatic AVS or severe AVS with dropped ejec-
tion fraction, as it carries low mortality and complication
rates with significant improvement in patient survival. How-
ever, transcutaneous aortic valve implantation is the treatment
of choice in high-risk surgical patients.
Mitral valve
The mitral valve apparatus is a complex three-dimensional
functional unit critical to unidirectional blood flow (44). The
mitral annulus (MA), the mitral valve leaflets, the chordae
Volume 7, July 2017
Valve Pathology Consequences
Figure 5 Mitral valve anatomy. Structure and nomenclature of mitral
valve. The mitral valve represents the gate between left atrium and left
ventricle. The perfect closure of the valve depends on cardiac papillary
muscles, which orchestrate the leaflet traction during systole through
chordae tendinae.
tendineae, and the LV wall along with its papillary muscles
(PM) compose it (Fig. 5).
Leaflets
Mitral valve contains two leaflets, one anterior (adjacent to
the AV) and one posterior (adjacent to the wall). Each leaflet is
separated by commissures, anterolateral, and posteromedial,
respectively (172). The anterior leaflet is the largest one and
attaches only to one third of the annulus, which is in continu-
ity with the AV. The posterior leaflet is smaller than anterior
one, but it attaches to two third of the annulus, and it contains
multiple indentations (referred as “clefts”), which forms 2 to
5 scallops (segments). Along the elongated free edge, triscal-
loped leaflet is the most common form. These indentations
should not extend all the way through the leaflet to the annu-
lus; if this is seen, then it is usually associated with patholog-
ical valve regurgitation (128). The posterior lateral segment,
referred as P1, is adjacent to the anterolateral commissure, P2
is central and varies in size, and the medial segment (P3) is
adjacent to the posteromedial commissure (32).
The leaflets are divided into several zones: “basal”, where
they are attached to the atrial-ventricular junction, clear, which
is the central portion, and “rough”, area where the chordae
insert and make up the coaptation area. For tight leaflet coap-
tation, a redundant leaflet tissue in the rough zone is criti-
cally important, allowing good apposition and preventing any
regurgitant volume.
Annulus
The junction between the LA and LV defines the MA and
it gives attachment to the mitral leaflets. It is a dynamic,
anatomically well-defined structure and, in three dimensions,
it has a nonplanar saddle shape (44,103,110,111). The anterior
portion of the MA is continuous with the aortic annulus. The
angle between MA-to-aortic annulus changes dynamically
over the cardiac cycle, with displacement coupled through
the fibrous continuum (198). For this reason, the anterior
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Valve Pathology Consequences
portion is fibrous and less prone to dilation. Beyond this point,
the remaining two-thirds of the annulus (posterior portion) are
mainly muscular, which makes this region more susceptible
to dilatation, as well as to pathological calcification (128).
Chordae tendinae
The chordae originate from PMs and attach to the leaflet in
a fan-like way. According to the attachment site, they are
divided into three types. Primary chords, involved in mitral
valve competence, which are attached to the free edge on the
“rough” zone. Secondary chords, involved in left ventricular
geometry and function, which are bound to the body of the
leaflet on the ventricular side. Tertiary chords, only on pos-
terior leaflet, which are attached directly to ventricular wall
(149).
Papillary muscles
There are two PMs arising from the LV free wall, one antero-
lateral and one postero-medial. The number of postero-medial
PM heads, in the general population, varies from one to three
(179). PMs are vital to mitral valve and LV correct functions
(124).
Structure and cell types
The mitral valve displays a structure consisting in three highly
conserved stratified layers like the AV. These three layers are
named as atrialis, which constitutes the flow side of the valve,
spongiosa in the middle of valve structure and fibrosa on
the opposite surface of flow side and structurally continuous
with the chordae tendineae (112). The atrialis layer presents
a conspicuous amount of elastin fibers in radially oriented
architecture, opposing to shear forces occurring on the flow
side of the leaflet when mitral valve is open. The fibrosa layer
is composed by collagen fibers, resisting to the strong tensile
forces acting when the valve is closed. The collagen structures
allow the flexure during valve opening/closing cycles. Finally,
the spongiosa layer is rich in proteoglycan and glycosamino-
glycan. It is able to resist the compressive forces on closed
valve and provides nutrients to the valve cells by promoting
fluid movement during flexure. Thus, the ECM composition
of all the three valve layers is crucial because supports the
leaflet mechanics. In the same manner, also the cellular com-
ponents of mitral valves are widely distributed, according
to their function in the valve structure. Therefore, the sur-
face of heart valves is covered by VECs, which are CD31
positive and perform functions similar to vascular endothe-
lial cells (153). Nonetheless, VECs are distinct from vascu-
lar endothelium for several features, such as the perpendic-
ular alignment in flow environments and the expression of
genes associated with chondrogenesis (23, 25). Smooth mus-
cle cells are specifically characterized by the expression of
smooth muscle myosin heavy chain and arranged as a thin
layer of cells near the flow side of the valve leaflet (13, 46).
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Comprehensive Physiology
While, VICs are fibroblastic-like cells originating by mes-
enchymal lineage. The principal function of VICs is the con-
tinuous synthesis and degradation of the ECM components,
thus maintaining correct valve ECM homeostasis. Since the
first step in valve degeneration is based on the ECM home-
ostasis alteration, VICs play a pivotal role in the cellular-
mediated mitral valve degeneration. Indeed, the transition of
VICs from a “quiescent” to an “activated” state has been
described as the principal event occurring in human myxo-
matous mitral valve degeneration (167). Activated VICs typ-
ically display myofibroblast-like features, such as expression
of cytoskeletal elements (i.e., α-SMA), desmin, markers of
mesenchymal activation (i.e., embryonic nonmuscle myosin,
SMemb) as well as increased expression of matrix catabolic
enzymes including matrix metalloproteinase (MMP)-1, -2, -9,
and -13, and elastases (i.e., cathepsin K). To date, activated
VIC phenotype is widely accepted as pathological trait in
heart valve impairment (114).
Mitral valve regurgitation
Abnormality in any part of mitral valve complex, preventing
normal leaflets cooptation, will result in mitral regurgitation
(MR). MR is now easily grouped and explained by Carpen-
tier’s classification according to leaflet movement: normal
leaflet movement with annular dilatation or leaflet perfora-
tion (type I); excessive leaflet movement (type II); restric-
tive leaflet movement (type III), diastolic restriction such as
rheumatic disease (IIIa) and systolic restriction as in func-
tional disease (IIIb) (31). Causes of MR are classified into
ischemic, due to coronary artery disease and nonischemic.
Most common nonischemic causes of MR are degenerative,
in developed countries, and rheumatic, in developing coun-
tries (86). Other nonischemic causes are infective endocardi-
tis, cardiomyopathies, inflammatory diseases, drug-induced,
traumatic, and congenital MR (21, 52).
Doppler echocardiography is the main method for assess-
ment of patients with MR. However, chest X-ray and electro-
cardiogram might give clues for MR diagnosis. Nonetheless,
2D echocardiography is considered the gold standard to con-
firm and quantify the lesion severity (Table 2), valve and
sub-valvular morphology (Fig. 6). 2D echocardiography is
able to define the possible causes and to assess the function,
giving the parameters for a good surgical repair.
Functional MR induced by annular dilatation, papillary
muscle displacement, and chordal tethering will cause LV
remodeling (1). Degenerative MR is usually related to mitral
valve prolapse (MVP) with leaflet thickening and chordae
elongation which could be restricted to only one segment,
fibroelastic deficiency, or more diffuse disease affecting multi-
segments, Barlow’s disease (2).
MVP is a common valve pathology and has an estimated
prevalence of 2% to 3% in the general population, affect-
ing more than 176 million people worldwide (45). Despite
the pathology was first described in the late 1800s (45), no
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Comprehensive Physiology Valve Pathology Consequences

Table 2 Echocardiography Criteria for the Identification of Mitral Since the mesenchymal cell response to altered mechanical
Valve Regurgitation stimuli lead to VIC activation, therefore causing ECM remod-
eling, VICs are the main cell type involved in the development
Mitral regurgitation and progression of mitral degeneration (167).
The biomechanical stimuli on heart valves are tension,
Qualitative shear stress, compression, and flexure (24, 180). These stim-
Valve morphology Flail leaflet/ruptured papillary uli may be associated with the initiation and extension of the
muscle/large coaptation defect valve regurgitation, however, little is known about VIC
Color flow regurgitant jet Very large central jet or eccentric mechanosensoring and mechanotransduction mechanisms
jet adhering, swirling, and and their associated signaling pathways.
reaching the posterior wall of the
left atrium The considerable role played by high tension in the patho-
logic process of degenerative valve diseases has been recently
CW signal of regurgitant jet Dense/triangular
highlighted. Hypertension or connective tissue disorders, such
Other Large flow convergence zonea as Marfan’s syndrome (19,189,216), are well-known risk fac-
Semi-quantitative tors for human MR. Because the increased tensile forces, in
Vena contracta width (mm) ≥7 (>8 for biplane)b hypertension, and impaired ECM homeostasis, in connective
tissue disorders, lead to VIC activation, causing their phe-
Upstream vein flow Systolic pulmonary vein flow
reversal notypic switch. In turn, this lead to increased glycosamino-
glycan synthesis, altered proteoglycan expression, as well
Inflow E-wave dominant ≥1.5 m/sc
as increased expression of α-SMA, embryonic SMMHC,
Other TVI mitral/TVI aortic >1.4 MMP-1, MMP-13, and cathepsin K (66, 106).
Quantitative Recently, in vivo experiments, on a sheep model, showed
Primary Secondaryd that mitral leaflet affected by turbulent flow easily under-
went myxomatous degeneration (195). Moreover, in vitro
EROA (mm2 ) ≥40 ≥20
cocultures, of VECs and VICs, suggested that nonpatholog-
R Vol (mL/beat) ≥60 ≥30 ical VECs are able to inhibits VICs activation and normal
+ enlargement of cardiac LV, LA “quiescent” VICs are able to limit VEC EndMT (22). How-
chambers/vessels ever, VICs are mostly reactive to tensile forces, due to their
association with the ECM, whereas VECs are responsive to
CW, continuous wave; EROA, effective regurgitant orifice area; LA, left shear stress.
atrium; LV, left ventricle; R Vol, regurgitant volume; TVI, time–velocity
integral.
a At a Nyquist limit of 50 to 60 cm/s.
Chemical stimuli
b For average between apical four- and two-chamber views.
c In the absence of other causes of elevated left atrial pressure and of
Overall, circulating plasmatic compounds and/or released
mitral stenosis. molecules from circulating cells represent the chemical stim-
d Different thresholds are used in secondary MR where an EROA
>20 mm2 and regurgitant volume >30 mL identify a subset of patients uli that are able to trigger any pathological response, when
at increased risk of cardiac events. find themselves in proximity to the valve structures. Among
these compounds, serotonin has been recently investigated as
major risk factor has been identified (190) and the initiating a possible stimulus for in vivo MR (156).
mechanisms driving MVP are not fully understood (192). Typically, circulating serotonin plasma levels are low
because of its rapid removal carried out either by platelet, by
liver or by lung cells. When serotonin synthesis overcomes
Cellular mechanisms these mechanisms of plasmatic clearance, circulating sero-
To date, the literature does not provide specific stimuli trig- tonin levels become sufficiently high to induce valvulopathy
gering mitral valve degeneration. The knowledge available (67). Gustafsson et al. (67) have shown high circulating sero-
provides only possible initiating signaling pathways concern- tonin levels in carcinoid syndrome, which in turn induced
ing mechanical and chemical stimuli, which in turn define the human myxomatous valvulopathy associated with exuber-
activation of specific transcription factors and downstream ant proteoglycans and glycosaminoglycans deposition. More-
gene expression modulation (Fig. 7). over, exogenous serotonin as well as other chemical stimuli
could be delivered to heart valves and provide a detrimental
chemical stimulus. A possible triggering effect, of degenera-
Mechanical stimuli
tive mitral valve pathology, is represented by VEC dysfunc-
As previously mentioned, degenerative mitral valve disease is tion that allows increased adherence of platelets to the surface
accounted for the dysregulation of ECM homeostasis. It is rea- of mitral leaflets (39, 194). VEC denudation resulting from
sonable that the abnormal biomechanical loading represents shear stress has been previously described and is commonly
the main mechanism involved in valve disease development. associated with MR (39, 194).

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Valve Pathology Consequences Comprehensive Physiology

Figure 6 Echocardiography of healthy and prolapsed mitral valve in systole. 2D-echocardiography of a healthy (left) and
regurgitant (central and right) mitral valve in long axis view. The left echocardiography image shows the correct alignment
(coaptation—red arrow) of healthy, thin mitral valve leaflets. The central echocardiography shows impaired closure (red
star). The right color-Doppler image shows the regurgitant volume toward the left atrium.

Molecular mediators distinct proteins, six of them belonging to the superfamily

The serotonin hypothesis
The serotonin formation is provided by two different isoforms
of the tryptophan hydroxylase (TPH-1 and -2), which is also
the rate-limiting step in the synthesis of this neurotransmitter
(205). The serotonin receptor family is constituted by seven
of Gq-protein-coupled receptors. The binding of serotonin
with its receptors activates phospholipase C (210), which in
turn activates the MAPK extracellular signaling-regulated
kinase (Erk) trigger the proliferative signaling (107). Several
studies in different cell types, including VICs, reported that
serotonin-mediated activation of phospho-Erk (p-Erk) and its

Figure 7 Cellular features of mitral valve regurgitation. The cartoon depicts the main cellular mechanisms involved
in the pathogenesis of mitral valve regurgitation. (A) The endothelial damage leading to EndMT of VECs into myofi-
broblas (MyoFB). (B) The fragmentation of elastin in the atrialis layer. (C) The differentiation of VICs into MyoFB
leading to fibrosis (collagen and glycosaminoglycans deposition depicted in blue in the spongiosa layer). (D) The
collagen fragmentation in the fibrosa layer. Adapted with permission from (109).

810 Volume 7, July 2017

Comprehensive Physiology
subsequent nuclear translocation lead to Rho kinase-mediated
mitogenesis (115, 210). As previously mentioned, high cir-
culating serotonin can induce valve disease, both in humans
and in murine models. Indeed, carcinoid syndrome patients,
displaying high level of plasmatic serotonin, are more prone
to develop valvulopathy (67). In particular, carcinoid-derived
valvulopathy is characterized by specific pathological
feature, such as myofibroblast proliferation, fibrosis, excess
deposition of ECM and myxomatous degeneration (67). Sim-
ilar valvulopathy occurs in rat model undergoing long-term
injections of serotonin (49, 68).
Several drugs upregulating serotonin levels (e.g., anorec-
tic drugs) or agonist of serotonin receptor (e.g., Parkinson’s
disease treatments) have been shown to be strictly implicated
in mitral valve disease (184,218). Strong evidence specifically
links the activation of the serotonin 2B receptor (5HT2BR)
in serotoninergic valve disease (54, 80, 177, 178). Constant
administration of serotonin in a rat model lead to an increased
expression of 5HT2BR and a parallel decreased level of sero-
tonin transporter (SERT) (51). Indeed, a mouse model lacking
SERT display severe mitral regurgitation, possibly due to the
decreased reuptake of serotonin (129).
In 2006, an important study on dogs, firstly reported an
increased 5HT2BR transcript level in the context of mitral
valve disease development and progression (155). More-
over, in this animal model, it was also shown a concomi-
tant decrease of SERT and an overactivation of Erk signaling
(48, 186). Furthermore, Disatian and colleagues reported not
only a several-fold increase in the expression of TPH type 1,
both in human and in a canine mitral valve disease model, but
also a raise in the expression of this enzyme already in the
early-stages of mitral valve disease (47, 48). Furthermore, it
has also been shown that inhibition of TPH type 1 or 5HT2BR
diminishes expression of strain-induced myxomatous proteins
in mitral valve models, supporting the serotonin implication in
the mechanisms involved in mitral valve degeneration (106).
Consistently, these important results support the hypoth-
esis of local serotonin as an initiating stimulus in mitral valve
degeneration.
TGF-𝛽 signaling
Among the growth factor superfamily, TGF-β1, -β2, -β3 iso-
forms, BMPs, inhibins and activins constitute the main sub-
families (84). In particular, the overexpression of TGF-β has
been demonstrated to be strongly involved in several cardiac
diseases based on fibrosis (100, 102), carcinoid heart disease
(204) and valvular pathologies such as AVS as well as MR
(61, 70). In particular, several studies have shown high lev-
els of TGF-β and overexpression of TGF-βR1 and 2 in MR,
both in humans (61, 70) and in vivo models (12, 48, 82, 150).
Indeed, these observation have been well characterized and
TGF-β has been recognized as driving factor of myofibrob-
lastic activation of VICs under tensile stress (130, 203).
Many studies have also reported a strong link between
serotonin and TGF-β, in vitro. It has been shown that
Volume 7, July 2017
Valve Pathology Consequences
serotonin induces TGF-β1 RNA expression in cultured ovine
aortic VICs by Gq-dependent signaling (90) and by Erk phos-
phorylation in mesangial cells (64).
All these results display a strong interplay between sero-
tonin signaling, TGF-β pathway, and the expression of effec-
tor genes and proteins involved in myxomatous mitral valve
degeneration.
Development-related gene pathways
Development-related pathways are highly conserved among
vertebrates, specifically, transcription factor signaling and
their downstream genes. However, valve development reg-
ulating pathways are also involved in other processes, such
as arteries, cartilage, bone, and tendon formation. Among
these, we will focus our attention especially on BMP2-Sox 9,
Wnt-periostin, and Notch 1 (28, 38, 126, 127).
BMPs are members of the TGF-β superfamily and their
principal effectors are SMAD proteins, which are activated
by phosphorylation and carry out their functions already
mentioned before (38, 53). Nevertheless, BMPs are consider-
ably involved in development and after-birth chondrogenesis
through sex determining region Y-box 9 (Sox 9) activation
(53, 215). The transcription factor Sox 9 mediates the expres-
sion of several structural genes including aggrecan, collagen
type II and N-cadherin, all important component of valve
ECM (215).
BMP2 is thought to play an important role in the devel-
opment of the spongiosa layer during heart valve formation
and ECM stratification (38, 112).
Experimental evidences showed both in animal models
and in human the colocalization of Sox 9 with aggrecan,
collagen type II and BMP2 in impaired mitral valves (122).
In addition, all these molecules are well known to be involved
in chondrogenesis process and have been found upregulated
in MVP human specimens (182).
Since chondrogenesis process and myxomatous valve dis-
play large morphologic similarities, BMP-Sox 9 signaling is a
potential candidate pathway to better understand degenerative
mitral valve disease and possibly identify potential therapeu-
tic targets.
Wnt signaling is highly involved in embryogenesis and is
activated by the specific binding of Wnt with Frizzled (Fz)
receptor and stabilized by low-density lipoprotein receptor-
related protein (Lrp)-5 or Lrp-6 (116, 123). The activation of
the Wnt canonical pathway leads to a reduced degradation of
β-catenin that translocates to the nucleus and activates the T
cell-specific transcription factor/lymphoid enhancer-binding
factor 1 (TCF/LEF). Wnt signaling regulates multiple path-
ways, in particular, development and postnatal chondrogenic
and osteogenic processes. Moreover, this axis is also involved
in endochondral bone formation and in maintaining bone den-
sity (72, 123). Interestingly, it has been recently shown that
Lrp-5 regulates bone formation by the inhibition of TPH type
1 and the synthesis of serotonin, which in turn directly inhibits
osteoclastogenesis (211). Furthermore, a deep implication of
811
Valve Pathology Consequences
Lrp-5-Wnt-β-catenin signaling in human aortic valve degen-
eration has been shown (28, 168). A similar osteogenic phe-
notypic switch of VICs, in aortic valve disease, through the
expression of bone phenotype markers such as osteopontin
and osteocalcin has also been shown in degenerative mitral
valve disease. In particular, the endochondral bone formation
has been hypothesized to be involved in myxomatous degen-
eration of mitral valves (28).
Given the fact that osteogenesis and chondrogenesis can-
not be active at the same time, it has been speculated that
Wnt signaling could reinforce a local (mitral valve) serotonin
synthesis, thus enhancing chondrogenesis (153).
The Notch signaling pathway, commonly shared by a
number of multicellular organisms, is based on the specific
binding of several ligands with transmembrane Notch recep-
tors (Notch 1-4), which, once activated, induced proteolytic
cleavage and subsequent release of its intracellular domain
(38). The intracellular domain translocates into the nucleus to
modulate downstream gene expression. Likewise Notch, also
its ligands are transmembrane proteins, thus this peculiar path-
way require cell-to-cell contact to be properly activated. For
this reason, Notch signaling is strongly related to the cellular
network formation and also to the epithelial-to-mesenchymal
transition that is crucial in the early stage of valve formation
(121, 199). In addition, localized activation of Notch signal-
ing has been shown to play a pivotal role in valve polarity
establishment and in mitral valve atrialis layer development
(38). This signaling pathway has also been demonstrated to
be susceptible to the shear stress (38). As previously men-
tioned, mutations in Notch gene are present in congenital
valve (i.e., bicuspid aortic valve) and in calcific aortic valve
disease, in human (59, 121). Unfortunately, to date, there are
no experimental evidence demonstrating the involvement of
Notch signaling in MR, thus further studies are necessary to
validate this hypothesis.
Other pathways
NO production, regulated by the enzyme family of NO syn-
thases (NOS), is well known to play a central role in a vari-
ety of biological functions including endothelium-dependent
vasodilation. The three NOS isoforms include eNOS, which
regulates endothelial NO signaling by calcium/calmodulin,
neuronal NOS (nNOS) and inducible NOS (iNOS), which is
activated in a variety of pathological processes (151). Release
of NO and its subsequent activation leads to cyclic guanosine
monophosphate (cGMP) generation, which in turn activates
cGMP-dependent protein kinase G that contribute to smooth
muscle relaxation, platelet function and cell division (151).
The implementation of a porcine model of mildly myxo-
matous mitral valve degeneration demonstrated the capability
to release NO by mitral VECs (136, 188) after calcium influx
and eNOS activation (202, 203). In addition, the activity of
ADP, bradykinin, and thrombin-induced NO levels increase
in myxomatous mitral valve (137).
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Comprehensive Physiology
AngII is a little peptide resulting from the enzymatic con-
version of angiotensin I and performed by angiotensin con-
verting enzyme (ACE). The binding of AngII with the AT1R
leads to the activation of phospholipase C, the generation of
the inositol trisphosphate (IP3), a second messengers. Con-
flicting evidence in the direct role for AngII in mitral valve
impairment have been collected. AngII was shown to strongly
stimulate collagen synthesis in human VICs, in vitro (69). It
has been reported a weak relation between AngII and ACE
inhibitor, in human mitral valves, contrarily to a strong asso-
ciation in rat mitral valves (141). Moreover, AngII has been
shown to increase the expression of TGF-β1 in human cardiac
fibroblasts (104) and that the ACE inhibition has been shown
to reduce TGF-β3 synthesis in VIC (150). Taken these results
together, a possible role for AngII in MR may be hypothe-
sized, but further investigation should unravel this issue.
Pathophysiology
MR can be divided into two different entities, acute and
chronic, which have different physiological consequences.
In addition, factors that affect the degree of MR are the lesion
severity (regurgitant orifice area), the driving force (LV sys-
tolic pressure) and the left atrial compliance (57). It is worth
mentioning that the principal determinant of MR is the regur-
gitant orifice area (Fig. 8).
Acute MR
In acute MR, there is no time for compensatory changes,
due to the regurgitant volume into a small non-compliant
LA. The regurgitation will cause a significant increase in LA
pressure and the pressure overload will be transmitted to the
pulmonary veins and subsequently to the pulmonary artery.
Eventually, patients with MR might develop acute pulmonary
edema when the LA and pulmonary pressure increase to a
point that exceeds oncotic pressure (17, 139). In worst cases,
edema is followed by hemorrhage because the pressure gra-
dient is high enough to permit the extravasation of erythro-
cytes. Another pathophysiological change could occur when
increase in cardiac output is needed (e.g., during exercise).
The rapid increase in LA pressure will lead the activation
of pulmonary vein baroreceptor, which will result in tissue
and cerebral acidosis, causing exertional dyspnea. Later on,
patients might develop dyspnea at mild exertion because of
chronic LA pressure overload (17, 139). At the same time,
MR will lead to volume overload in the LV, increasing its
distension and wall stress. Finally, LV failure occurs as a
result of increase wall stress with no time for compensatory
hypertrophy resulting in peripheral vascular collapse or acute
pulmonary edema, or both (174).
Chronic MR
In chronic MR, significant regurgitant volume, from LV to LA
during systole, increases LA pressure subsequently leading
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Comprehensive Physiology
Figure 8 Mitral valve regurgitation pathophysiology. Phenotype of regurgitant mitral valve and its involvement in cardiac diseases. In
the left side pictures of normal and regurgitant mitral valve in systole and diastole. In the closed regurgitant mitral valve picture is clear
the improper coaptation of valve leaflets. In the right side of the figure are mentioned the main cardiac pathologies caused by mitral valve
regurgitation.
to LA dilatation to accommodate the excessive volume. The
early cardiac output compensation, in the acute phase, will be
gradually replaced by LV dilatation and eccentric hypertro-
phy to increase LV compliance and volume. This will cause
an increase in the afterload. Such compensatory changes hap-
pen through remodeling of the extramyocardial matrix. In
particular, collagen fiber disruption allows the rearrangement
and slippage of myocardial fibers causing chamber dilata-
tion. Eccentric hypertrophy develops by accumulating new
sarcomeres in series in the LV, which increases the length of
cardiomyocytes without increasing length of sarcomere pre-
serving the effect of preload (57). Myocardial contractility
will also be affected by the dilatation, due to the increase in
wall stress, leading to ejection fraction drop (57). Elevation
of LA pressure, through abnormal diastolic pressure gradient,
will lead to pulmonary venous hypertension and reduction in
cardiac output inducing pulmonary vascular resistance. This
will cause a further reduction of cardiac output, due to the
secondary obstruction caused by the developed pulmonary
hypertension, which will lead to RV pressure overload and
eventually an overt RV failure (87, 101). Both diastolic and
systolic dysfunction will cause eventually heart failure, which
conveys a decrease in survival and require immediate surgical
intervention.
Management of MR
Acute MR
Mortality rates are extremely high without surgical inter-
vention. However, patient with acute MR will also benefit
from hemodynamically stabilization by afterload reduction
accomplished either by pharmacological (e.g., vasodilators)
or by mechanical treatments (e.g., intra-aortic balloon
pump).
Volume 7, July 2017
Valve Pathology Consequences
Chronic MR
Mild and moderate MR will only require a series of clini-
cal examination and echocardiography follow up to evaluate
patient status and only if symptoms occur surgical interven-
tion will be evaluated. On the other hand, patient with mod-
erate to severe MR will require a closer attention, examining
these patients yearly. Unfortunately, like all other valve dis-
eases, there is no medical therapy that could slow the pro-
gression of MR. However pharmacological therapy is used
with functional MR associated with LV dysfunction. In this
case, angiotensin-converting enzyme inhibitors, β-blockers,
and biventricular pacing have been shown to improve sur-
vival rates reducing the degree of MR (20, 29, 113). In any
event, when patients develop severe MR and symptoms occur,
only surgical intervention will completely restore normal life
expectancy.
Conclusion
Calcific and degenerative valve pathologies are an under-
recognized heath problem. The aging population and the lack
of effective pharmacological therapies will increase drasti-
cally the burden on the global health system over the next
decades. Up to now, only clinical examination and cardiac
imaging recognize these pathologies and, most of the time,
these diseases are recognized only at advanced stages due to
symptoms occurrence. Once patients experience any symp-
toms, the only available options are based on surgical interven-
tion, from percutaneous to open heart surgery. However, these
procedures do not cure the diseases, but treat only the symp-
toms, leaving the underling pathological processes untouched.
Further studies that will establish innovative, low-cost screen-
ing procedure will allow the identification of affected patients
813
Valve Pathology Consequences
at earlier stages and, in turn, the possibility to find and inter-
vene on the triggers of these debilitating pathologies.
Acknowledgements
The authors thank the Fondazione Gigi and Pupa Ferrari
ONLUS for the financial support. The authors thank Giovanni
Fumagalli.
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Volume 7, July 2017