The pathogenesis of aortic stenosis: not just a matter of wear and tear

Abstract

Aortic valve stenosis (AS) is the commonest form of valvular heart disease in the Western world. Its prevalence increases exponentially with age and it is present in 2-7% of all patients over 65 years of age. In view of the considerable cardiovascular morbidity and mortality associated not only with AS, but even its earlier stage, aortic sclerosis, many investigations have been directed towards better understanding of its pathogenesis, with the ultimate objective of developing strategies to retard its progression. Although risk factors and downstream mediators appear similar for AS and atherosclerosis (older age, male sex, hypertension, smoking, hypercholesterolemia, and diabetes, as many as 50% of patients with AS do not have clinically significant atherosclerosis. On the basis both of recent experimental evidence and clinical trials, it appears that atherogenesis is not pivotal to the pathogenesis of AS. On the other hand, there is increasing evidence of active involvement of aortic valve fibroblasts with resultant increased production of reactive oxygen species, active pro-inflammatory and pro-fibrotic processes culminating in calcification. We also discuss the evidence of involvement of the nitric oxide system in the pathogenesis of AS. The renin-angiotensin system has also emerged as a major player in the pathogenesis of AS. Histologically, there is increased ACE expression and elevated angiotensin II levels in stenotic valves, while we have just demonstrated amelioration of AS with the use of ACE inhibitors in an animal model. We further discuss intervention studies aimed at retarding AS progression, including recent failures of statins to retard progression of AS in large randomized clinical studies. Finally, we discuss the special case of bicuspid aortic valve, including its genetics and unique associated features.

Full text

Introduction
Aortic stenosis (AS) may be defined as narrow-
ing of the aortic valve, due primarily to a combi-
nation of progressive fibrosis and calcification
of the matrix, with consequent increase in valve
stiffness, progressive reductions in valve area
and concomitant increases in left ventricular
afterload and work. The earliest stages of AS
have been designated aortic valve sclerosis
(ASc), implying disordered valve morphology
(including potential calcification as well as fibro-
sis) in the absence of marked obstruction to left
ventricular outflow.
AS is currently the most common form of valvu-
lar heart disease in the Western world [1], in
large part because the most frequently occur-
ring form of AS develops predominantly in indi-
viduals of advancing age. For example, in the
Helsinki Ageing Study [2], the proportion of indi-
viduals with detectable valve calcification in-
creased from approximately 40% to 75% be-
tween ages of 65 and 85 years, while approxi-
mately 3% of subjects over 75 years of age had
severe AS.
Given that the only proven therapy for severe AS
is aortic valve replacement, and that there is
currently no definitive evidence that any treat-
ment can retard progression of the disease, the
development of severe symptomatic AS in eld-
erly individuals presents an increasing medical
and health economic dilemma. On the one
hand, severe AS rarely remains asymptomatic
for any great length of time: patients classically
develop variable components of exertional dysp-
noea, angina (irrespective of the presence or
Am J Cardiovasc Dis 2011;1(2):185-199
www.AJCD.us /ISSN: 2160-200X/AJCD1107002
Review Article
Pathogenesis of aortic stenosis: not just a matter of wear
and tear
Aaron L Sverdlov, Doan TM Ngo, Matthew J Chapman, Onn Akbar Ali, Yuliy Y Chirkov, John D Horowitz
The Queen Elizabeth Hospital, University of Adelaide, South Australia, Australia
Received July 3, 2011; accepted July 20, 2011; Epub July 28, 2011; published August 15, 2011
Abstract: Aortic valve stenosis (AS) is the commonest form of valvular heart disease in the Western world. Its preva-
lence increases exponentially with age and it is present in 2-7% of all patients over 65 years of age. In view of the
considerable cardiovascular morbidity and mortality associated not only with AS, but even its earlier stage, aortic
sclerosis, many investigations have been directed towards better understanding of its pathogenesis, with the ultimate
objective of developing strategies to retard its progression. Although risk factors and downstream mediators appear
similar for AS and atherosclerosis (older age, male sex, hypertension, smoking, hypercholesterolemia, and diabetes,
as many as 50% of patients with AS do not have clinically significant atherosclerosis. On the basis both of recent ex-
perimental evidence and clinical trials, it appears that atherogenesis is not pivotal to the pathogenesis of AS. On the
other hand, there is increasing evidence of active involvement of aortic valve fibroblasts with resultant increased
production of reactive oxygen species, active pro-inflammatory and pro-fibrotic processes culminating in calcification.
We also discuss the evidence of involvement of the nitric oxide system in the pathogenesis of AS. The renin-
angiotensin system has also emerged as a major player in the pathogenesis of AS. Histologically, there is increased
ACE expression and elevated angiotensin II levels in stenotic valves, while we have just demonstrated amelioration of
AS with the use of ACE inhibitors in an animal model. We further discuss intervention studies aimed at retarding AS
progression, including recent failures of statins to retard progression of AS in large randomized clinical studies. Fi-
nally, we discuss the special case of bicuspid aortic valve, including its genetics and unique associated features.
Keywords: Aortic valve stenosis, bicuspid aortic valve, nitric oxide, oxidative stress, inflammation, fibrosis, calcifica-
tion, intervention studies

Pathogenesis of aortic stenosis
186 Am J Cardiovasc Dis 2011;1(2):185-199
absence of epicardial coronary artery disease)
and atrial or ventricular arrhythmias, resulting in
poor quality of life, increased rates of hospitali-
zation and increased mortality rates [3]. Al-
though the precise natural history of severe AS
in the elderly is uncertain, a number of studies
suggested that mortality is high in the some-
what selected subgroup of individuals who do
not undergo aortic valve replacement [4-7]. Ad-
ditionally, both ASc and AS may be associated
with cardiovascular problems which are appar-
ently remote from the valve itself. For example,
there is considerable evidence that ASc is an
independent marker of increased risk of cardio-
vascular events [8, 9], while severe AS is associ-
ated with increased risk of haemorrhage, espe-
cially into the gastrointestinal tract, primarily
because of the development of acquired von
Willebrand's factor deficiency [10, 11].
It should also be stated at the outset that AS is
a substantially heterogeneous disease, but with
only two "common causes". These are AS devel-
oping in a previously normal trileaflet valve, and
AS associated with congenitally bicuspid aortic
valves (BAV). These and other, rarer, causes of
AS are summarized in Table 1.
Given that AS frequently develops in otherwise
normal valves in aged individuals, this has been
regarded as degenerative AS, implying a rela-
tionship to the normal process of "wear and
tear" within the valve [12]. The purpose of this
review is to demonstrate that AS is not primarily
a "degenerative" process, but rather the result
of a progressive inflammatory process within
the valve matrix. Furthermore, we will present
evidence that the development and progression
of AS, rather than occurring with the implied
inevitability of a purely degenerative disease,
should theoretically be amenable to pharmaco-
therapy.
Aortic valve anatomy and physiology: what are
the homeostatic determinants?
The normal aortic valve consists of several lay-
ers of fibroblast-rich tissue, containing both col-
lagen and elastin fibres, covered by a
monolayer of endothelial cells [13]. There are
also minute intravalvular blood vessels [14],
consistent with a substantial oxygen demand by
the other matrix cells.
The normal aortic valve interstitial cells are
probably mainly fibroblasts, but some smooth
muscle cells have been identified [15], raising
the issue of potential variability in tone of the
valve, an area which has been pursued in a
number of experimental studies. For example,
Pompilio et al [16] showed that intact porcine
aortic valve contracted in response to
phenylephrine, and also exhibited (endothelium-
dependent) relaxation with acetylcholine. Fur-
thermore, normal aortic valve interstitial cells
(presumably including smooth muscle cells)
have been shown to contract in response to 5-
hydroxytryptamine, endothelin-1 and norepi-
nephrine [17]. An important, but incompletely
resolved issue relates to the prevalence and
function of myofibroblasts, which exhibit some
contractile properties, within normal aortic
valves. The role of the myofibroblasts will be
discussed more extensively in the context of
valve pathology.
The physiological role of the aortic valve endo-
thelium has attracted considerable attention
over the past 15 years, although its role is still
incompletely understood. A number of studies
suggest that valve endothelium behaves in a
qualitatively similar manner to vascular endo-
thelium, for example releasing nitric oxide (NO)
in response to stimuli such as acetylcholine
[16], or 5-hydroxytryptamine [18].
Table 1. Causes of AS. Wide frequency range generally reflects the age group(s) assessed by individual
studies as well as population subgroups studied [79, 115-117].
Etiology Approximate frequency (%) Associated features
Aging/calcific 50 - 70 Increased risk of coronary events.
Bicuspid aortic valve 6 - 40 Dilatation or dissection of the aorta, involving the aortic root,
ascending aorta, or aortic arch.
Rheumatic 2 - 11 Mitral valve almost always affected as well
Unicuspid aortic valve <1 Dilatation or dissection of the aorta, involving the aortic root,
ascending aorta, or aortic arch.
Post-endocarditis <1 Extra-cardiac embolic phenomena.

Pathogenesis of aortic stenosis
187 Am J Cardiovasc Dis 2011;1(2):185-199
A critical but unresolved issue is what is the
physiological response of the valve endothelium
to shear stress, a normal stimulus for NO re-
lease, but also potentially for activation of endo-
thelial NAD(P)H oxidase [19, 20], which in turn
would lead to release of superoxide anion (O2-)
and "scavenging" of NO. Despite ongoing uncer-
tainties, the differences between normal aortic
valve and vascular endothelial cells regarding
responses to shear stress were reviewed by
Butcher et al [21], who demonstrated that the
changes in gene expression in response to
shear stress varied substantially.
Finally, little work has been done regarding
other endothelial autocoids within the valve.
Notably, little is known about the physiology of
prostacyclin release from the valve, although it
has been detected [22]. No studies have prop-
erly addressed the issue of tissue penetration of
autocoids released from valvular endothelium,
other than the obvious implications of studies
measuring valve contraction. It is therefore un-
certain to what extent endothelial NO and
prostacyclin might modulate the function of
valve interstitial cells, a physiologically impor-
tant issue given evidence that in AS valve endo-
thelium is dysfunctional or absent, as discussed
below.
Cellular histopathology of AS
Features of stenotic aortic valve lesions
Histopathologic studies have demonstrated that
development and progression of calcific AS are
based on an active process that shares some
similarities with atherosclerosis. It has been
suggested that aortic valve lesions begin with
disruption of valve endothelium predominantly
on the aortic side due to high shear stress [23-
25].
Inflammation and lipid deposition
Aortic valve lesions typically present with areas
of subendothelial thickening, which represents
the early stage of aortic stenosis. Increased
thickening of aortic valve leaflets is character-
ized by accumulation of inflammatory infiltrates
of predominantly macrophages and T-
lymphocytes, lipids, oxidized lipids, (summarized
in Figure 1) [25-28] all of which potentially acti-
vate a host of pro-fibrotic and pro-inflammatory
markers. Macrophages and T-lymphocytes have
been detected and tend to be located near the
surface of the lesion [25-28]. Immunohisto-
chemical studies have found co-localization of
apolipoproteins (apo) B, apo (a), apoE with lipid
laden foam cells and macrophages [29] as well
as oxidative modification of residential low den-
sity lipoproteins (LDLs) in early stenotic aortic
valve lesions [30].
Valvular matrix remodelling and fibrosis
The presence of macrophages and T-
lymphocytes, along with oxidized LDL and apoli-
poprotein accumulation activate several pro-
fibrotic and pro-inflammatory cytokines which
may modulate aortic valve remodelling and sub-
sequent calcification. Transforming growth fac-
tor β1 (TGF-β1) [31] and interleukin-1β [32]
have been found in valve matrix and are associ-
ated with increased local production of matrix
metalloproteinases I and II (MMP-1 and MMP-
2). All of these contribute to cell apoptosis, ex-
tracellular matrix formation, remodelling and
consequently predispose to calcification. In ad-
dition to TGF-β1 and interleukin-1β, tumour ne-
crosis factor-α (TNFα), another pro-inflammatory
cytokine commonly responsible for immune
regulation, inflammation and tissue remodel-
ling, is co-localized with MMP-1 [33]. Further-
more, tenascin C, an extracellular matrix glyco-
protein implicated in cell proliferation, migra-
tion, differentiation and apoptosis, which is in-
volved in stimulation of bone formation and
mineralization, is co-localized with MMP-2 in
calcified aortic valve leaflets [34], and is associ-
ated with progression of AS [35].
Angiotensin II (Ang II), an important mediator of
inflammation and fibrosis, could be formed by
angiotensin converting enzyme (ACE) as well as
the mast cell (MC)-derived neutral protease,
chymase [36]. ACE has been identified in
stenotic but not in normal aortic valves [37]. It
has been shown that MC-derived chymase is
also upregulated in stenotic valves, providing
further evidence for local production of Ang II
[38]. In addition, cathepsin G, another neutral
protease also capable of generating Ang II, is
present in increased concentrations throughout
human stenotic aortic valves compared to nor-
mal valves [39]. These findings provide a poten-
tial basis for a role of angiotensin II in aortic
valve remodelling along with other pro-fibrotic
and pro-inflammatory mechanisms.
As summarized in Figure 1, current concepts of
the pathogenesis of AS centre histologically on

Pathogenesis of aortic stenosis
188 Am J Cardiovasc Dis 2011;1(2):185-199
inflammation and lipid deposition, and bio-
chemically on activation of cytokines and matrix
metalloproteinases, together with generation of
angiotensin II. These processes are postulated
to induce injury of all valve components, leading
to fibrosis and calcification.
Calcification
Calcification of aortic valve leaflets tends to
occur more predominantly in the later stages of
AS, and is located deeper in the lesion [25].
Active calcification is a major factor in reducing
valvular mobility in severe AS. Early lesions of
aortic valves show fine stippled mineralization,
progressing to active bone formation resulting
in gross calcification at later stages of the dis-
ease (Figure 1).
The process of calcification (and sometimes
Figure 1. Schema of postulated mechanisms underlying aortic valve lesion formation. Inflammatory infiltrations of T-
lymphocytes and macrophages, along with lipid accumulation, is primarily responsible for early thickening of aortic
valves. Interactions between chemical stimuli and disruption of valvular homeostasis: pro- and anti- fibrotic mecha-
nisms. Later stages of aortic stenosis: - cytokine release and angiotensin II promote extracellular matrix proteins se-
cretion at early stages of mineralization which in turns begin the processes of bone formation. This process occurs
largely at the end stage of aortic stenosis where aortic valves mobility is significantly reduced due to a build up of
bone-like calcific nodules.

Pathogenesis of aortic stenosis
189 Am J Cardiovasc Dis 2011;1(2):185-199
ossification) of aortic valve leaflets resembles
that associated with atheroma formation. The
presence of inflammation, fatty streak forma-
tion from lipid depositions, cytokine release,
metalloproteinases, ACE, Ang II, all possibly con-
tribute to the production of an extracellular ma-
trix, and matrix vesicles that initiate mineraliza-
tion.
Co-localization of macrophages and oxidized
LDLs with osteopontin, a protein needed in
bone formation, has been found and thought to
be involved in extracellular matrix production in
human stenotic aortic valves [40, 41]. Further-
more, Mohler et al [42] described an active
process of calcification using immunohisto-
chemistry in an extensive study of 347 human
stenotic aortic valves. In addition to active os-
teoblasts and osteoclasts, this and other inves-
tigators [43] detected bone morphogenic pro-
teins 2- and 4- (BMP-2, BMP-4). BMPs stimulate
osteoblastic differentiation with subsequent
calcification. In agreement with previous studies
[25, 41], Mohler et al [42] also found that
macrophages and lymphocytes accumulate in
areas of calcification.
There is also evidence of angiogenesis, which is
essential for longitudinal bone growth in
stenotic valves. T-lymphocytes aggregates tend
to co-localize with sites of neoangiogenesis
within ossified valves [42]. This and the pres-
ence of heat shock protein 60 (hsp60) [44],
commonly expressed by cells under stress con-
ditions, together indicate a highly active, and
chronic immunomediated process from stress
to inflammation to calcification.
It has also been shown that the calcification
process of aortic valves may also be regulated
by receptor activator of nuclear factor κB, its
ligand (RANK, and RANKL), and the soluble re-
ceptor osteoprotegerin (OPG) [45]. The study
detected increased concentrations of RANKL in
calcified compared to control valves. Further-
more, there was a significant reduction of OPG
positive cells in aortic stenotic valves compared
to controls. It has been shown that in mice defi-
cient for OPG, vascular calcification was associ-
ated with increased expression of RANKL [46].
Additionally, in Kaden et al [45] shown that long
-term cell culture of stenotic aortic valves in the
presence of RANKL induced significant increase
in matrix calcium deposition and the formation
of cell nodules compared to controls. Thus, the
RANKL/OPG pathway may also be involved in
the calcific process of aortic valve stenosis.
A recent study [47] also revealed an association
between presence of aortic stenosis and low
serum levels of the anti-calcific protein fetuin-A.
Furthermore, there was evidence that fetuin-A
was deposited in calcific aortic valves. Never-
theless, the relative importance of these find-
ings to the overall pathogenesis of aortic steno-
sis remains uncertain.
Matrix Gla-protein (MGP), another anti-calcific
protein implicated in development of AS, is syn-
thesised in different tissues and undergoes vita-
min K dependent γ-carboxylation (reviewed by
[48]. The γ-carboxylated form of MGP is involved
in inhibition of tissue calcification possibly by
preventing differentiation of vascular smooth
muscle cells into chondrocyte-like cells or by
blocking BMP function [48]. Patients with aortic
valve calcification were found to have lower
concentrations of uncarboxylated MGP com-
pared with normal controls [49]; additionally
both renal dysfunction and oral warfarin therapy
were predictive of low MGP levels. Interestingly,
another study found elevated levels of both un-
carboxylated and carboxylated de-phosporylated
forms of MGP in patients with severe AS [50].
The exact biological significance of these find-
ings is still unclear.
Oxidative stress
A number of studies have documented in-
creased intravalvular content of a variety of pro-
oxidants in models of AS and in clinical samples
[23, 51-54]. Importantly, this evidence of in-
creased oxidative stress was not specifically
associated with extent of local atherogenesis in
any study.
Thioredoxin, thioredoxin reductase, NADPH oxi-
dase and thioredoxin binding protein (TXNIP)
are components of a ubiquitous system, which
regulates intracellular redox stress [55]. TXNIP
in particular is a fundamental mediator of in-
creased redox stress as it binds to, and inacti-
vates thioredoxin [56]. Increased expression of
TXNIP is associated with activation of apoptosis
signalling pathways [57], and is suppressed by
endothelial NO release [58]. We have recently
demonstrated in a rabbit model of mild AS with
histological features similar to that of human
disease, increased intravalvular concentration

Pathogenesis of aortic stenosis
190 Am J Cardiovasc Dis 2011;1(2):185-199
of TXNIP compared to control animals [53]. Fur-
thermore, co-treatment with ramipril retarded
the development of AS in this model, as meas-
ured by reduction both in transvalvular velocity
and valve echogenicity on echocardiography
[59]. This retardation further correlated with
reduction in valvular calcium and macrophage
infiltration and a reduction in TXNIP accumula-
tion within the valve matrix. This finding further
underscores the importance of ACE - Ang II sys-
tem in pathogenesis of AS.
Evidence of the role of nitric oxide in the patho-
genesis of aortic stenosis
AS, even in its early phases is associated with
the pathogenesis of acute coronary syndromes
(ACS) [8, 60, 61], which are paralleled by plate-
let hyper-aggregability [62]. Stenotic aortic
valves constitute a pro-aggregatory milieu, po-
tentially contributing to thromboembolism [24,
63]. It has been shown that patients with AS
exhibit increased platelet reactivity [64], and
thrombus formation has been documented on
severely stenosed valves [65]. AS has recently
been linked to the phenomenon of nitric oxide
(NO) resistance, even at its earliest stages [66,
67].
NO is a physiological modulator of both vasomo-
tor tone and platelet aggregation. These effects
of NO are predominantly mediated by cyclic
guanosine-3,'5'-monophosphate (cGMP), via
activation of soluble guanylate cyclase (sGC).
However, in patients with ischemic heart dis-
ease, platelets and coronary/peripheral arteries
respond poorly to the anti-aggregatory and vaso-
dilator effects of NO donors (e.g. nitroglycerin)
[68]. This “NO resistance” represents a multi-
faceted disorder at sites of abnormal NO-driven
physiology, and as such may contribute to the
increased risk of ischemic events. NO resis-
tance results from a combination of
“scavenging” of NO by superoxide radical (O2
−)
and of inactivation of sGC. The haem moiety of
sGC is a “receptor” for NO and a mediator of NO
-dependent activation. However, reactive oxygen
species, and O2-in particular, diminish sGC sen-
sitivity to NO because of the oxidation of the
enzyme-bound haem and its subsequent loss
(haem-deficient sGC).
We have investigated [66] whether AS, either
with or without concomitant coronary artery dis-
ease, is associated with impaired NO respon-
siveness. Two major abnormalities of platelet
function were documented in patients with AS.
First, platelets manifested increased aggregabil-
ity in response to ADP. Second, the anti-
aggregating effects of NO donor SNP were sig-
nificantly reduced, thus representing NO resis-
tance at the platelet level. However, presence of
the former abnormality did not account for the
occurrence of the latter.
The severity of NO resistance was independent
of presence/absence of hemodynamically sig-
nificant coronary artery disease. These results
indicate therefore that AS represents an addi-
tional "marker" of platelet NO resistance. Inter-
estingly, there was no correlation between AVS
severity and the extent of platelet NO resis-
tance. This suggests that platelet abnormalities
and impairment of NO-related mechanisms may
appear early in the clinical course of AVS.
Indeed, in our recent cross-sectional population
study evaluating biochemical and physiological
correlates of the presence of aortic valve sclero-
sis (ASc), a precursor to AS, in aging individuals
[67] we have documented that ASc was also
associated with impaired platelet responsive-
ness to NO. In that study, the extent of ASc
manifestation was strongly associated with the
extent of platelet resistance to NO. This finding
is of potential relevance to the association be-
tween ASc and thrombotic events [8]. Further-
more, tissue resistance to NO per se might also
contribute to the calcification process [69].
Asymmetric dimethylarginine (ADMA) is an en-
dogenous competitive inhibitor of NO synthase
(eNOS) and a marker and mediator of endothe-
lial dysfunction [70-72]. We have also demon-
strated that plasma concentrations of ADMA are
elevated in patients with AS, compared with
controls [73]. Thus, this finding provides further
support to the suggestion that NO generation is
impaired in AS.
A critical question which arises is whether the
pathogenesis of AS is fundamentally related to
dysfunction of the valve endothelium. NO resis-
tance in AS may be paralleled by impairment of
aortic valve endothelial function. Previous stud-
ies [63, 74, 75] have demonstrated that even

Pathogenesis of aortic stenosis
191 Am J Cardiovasc Dis 2011;1(2):185-199
early aortic valve calcification is associated with
some decrease in endothelial function and even
loss of valvular endothelium. Narrowing of the
aortic valve orifice in AS, together with deforma-
tion of leaflets and increasingly rough surface of
the valve, contributes to local turbulence in
blood flow which creates shear stress, affecting
both valve endothelium and passing platelets
[76]. Furthermore, loss of aortic valve endothe-
lium may predispose towards calcification of the
aortic valve leaflets [69, 77, 78]. Potentially,
aortic valve endothelium plays a critical role in
maintaining normal aortic valve function.
In isolated porcine and canine aortic valves, it
was shown that the aortic valve endothelium
releases NO and prostacyclin, which both exert
local anti-thrombotic effects and reduce leaflet
tone [16, 18, 63]. The main "luminal" implica-
tion of these studies was that the stenotic aortic
valve constitutes a pro-aggregatory milieu: - the
clinical consequences of a loss of valve anti-
aggregatory function must be considered to-
gether with platelet hyperaggregability and
platelet resistance to NO occurring in AVS [66].
However, it is equally possible that disordered
valve endothelial function contributes to devel-
opment of valve matrix pathology. In our recent
studies [69] with porcine aortic valve interstitial
cell cultures, calcific nodule formation and asso-
ciated redox stress were inhibited by NO donors.
This suggests that NO has a direct protective
effect extending to the inhibition of the calcifica-
tion processes in aortic valve cells.
Bicuspid aortic valve (BAV): a special case?
BAV represents the most common congenital
cardiac abnormality, with estimates of its preva-
lence ranging from 0.5 to 2% of the general
population [79]. Structurally, BAV is heterogene-
ous, depending on the pattern of valve leaflet
fusion, and its association with extravalvular
disease, as summarized in Table 2. However,
the most common pattern, accounting for about
75% of BAV cases, is fusion of the right and left
(R-L: Type I) valve leaflets, while Type II BAV
(fusion of the right and non-coronary cusps)
accounts for virtually all other cases.
A fundamental and unique issue with BAV is the
association with dilatation of the aortic root and
arch, which is a particularly prominent feature
of Type I BAV, and which is associated with risk
of aortic dissection and rupture. On the other
hand, progression of AS appears to be more
rapid in Type II [79], where most patients re-
quire eventual aortic valve replacement.
From a pathophysiological point of view, BAV is
unique and intriguing because it presents as a
basis for a rapidly progressive form of AS and/or
aortopathy, and because of the possible insight
it provides regarding the pathogenesis of AS.
As regards the rapid progression, in a retrospec-
tive study of 156 adult patients with BAV, Tha-
nassoulis et al [80] have demonstrated a mean
increase in diameter of ascending aorta of
0.37mm/year and of aortic sinus of Valsava of
0.17mm/year. Tzemos et al [81] documented
increase in aortic valve gradient of 0.7 mmHg
per annum in a prospective cohort of 642 sub-
jects with BAV.
As regards available data on the pathogenesis
of BAV, despite a very large number of recent
investigations, both basic and clinical, no coher-
ent picture has emerged. Rather, it needs to be
emphasised that there is increasing evidence of
heterogeneity of pathogenesis, notably between
Type I and Type II BAV, with increasing evidence
of redox stress and inflammatory activation for
Type I, and of predominant endothelial dysfunc-
tion for Type II [82]. Furthermore, there appears
to be a substantial genetic component to BAV
Table 2. Subtypes of BAV by anatomical structure and associated features [118].
Subtype of BAV Valvular anatomical features Associated features
Type I (70-75%) Fusion of the right and left coronary cusp
resulting in anterior – posterior commissural
orientation
More common in males.
Aortic root dilatation and higher
prevalence of aortic coarctation.
Type II (25-30%) Fusion of the right and non-coronary cusp
result in right and left commissural orienta-
tion.
More common in females.
Higher association with progression
to aortic stenosis and regurgitation
Type III (~1%) Fusion of the non-and left coronary cusp.

Pathogenesis of aortic stenosis
192 Am J Cardiovasc Dis 2011;1(2):185-199
pathogenesis.
As regards the issue of inheritance of BAV, al-
though all series show a predominance of male
patients, the overall thrust of genetic investiga-
tions is to suggest autosomal dominant inheri-
tance in most cases. A number of studies reveal
multiple cases within kindred [83-85]. The most
extensively documented mutations underlying
BAV occur in the NOTCH1 gene [86], which
would also predispose towards valve calcifica-
tion, given that NOTCH signalling has been
linked to expression of osteogenic genes osteo-
pontin and osteocalcin [87]. However the rele-
vant signal transduction pathways for AS devel-
opment in BAV remain uncertain [88] and there
is also evidence of multiple mutations associ-
ated with BAV [85].
The issue of association of BAV with endothelial
dysfunction and possibly with impaired eNOS
signalling has attracted considerable attention
since it was observed that mice lacking the
eNOS gene frequently also had BAV (but not
other congenital cardiovascular abnormalities)
[89]. However, Fernandez et al [82] have more
recently clarified this observation: the abnormal-
ity is actually Type II BAV (right - non-coronary
cusp fusion). Interestingly, these investigators
documented that Type I BAV is present in inbred
Syrian hamsters, without any known association
with eNOS deficiency
While no other endothelial function studies
have specified BAV subtype, there is substantial
additional evidence that BAV may indeed be
associated with eNOS deficiency and/or endo-
thelial dysfunction. Aicher et al [90] quantitated
eNOS protein expression in the aortic wall (but
not the valve) at the time of surgical valve re-
placement and observed lower levels of eNOS in
BAV versus tricuspid ("normal") valves in the
presence of AS. Furthermore, eNOS expression
was negatively correlated with aortic diameter.
A further study has examined brachial flow-
mediated dilatation (FMD): - in a group of men
with BAV, there was an association between
aortic dilatation and impaired FMD [91], consis-
tent with findings of Archer el al [90]. A number
of other studies have evaluated the association
between AS and NO deficiency, but not always
in the context of BAV: these findings will be dis-
cussed later.
The concept of inflammatory change and oxida-
tive stress is a relevant component of the patho-
genesis of BAV, and is in a sense linked to that
of NO deficiency. For example, Tzemos et al [91]
observed that aortic dilatation in BAV was asso-
ciated not only with impaired FMD, but also with
increased plasma levels of matrix metallopro-
teinase 2 (MMP-2), which might have contrib-
uted to the dilatation process. Furthermore,
Phillippi et al [92] demonstrated that suscepti-
bility to oxidative stress was increased, and re-
sponsive expression of metallothionein de-
creased in BAV aortae. While these findings
implicate oxidative stress and inflammatory
activation, there is also evidence that endothe-
lial progenitor cell mobilization in response to
these changes may be abnormal [93], contribut-
ing to perturbation of aortic and valvular endo-
thelial function.
Intervention studies to retard the progression of
AS
Statins
Statins were the first commercially available
drugs studied in aortic stenosis given their es-
tablished benefit in primary and secondary pre-
vention of cardiovascular diseases, as well as
evidence of association of lipid infiltration of
aortic valve in AS [25, 29, 30]. Whilst there are
many "dyslipidemic" animal models of AS (see
animal model section), only one model so far
has shown benefit of statin therapy [94].
There were a number of very encouraging hu-
man retrospective studies of statins in AS, sug-
gesting significant reduction in the rate of pro-
gression of AS with statin use [95-99]. Yet, only
one small prospective open-label non-
randomized observational study of rosuvastatin
in patients with moderate AS showed slowing of
haemodynamic progression of AS [100]. Three
large prospective double-blind randomized pla-
cebo control trials of statins in AS failed to show
any retardation of AS progression [101-103].
ACE-inhibitors (ACEI)/Angiotensin receptor
blockers (ARB)
Numerous studies have demonstrated in-
creased ACE activity/expression and Ang II pres-
ence in stenotic valves [37-39], providing a ra-
tionale for investigation of benefits of ACEI/ARB
therapy in AS. Treatment of cholesterol-fed rab-
bits with angiotensin receptor-1 blocker olme-

Pathogenesis of aortic stenosis
193 Am J Cardiovasc Dis 2011;1(2):185-199
sartan was associated with decreased macro-
phage infiltration and reductions in osteopontin
and ACE in aortic valves [104]. Unfortunately,
no hemodynamic valvular measures of AS pro-
gression were performed. We have demon-
strated hemodynamic retardation of AS, con-
comitantly with reduction in calcification, macro-
phage infiltration, redox stress and improve-
ment in endothelial function, with ACEI ramipril
treatment in a rabbit model of AS [59].
As regards human studies, no prospective trials
of either class of agents have been published to
date. Two main retrospective evaluations of
population studies have provided conflicting
data: - Rosenhek et al [98], utilizing echocardo-
graphic parameters, found no significant effect
of ACE-I therapy on AS progression (albeit with a
trend to slower progression), while O'Brien et al
[105], in a study using CT-based calcification
assessment, found lower rates of AS calcifica-
tion, after correction for comorbidities.
Bisphosphonates
Bisphosphonates, usually used for treatment of
osteoporosis in humans, inhibit bone resorption
as they cause osteoclast apoptotosis [106,
107]. They also inhibit an enzyme in the choles-
terol synthesis pathway, which causes abnor-
malities in the cytoskeleton in the osteoclast,
thus reducing bone resorption [108]. Thus,
bisphosphonates may directly reduce valvular
calcification via their osteoblast action, as well
as indirectly via inhibition of inflammation and
resultant fibrosis. A study in a rat model of dialy-
sis suggested that etidronate, now rarely used
bisphosphanate, limited aortic calcification
[109].
Two small retrospective human studies of
bisphophonates demonstrated reduction in the
AS progression rate [110, 111]. However the
small size and observational nature of these
studies make the results hypothesis generating
at best.
Aldosterone blockade
Aldosterone has been implicated in animal stud-
ies in vascular inflammation and myocardial
fibrosis, and these were ameliorated by aldos-
terone blockade [112]. Eplerenone, an aldoster-
one-receptor antagonist, shown to improve out-
comes in heart failure patients [113], was tri-
alled in a randomized double-blind placebo-
controlled study in patients with moderate-
severe AS [114]. This small trial failed to show
reduction in rate of progression of valve steno-
sis.
Conclusion
The major objective of this review is to present
the case that pathogenesis of AS is an active
process that involves a combination of inflam-
matory activation, increased oxidative stress,
fibrosis and calcification, which should be ame-
nable to therapeutic intervention.
AS is the commonest form of valvular heart dis-
ease and its prevalence is rising due to increas-
ing longevity, especially in Western world. We
present evidence that AS, previously thought to
be "degenerative" disorder of aging, is a com-
plex active process, involving valvular endothe-
lium, fibroblasts and extracellular matrix. The
process is characterized by inflammatory activa-
tion and lipid deposition within valve lesions.
There is extensive valvular matrix remodelling
and fibrosis with increased production of MMP-
1 and -2, TGF-β1, interleukin-1β and TNFα.
There is extensive evidence for increased pro-
duction of Ang II, a major pro-inflammatory and
pro-fibrotic mediator, within stenotic valves. This
would lead to further fibrosis and calcification.
Impaired activation of anti-calcific modulators,
such as fetuin-A and MGP, is alos important in
AS. There is concurrent increased in oxidative
stress and evidence of impairment of the nitric
oxide system as well as associated systemic
endothelial dysfunction.
In BAV, representing the most common congeni-
tal cardiac abnormality, valvular inflammation is
combined with aortopathy. BAV illustrates
unique interplay of genetically driven inflamma-
tory activation and, in some cases, deficiency of
nitric oxide formation.
The most promising targets for pharmacological
interventions have been thought to be lipid infil-
tration and atheroma formation. However, re-
sults of all randomized double-blind prospective
studies of statins have been disappointing [101
-103]. ACE-I/ARB therapy, on the other hand,
shows promising results. Although post-hoc
clinical data are limited and inconclusive, we
have recently demonstrated in an animal model
that ACE-I ramipril retards progression of mild

Pathogenesis of aortic stenosis
194 Am J Cardiovasc Dis 2011;1(2):185-199
AS [59]. In a separate study, olmesartan treat-
ment was also associated with reductions in
macrophage infiltration and ACE in aortic valves
in a rabbit model [104].
Therefore AS represents the result of a pro-
longed inflammatory process leading to valve
calcification and ossification, which represents
a potential target for therapeutic interventions.
Acknowledgements
This work is supported in part by research
grants from the National Health and Medical
Research Council of Australia (NHMRC) and
Cardiovascular Lipid Research Grants
(Australia).
Address correspondence to: John D Horowitz, Cardiol-
ogy Unit, The Queen Elizabeth Hospital, University of
Adelaide, 28 Woodville Road, Woodville, SA 5011,
Australia. Tel: +61 8 82226000; Fax: +61 8
82227201, E-mail: john.horowitz@adelaide.edu.au
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