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Aortic Stenosis
A General Overview of Clinical, Pathophysiological and Therapeutic Aspects
Finn Akerstrm, Maria G Barderas, Luis Rodrguez-Padial Expert Rev Cardiovasc Ther. 2013;11(2):239-250.

Abstract
Aortic stenosis is the most prevalent valve pathology and calcific aortic valve disease (CAVD) is its most frequent etiology in developed countries. There is extensive evidence that CAVD represents an active disease process similar to that of atherosclerosis with similar classical cardiovascular risk factors and pathological mechanisms. Given that in the vast majority of situations the only treatment available is valve replacement there is a need to develop pharmacological therapies that retard the disease progression. Lipid-lowering therapies have been the focus of research, however, so far with negative results. Future studies, including animal models, shall provide an opportunity to further evaluate the disease mechanisms of CAVD and to discover potential disease biomarkers and pharmacological interventions that can reduce the need for valve replacement.

Clinical Aspects of Aortic Stenosis

Prevalence & Etiology

Aortic stenosis (AS) is the most prevalent valve pathology and the third most common cardiovascular disease (CVD) after hypertension and coronary artery disease. Since it commonly affects older individuals, with an aging population, its prevalence is expected to further increase.[1] For example, in the Cardiovascular Health Study which included 5201 men and women aged 65 years and above, 26% of the study participants had aortic sclerosis, an early stage of AS without hemodynamic obstruction and this increased to 48% in those older than 85 years. In the same way, AS was detected in 2% of the study population and in those above 85 years of age this was increased to 4%.[2] Three main etiologies cause AS in the adult: rheumatic heart disease and calcific aortic valve disease (CAVD) of a normal trileaflet valve or of a congenital bicuspid valve (CAVD is sometimes referred to as degenerative or senile AS). CAVD has previously been considered a passive degenerative 'wear and tear' disease that comes with increasing age. However, during the last two decades, a large body of evidence has surfaced, which supports an active disease process that shares both pathophysiological features and risk factors with that of atherosclerosis.[3] Bicuspid congenital aortic valve (AV) is present in about 12% of babies born and is the most common cardiac congenital abnormality. Most individuals are men (7080%), the most prevalent anatomy for a bicuspid valve is fusion of the right and left coronary cusps and it is frequently associated with other cardiovascular abnormalities, such as dilatation of the aortic root and arch. The valve abnormality commonly confers a normal valve and cardiac function until later on in life when degenerative calcification develops, a pathological process that is similar to that of the aortic trileaflet valve, although congenitally abnormal valves are associated with earlier valvular calcification.[4,5] This is supported by the data from a registry 646 patients who received AV replacement (AVR) at the Mayo Clinic between 1981 and 1985 (children and concomitant mitral disease were included), that reported a majority of bicuspid valves in those below 70 years of age and a majority of calcified trileaflet valves in older patients. In developed countries, rheumatic fever is nowadays infrequent and rheumatic AS is becoming an increasingly rare disease in this part of the world. Nevertheless, in developing countries and thus worldwide, rheumatic heart disease is the most common cause of AS and with an immigrant population this etiology still needs attention. Importantly and in contrast to CAVD, rheumatic AS invariably accompanies mitral valve involvement and the echocardiographic examination commonly reveals commissural fusion.[4] For the purpose of this review, rheumatic AS will not be further discussed, and the authors will here on concentrate on CAVD.
Clinical Course

The narrowing of the AV effective orifice due to progressive leaflet calcification does not cause a significant pressure gradient until more than half of its normal area (3 cm2) is lost.[6] At this point the average rate of progression has been reported as a decrease in valve area of 0.1 cm2 per year.[7] Severe AS is defined by echocardiography as a valve area less than 1.0 cm2, mean gradient above 40 mmHg, or jet velocity greater than 4.0 m/s.[8,9] Nonetheless, it is worth noting that the disease process represents a continuum and this makes it difficult to define its severity by single values. With time, the valve area is progressively decreased and obstruction gradually progresses, which results in increased wall stress (afterload) of the left ventricle (LV). As a result, concentric LV hypertrophy develops in proportion to the increased intraventricular pressure, which permits wall stress to remain normal.[10] This compensatory process normally maintains the LV wall-stress and ejection fraction within normal limits and the patient remains asymptomatic for years. Eventually, because of the gradually reduced valve area, the hypertrophic process is not able to compensate the elevated intraventricular pressure and wall stress and thus the LV ejection decreases. However, LV hypertrophy is not an ideal compensatory mechanism since it usually reduces the diastolic function, which increases end diastolic pressures. Therefore, dyspnea in AS may be the result of diastolic and/or systolic LV dysfunction.[1] Once the individual develops symptoms (dyspnea, angina or syncope), commonly only observed on exertion, AVR is indicated since average survival in this patient group is 23 years in the absence of surgery.[8,9] In a selected patient subgroup with high perioperative risk, percutaneous transcatheter AV implantation is currently an established therapeutic option. The management of patients with asymptomatic severe AS remains a dilemma for cardiologists and surgeons. Pellikka et al. published in 1995[11] and 2005[12] results from two patient registries with asymptomatic AS. The first registry showed that no patient had sudden death or cardiac death without preceding symptoms, however the second report, which included more patients and a longer follow-up (5 years), revealed a rate of sudden death that was not preceded by symptoms of approximately 1%. In 2008 Brown et al. reported a significant survival benefit in the same type of patients (asymptomatic severe AS) who underwent AVR, when compared with those who remained asymptomatic and did not have AVR. [13] Nevertheless, it should be noted that the three studies are retrospective analyses, all from the Mayo Clinic with a reported relatively low surgical mortality for AVR. Hence, there might not be a survival benefit from AVR for a patient with asymptomatic severe AS at a center with a lower operative volume, less experience and subsequently higher surgical mortality rate. Furthermore, there might have be patients who developed symptoms and did not inform their physicians or, owing to their sometimes subtle nature, did not notice them. At present, clinical guidelines do not indicate AVR in patients with asymptomatic AS (with the exception of LV dysfunction or an abnormal exercise stress test) and recommend close clinical follow-up since most will develop symptoms within 5 years.[14]

Pathogenesis of Calcific AV Disease: An Active Process

Normal AV Anatomy & Function

The heart valves are composed of a specialized set of cells, namely valvular endothelial cells (VECs) and valvular interstitial cells (VICs) and an extracellular matrix that includes collagen, elastin and glycosaminoglycans (Figure 1). A typical layered composition of cellular and extracellular compartments can be found in all four valves. It represents a spatial and differential arrangement that provides the strength and flexibility needed for a unidirectional, unobstructed blood flow and to withstand mechanical stress and strain on valve closure. The VECs line the blood-containing surfaces of the valves and the VICs are present in all three layers of the valve: fibrosa, spongiosa and ventricularis. The fibrosa, close to the aortic (outflow) side, contains a layer of collagen that provides strength and the ventricularis is next to the ventricular (inflow) side of the valve and is rich in elastin. The spongiosa is the central layer and contains loose connective tissue made up of glycosaminoglycans that permits a necessary rearrangement of the collagen and elastin layers throughout the cardiac cycle.[3]

Figure 1.

Cellular architecture of the aortic valve. GAGs: Glycosaminoglycans; VIC: Valve interstitial cells. Reproduced with permission from [3]. The VICs have fibroblast-like characteristics but experimental studies have shown that they constitute a heterogeneous, dynamic and highly plastic population of cells.[15] A division into a family of five phenotypes has been proposed: embryonic progenitor endothelial/mesenchymal cells, quiescent VICs (qVICs), activated VICs, progenitor VICs and osteoblast VICs (). The embryonic progenitor endothelial/mesenchymal cells, located in the embryonic cardiac cushions, are involved in the valve formation during embryogenesis and undergo endothelial-to-mesenchymal transformation. The qVICs are at rest during normal adult valve physiology and the activated VICs regulate stress related responses in relation to valve injury and disease. The progenitor VICs are valvular stem cells and are derived from bone marrow-derived cells, circulating cells and resident valvular progenitor cells. The osteoblast VICs are referred to as the VICs that undergo osteoblastic

differentiation and therefore involved in valve calcification.[16] The different VICs subpopulations present a separate morphology and synthesize a unique extracellular matrix, which permits autocrine and paracrine regulation of cell function and differentiation from one form to another and, in disease, to other cell types such as myofibroblast- and osteoblast-like cells.[17] This VICs subgroup and the extracellular matrix interplay, appears to be a crucial piece in the pathogenesis of calcification and fibrosis that leads to valve obstruction.
Table. Proposed classification of valve interstitial cells phenotype. Cell type Location Function

Embryonic progenitor endothelial/mesenchymal cells qVICs

Give rise to resident qVICs, possibly through an activated Embryonic stage. EMT can be detected by the loss of endothelial and cardiac cushions the gain of mesenchymal markers Heart valve leaflet Maintain physiologic valve structure and function and inhibit angiogenesis in the leaflets

pVICs

Bone marrow, Enter valve or are resident in valve to provide aVICs to circulation and/or repair the heart valve, may be CD34, CD133, and/or S100heart valve positive leaflet A-SMA-containing VICs with activated cellular repair processes including proliferation, migration and matrix remodeling. Respond to valve injury attributable to pathological conditions and abnormal hemodynamic/mechanical forces Calcification, chondrogenesis and osteogenesis in the heart valve. Secrete alkaline phosphatase, osteocalcin, osteopontin, bone sialoprotein

aVICs

Heart valve leaflet

obVICs

Heart valve leaflet

A-SMA: alpha-smooth muscle actin; aVICs: activated VICs; EMT: Epithelial-mesenchymal transition; obVICs: osteoblast VICs; pVICs: progenitor VICs; qVICs: quiescent VICs; VIC: Valvular interstitial cells. Reproduced with permission from [16]. The VECs are similar to other vascular endothelial cells although phenotypically different.[18] They are thought to interact with VICs in order to regulate normal valve function and may therefore be disease mediators. There are also differences between those VECs lining the aortic side and the ventricular side of the AV and this may explain the side-specificity of early AV calcification to the aortic side of the valve. Although not fully elucidated, the AV endothelium is thought to have similar functions to that of the vascular system, namely an important regulator of physiological and pathological processes, especially atherogenesis.[19]
Calcific AV Disease is an Active Process

Histopathological studies of early lesions in CAVD showed presence of chronic inflammatory cell infiltrate, basement membrane disruption, lipid accumulation in the adjacent fibrosa, thickening of fibrosa and calcification.[20] Interestingly, abnormalities related to aging (presence of adipose cells interposed between the fibrosa and ventricualris layer of the leaflet, nonspecific thickening of the leaflet tip and decrease thickness and length of the spongiosa) were different from those linked with CAVD. The findings indicated that CAVD is an active disease process, and not a passive age-related degeneration, that occurs in a subset of patients, and displays similarities with the pathogenesis of atherosclerosis. Indeed, epidemiological studies have confirmed that AS and atherosclerosis share common clinical cardiovascular risk factors such as male gender, age, arterial hypertension, smoking and elevated LDL cholesterol (LDLc) levels.[21] Furthermore, although the prevalence of CAVD is clearly related to aging, the majority of elderly patients to not develop macroscopic AV leaflet abnormalities.[22] Age could therefore, in most situations, be considered a prerequisite for the development of CAVD that occurs in the presence of an accumulation of certain risk factors, some of which are not currently known.
The Effect of External Hemodynamic Forces: Elevated Stretch & Shear Stress

The initial events of the CAVD process may, in part, be represented by the presence of abnormal hemodynamic forces that exert increased stretch and shear stress of the AV cusps, producing inflammation and extracellular matrix remodeling. Ex vivo studies have demonstrated that supraphysiological stretch, which deform the AV cusps during the cardiac cycle, induces pathological processes similar to what has been observed in CAVD, namely, increased expression by VICs of remodeling enzymes, proinflammatory proteins, bone morphogenetic proteins and ALP.[23,24] These changes are observed mainly on the aortic side of the AV cusps (fibrosa layer), probably owing to increased deformation of the fibrosa layer than the ventricularis as well as different transcriptional profiles expressed by the VECs on the opposite faces of the AV cusps. [19] Furthermore, in vitro VECs have been found to respond to supraphysiological stretch through increased expression of adhesion molecule.[25] Additionally, Xing et al. found that increased shear stress, as represented by increased cyclic pressures, led to increased collagen and sulfated glycosaminoglycan synthesis, in vitro.[26] Thus, abnormal mechanical forces regulate the AV pathogenesis through increased stretch and shear stress may explain the increased prevalence of CAVD in patients with hypertension and the fact that patients with bicuspid AV (with a different hemodynamic profile) present a more rapid progression of AS than those with trileaflet AV.[5]
Lipid Accumulation

During the early phases of the CAVD process there is increased accumulation of LDLc in the subendothelial space and in the deep layer of fibrosa.[20,27] Apolipoproteins B, (a), and E, have all been identified near the lipidrich regions, which indicates that the accumulating lipids are derived from plasma lipoproteins. The triggers responsible for the increased lipid accumulation are not completely known although enhanced endothelial permeability and/or retention of LDLc by extracellular matrix have been suggested as potential mechanisms.[27] Oxidized LDLc are taken up by macrophages and become foam cells and together with activated T lymphocytes[28] they activate several proinflammatory cytokines such as TGF-1[29] and IL-1 that in turn recruit local production of matrix metalloproteinases, all of which contribute to cell apoptosis, extracellular matrix remodeling with fibrosis and local calcification.[30] Furthermore, TNF-, another important regulator of inflammation and tissue remodeling, has been found to colocalize with MMP-1.[31]
Oxidative Stress

Abnormalities in oxidative stress seem to be crucial in many cardiovascular pathologies, namely atherosclerosis, arterial hypertension and thrombosis,[32] and there is evidence that reactive oxygen species (ROS) could also be a key player in the initiating events of the pathogenesis of AS. Calcified AVs have been found to exhibit increased levels of superoxide and hydrogen peroxide in humans, [33] and in mice ROS were found to be increased before the development of hemodynamically significant valve stenosis, indicating that it is not merely a result of valve stress.[34] Furthermore, ROS have been shown to be implicated in several signaling pathways in AS such as TGF-1.[35]
ReninAngiotensinAldosterone System

The reninangiotensinaldosterone system (RAAS) is probably another important signaling pathway in the pathogenesis of AS. Angiotensin-converting enzyme (ACE)[36] and angiotensin II receptors are found in stenotic AVs.[37] Angiotensin II is known to cause inflammation and fibrosis and may be formed by ACE as well as mast cell-derived neutral protease chymase.[37] Furthermore, and discussed later on in this review, there is some clinical evidence that inhibition of the RAS pathway may slow AV disease progression.[38]
AV Calcification

The progress of continuous calcification of the AV is what primarily conditions the hemodynamic obstruction in AS, thus making it a final common pathway in CAVD. At the beginning of the AV calcification process, nodules are formed near the margins of attachments of the valvular cusps and there is as yet no hemodynamic obstruction (sclerosis). As the disease advances, these calcified nodules extend throughout the cusps towards the outflow surfaces leading to hemodynamic obstruction (stenosis). Cardiovascular calcification is characterized by the presence of hydroxyapatite deposited on a bone-like matrix of collagen, osteopontin and other bone matrix proteins (BMP).[39] Histological studies of calcified AVs from surgeries have confirmed the presence of osteoblast bone formation;[40,41] the presence of BMP 2 and 4, which are potent osteogenic morphogens;[40] and the expression of bone extracellular matrix molecules like alkaline phosphatase, osteocalcin, bone sialoprotein and osteopontin.[41] Interestingly, accumulation (adsorption) of bone related proteins such as osteopontin has also been suggested as a mechanism in bioprosthetic AV calcification and dysfunction.[42]

The AV calcification is mediated by VICs differentiation and proliferation into an ossification phenotype induced by an interplay of several mechanisms that includes upregulation of procalcific markers (e.g., osetopontin), biomechanical forces (elevated AV cusp stretch,[23] shear stress[26] or altered ECM stiffness[43]), ROS,[33] inflammatory markers and growth factors.[29] Hence, osteoblasts and myofibroblasts, responsible for the bone formation within the AV, probably originate from native qVICs. In addition, endothelial cell transition[44] and a small number of circulating progenitor cells[45] may also contribute to the osteoblast and myofibroblast population. The importance of VIC proliferation and differentiation in CAVD is supported by the fact that inhibition of VIC proliferation significantly reduces calcification. For instance, the use of ERK pathway antagonists showed reduced VIC proliferation, apoptosis, nodule formation leading and maintained the cells in the quiescent phenotype,[46] and the delivery of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) that reduced VIC proliferation showed decreased apoptosis and calcification.[47,48] On a separate note, the previously mentioned altered expression of markers of bone turnover has led to the evaluation of a potential association between the pathophysiology of CAVD, altered calcium metabolism and bone mineralization. An experimental study in apoE/ mice confirmed an inverse relationship between bone mineralization and AV calcification, driven by systemic and local inflammation.[49] In humans, AV calcification is accelerated by low serum calcium levels,[50] chronic kidney disease[51] and elevated parathyroid hormone and low vitamin D levels.[52] Furthermore, multivariable analyses have reported an independent association between low bone mineral density (osteoporosis) and cardiovascular ectopic calcification [53] including progression of AV calcification.[54] This contradictory association (low bone mineralization and cardiovascular ectopic calcification) has been referred to as the calcification paradox.[55]
Genetics of Calcified AS

Epidemiological studies have shown that bicuspid AVs is not merely a sporadic event but present patterns of genetic inheritance, occurring more frequently in first-degree relatives of affected patients.[56] Geographical clustering and familial aggregation has also been found in individuals with trileaflet CAVD. [57] During recent years, different groups have evaluated the genetic variants associated with the development of CAVD. Probably one of the most significant findings is the association between the vitamin D receptor gene polymorphism of the B allele and prevalence of CAVD in a casecontrol comparison.[58] The B allele of the vitamin D receptor is known to be associated with a faster bone loss with aging, [59] and as a result, the study results provide further evidence that abnormal bone metabolism is an important factor in CAVD pathogenesis (see discussion on the calcification paradox in the previous section). Another interesting study reported that a specific mutation in the NOTCH1 gene was found to be associated with severe AV calcification in seven patients, with both bicuspid and trileaflet AVs, but free from other cardiac malformation. A proposed involvement of the Notch signaling pathway in CAVD is the promotion of osteoblast differentiation by upregulation of osteoblast-specific genes like osteopontin and osteocalcin.[60] Other important discoveries include lipoprotein abnormalities[61] and polymorphism in the estrogen receptor.[62] Although this field of research is at an early stage compared with other study platforms, its recent technological advances now make it possible to thoroughly analyze the human genome in the search for suspected and unsuspected gene alterations in CAVD. Finally, additional signaling pathways, such as TNF-, NF-B and Wnt/-catenin signaling, as well as calcification-specific transcription factors like MSX2, RUNX2 and SOX9, have been shown to regulate the pathogenesis of CAVD. Their discussion is nonetheless beyond the scope of this article and the reader is therefore referred to an excellent and detailed review of the CAVD pathogenesis by Miller et al. [32] Figure 2 provides a summary of the main aspects of the CAVD pathogenesis.

Figure 2.

Summary of the currently considered essential components of the pathogenesis of calcific aortic valve disease that have been schematically separated into early, intermediate and late stages. Aging can generally be regarded as a prerequisite to the initiation of the disease process that is augmented by the presence of risk factors (abnormal calcium metabolism, classical cardiovascular risk factors, genetic predisposition and hemodynamic forces). Together these produce endothelial injury of the AV leaflet endothelium which results in subendothelial lipid accumulation, infiltration of activated inflammatory cells and release of a cascade of proinflammatory cytokines, which induces ECM remodeling and VICs osteogenic differentiation and

proliferation. As a result there is upregulation of procalcific mediators (BMP-2 and 4, osteopontin, osteocalcin and osteonectin), which promotes AV calcification and eventually hemodynamic obstruction. AV: Aortic valve; BMP: Bone matrix protein; ECM: Extracellular matrix; LDLc: LDL cholesterol; ROS: Reactive oxygen species; VICs: Valve interstitial cells.

Medical Therapies in Calcific AV Disease: Evidence From Experimental & Clinical Studies
Currently the only treatment for CAVD is surgical or percutaneous valve replacement. Given its high prevalence, long disease course and increased knowledge of its pathogenesis, with several potential therapeutic targets, medical therapies have been evaluated in CAVD both in experimental and clinical studies. The following section will deal with three principal lines of investigations, lipid-lowering therapies, RAAS inhibition and bisphosphonates. Figure 3 gives an overview of the natural progression of CAVD with its current treatment options and novel potential therapeutic targets that could slow or reverse the disease course.

Figure 3.

Schematic overview of the natural progression of calcific aortic valve disease with its current treatment options and novel potential therapeutic targets that may slow or reverse the disease course. ACE: Angiotensinconverting; CAVD: calcific aortic valve disease; ECM: Extracellular matrix; TAVI: Transcatheter aortic valve implantation; VICs: Valve intersticial cells. Data taken from [77].
Lipid-lowering Therapies

Lipids play a crucial role in atherosclerosis and, as previously discussed, also in CAVD where plasma LDLc has been found to deposit in the human AV subendothelial space, oxidize and initiate several pathogenic processes.[27] It is therefore of no surprise that patients with homozygous familial hypercholesterolemia, with very high LDLc levels, develop CAVD.[63] Furthermore, apolipoprotein E and LDLc receptor deficient mice have been used in several in vivo models to produce pathological changes of the AVs, similar to that of human AV sclerosis and stenosis.[64] In studies with rabbits fed a cholesterol-high diet, atorvastatin reduced the expression of markers of bone matrix and calcification.[6567] In humans, epidemiological data show that statin treatment is linked with slowing of the hemodynamic progression of CAVD.[68] Despite such promising results, three randomized clinical trials (SALTIRE,[69] SEAS[70] and ASTRONOMER[71]) failed to show the slowing of CAVD progression in patients with predominantly mild-to-moderate AV stenosis that received lipid-lowering therapy (atorvastatin, simvastatin combined with ezetemibe and rosuvastatin, respectively). It is worth noting that, the SEAS trial had a relatively large sample size (1873 patients) and long follow-up (median of 52.2 months).[70] Only one prospective study (RAAVE),[72] showed a small but significant decrease in AV area in patients receiving rosuvastatin. Importantly, in this study, the patient groups were not randomized and the treatment drug was only given to those with elevated LDLc. Still, the negative results may seem perplexing since a large body of preclinical evidence and human epidemiological data support a treatment benefit of lipid-lowering therapies. One explanation may be that treatment with lipid-lowering drugs may still slow the progression of CAVD, but their effect is less, or nonexistent, at later stages of the disease process. As previously discussed, pathological changes are present in AV without any significant hemodynamic obstruction.[20] As suggested by Helske and Otto, certain pharmacological therapies are only effective at one point in a disease process.[73] Hence, in the case of CAVD, lipid-lowering therapies may only work before pathological VICs phenotype transition has occurred and not when osteoclast cell transition and calcification is established. This hypothesis, that reduction in lipid levels in the early stages of the CAVD pathogenesis, is supported by a recent study in Reversa mice where normalization of blood cholesterol was achieved by a 'genetic switch' at 6 months. [34] At this stage, only markers of CAVD, such as elevated superoxide activation, lipid deposition and calcification, were present. After another 6 months, these pathological changes were reversed in the group with normalization of cholesterol levels. The nonreversed group, with continuous high cholesterol levels, presented more pronounced AV calcification and reduced cusp mobility. In line with this, a retrospective study found statin therapy to be related to a slower progression of CAVD in patients with AV sclerosis and mild-stenosis but not in moderate AV stenosis.[74] Another possibility is that, although CAVD is found in individuals with normal lipid levels, those who benefit the most from lipid-lowering therapy are those with hyperlipidemia. These patients were systematically excluded from the three negative randomized clinical trials since their inclusion would have been unethical. Indeed, in the positive RAAVE trial,[72] patients who received rosuvastatin had high baseline LDLc levels and the animal models of CAVD were provoked by very high cholesterol levels.[6567] Nevertheless, this issue has no real effect on clinical practice since patients with hypercholesterolemia should receive lipid-lowering treatment based on current clinical guidelines regardless of coexisting CAVD.[75] Finally, given the complex CAVD pathogenesis, involving several molecular pathways, preventing lipid accumulation just may not be enough. Therefore, therapies that target several pathological mechanisms simultaneously might be more effective.

RAAS Inhibition
Calcific AV show local presence of ACE and AT II receptors and two animal studies with an angiotensin receptor 1 blocker (olmesartan)[76] and an ACE inhibitor (ramipril),[77] respectively, showed a reduction in markers of CAVD process[76,77] and hemodynamic retardation of AV stenosis.[77] However, retrospective clinical studies have evaluated the effect of RAAS inhibition in humans with CAVD but with conflicting results.[68,78] To date, no prospective studies in this area have been made, making the clinical implications of RAAS blockage an area of uncertainty.

Proteomics: A Novel Approach to Study Calcific AV Disease Pathogenesis & to Discover Potential Biomarkers & Therapeutic Targets
Even though preclinical studies have brought significant advances in the study of the pathogenesis of CAVD, many of its pathological mechanisms need further analysis. Additionally, like in other areas of cardiology, new biomarkers are needed to provide improved diagnostic accuracy at the early stages of disease. In CAVD this is of special interest since it presents a relatively long asymptomatic phase and pharmacological treatment may be more effective at these early stages of the disease.[34,73] One of the limitations of biomarker research is that, until now, each study may analyze data from thousands of patients but only focuses on a small number of proteins. The new proteomic approaches will probably represent a considerable advance by providing the investigators with the possibility of exploring hundreds of proteins at once and identifying new unforeseen biomarkers.[79,80] Proteomics has been defined as a global large-scale qualitative and quantitative dynamic study of the proteins expressed in one biological system under given conditions and at a specific point in time. [81] Until recently, the usual approach for the study of CVD has been to study the role of a candidate protein supposedly involved in the formation or progression of the disease. However, with the appearance of the new proteomic techniques of protein separation (2D gel electrophoresis, 2D difference gel electrophoresis, multidimensional liquid chromatography), and their identification by mass spectrometry, the evaluation of thousands of proteins at once is now possible. This is nicely exemplified by a recent study by Mebazaa et al. [82] where they confirmed the incremented diagnostic value of a novel candidate diagnostic biomarker, the protein QSOX-1, on top of brain natriuretic peptide in patients with acute decompensated heart failure. By contrast to other biomarkers used in clinical practice in cardiology, like brain natriuretic peptide and troponin, the discovery of QSOX-1 is the result of an unbiased hypothesis-free approach and not based on currently known pathophysiological mechanisms. Therefore, in a hypothesis-driven approach, QSOX-1 probably would not have been identified since its role in acute decompensated heart failure is not well understood. The AV probably contains thousands of proteins, some of whose expression is modulated in pathological situations like CAVD. Their alteration should help us to better understand the pathogenesis of CAVD and facilitate the discovery of potential biomarkers and therapeutic targets. A potential biomarker that could detect the initial stages of CAVD is of special interest, since there is evidence that pharmacological intervention at this early stage may halt the disease progresses.[34,74] Our group have studied the human calcified AV by proteomic techniques and identified several alterations in the abundance of proteins, some of which are known to be implicated in other CVD process such as atherosclerosis. The authors compared the proteome of 20 patients with CAVD, mainly severe stenosis, with a control group consisting of normal AV from necropsies (Figure 4).[83] Through differential electrophoresis (DIGE) and matrixassisted laser desorption/ionization time of flight we detected altered abundances in 43 protein species in calcific AV compared with healthy AV (35 and eight proteins presented increased and decreased abundances, respectively). All of which corresponded to biological systems implicated in CVD like inflammation, fibrosis, lipid metabolism and transport and coagulation. The abundance of inflammatory proteins, such as -1-antitrypsin and serum amyloid P-component, confirms the implication of this biological process in CAVD and also its link to atherosclerosis, since inflammation is accepted as a central part of the pathogenesis. As detailed previously, oxidative stress seems to be a significant factor in the initiation and progression of CVD including CAVD. [33] The authors found increased expression of two proteins, glutathione S-transferase and superoxide dismutase, involved in the regulation of oxidative stress that have been found to increase cell resistance to oxidative vascular injury.[84] Therefore, these proteins may exert a protective role in the pathogenesis of CAVD, at least at some point in the disease process.

Figure 4.

Schematic representation of the study protocol. Normal and stenotic arotic valve samples where homogenized and the protein extract where analyzed by 2D-DIGE, revealing increased and decreased abundances of 35 and eight proteins, respectively. The proteins where validated by IHC and WB and identified by mass spectrometry. 2D-DIGE: 2D difference gel electrophoresis; C: Control; IHC: Immunohistochemistry; P: Patients; WB: Western blot. Adapted with permission from [83]. In another study the authors analyzed the plasma of 24 patients with severe calcific AV stenosis and compared it with a control group.[85] Plasma is a useful clinical sample owing to its noninvasive acquisition and simple and inexpensive technique of sample collection.[86] Moreover, the plasma proteome should theoretically represent every organ through their interaction with the blood circulation. A total of 36 proteins with altered expressions

were observed, again corresponding to the chief biological process implicated in the CAVD disease process, as described previously. Unexpected proteins with unknown functions in CAVD included 2 -HS glycoprotein or fetuin, and ceruoplasmin (cp). Interestingly, studies of 2-HS glycoprotein have reported a reduced activity in atherosclerosis,[87] an inverse relationship with AV calcification[88] and an inhibitory role of ectopic calcification.[89] This provides a rationale for its decreased activity, and implication in AV calcification, as observed in our study. Cp may be involved in copper transport, angiogenesis, coagulation and protection against oxidant stress. Epidemiological data suggest that serum cp could be a risk factor for CVD and it is a potent catalyst of LDLc oxidation in vitro.[90] Therefore, the high levels of cp observed may represent an oxidative state of the calcified AV. Although our work constitutes the initial phase of proteomic research in CAVD (no other proteomic studies in human AV have so far been published), its unbiased approach and high analytic capacity represent important advantages. The authors therefore believe that the discipline will grow and that future work will prove to be fruitful, enhancing our understanding of CAVD pathogenesis, with the final aim of developing potential biomarkers and therapeutic targets.

Expert Commentary
AS is an increasingly prevalent CVD, with CAVD being the principal cause in the developed world. Throughout the last two decades, experimental and clinical studies have demonstrated that CAVD is not a passive degenerative disease process as once thought. Instead, it represents a highly complex interplay of various signaling pathways involving multiple molecular effectors. It encompasses: endothelial dysfunction; subsequent matrix remodeling with lipid accumulation; inflammation and fibrosis; and calcification and hemodynamic obstruction as final common disease processes. Classical cardiovascular risk factors, such as arterial hypertension, smoking and elevated LDLc and oxidative stress provoke endothelial dysfunction, which seems to be one of the initiating events. During recent years, and as a result of the development of hypercholesterolemic animal models, increased lipid levels has been recognized as an important contributor to the pathogenesis of CAVD and has therefore been thought of as a potential pharmaceutical target. Despite positive epidemiological data and animal studies with lipid-lowering therapies, prospective clinical studies have so far been disappointing and valve replacement is the only approved treatment.

Five-year View
Although significant progress has been made in the understanding of the pathogenesis of CAVD, there are still many uncertainties. Current knowledge of the precise interplay of genetics, clinical risk factors, initiating cellular events and subsequent signaling pathways, VICs phenotype transition and calcification, is unavailable. During the next five years, the hypercholesterolemic animal models should have, at least to some extent, allowed clarification some of these disease mechanisms. Furthermore, future genetics studies should provide increased understanding of the relationships between the CAVD phenotype and genotype. Proteomic studies provide an unbiased nonhypothesis driven study approach and we believe that the future will confirm its usefulness as a novel platform in CAVD pathogenesis research. With better understanding of the pathological process of CAVD, we shall be able to more accurately elucidate the key features involved in disease initiation, progression and calcification. Thus, providing us with an opportunity to discover potential biomarkers and develop pharmacological intervention that slows or reverses the disease process, reducing the need for valve replacement.

Sidebar
Key Issues

Aortic stenosis (AS) is the most prevalent valve pathology, and calcific valve disease (CAVD) is the most frequent etiology in developed countries. Hemodynamic obstruction (stenosis) appears when more than half of its normal area is lost and clinical symptoms (exertional angina, dyspnea and syncope) usually appear when severe stenosis is produced (jet velocity >4.0 m/s; mean gradient >40 mmHg; valve area <1 cm2). The current treatment of AS consists of surgical valve replacement in symptomatic severe AS. In selected subgroups of patients with high operative risk, percutaneous valve insertion is an alternative option.

During the last two decades, a large body of experimental and clinical evidence has demonstrated that CAVD represents an active disease process, and not just passive 'wear and tear' degeneration as once thought. The pathogenesis is similar to that of atherosclerosis since it shares common classical risk factors (arterial hypertension, smoking and hypercholesterolemia) and pathological components (endothelial dysfunction, lipid accumulation and inflammation). The pathogenesis of CAVD is thought to be initiated by endothelial damage through hemodynamic forces (arterial hypertension, elevated stretch and shear stress) and increased oxidative stress. Hypercholesterolemia seems to be an important part of that CAVD process, leading to LDL cholesterol subendothelial accumulation and initiation of several inflammatory processes. This triggers, the differentiation of valve interstitial cells into an osteoblast phenotype, which causes aortic valve fibrosis and calcification (sclerosis) and eventually hemodynamic obstruction (stenosis). Genetic studies have found CAVD to be associated with vitamin D receptor gene polymorphism of the B allele, a specific mutation in the NOTCH 1gene, polymorphism in the estrogen receptor and lipoprotein abnormalities. Given the important role of hypercholesterolemia in the pathogenesis of CAVD, both animal and clinical studies have evaluated the effect of lipid-lowering therapies in CAVD. Initial data from animal studies have been promising, however, three randomized clinical trials with lipid-lowering drugs including statins and ezetimibe, failed to show reduction of the disease progression of CAVD. The contradictory findings could be explained by lipid-lowering therapy only being effective at the early stages of CAVD or in those with already high lipid levels who were systematically excluded from the trials for ethical reasons. Future studies, including animal models and proteomic studies, shall provide an opportunity to further evaluate the disease mechanisms of CAVD. This will provide us with an opportunity to more easily discover potential disease biomarkers and develop pharmacological interventions that could reduce the need for valve replacement.

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