Pathophysiologic mechanisms of calcific aortic stenosis

Abstract

Calcific aortic stenosis (CAS) comprises the leading indication for valve replacement in the Western world. Until recently, progressive calcification was considered to be a passive process. Emerging evidence, however, suggests that degenerative aortic stenosis constitutes an active process involving stimulation of several pathophysiologic pathways such as inflammation and osteogenesis. In addition, CAS and atherosclerosis share common features regarding histopathology of lesions. These novel data raise a new perspective on the prevention and treatment of disease. The current article reviews the most important pathophysiologic mechanisms of senile aortic stenosis.

Keywords

aortic calcific pathophysiologic stenosis

Introduction

Aortic stenosis is the most common valvular lesion in Europe and North America [Ramaraj and Sorrell, 2008]. As the incidence of acute rheumatic fever has declined, calcific aortic stenosis (CAS) has become the most common indication for surgical valve replacement in the United States [Rajamannan, 2009]. Regarding population aged >65 years, its incidence is 2–7% [Ramaraj and Sorrell, 2008]. Interestingly, aortic sclerosis (aortic valve calcification without hemodynamic compromise) is present in more than 25% of patients older than age 65 years [Boss et al. 2008].

Recent studies provide evidence that atherosclerosis and CAS share common features in relation to risk factors and histopathologic lesions [Rajamannan, 2009]. Moreover, histopathologic evidence suggests that early lesions in CAS are not just a result secondary to aging, but an active cellular process. Recent research implies that the classical ‘response to injury hypothesis’, initially described in atherosclerosis, seems to represent the cornerstone of pathophysiology [Yetkin and Waltenberger, 2009].

Anatomy–histology

The internal collagen framework of aortic valve cusps is arranged in three layers: fibrosa, spongiosa and ventricularis. This structure provides strength, a cushion for the mechanical forces and elasticity for changes of shape during opening and closing. All three layers of the aortic valve are avascular and are innervated by adrenergic and cholinergic neural networks [Xu et al. 2010].

Valve integrity is determined by the extracellular matrix. The quality and quantity of extracellular matrix components are dependent on the function of valvular interstitial cells (VICs) [Schoen, 2005]. VICs are elongated, spindle-like cells and most of them are located in fibrosa. They display morphological and functional characteristics of fibroblasts, smooth muscle cells and myofibroblasts [Taylor et al. 2003].

Five distinct phenotypes of VICs have been recognized:

embryonic progenitor endothelial/mesenchymal VICs;

quiescent VICs;

activated VICs;

adult progenitor VICs;

osteoblastic VICs.

These cells present plasticity and are capable of changing their phenotype under certain circumstances (Figure 1) [Alexopoulos et al. 2011b; Liu et al. 2007].

Figure 1.

Subtypes of valvular interstitial cells (VICs).

Not surprisingly, age-related reduction of this cellular population is accompanied by fibrous degeneration [Taylor et al. 2003].

Risk factors

Many of the risk factors for atherosclerosis are also associated with aortic valve sclerosis, which has led to the suggestion that calcific aortic valve disease is an atherosclerotic-like process. Clinical factors identified by multivariate analysis are older age, male sex, elevated serum lipoprotein (a) and low-density lipoprotein (LDL) levels, hypertension, smoking status, and shorter stature. Renal dysfunction and abnormalities in calcium and phosphate metabolism contribute also to the burden of disease. It remains unclear whether clinical risk factors associated with the presence of aortic stenosis also predict disease progression [Freeman and Otto, 2010].

Pathophysiology of CAS

Several factors lead to the activation of endothelium with subsequent expression of significant factors such as cytokines and adhesion molecules. Subendothelial accumulation of lipids and inflammatory cells comprise the early lesions which trigger a ‘response to injury’. This phase includes remodeling of extracellular matrix and transformation of quiescent VICs to activated VICs that consequently gain osteoblastic phenotype (Figure 2) [Alexopoulos et al. 2011b].

Figure 2.

Schematic illustration of pathophysiology of calcific aortic stenosis (ox-LDL, oxidized low-density lipoprotein; ang II, angiotensin II; qVIC, quiescent valvular interstitial cell; aVIC, activated valvular interstitial cell).

Expression of bone regulatory factors is related to formation of calcified nodules, lesions which represent later stages of aortic stenosis [Rajamannan, 2010].

Mechanical forces

Aortic valve cusps are subjected to the unceasing influence of severe mechanical forces during the whole lifetime. It is believed that the attachment area of the aortic leaflets to the aortic root encounters the strongest mechanical influence, which may provoke endothelial dysfunction [Rajamannan, 2009]. Preliminary studies suggested the ‘wear and tear’ theory in order to underline significance of mechanical stress in pathogenesis of aortic stenosis [Yetkin and Waltenberger, 2009].

However, mechanical stress does not necessarily hold the primary role in pathogenesis. Genetically predisposed individuals in atherosclerosis and aortic valve sclerosis are very prone to mechanical forces that trigger effortlessly key molecular signaling pathways in both diseases [Parolari et al. 2009].

Endothelium

Abnormal activation of the aortic valve endothelium was observed initially in experimental hypercholesterolemia rabbits [Rajamannan, 2009]. Recent research has shown increased E-selectin plasma levels in patients with severe CAS which normalize after aortic valve surgery. In addition, high levels of endothelial microparticles have been found in these patients. Endothelial microparticles are small vesicles that consist of a plasma membrane surrounding a small amount of cytosol. Inflammatory infiltration in calcified aortic valves has been related to circulating levels of endothelial microparticles. Several endothelial markers such as CD31, CD34 and von Willebrand factor were markedly expressed in tissue samples of human aortic valves derived from patients undergoing valve replacement for severe calcific, nonrheumatic aortic stenosis. Remarkably, decreased availability of nitric oxide and prostacyclin was noted in these specimens [Parolari et al. 2009].

Lipids

Initially, Otto and colleagues noted the association of lipid metabolism with CAS. Accumulation of intracellular and extracellular lipids was a constant finding in pathologic specimens [Otto et al. 1994].

Involvement of the low-density lipoprotein receptor related proteins 5/6 (Lrp5/6) in valve calcification has been implicated in several studies [Rajamannan, 2009]. The Lrp5/Wnt signaling pathway has great importance in bone remodeling. Wnt3a protein is secreted by endothelial cells and has the ability to bind to LDL receptor-related proteins Lrp5 or Lrp6 complex on the myofibroblast extracellular membrane [Rajamannan et al. 2005]. This signal results in cytoplasmic accumulation of catenin which subsequently enters nucleus and interacts with proteins of the T-cell factor/lymphoid-enhancer factor-1 family affecting expression of target genes such as cyclin D, Runx2/Cbfa1, and Sox9 (Figure 3) [Alexopoulos et al. 2011b].

Figure 3.

Effects of the LRP5/Wnt signaling pathway on gene expression.

Recent research has shown that mutations in the EGFP domain of the Lrp6 receptor impairs cellular LDL clearance. In addition, double knockout mice lacking both apolipoprotein E (apoE) and Lrp5 present approximately 60% higher cholesterol levels compared with the age-matched apoE knockout mice [Rajamannan, 2011]. This finding may be associated with an alternative pathway for cholesterol catabolism mediated by Lrp5. Lrp5 plays a significant role in extracellular matrix production and differentiation process of aortic VICs into osteoblasts, providing another link between lipid metabolism and aortic valve calcification [Rajamannan, 2011; Caira et al. 2006].

Inflammation

Intense inflammatory infiltration has been reported in fibromyxomatous and calcified areas of degenerated aortic cusps [Alexopoulos et al. 2010]. Recent evidence suggests that inflammatory cells express pro-inflammatory cytokines and neoangiogenic factors contributing to increased thermal heterogeneity in aortic valve stenosis. Temperature difference can be recorded by specially designed thermographic catheters with a sensitive thermistor at the distal tip [Toutouzas et al. 2008].

As we mentioned above, several inflammatory mediators have been observed in diseased valves such as terminal complement complex C5b-9, interleukins (IL-1b, IL-6, IL-8), tumor necrosis factor-alpha (TNF-α) and Heat Shock Protein-60 (HSP-60) [Parolari et al. 2009]. In addition, high serum levels of soluble endothelial adhesion molecules have been found in patients with severe aortic stenosis who had no history of coronary artery disease [Yetkin and Waltenberger, 2009]. There is also intense expression of transforming growth factor-beta1 (TGF-β1) which binds to specific proteins of extracellular matrix and promotes cellular migration and aggregation as well as apoptosis of VICs [Parolari et al. 2009; Mohler, 2004].

Toll-like receptors (TLRs) on interstitial cells seem to play critical role in inflammatory response. In vitro studies have demonstrated that stimulation of TLRs activates the NF-κB pathway with subsequent expression of cytokines and bone-related factors [Meng et al. 2008].

Recent research has shown that aortic VICs in areas containing calcific deposits showed significantly higher nuclear factor of activated T-cells (NFATc1) nuclear immunolocalization compared with noncalcified fibrocellular regions. transforming growth factor-beta1 (TGF-β1) is a member of a multigene family of transcription factors that belong to the Rel group and control T lymphocyte activation and differentiation [Alexopoulos et al. 2010, 2011a].

Extracellular matrix remodeling

Extracellular matrix proteins with lytic activity may exert favorable effects in a normal repair process. Nevertheless, overexpression of these factors or defective inhibitory mechanisms are responsible for valve injury [Yetkin and Waltenberger, 2009].

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that are capable of degrading all kinds of extracellular matrix proteins, but also can process a number of bioactive molecules. MMP-1 colocalizes with TNF-a suggesting an association between extracellular matrix remodeling and inflammation [Kaden et al. 2005]. In addition, MMP-12 contributes actively to valve mineralization by degradation of elastic fibers and subsequent calcium deposition onto fragmented elastin. Moreover, fragmented elastin not only acts as a potential nidus for mineral deposition but is also implicated in activation of osteochondrogenic pathways in VICs [Perrotta et al. 2011].

It is well known that the activity of MMPs is dependent on their respective tissue inhibitors (TIMPs). There is much confusion about role of TIMPs in pathophysiology of aortic stenosis. As previous studies have shown conflicting results, further research is needed in order to unravel a possible connection with valve remodeling [Kaden et al. 2005].

Apart from MMPs, several other factors with degrading activity such as cathepsins S, K and V overwhelm calcified lesions. Cathepsin S expression is more prominent in severely calcified areas while cathepsin V is related to endothelial cells [Helske et al. 2006b]. Interestingly, intense elastolysis and inflammation are associated with high levels of cathepsin S whereas valve mineralization is diminished in cathepsin S-deficient mice [Aikawa et al. 2009]. Moreover, colocalization of cathepsin G and TGF-β1 in mast cells lends further support to hypothesis that inflammation is the underlying cause of calcification [Helske et al. 2006a].

Another extracellular matrix glycoprotein, tenascin-C, presents abundant expression in calcified valves. Tenascin-C is a highly conserved, multifunctional protein implicated in cell proliferation, migration, differentiation and apoptosis [Wallner et al. 1999]. Emerging evidence suggests that tenascin-C enhances alkaline phosphatase (ALP) activity and expression of MMPs promoting calcium deposition in degenerative lesions [Yetkin and Waltenberger, 2009; Mohler, 2004].

Upregulation of cystatin C, a cysteine protease inhibitor, has been found in calcific human aortic valves. Its presence must not be considered circumstantial as a previous study reported increased expression in mature osteoblasts [Huh et al. 1995].

Valvular interstitial cells

Osteoblastic phenotype of VICs implies expression of bone proteins exhibiting regulatory or structural role. VICs in calcified lesions show significantly higher expression of bone regulatory factors such as bone morphogenetic protein (BMP), Runx2/Cbfa1 and Osterix in relation to noncalcified areas [Alexopoulos et al. 2010; Rajamannan, 2009]. Sox9, a critical regulator of both early and late stages of chondrogenesis is overexpressed in stenotic aortic valves [Alexopoulos et al. 2010]. Several lines of evidence suggest an important role of the OPG/RANKL/RANK (osteoprotegerin/receptor activator of NF-κB ligand/receptor activator of NF-κB) axis in valve pathology [Mallat and Tedgui, 2004]. Specifically, in cultured human aortic valve myofibroblasts, RANKL enhances the expression of osteoblast-related genes promoting calcium deposition in extracellular matrix [Kaden et al. 2004]. This phenotype of interstitial cells was also found to be related with high levels of TLRs 2 and 4. Activation of these receptors may lead to increased expression of cytokines and osteogenic factors such as BMP-2 and Runx2/Cbfa1 [Yang et al. 2009].

Genetic influence

Activation of signaling pathways is dependent on the expression of inhibitory proteins. Recent evidence has shown that BMP is inhibited by several proteins such as noggin, chordin, follistatin, gremlin and Smad proteins. Relative deficiency of these factors could be responsible for aortic valve ossification [Mohler, 2004].

Determination of allelic variants of lipoproteins in patients suffering from aortic stenosis comprises an interesting research topic. Although a higher prevalence of apoE2 and apoE4 has been observed in some studies, these results have not been affirmed by other researchers [Novaro et al. 2003].

Inflammation is a prerequisite for aortic valve calcification. Polymorphisms of the IL-10 gene promoter as well as simultaneous presence of the rare chemochine receptor 5 and connective tissue growth factor alleles are associated with severe calcium burden in patients with aortic stenosis [Parolari et al. 2009].

Regarding bone metabolism genomics, vitamin D receptor genetic polymorphism has been extensively investigated [Ortlepp et al. 2001]. High incidence of the B allele has been found in aortic stenosis and is related with reduced calcium absorption, bone resorption and increased expression of parathormone [Parolari et al. 2009; Rajamannan, 2009]. Targeted loss of Sox9 function during valve development is related to predisposition for calcific valve phenotype in adulthood [Peacock et al. 2010]. Runx2/Cbfa1, a master transcription factor for bone formation, induces transcription of osteoblast-related genes such as osteocalcin gene [Alexopoulos et al. 2010, 2011a]. Transcriptional activity of Runx2/Cbfa1 and Sox9 may be affected by Notch1 signaling. Recent research demonstrated that mutations of the Notch1 gene are associated with enhanced calcium deposition in aortic valves probably via enhanced Runx2/Cbfa1 and attenuated Sox9 expression [Meier-Steigen et al. 2010; Mead and Yutzev, 2009; Garg et al. 2005].

Finally, a polymorphism of the alpha estrogen receptor in postmenopausal women is related to increase of cholesterol levels and predisposition to aortic valve calcification [Nordstrom et al. 2003].

Other factors

High levels of angiotensin-converting enzyme have been observed in fibromyxomatous lesions presenting increased LDL and apoB concentration. It is speculated that plasma lipoproteins promote retention of angiotensin-converting enzyme [Parolari et al. 2009]. Lisinopril managed to attenuate the presence of angiotensin-converting enzyme in experimental models [Sun et al. 1995]. Members of the renin–angiotensin system are implicated in mechanisms of repair of the aortic valve as a normal response to injury. However, hyperactivation of the renin–angiotensin system exerts deleterious effects leading to pathologic fibrosis [Mohler, 2004].

Several studies have confirmed neovessel formation in calcified valves [Soini et al. 2003]. Angiogenesis may be implicated in calcification process by various ways including recruitment of inflammatory cells, transdifferentiation of pericytes of the neovessel wall and secretion of cytokines from activated endothelial cells [Alexopoulos et al. 2010]. The whole process is related with increased levels of endothelial nitric oxide synthase, vascular endothelial growth factor (VEGF) and its receptors Flt-1 and Flk-1 [Parolari et al. 2009]. Recent research has demonstrated high levels of periostin in areas of neoangiosis in degenerated valves. Periostin is expressed by VICs and inflammatory cells and not only exerts angiogenic activity but also enhances secretion of MMPs [Hakuno et al. 2010]. Angiogenesis comprises an attractive diagnostic and therapeutic target. ανβ3-integrin targeted nanoparticles bearing gadolinium chelates can be used to image neovessels in atherosclerotic lesions with proton MRI [Waters et al. 2008]. Notably, low-grade lesions are characterized by greater neoangiosis relatively to severe lesions suggesting a temporal pattern of the phenomenon in development of aortic stenosis [Soini et al. 2003]. Considering the fact that neoangiosis is characterized by a short time window as it takes place in days or months, potent therapeutic intervention must be timely and targeted [Parolari et al. 2009].

Great effort has been made by several researchers in order to relate infectious agents with aortic stenosis but data are conflicting. Recently, the presence of nanobacteria, growing self-replicating calcifying nanoparticles that potentially represent new pathogens, was indicated in aortic valve specimens collected at surgery. Their expression has been noted already in carotid disease and abdominal aorta aneurysms [Bratos-Perez et al. 2008].

Treatment effects on pathophysiologic mechanisms

Statins

The role of statins is not limited to cholesterol-lowering effects. The JUPITER (Justification for the Use of statins in Primary prevention: an Intervention Trial Evaluating Rosuvastatin) trial showed that statin therapy in patients with normal cholesterol levels but elevated C-reactive protein is related with reduced cardiovascular morbidity and mortality. These findings suggest that statins possess beneficial anti-inflammatory and anticoagulant properties [Parolari et al. 2011]. In contrast to mild forms of disease, findings are inconsistent in patients with moderate to severe degrees of aortic stenosis [Parolari et al. 2009].

Previous reports suggest that expression of MMPs is reduced by VICs and macrophages under the effect of statin treatment [Mohler, 2004]. TGF-β is an important regulator of cellular proliferation and differentiation and modulator of inflammation and extracellular matrix remodeling. Several studies provide evidence that levels of TGF-β in human VICs are reduced with implementation of statin treatment in initial stages of disease. This results in attenuated presence of ALP and osteocalcin in calcified lesions [Osman et al. 2006]. However, these findings were not confirmed in late stages of disease indicating the narrow therapeutic time window of statins [Anger et al. 2009].

Statins are shown to attenuate leukocyte infiltration and expression of bone regulatory factors in aortic valves of experimental models with hypercholesterolemia [Rajamannan, 2009]. These effects are attributed to modulation of Lrp5 pathway and endothelial nitric oxide synthase [Kaden et al. 2004]. They also reduce the expression of regulators of G protein-mediated signaling proteins (RGS) in calcified valves triggering activation of extracellular-regulated kinases that enhance proliferation of myofibroblasts [Parolari et al. 2009].

On the other hand, increased expression of BMP-2 has been observed in experimental models treated with HMG-CoA reductase inhibitors [Anger et al. 2009]. Atorvastatin also extenuates the activity of ALP in cultured interstitial aortic cells in contrast to bone tissue where it exerts an opposite effect [Miller et al. 2008]. These contradictory findings, called the ‘statin paradox’, suggest that the beneficial impact could be time-dependent and beyond this time window statins exhibit neutral or even harmful effects. A possible explanation is that different cell populations prevail during several stages of pathophysiologic process resulting in variable response to statin treatment. This could be related to the results of several trials that failed to demonstrate positive therapeutic effects [Parolari et al. 2009]. In the SALTIRE (Scottish Aortic Stenosis and Lipid Lowering Trial, Impact on Regression), SEAS (Simvastatin and Ezetimibe in Aortic Stenosis study) and ASTRONOMER (Aortic Stenosis Progression Observation: Measuring Effects of Rosuvastatin) studies, statin therapy had no effect on the rate of progression of aortic stenosis. Further projects such as STOP AS (Therapy on the Progression of Calcific Valvular Aortic Stenosis), AORTICA (Efficacy of Fluvastatin on Inflammatory Markers in the Hemodynamic Progression of Degenerative Aortic Stenosis) and STAAT (Statin Therapy in Asymptomatic Aortic Stenosis) are under way to try to define the role of statins in pathophysiologic process [Salas et al. 2011]. Meanwhile, there is no indication for statin use specific to aortic stenosis [Yetkin and Waltenberger, 2009].

Other treatment modalities

As we discussed earlier, angiotensin-converting enzyme exhibits intense presence in diseased valves. Preliminary reports suggest that olmesartan, an angiotensin type 1 receptor antagonist, preserves endothelial integrity and inhibits transdifferentiation of VICs into myofibroblasts or osteoblasts [Arishiro et al. 2007]. Nevertheless, there is no current recommendation for use of angiotensin-converting enzyme inhibitors in order to delay progression of calcific aortic stenosis. The current trial ROCK-AS (The Potential of Candesartan to Retard the Progression of Aortic Stenosis) may provide more evidence regarding involvement of angiotensin-converting enzyme in inflammation, calcification, lipid accumulation and fibrosis [Salas et al. 2011].

Bisphosphonates inhibit bone resorption and are widely used for the prevention and treatment of osteoporosis. Previous studies have shown that cardiovascular calcification is associated with decreased bone density suggesting complex pathophysiologic mechanisms. Interestingly, progression of aortic valve disease is slower in patients receiving bisphosphonates for osteoporosis but further research is needed in order to confirm these findings [Salas et al. 2011].

Aortic valve replacement comprises mainstay of treatment in severe disease but many patients are not eligible as they suffer from several comorbidities. Transcatheter aortic valve implantation (TAVI) is an alternative method that offers substantial improvements in symptoms. Balloon aortic valvuloplasty (BAV), the least invasive percutaneous option, is associated with early restenosis. However, novel improvements of technique, such as use of drug eluting balloons, promise long-lasting results [Spargias et al. 2009].

Conclusions

Age-related aortic valve calcification comprises a complex process involving activation of major molecular pathways. Several risk factors trigger a response to injury with subsequent inflammatory infiltration. Enhanced expression of inflammatory mediators is associated with activation of VICs that mediate valve mineralization. Remodeling of extracellular matrix and neoangiosis are prominent features in aortic valve lesions. Currently, the only effective treatment for severe symptomatic aortic stenosis is valve replacement and further elucidation of underlying mechanisms is needed in order to achieve integrated prevention and treatment of disease.

Footnotes

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

The authors declare no conflicts of interest in preparing this article.

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