Activation of TLR3 Induces Osteogenic Responses in Human Aortic Valve Interstitial Cells through the NF-κB and ERK1/2 Pathways

Calcific aortic valve disease (CAVD) is characterized by chronic inflammation and progressive calcification in valve leaflets. Aortic valve interstitial cells (AVICs) play a critical role in the pathogenesis of CAVD. Previous studies show that stimulation of Toll-like receptor (TLR) 2 or TLR4 in AVICs in vitro up-regulates the expression of osteogenic mediators. Double-stranded RNA (dsRNA) can activate pro-inflammatory signaling through TLR3, the NLRP3 inflammasome and RIG-I-like receptors. The objective of this study is to determine the effect of dsRNA on AVIC osteogenic activities and the mechanism of its action. Methods and results: AVICs isolated from normal human valves were exposed to polyinosinic-polycytidylic acid [poly(I:C)], a mimic of dsRNA. Treatment with poly(I:C) increased the production of bone morphogenetic protein-2 (BMP-2), transforming growth factor beta-1 (TGF-β1) and alkaline phosphatase (ALP), and resulted in calcium deposit formation. Poly(I:C) induced the phosphorylation of NF-κB and ERK1/2. Knockdown of TLR3 essentially abrogated NF-κB and ERK1/2 phosphorylation, and markedly reduced the effect of poly(I:C) on the production of BMP-2, TGF-β1 and ALP. Further, inhibition of either NF-κB or ERK1/2 markedly reduced the levels of BMP-2, TGF-β1 and ALP in cells exposed to poly(I:C). Conclusion: Poly(I:C) up-regulates the production of BMP-2, TGF-β1 and ALP, and promotes calcium deposit formation in human AVICs. The pro-osteogenic effect of poly(I:C) is mediated primarily by TLR3 and the NF-κB and ERK1/2 pathways. These findings suggest that dsRNA, when present in aortic valve tissue, may promote CAVD progression through up-regulation of AVIC osteogenic activities.     

Oxidative-mechanical stress signals stem cell niche mediated Lrp5 osteogenesis in eNOS(-/-) null mice

Calcific aortic valve disease (CAVD) is the most common indication for valve surgery in the USA. This study hypothesizes that CAVD develops secondary to Wnt3a/Lrp5 activation via oxidative‐mechanical stress in eNOS null mice. eNOS^?/? mice were tested with experimental diets including a control (n = 20), cholesterol (n = 20), cholesterol + Atorvastatin (n = 20). After 23 weeks the mice were tested for the development of aortic stenosis by Echo, Histology, MicroCT, and RTPCR for bone markers. In vitro studies measured Wnt3a secretion from aortic valve endothelial cells and confirmed oxidative stress via eNOS activity. Anion exchange chromatography was performed to isolate the mitogenic protein. Myofibroblast cells were tested to induce bone formation. Cholesterol treated eNOS mice develop severe stenosis with an increase in Wnt3a, Lrp5, Runx2 (threefold increase (P < 0.0001) in the bicuspid versus tricuspid aortic valves. Secretion of Wnt3a from aortic valve endothelium in the presence of abnormal oxidative stress was correlated with diminished eNOS enzymatic activity and tissue nitrite levels. Initial characterization of the architecture for a stem cell nice was determined by protein isolation using anion‐exchange chromatography and cell proliferation via thymidine incorporation. Osteoblastogenesis in the myofibroblast cell occurred via Lrp5 receptor upregulation in the presence of osteogenic media. Targeting the Wnt3a/Lrp5 pathway in valve calcification and activation of osteogenesis is via an oxidative‐mechanical stress in CAVD. These findings provide a foundation for treating this disease process by targeting the cross talk mechanism in a resident stem cell niche. J. Cell. Biochem. 113: 1623–1634, 2012. © 2011 Wiley Periodicals, Inc.     

In‐vitro analysis of early calcification in aortic valvular interstitial cells using Laser‐Induced Breakdown Spectroscopy (LIBS)

Calcific aortic valve disease (CAVD) is a major cardiovascular disorder caused by osteogenic differentiation of valvular interstitial cells (VICs) within aortic valves. Conventional methods like colorimetric assays and histology fail to detect small calcium depositions during in‐vitro VIC cultures. Laser‐induced breakdown spectroscopy (LIBS) is a robust analytical tool used for inorganic materials characterizations, but relatively new to biomedical applications. We employ LIBS, for the first time, for quantitative in‐vitro detection of calcium depositions in VICs at various osteogenic differentiation stages. VICs isolated from porcine aortic valves were cultured in osteogenic media over various days. Colorimetric calcium assays based on arsenazo dye and Von Kossa staining measured the calcium depositions within VICs. Simultaneously, LIBS signatures for Ca I (422.67 nm) atomic emission lines were collected for estimating calcium depositions in lyophilized VIC samples. Our results indicate excellent linear correlation between the calcium assay and our LIBS measurements. Furthermore, unlike the assay results, the LIBS results could resolve calcium signals from cell samples with as early as 2 days of osteogenic culture. Quantitatively, the LIBS measurements establish the limit of detection for calcium content in VICs to be ～0.17±0.04 μg which indicates a 5‐fold improvement over calcium assay. Picture : Quantitative LIBS enables in‐vitro analysis for early stage detection of calcium deposition within aortic valvular interstitial cells (VICs).

     

End stage renal disease‐induced hypercalcemia may promote aortic valve calcification via Annexin VI enrichment of valve interstitial cell derived‐matrix vesicles

Patients with end‐stage renal disease (ESRD) have elevated circulating calcium (Ca) and phosphate (Pi), and exhibit accelerated progression of calcific aortic valve disease (CAVD). We hypothesized that matrix vesicles (MVs) initiate the calcification process in CAVD. Ca induced rat valve interstitial cells (VICs) calcification at 4.5 mM (16.4‐fold; p < 0.05) whereas Pi treatment alone had no effect. Ca (2.7 mM) and Pi (2.5 mM) synergistically induced calcium deposition (10.8‐fold; p < 0.001) in VICs. Ca treatment increased the mRNA of the osteogenic markers Msx2, Runx2, and Alpl (p < 0.01). MVs were harvested by ultracentrifugation from VICs cultured with control or calcification media (containing 2.7 mM Ca and 2.5 mM Pi) for 16 hr. Proteomics analysis revealed the marked enrichment of exosomal proteins, including CD9, CD63, LAMP‐1, and LAMP‐2 and a concomitant up‐regulation of the Annexin family of calcium‐binding proteins. Of particular note Annexin VI was shown to be enriched in calcifying VIC‐derived MVs (51.9‐fold; p < 0.05). Through bioinformatic analysis using Ingenuity Pathway Analysis (IPA), the up‐regulation of canonical signaling pathways relevant to cardiovascular function were identified in calcifying VIC‐derived MVs, including aldosterone, Rho kinase, and metal binding. Further studies using human calcified valve tissue revealed the co‐localization of Annexin VI with areas of MVs in the extracellular matrix by transmission electron microscopy (TEM). Together these findings highlight a critical role for VIC‐derived MVs in CAVD. Furthermore, we identify calcium as a key driver of aortic valve calcification, which may directly underpin the increased susceptibility of ESRD patients to accelerated development of CAVD.     

The pathophysiological basis of pharmacological interventions in CAVD

Calcific aortic valve disease (CAVD) results in aortic valve stenosis and is one of the most common cardiac diseases in both Western and developing countries. The burden of this disease is expected to increase rapidly in the future, but there are still no relevant pharmacological therapies available and aortic valve replacement remains the sole definite therapy. This review presents an overview of the most common causes of CAVD, followed by current debates and trials related to the onset and progression of this disease. Several differences and similarities between the different causes of CAVD are presented. Additionally, stages of CAVD are compared with stages in atherosclerosis. Finally, future directions for research on CAVD will be discussed.     

The new insight into extracellular NAD+ degradation-the contribution of CD38 and CD73 in calcific aortic valve disease

Nicotinamide adenine dinucleotide (NAD⁺) is crucial for cell energy metabolism and many signalling processes. Recently, we proved the role of ecto-enzymes in controlling adenine nucleotide–dependent pathways during calcific aortic valve disease (CAVD). This study aimed to investigate extracellular hydrolysis of NAD⁺ and mononucleotide nicotinamide (NMN) in aortic valves and aorta fragments of CAVD patients and on the inner aortic surface of ecto-5′-nucleotidase knockout mice (CD73−/−). Human non-stenotic valves (n = 10) actively converted NAD⁺ and NMN via both CD73 and NAD⁺-glycohydrolase (CD38) according to our analysis with RP-HPLC and immunofluorescence. In stenotic valves (n = 50), due to reduced CD73 activity, NAD⁺ was degraded predominantly by CD38 and additionally by ALP and eNPP1. CAVD patients had significantly higher hydrolytic rates of NAD⁺ (0.81 ± 0.07 vs 0.56 ± 0.10) and NMN (1.12 ± 0.10 vs 0.71 ± 0.08 nmol/min/cm²) compared with controls. CD38 was also primarily engaged in human vascular NAD⁺ metabolism. Studies using specific ecto-enzyme inhibitors and CD73−/− mice confirmed that CD73 is not the only enzyme involved in NAD⁺ and NMN hydrolysis and that CD38 had a significant contribution to these pathways. Modifications of extracellular NAD⁺ and NMN metabolism in aortic valve cells may be particularly important in valve pathology and could be a potential therapeutic target.     

The elastic properties of valve interstitial cells undergoing pathological differentiation

Increasing evidence indicates that the progression of calcific aortic valve disease (CAVD) is influenced by the mechanical forces experienced by valvular interstitial cells (VICs) embedded within the valve matrix. The ability of VICs to sense and respond to tissue-level mechanical stimuli depends in part on cellular-level biomechanical properties, which may change with disease. In this study, we used micropipette aspiration to measure the instantaneous elastic modulus of normal VICs and of VICs induced to undergo pathological differentiation in vitro to osteoblast or myofibroblast lineages on compliant and stiff collagen gels, respectively. We found that VIC elastic modulus increased after subculturing on stiff tissue culture-treated polystyrene and with pathological differentiation on the collagen gels. Fibroblast, osteoblast, and myofibroblast VICs had distinct cellular-level elastic properties that were not fully explained by substrate stiffness, but were correlated with α-smooth muscle actin expression levels. C-type natriuretic peptide, a peptide expressed in aortic valves in vivo, prevented VIC stiffening in vitro, consistent with its ability to inhibit α-smooth muscle actin expression and VIC pathological differentiation. These data demonstrate that VIC phenotypic plasticity and mechanical adaptability are linked and regulated both biomechanically and biochemically, with the potential to influence the progression of CAVD.     

Augmented Osteogenic Responses in Human Aortic Valve Cells Exposed to oxLDL and TLR4 Agonist: A Mechanistic Role of Notch1 and NF-κB Interaction

Aortic valve calcification causes the progression of calcific aortic valve disease (CAVD). Stimulation of aortic valve interstitial cells (AVICs) with lipopolysaccharide (LPS) up-regulates the expression of osteogenic mediators, and NF-κB plays a central role in mediating AVIC osteogenic responses to Toll-like receptor 4 (TLR4) stimulation. Diseased aortic valves exhibit greater levels of oxidized low-density lipoprotein (oxLDL). This study tested the hypothesis that oxLDL augments the osteogenic responses in human AVICs through modulation of NF-κB and Notch1 activation. AVICs isolated from normal human aortic valves were treated with LPS (0.1 µg/ml), oxLDL (20 µg/ml) or LPS plus oxLDL for 48 h. OxLDL alone increased cellular bone morphogenetic protein-2 (BMP-2) levels while it had no effect on alkaline phosphatase (ALP) levels. Cells exposed to LPS plus oxLDL produced higher levels of BMP-2 and ALP than cells exposed to LPS alone. Further, LPS plus oxLDL induced greater NF-κB activation, and inhibition of NF-κB markedly reduced the expression of BMP-2 and ALP in cells treated with LPS plus oxLDL. OxLDL also induced Notch1 activation and resulted in augmented Notch1 activation when it was combined with LPS. Inhibition of Notch1 cleavage attenuated NF-κB activation induced by LPS plus oxLDL, and inhibition of NF-κB suppressed the expression of BMP-2 and ALP induced by the synergistic effect of Jagged1 and LPS. These findings demonstrate that oxLDL up-regulates BMP-2 expression in human AVICs and synergizes with LPS to elicit augmented AVIC osteogenic responses. OxLDL exerts its effect through modulation of the Notch1-NF-κB signaling cascade. Thus, oxLDL may play a role in the mechanism underlying CAVD progression.     

Calcific nodule morphogenesis by heart valve interstitial cells is strain dependent

Calcific aortic valve disease (CAVD) results in impaired function through the inability of valves to fully open and close, but the causes of this pathology are unknown. Stiffening of the aorta is associated with CAVD and results in exposing the aortic valves to greater mechanical strain. Transforming growth factor β1 (TGF-β1) is enriched in diseased valves and has been shown to combine with strain to synergistically alter aortic valve interstitial cell (AVIC) phenotypes. Therefore, we investigated the role of strain and TGF-β1 on the calcification of AVICs. Following TGF-β1 pretreatment, strain induced intact monolayers to aggregate and calcify. Using a wound assay, we confirmed that TGF-β1 increases tension in the monolayer in parallel with α-smooth muscle actin (αSMA) expression. Continual exposure to strain accelerates aggregates to calcify into mature nodules that contain a necrotic core surrounded by an apoptotic ring. This phenotype appears to be mediated by strain inhibition of AVIC migration after the initial formation of aggregates. To better interpret the extent to which externally applied strain physically impacts this process, we modified the classical Lamé solution, derived using principles from linear elasticity, to reveal strain magnification as a novel feature occurring in a mechanical environment that supports nodule formation. These results indicate that strain can impact multiple points of nodule formation: by modifying tension in the monolayer, remodeling cell contacts, migration, apoptosis, and mineralization. Therefore, strain-induced nodule formation provides new directions for developing strategies to address CAVD.     

Deficiency in the anti‐aging gene Klotho promotes aortic valve fibrosis through AMPKα‐mediated activation of RUNX2

Fibrotic aortic valve disease (FAVD) is an important cause of aortic stenosis, yet currently there is no effective treatment for FAVD due to its unknown etiology. The purpose of this study was to investigate whether deficiency in the anti‐aging Klotho gene (KL) promotes high‐fat‐diet‐induced FAVD and to explore the underlying molecular mechanism. Heterozygous Klotho‐deficient (KL^+/?) mice and WT littermates were fed with a high‐fat diet (HFD) or normal diet for 13 weeks, followed by treatment with the AMPKα activator (AICAR) for an additional 2 weeks. A HFD caused a greater increase in collagen levels in the aortic valves of KL^+/? mice than of WT mice, indicating that Klotho deficiency promotes HFD‐induced aortic valve fibrosis (AVF). AMPKα activity (pAMPKα) was decreased, while protein expression of collagen I and RUNX2 was increased in the aortic valves of KL^+/? mice fed with a HFD. Treatment with AICAR markedly attenuated HFD‐induced AVF in KL^+/? mice. AICAR not only abolished the downregulation of pAMPKα but also eliminated the upregulation of collagen I and RUNX2 in the aortic valves of KL^+/? mice fed with HFD. In cultured porcine aortic valve interstitial cells, Klotho‐deficient serum plus cholesterol increased RUNX2 and collagen I protein expression, which were attenuated by activation of AMPKα by AICAR. Interestingly, silencing of RUNX2 abolished the stimulatory effect of Klotho deficiency on cholesterol‐induced upregulation of matrix proteins, including collagen I and osteocalcin. In conclusion, Klotho gene deficiency promotes HFD‐induced fibrosis in aortic valves, likely through the AMPKα–RUNX2 pathway.     

A strain-based finite element model for calcification progression in aortic valves

Calcific aortic valve disease (CAVD) is a serious disease affecting the aging population. A complex interaction between biochemicals, cells, and mechanical cues affects CAVD initiation and progression. In this study, motivated by the progression of calcification in regions of high strain, we developed a finite element method (FEM) based spatial calcification progression model. Several cardiac cycles of transient structural FEM simulations were simulated. After each simulation cycle, calcium deposition was placed in regions of high circumferential strain. Our results show the radial expansion of calcification as spokes starting from the attachment region, agreeing very well with the reported clinical data.     

Ligation of ICAM-1 on human aortic valve interstitial cells induces the osteogenic response: A critical role of the Notch1-NF-κB pathway in BMP-2 expression

Calcific aortic valve disease (CAVD) is a chronic inflammatory condition and affects a large number of elderly people. Aortic valve interstitial cells (AVICs) occupy an important role in valvular calcification and CAVD progression. While pro-inflammatory mechanisms are capable of inducing the osteogenic responses in AVICs, the molecular interaction between pro-inflammatory and pro-osteogenic mechanisms remains poorly understood. This study tested the hypothesis that intercellular adhesion molecule-1 (ICAM-1) plays a role in mediating pro-osteogenic factor expression in human AVICs. AVICs were isolated from normal human aortic valves and cultured in M199 medium. Treatment with leukocyte function-associated factor-1 (LFA-1, an ICAM-1 ligand) up-regulated the expression of bone morphogenetic protein-2 (BMP-2) and resulted in increased alkaline phosphatase activity and formation of calcification nodules. Pre-treatment with lipopolysaccharide (LPS, 0.05 μg/ml) increased ICAM-1 levels on cell surfaces and exaggerated the pro-osteogenic response to LFA-1, and neutralization of ICAM-1 suppressed this response. Further, ligation of ICAM-1 by antibody cross-linking also up-regulated BMP-2 expression. Interestingly, LFA-1 elicited Notch1 cleavage and NF-κB activation. Inhibition of NF-κB markedly reduced LFA-1-induced BMP-2 expression, and inhibition of Notch1 cleavage with a γ-secretase inhibitor suppressed LFA-1-induced NF-κB activation and BMP-2 expression. Ligation of ICAM-1 on human AVICs activates the Notch1 pathway. Notch1 up-regulates BMP-2 expression in human AVICs through activation of NF-κB. The results demonstrate a novel role of ICAM-1 in translating a pro-inflammatory signal into a pro-osteogenic response in human AVICs and suggest that ICAM-1 on the surfaces of AVICs contributes to the mechanism of aortic valve calcification.     

Osteogenesis inducers promote distinct biological responses in aortic and mitral valve interstitial cells

Both aortic and mitral valves calcify in pathological conditions; however, the prevalence of aortic valve calcification is high whereas mitral valve leaflet calcification is somewhat rare. Patterns of valvular calcification may differ due to valvular architecture, but little is known to that effect. In this study, we investigated the intrinsic osteogenic differentiation potential of aortic versus mitral valve interstitial cells provided minimal differentiation conditions. For the assessment of calcification at the cellular level, we used classic inducers of osteogenesis in stem cells: β-glycerophosphate (β-Gly), dexamethasone (Dex), and ascorbate (Asc). In addition to proteomic analyses, osteogenic markers and calcium precipitates were evaluated across treatments of aortic and mitral valve cells. The combination of β-Gly, Asc, and Dex induced aortic valve interstitial cells to synthesize extracellular matrix, overexpress osteoblastic markers, and deposit calcium. However, no strong evidence showed the calcification of mitral valve interstitial cells. Mitral cells mainly responded to Asc and Dex by cell activation. These findings provide a deeper understanding of the physiological properties of aortic and mitral valves and tendencies for calcific changes within each valve type, contributing to the development of future therapeutics for heart valve diseases.     

Defining the Role of Fluid Shear Stress in the Expression of Early Signaling Markers for Calcific Aortic Valve Disease

Calcific aortic valve disease (CAVD) is an active process presumably triggered by interplays between cardiovascular risk factors, molecular signaling networks and hemodynamic cues. While earlier studies demonstrated that alterations in fluid shear stress (FSS) on the fibrosa could trigger inflammation, the mechanisms of CAVD pathogenesis secondary to side-specific FSS abnormalities are poorly understood. This knowledge could be critical to the elucidation of key CAVD risk factors such as congenital valve defects, aging and hypertension, which are known to generate FSS disturbances. The objective of this study was to characterize ex vivo the contribution of isolated and combined abnormalities in FSS magnitude and frequency to early valvular pathogenesis. The ventricularis and fibrosa of porcine aortic valve leaflets were exposed simultaneously to different combinations of sub-physiologic/physiologic/supra-physiologic levels of FSS magnitude and frequency for 24, 48 and 72 hours in a double cone-and-plate device. Endothelial activation and paracrine signaling were investigated by measuring cell-adhesion molecule (ICAM-1, VCAM-1) and cytokine (BMP-4, TGF-β1) expressions, respectively. Extracellular matrix (ECM) degradation was characterized by measuring the expression and activity of the proteases MMP-2, MMP-9, cathepsin L and cathepsin S. The effect of the FSS treatment yielding the most significant pathological response was examined over a 72-hour period to characterize the time-dependence of FSS mechano-transduction. While cytokine expression was stimulated under elevated FSS magnitude at normal frequency, ECM degradation was stimulated under both elevated FSS magnitude at normal frequency and physiologic FSS magnitude at abnormal frequency. In contrast, combined FSS magnitude and frequency abnormalities essentially maintained valvular homeostasis. The pathological response under supra-physiologic FSS magnitude peaked at 48 hours but was then maintained until the 72-hour time point. This study confirms the sensitivity of valve leaflets to both FSS magnitude and frequency and suggests the ability of supra-physiologic FSS levels or abnormal FSS frequencies to initiate CAVD mechanisms.     

Interactions between Chlamydia pneumoniae and trace elements: a possible link to aortic valve sclerosis

An association between Chlamydia pneumoniae and atherosclerotic cardiovascular diseases has been suggested. However, other factors may interact in the pathogenesis of valve sclerosis. Therefore, trace elements important for C. pneumoniae growth and host defense and markers of C. pneumoniae infection were studied in sclerotic valves and serum. Forty-six patients undergoing surgical valve replacement due to advanced aortic sclerosis were prospectively studied. Valves from 15 forensic cases with no heart valve disease and plasma from 46 healthy volunteers served as controls. C. pneumoniae was detected in 16/46 (34.8 %) sclerotic valves and in 0/15 forensic controls. IgG and IgA antibodies to C. pneumoniae were present in 54.3% and 26.1 % patients, respectively. In the patients’ valves, iron, magnesium, and zinc each correlated to calcium, a marker of the histopathological severity of disease. Patients showed 10- to 70-fold increases of these trace elements in valves and an increased copper/zinc ratio in serum. In a majority of aortic sclerosis patients, one of several markers of C. pneumoniae infection were detected and all patients had a disturbed trace element balance in valves and serum suggestive of active immune process and infection. The pattern of trace element changes was essentially similar regardless of positive makers of C. pneumoniae, suggesting a similar etiopathogenesis in both subgroups. The 20-fold increase in iron, essential for C. pneumoniae growth, in sclerotic valves suggests a new possible link to this infection in aortic sclerosis.     

Ex Vivo Evidence for the Contribution of Hemodynamic Shear Stress Abnormalities to the Early Pathogenesis of Calcific Bicuspid Aortic Valve Disease

The bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly and is frequently associated with calcific aortic valve disease (CAVD). The most prevalent type-I morphology, which results from left-/right-coronary cusp fusion, generates different hemodynamics than a tricuspid aortic valve (TAV). While valvular calcification has been linked to genetic and atherogenic predispositions, hemodynamic abnormalities are increasingly pointed as potential pathogenic contributors. In particular, the wall shear stress (WSS) produced by blood flow on the leaflets regulates homeostasis in the TAV. In contrast, WSS alterations cause valve dysfunction and disease. While such observations support the existence of synergies between valvular hemodynamics and biology, the role played by BAV WSS in valvular calcification remains unknown. The objective of this study was to isolate the acute effects of native BAV WSS abnormalities on CAVD pathogenesis. Porcine aortic valve leaflets were subjected ex vivo to the native WSS experienced by TAV and type-I BAV leaflets for 48 hours. Immunostaining, immunoblotting and zymography were performed to characterize endothelial activation, pro-inflammatory paracrine signaling, extracellular matrix remodeling and markers involved in valvular interstitial cell activation and osteogenesis. While TAV and non-coronary BAV leaflet WSS essentially maintained valvular homeostasis, fused BAV leaflet WSS promoted fibrosa endothelial activation, paracrine signaling (2.4-fold and 3.7-fold increase in BMP-4 and TGF-β1, respectively, relative to fresh controls), catabolic enzyme secretion (6.3-fold, 16.8-fold, 11.7-fold, 16.7-fold and 5.5-fold increase in MMP-2, MMP-9, cathepsin L, cathepsin S and TIMP-2, respectively) and activity (1.7-fold and 2.4-fold increase in MMP-2 and MMP-9 activity, respectively), and bone matrix synthesis (5-fold increase in osteocalcin). In contrast, BAV WSS did not significantly affect α-SMA and Runx2 expressions and TIMP/MMP ratio. This study demonstrates the key role played by BAV hemodynamic abnormalities in CAVD pathogenesis and suggests the dependence of BAV vulnerability to calcification on the local degree of WSS abnormality.