Epigenetic Regulation in calcific aortic valve disease: Mechanisms and therapeutic potential.

The involvement of gut microbiota in calcific aortic valve disease (CAVD) pathogenesis remains underexplored. Here, we provide evidence for a strong association between the gut microbiota and CAVD development. ApoE−/− mice were stratified into easy‐ and difficult‐ to calcify groups using neural network and cluster analyses, and subsequent faecal transplantation and dirty cage sharing experiments demonstrated that the microbiota from difficult‐to‐calcify mice significantly ameliorated CAVD. 16S rRNA sequencing revealed that reduced abundance of Faecalibacterium prausnitzii (F. prausnitzii) was significantly associated with increased calcification severity. Association analysis identified F. prausnitzii‐derived butyric acid as a key anti‐calcific metabolite. These findings were validated in a clinical cohort (25 CAVD patients vs. 25 controls), where serum butyric acid levels inversely correlated with disease severity. Functional experiments showed that butyric acid effectively hindered osteogenic differentiation in human aortic valve interstitial cells (hVICs) and attenuated CAVD progression in mice. Isotope labeling and 13C flux analyses confirmed that butyric acid produced in the intestine can reach heart tissue, where it reshapes glycolysis by specifically modifying GAPDH. Mechanistically, butyric acid‐induced butyrylation (Kbu) at lysine 263 of GAPDH competitively inhibited lactylation (Kla) at the same site, thereby counteracting glycolysis‐driven calcification. These findings uncover a novel mechanism through which F. prausnitzii and its metabolite butyric acid contribute to the preservation of valve function in CAVD, highlighting the gut microbiota‐metabolite‐glycolysis axis as a promising therapeutic target.

Introduction: Calcific aortic valve disease (CAVD) is the most common aetiology of acquired aortic valve disease. Initially considered a passive and degenerative process, the current view has redefined CAVD as an athero-inflammatory process in early stages that then progresses into a more complex condition. Supporting the role of inflammation-induced valve calcification are growing evidences on the induction of inflammation and subsequent pro-calcifying responses in valve interstitial (VIC) and endothelial cells (VEC) by immune mediators such as Toll-like receptor (TLR) ligands and tumor necrosis factor-α (TNF-α). Objectives: Prompted by evidences of a constitutive interferon (IFN) activity associated to ectopic calcification in a rare disease, the Singleton-Merten syndrome, and the infiltration of immune cells secreting IFN in diseased valves, we aimed to explore the role of these cytokines and Janus kinases (JAK)/Signal transducers and activators of transcription (STAT) pathways on CAVD pathogenesis. Materials and methods: Human aortic valve cells explanted from patients with no valve disease were used as a model for studying CAVD. Cells were exposed to recombinant IFN-α and IFN-γ combined or not with TLR ligands (in VIC) or TNF-α (in VEC). Inflammation, calcification and angiogenic responses as well as cell proliferation and apoptosis were studied using different cellular and molecular biology techniques such as Western Blot, ELISA, qPCR, flow cytometry, immunofluorescence and calcification assays. In addition, total protein and RNA were extracted from both non-mineralized and calcified valves for gene and protein expression analysis. Results: IFN activate several signalling pathways and promote inflammatory responses in VIC, characterized by adhesion molecule expression and nuclear factor-κB activation. In addition, IFN trigger VIC differentiation towards a pro-osteogenic phenotype and promote calcific nodule formation in high-phosphate conditions. Strikingly, we found an IFN-Lipopolysaccharide (a TLR4 ligand) interplay that further potentiates the pro-inflammatory, pro-angiogenic and pro-osteogenic responses. Significant findings include the blockade of the responses by Jakinibs, which are JAK inhibitors currently used in clinics. Additionally, this study provides several new insights about sex-differences in CAVD, with unexpected greater responses to IFN and LPS in male cells and identified some of the underlying molecular mechanisms such as larger protein kinase B/Akt activation in cells from females, and larger osteogenic signalling, hypoxia-inducible factor (HIF)-1α and extracellular signal-regulated kinases activation in male cells. We also found sex-differences in gene expression profile in calcified tissue that correlate with lower valve calcification in female patients. Furthermore, our work demonstrates a novel role for type I IFN signalling on TLR3-mediated effects in VIC. Finally, data unveiled differences on monocyte adhesion to side-specific VEC monolayers in response to IFN-γ and TNF-α with potential relevance in CAVD pathogenesis. Conclusions: Our findings highlight that IFN act as pro-inflammatory and pro-osteogenic cytokines in VIC and support the model of inflammation-induced calcification, the concept of additional pro-angiogenic mechanisms beyond valve thickening and hypoxia, and the notion of CAVD as a sex-divergent disease from the early inflammatory stages. Clinically relevant findings include the blockade of IFN-induced responses by Jakinibs, and by a HIF-1α inhibitor. JAK/STAT and HIF-1α pathways emerge as potential therapeutic targets for CAVD.

Although often studied independently, little is known about how aortic valve endothelial cells and valve interstitial cells interact collaborate to maintain tissue homeostasis or drive valve calcific pathogenesis. Inflammatory signaling is a recognized initiator of valve calcification, but the cell-type-specific downstream mechanisms have not been elucidated. In this study, we test how inflammatory signaling via NFκB (nuclear factor κ-light-chain enhancer of activated B cells) activity coordinates unique and shared mechanisms of valve endothelial cells and valve interstitial cells differentiation during calcific progression. Approach and Results: Activated NFκB was present throughout the calcific aortic valve disease (CAVD) process in both endothelial and interstitial cell populations in an established mouse model of hypercholesterolemia-induced CAVD and in human CAVD. NFκB activity induces endothelial to mesenchymal transformation in 3-dimensional cultured aortic valve endothelial cells and subsequent osteogenic calcification of transformed cells. Similarly, 3-dimensional cultured valve interstitial cells calcified via NFκB-mediated osteogenic differentiation. NFκB-mediated endothelial to mesenchymal transformation was directly demonstrated in vivo during CAVD via genetic lineage tracking. Genetic deletion of NFκB in either whole valves or valve endothelium only was sufficient to prevent valve-specific molecular and cellular mechanisms of CAVD in vivo despite the persistence of a CAVD inducing environment. Our results identify NFκB signaling as an essential molecular regulator for both valve endothelial and interstitial participation in CAVD pathogenesis. Direct demonstration of valve endothelial cell endothelial to mesenchymal transformation transmigration in vivo during CAVD highlights a new cellular population for further investigation in CAVD morbidity. The efficacy of valve-specific NFκB modulation in inhibiting hypercholesterolemic CAVD suggests potential benefits of multicell type integrated investigation for biological therapeutic development and evaluation for CAVD.

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.

Alterations in lipid metabolism and inflammatory processes are well established as potential risk factors in the development and progression of cardiovascular disease.1 However, with complications ranging from valve dysfunction to arrhythmia to myocardial infarction and stroke, the underlying mechanisms may be as varied as cardiovascular disease itself. On the other hand, the reoccurrence of common molecular and cellular pathways identified in the collective body of cardiovascular research could suggest shared initiators or mechanistic nodes between seemingly divergent processes, including lipid metabolism and inflammation. One area where this may hold true is cardiovascular calcification, in which dysregulated mineral metabolism in cardiovascular tissues leads to increased morbidity and mortality. Article see p 677 Calcification of soft tissues results from the deposition of calcium, largely in the form of hydroxyapatite, in the vascular wall or valve leaflets. Previously thought to be a passive degenerative process, it has become increasingly apparent that cardiovascular calcification is an active process initiated by many triggers. Recent studies have demonstrated variation in the LPA gene, which determines the plasma concentration of lipoprotein(a) [Lp(a); pronounced “L P little a”] to be associated with calcific aortic valve disease (CAVD).2,3 Lp(a) consists of a low-density lipoprotein (LDL)–like particle in which apolipoprotein(a) is covalently bound to apolipoprotein B. Additionally, Lp(a) is a genetic risk factor for atherosclerotic events.4 As in atherosclerosis, calcifications in CAVD localize to areas with lipoprotein accumulation and inflammatory cell infiltration, suggesting a shared disease process.5 However, some noticeable differences exist, including increased mechanical stresses and calcification-involved valve obstruction in CAVD as opposed to microcalcifications leading atherosclerosis plaque rupture.6 In this issue of Circulation , Bouchareb et al7 propose a highly plausible mechanistic pathway through which Lp(a) and valve interstitial cell (VIC)–derived autotaxin may induce valve calcification by regulating inflammation-induced bone …

Calcific aortic valve disease (CAVD) is one of the leading causes of cardiovascular death in the elderly population worldwide. MicroRNAs (miRNAs) are highly dysregulated in patients with CAVD undergoing surgical aortic valve replacement. However, the miRNA-dependent mechanisms regulating inflammation and calcification or miRNA-mediated cell-cell crosstalk during the pathogenesis of CAVD remain poorly understood. Here, we investigated the role of extracellular vesicle (EV)-associated miR-145-5p, which we showed to be strongly upregulated in CAVD in mice and humans during valve calcification. Human TaqMan arrays identified dysregulated miRNAs in CAVD tissue explants from patients compared to non-calcified (patients with aortic insufficiency) heart valve explants from patients undergoing SAVR. Echocardiography was used in conjunction with the quantification of dysregulated miRNAs in a murine CAVD model. In vitro calcification experiments were performed to investigate the effects of EV-miR-145-5p on calcification and crosstalk in heart valve cells. Integrated OMICS analysis were performed to analyze molecular miRNA signatures and their effects on signaling pathways-associated with CAVD. RNA sequencing, high-throughput transcription factor activity assays, osteogenesis and proteomic arrays revealed that a number of genes, miRNAs, TFs and proteins are critical for calcification and apoptosis involved in the pathogenesis of CAVD. Among several miRNAs dysregulated in valve explants from CAVD patients, miR-145-5p was the most highly sex-independently upregulated miRNA (AUC, 0.780, p-value, 0.01) in patient-plasma. large EV population (170-800 nm) isolated from aortic valve tissues explanted from patients with CAVS after SAVR demonstrated a significantly higher level of miR-145-5p expression in comparison to control (vesicle-free plasma). MiRNA arrays utilizing with aortic stenosis samples from patients and mice showed that the expression of miR-145-5p is significantly upregulated and positively correlated with cardiac function based on echocardiography. In vitro experiments confirmed that miR-145-5p is encapsulated in EVs and transported into interstitial cells of the aortic valve. The results of integrated OMICs show that miR-145-5p is related to markers of inflammation, calcification, and apoptosis. In vitro calcification experiments demonstrated that miR-145-5p regulates the ALPL gene, a hallmark of calcification in vascular and heart valve cells. Mechanistically, EV-mediated shuttling of miR-145-5p suppressed the expression of ZEB2, a negative regulator of the ALPL gene, by binding to its 3' untranslated region to inhibit its translation, thereby diminishing the calcification of VIC. Elevated levels of pro-calcific and pro-apoptotic EV-associated miR-145-5p contribute to the progression of CAVD via the ZEB2-ALPL axis, which could potentially be therapeutically targeted to minimize the burden of CAVD.

Celastrol Alleviates Aortic Valve Calcification Via Inhibition of NADPH Oxidase 2 in Valvular Interstitial Cells

Calcific aortic valve disease (CAVD) is one of the leading causes of cardiovascular death in the elderly population worldwide. MicroRNAs (miRNAs) are highly dysregulated in patients with CAVD undergoing surgical aortic valve replacement (SAVR). However, the miRNA-dependent mechanisms regulating inflammation and calcification or miRNA-mediated cell–cell crosstalk during the pathogenesis of CAVD remain poorly understood. Here, we investigated the role of extracellular vesicle (EV)-associated miR-145-5p, which we showed to be strongly upregulated in CAVD in mice and humans during valve calcification. Human TaqMan miRNA arrays identified dysregulated miRNAs in CAVD tissue explants from patients compared to non-calcified (patients with aortic insufficiency) heart valve tissue explants from patients undergoing SAVR. Echocardiographic parameters were measured in conjunction with the quantification of dysregulated miRNAs in a murine CAVD model. In vitro calcification experiments were performed to investigate the effects of EV-miR-145-5p on calcification and crosstalk in heart valve cells. Integrated OMICS analyses were performed to analyze molecular miRNA signatures and their effects on signaling pathways-associated with CAVD. RNA sequencing, high-throughput transcription factor (TF) activity assays, and osteogenesis arrays revealed that a number of genes, miRNAs, TFs are critical for calcification and apoptosis involved in the pathogenesis of CAVD. Among several miRNAs dysregulated in valve explants from CAVD patients, miR-145-5p was the most highly sex-independently upregulated miRNA (AUC, 0.780, p value, 0.01) in patient plasma. Large EV population (170–800 nm) isolated from aortic valve tissues explanted from patients with CAVS (calcific aortic valve stenosis) after SAVR demonstrated a significantly higher level of miR-145-5p expression in comparison to control (vesicle-free plasma). MiRNA arrays utilizing with aortic stenosis samples from patients and mice showed that the expression of miR-145-5p is significantly upregulated and positively correlated with cardiac function based on echocardiography. In vitro experiments confirmed that miR-145-5p is encapsulated in EVs and transported into interstitial cells of the aortic valve. The results of integrated OMICs show that miR-145-5p is related to markers of inflammation, calcification, and apoptosis. In vitro calcification experiments demonstrated that miR-145-5p regulates the ALPL gene, a hallmark of calcification in vascular and heart valve cells. Mechanistically, EV-mediated shuttling of miR-145-5p suppressed the expression of ZEB2, a negative regulator of the ALPL gene, by binding to its 3' untranslated region to inhibit its translation, thereby diminishing the calcification of valvular interstitial cells. Elevated levels of pro-calcific and pro-apoptotic EV-associated miR-145-5p contribute to the progression of CAVD via the ZEB2-ALPL axis, which could potentially be therapeutically targeted to minimize the burden of CAVD.Graphical abstractSupplementary InformationThe online version contains supplementary material available at 10.1007/s00395-025-01133-w.

The progression of calcific aortic valve disease through injury, cell dysfunction, and disruptive biologic and physical force feedback loops

Calcific aortic valve disease (CAVD) represents a spectrum of disease spanning from milder degrees of calcification of valve leaflets, i.e., aortic sclerosis, to severe calcification i.e., aortic stenosis (AS) with hemodynamic instability. The prevalence of CAVD is increasing rapidly due to the aging of the population, being up to 2.8% among patients over 75 years of age. Even without significant aortic valve stenosis, aortic sclerosis is associated with a 50% increased risk of myocardial infarction and death from cardiovascular causes. To date, there is no pharmacological treatment available to reverse or hinder the progression of CAVD. So far, the cholesterol-lowering therapies (statins) and renin–angiotensin system (RAS) blocking drugs have been the major pharmacological agents investigated for treatment of CAVD. Especially angiotensin receptor blockers (ARB)s and angiotensin convertase enzyme inhibitors (ACEI)s, have been under active investigation in clinical trials, but have proven to be unsuccessful in slowing the progression of CAVD. Several studies have suggested that other vasoactive hormones, including endothelin and apelin systems are also associated with development of AS. In the present review, we discuss the role of vasoactive factors in the pathogenesis of CAVD as novel pharmacological targets for the treatment of aortic valve calcification.Key messagesVasoactive factors are involved in the progression of calcific aortic valve disease.Endothelin and renin–angiotensin systems seem to be most prominent targets for therapeutic interventions in the view of valvular pathogenesis.Circulating vasoactive factors may provide targets for diagnostic tools of calcified aortic valve disease.

Mitochondrial dysfunction and immune cell infiltration play crucial yet incompletely understood roles in the pathogenesis of calcific aortic valve disease (CAVD). This study aimed to identify immune-related mitochondrial genes critical to the pathological process of CAVD using multiomics approaches. The CIBERSORT algorithm was employed to evaluate immune cell infiltration characteristics in CAVD patients. An integrative analysis combining weighted gene coexpression network analysis (WGCNA), machine learning, and summary data-based Mendelian randomization (SMR) was performed to identify key mitochondrial genes implicated in CAVD. Spearman’s rank correlation analysis was also performed to assess the relationships between key mitochondrial genes and infiltrating immune cells. Compared with those in normal aortic valve tissue, an increased proportion of M0 macrophages and resting memory CD4 T cells, along with a decreased proportion of plasma cells and activated dendritic cells, were observed in CAVD patients. Additionally, eight key mitochondrial genes associated with CAVD, including PDK4, LDHB, SLC25A36, ALDH9A1, ECHDC2, AUH, ALDH2, and BNIP3, were identified through the integration of WGCNA and machine learning methods. Subsequent SMR analysis, incorporating multiomics data, such as expression quantitative trait loci (eQTLs) and methylation quantitative trait loci (mQTLs), revealed a significant causal relationship between ALDH9A1 expression and a reduced risk of CAVD. Moreover, ALDH9A1 expression was inversely correlated with M0 macrophages and positively correlated with M2 macrophages. These findings suggest that increased ALDH9A1 expression is significantly associated with a reduced risk of CAVD and that it may exert its protective effects by modulating mitochondrial function and immune cell infiltration. Specifically, ALDH9A1 may contribute to the shift from M0 macrophages to anti-inflammatory M2 macrophages, potentially mitigating the pathological progression of CAVD. In conclusion, ALDH9A1 represents a promising molecular target for the diagnosis and treatment of CAVD. However, further validation through in vivo and n vitro studies is necessary to confirm its role in CAVD pathogenesis and therapeutic potential.

Background Calcific aortic valve disease (CAVD) is the most common native valve disease. Valvular interstitial cell (VIC) osteogenic differentiation and valvular endothelial cell (VEC) dysfunction are key steps in CAVD progression. Circular RNA (circRNAs) is involved in regulating osteogenic differentiation with mesenchymal cells and is associated with multiple disease progression, but the function of circRNAs in CAVD remains unknown. Here, we aimed to investigate the effect and potential significance of circRNA-miRNA-mRNA networks in CAVD. Methods Two mRNA datasets, one miRNA dataset, and one circRNA dataset of CAVD downloaded from GEO were used to identify DE-circRNAs, DE-miRNAs, and DE-mRNAs. Based on the online website prediction function, the common mRNAs (FmRNAs) for constructing circRNA-miRNA-mRNA networks were identified. GO and KEGG enrichment analyses were performed on FmRNAs. In addition, hub genes were identified by PPI networks. Based on the expression of each data set, the circRNA-miRNA-hub gene network was constructed by Cytoscape (version 3.6.1). Results 32 DE-circRNAs, 206 DE-miRNAs, and 2170 DE-mRNAs were identified. Fifty-nine FmRNAs were obtained by intersection. The KEGG pathway analysis of FmRNAs was enriched in pathways in cancer, JAK-STAT signaling pathway, cell cycle, and MAPK signaling pathway. Meanwhile, transcription, nucleolus, and protein homodimerization activity were significantly enriched in GO analysis. Eight hub genes were identified based on the PPI network. Three possible regulatory networks in CAVD disease were obtained based on the biological functions of circRNAs including: hsa_circ_0026817-hsa-miR-211-5p-CACNA1C, hsa_circ_0007215-hsa-miR-1252-5p-MECP2, and hsa_circ_0007215-hsa-miR-1343-3p- RBL1. Conclusion The present bionformatics analysis suggests the functional effect for the circRNA-miRNA-mRNA network in CAVD pathogenesis and provides new targets for therapeutics.

Rationale: Calcific aortic valve disease (CAVD) is a progressive disorder characterized by aortic valve (AV) calcification and fibrosis. Despite advances in our understanding of CAVD pathogenesis, no drug has proven effective in preventing AV calcification. The aim of this study was to identify the key pathogenic genes in CAVD and elucidate mechanisms that may guide development of new targeted therapies. Methods: A CAVD model was established in ApoE-/- mice by administering a high-cholesterol diet for 24 weeks. An adeno-associated virus was used to induce alpha-1-microglobulin/bikunin precursor (AMBP) overexpression. RNA sequencing, quantitative real-time polymerase chain reaction, western blotting, immunofluorescence, histopathology, and echocardiography were performed to assess AV function. The mechanism of interaction between AMBP and four-and-a-half LIM domain protein 3 (FHL3) was explored using bioinformatics analyses, co-immunoprecipitation, and AlphaFold3-based simulations of crystal structures. Results: RNA sequencing identified AMBP as a key regulator of CAVD. AMBP was increased in calcified AV from CAVD patients and high cholesterol diet (HCD)-induced ApoE-/- mice. In vivo, AMBP overexpression significantly reduced HCD-induced AV calcification and fibrosis. In vitro, AMBP knockdown elevated osteogenic markers, RUNX2 and OSTERIX, and promoted calcium deposition in valvular interstitial cells induced by osteogenic medium (OM), whereas AMBP overexpression reversed these effects. Mechanistically, AMBP inhibited OM-induced phosphorylation of ERK1/2 (P-ERK1/2) and JNK (P-JNK) by competitively binding to the zinc finger domain of FHL3. This interaction disrupted the protective role of FHL3 in preventing ubiquitin-proteasome-mediated degradation of P-ERK1/2 and P-JNK. P-ERK1/2 and P-JNK inhibitors and agonists confirmed that the protective effects of AMBP against CAVD were mediated via these pathways in vivo and in vitro. Conclusions: AMBP protects valvular interstitial cells from osteoblastic differentiation and calcium deposit accumulation, thereby alleviating AV calcification. This study sheds additional light on the pathogenesis of CAVD and potential new therapeutic approaches.

Calcific aortic valve disease (CAVD) has been associated with activation of molecular pathways involved in both heart valve and bone development. There is increasing evidence that developmental pathways are reactivated during disease and also that the process of calcification during CAVD may mimic that of normal endochondral bone formation. A number of different signaling pathways and transcription factors play essential roles both in heart valve development and bone formation. Notably, these mechanisms have also been implicated in the pathogenesis of CAVD. Although studies in human, mouse, and porcine models demonstrate conserved mechanisms in CAVD, the individual contributions to pathogenesis and requirements of each pathway for the development of aortic valve calcification are not fully understood. Furthermore, the crosstalk among these signaling pathways, although hypothesized, remains unknown. The goal of this chapter is to review pathways involved in development of both heart valves and bone and their reactivation in CAVD.

INTRODUCTION: Calcific aortic valve disease (CAVD) is an active process presumably triggered by interplays between atherogenic risk factors, molecular signaling networks and hemodynamic cues. While our earlier work demonstrated that progressive alterations in fluid wall-shear stress (WSS) on the fibrosa could trigger leaflet inflammation, the mechanisms of CAVD pathogenesis secondary to side-specific WSS abnormalities are poorly understood. HYPOTHESIS: Supported by our previous studies, we hypothesize that valve leaflets are sensitive to both WSS magnitude and pulsatility and that abnormalities in either promote CAVD development. OBJECTIVE: This study aims at elucidating ex vivo the contribution of isolated and combined alterations in WSS magnitude and pulsatility to valvular calcification. METHODS: The fibrosa and ventricularis of porcine leaflets were subjected simultaneously to different combinations of WSS magnitude and pulsatility (i.e., physiologic, sub- and supra-physiologic levels) for 48 hours in a double-sided shear stress bioreactor. Endothelial activation (ICAM-1, VCAM-1), paracrine expression (TGF-β and BMP-4), and proteinase/collagenase expression (MMP-2, cathepsin L) were detected by immunohistochemistry, while osteogenic differentiation (α-SMA) was assessed via western blot. RESULTS: Regardless of the magnitude or frequency, non-physiologic WSS conditions did not result in endothelial activation. Tissue exposure to either supra-physiologic WSS magnitude or pulsatility significantly upregulated paracrine (74-fold increase), proteinase (4-fold increase), collagenase (5-fold increase) and α-SMA (23-fold increase) expressions relative to the levels measured under physiologic WSS. In contrast, combined alterations in WSS magnitude and pulsatility downregulated those responses. CONCLUSION: This study demonstrates the sensitivity of aortic valve leaflets to both WSS magnitude and pulsatility and the ability of supra-physiologic WSS magnitude or pulsatility to trigger events involved in early CAVD pathogenesis. The results provide new potential insights into the mechanisms of CAVD secondary to hypertension and Paget’s disease, which are associated with abnormal blood flow and leaflet WSS.