Human Aortic Valve Research Articles

Upregulation of NOR-1 in calcified human vascular tissues: impact on osteogenic differentiation and calcification

Cardiovascular calcification is a significant public health issue whose pathophysiology is not fully understood. NOR-1 regulates critical processes in cardiovascular remodeling, but its contribution to ectopic calcification is unknown. NOR-1 was overexpressed in human calcific aortic valves and calcified atherosclerotic lesions colocalizing with RUNX2, a factor essential for osteochondrogenic differentiation and calcification. NOR-1 and osteogenic markers were upregulated in calcifying human valvular interstitial cells (VICs) and human vascular smooth muscle cells (VSMCs). Gain- and loss-of-function approaches demonstrated that NOR-1 negatively modulates the expression of osteogenic genes relevant for the osteogenic transdifferentiation (RUNX2, IL-6, BMP2, and ALPL) and calcification of VICs. VSMCs from transgenic mice overexpressing NOR-1 in these cells (TgNOR-1VSMC) expressed lower basal levels of osteogenic genes (IL-6, BMP2, ALPL, OPN) than cells from WT littermates, and their upregulation by a high-phosphate osteogenic medium (OM) was completely prevented by NOR-1 transgenesis. Consistently, this was associated with a dramatic reduction in the calcification of both transgenic VSMCs and aortic rings from TgNOR-1VSMC mice exposed to OM. Atherosclerosis and calcification were induce in mice by the administration of AAV-PCSK9D374Y and a high-fat/high-cholesterol diet. Challenged-TgNOR-1VSMC mice exhibited decreased vascular expression of osteogenic markers, and both less atherosclerotic burden (assessed in whole aorta and lesion size in aortic arch and brachiocephalic artery) and less vascular calcification (assessed either by near-infrared fluorescence imaging or histological analysis) than WT mice. Our data indicate that NOR-1 negatively modulates the expression of genes critically involved in the osteogenic differentiation of VICs and VSMCs, thereby restraining ectopic cardiovascular calcification.

Identification of hub genes and key signaling pathways by weighted gene co-expression network analysis for human aortic stenosis and insufficiency.

Human aortic valve stenosis (AS) and insufficiency (AI) are common diseases in aging population. Identifying the molecular regulatory networks of AS and AI is expected to offer novel perspectives for AS and AI treatment. Highly correlated modules with the progression of AS and AI were identified by weighted genes co-expression network analysis (WGCNA). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed by the clusterProfiler program package. Differentially expressed genes (DEGs) were identified by the DESeqDataSetFromMatrix function of the DESeq2 program package. The protein-protein interaction (PPI) network analyses were implemented using the STRING online tool and visualized with Cytoscape software. The DEGs in AS and AI groups were overlapped with the top 30 genes with highest connectivity to screen out ten hub genes. The ten hub genes were verified by analyzing the data in high throughput RNA-sequencing dataset and real-time PCR assay using AS and AI aortic valve samples. By WGCNA algorithm, 302 highly correlated genes with the degree of AS, degree of AI, and heart failure were identified from highly correlated modules. GO analyses showed that highly correlated genes had close relationship with collagen fibril organization, extracellular matrix organization and extracellular structure organization. KEGG analyses also manifested that protein digestion and absorption, and glutathione metabolism were probably involved in AS and AI pathological courses. Moreover, DEGs were picked out for 302 highly correlated genes in AS and AI groups relative to the normal control group. The PPI network analyses indicated the connectivity among these highly correlated genes. Finally, ten hub genes (CD74, COL1A1, TXNRD1, CCND1, COL5A1, SERPINH1, BCL6, ITGA10, FOS, and JUNB) in AS and AI were found out and verified. Our study may provide the underlying molecular targets for the mechanism research, diagnosis, and treatment of AS and AI in the future.

Abstract P1039: Soluble Extracellular Matrix Protein Induces Senescence In Aortic Valve Interstitial Cells To Up-regulate Valvular Osteogenic Activity

Background: Calcific aortic valve disease (CAVD) is the most prevalent heart valve disease in the elderly, and elevated osteogenic activity of aortic valve interstitial cells (AVICs) play a critical role in CAVD progression to severe aortic valve stenosis. While soluble extracellular matrix (ECM) proteins, including biglycan and matrilin 2, have been found to up-regulate the osteogenic activity in human AVICs, the mechanism underlying the cellular and molecular mechanisms remain unclear. As greater densities of senescent AVICs were observed in human calcified aortic valves, we tested the hypothesis that soluble ECM protein induces senescence in AVICs to up-regulate valvular osteogenic activity. Methods and Results: Human AVICs were isolated from normal and CAVD aortic valves. Levels of P16 and P21 were assessed by immunoblotting, and senescent changes is identified using senescence-associated β-galactosidase (SA-β-gal) staining. AVICs from diseased valves displayed higher levels of P16 and P21, greater SA-β-gal staining and active calcium deposition. Soluble matrilin 2 (2.0 μg/ml) up-regulated the expression of P16 and P21, and enhanced SA-β-gal staining in normal AVICs, leading to greater calcium deposition. Further, knockdown of P16 markedly reduced calcium deposition in AVICs exposed to soluble matrilin 2. The impact of soluble matrlin 2 is associated with activation of the M6PR-ERK1/2 pathway. Blocking or knockdown of M6PR (M6P, 10 mM or siRNA, 1.0 nM), or inhibition of ERK1/2 (PD98059, 25 μM) attenuated the effect of soluble matrilin 2 on AVIC senescence and resultant up-regulation of osteogenic activity. Conclusion: AVICs from aortic valves of patients with CAVD have higher levels of senescence activity. Soluble matrilin 2, via the M6PR-ERK1/2 pathway, induces AVIC senescence to promote the osteogenic activity. Targeting the D-M6PR-ERK1/2 pathway may suppress AVIC senescence and attenuate aortic valve calcification associated with CAVD progression.

Abstract P3198: Spermidine Ameliorates Aortic Valve Degenerations By Improving Mitochondrial Biogenesis

Aortic stenosis is the most common heart valve disease in the elderly, and aging is a leading factor. However, there is no medical treatment yet except for an operation. We investigated that mitochondrial function deteriorated in isolated human aortic valve interstitial cells (hAVICs) from the patients who had aortic stenosis (Figure A, B). We examined the therapeutic effect of spermidine (SPD) which is known to enhance mitochondrial function in the treatment of diseased hAVICs. As a result, we showed the expression of fibrosis, inflammation, and calcification markers decreased after the SPD treatment. Using the LDLR knockout mice model fed a high-fat diet, we found that the mitochondrial function in mouse AVICs improved after the SPD treatment, and the thickening, inflammation, and calcification of aortic valve leaflets significantly decreased in the SPD-fed mice (Figure C, D). Ingenuity pathway analysis from proteomic data in this animal model suggested the IRS2-AKT-TP53-SIRT1-DNMT1 pathway as a treatment mechanism and we could confirm that TP53, SIRT1, and PPARG increased while IRS2, AKT and DNMT1 were inhibited by the treatment of SPD in ex-vivo and in vitro experiments. In addition, we found that 5-azacytidine, as a DNMT1 inhibitor, induces the mitochondrial biogenesis-related genes by lowering mtDNA hypermethylation in hAVICs. In conclusion, spermidine ameliorates the mitochondrial function through the IRS-AKT-TP53-SIRT1-DNMT1 pathway in AVICs and the inhibition of DNMT1 and mtDNA hypermethylation improves mitochondrial biogenesis which can be a treatment mechanism for aortic stenosis.

Multiomics of Tissue Extracellular Vesicles Identifies Unique Modulators of Atherosclerosis and Calcific Aortic Valve Stenosis.

Fewer than 50% of patients who develop aortic valve calcification have concomitant atherosclerosis, implying differential pathogenesis. Although circulating extracellular vesicles (EVs) act as biomarkers of cardiovascular diseases, tissue-entrapped EVs are associated with early mineralization, but their cargoes, functions, and contributions to disease remain unknown. Disease stage-specific proteomics was performed on human carotid endarterectomy specimens (n=16) and stenotic aortic valves (n=18). Tissue EVs were isolated from human carotid arteries (normal, n=6; diseased, n=4) and aortic valves (normal, n=6; diseased, n=4) by enzymatic digestion, (ultra)centrifugation, and a 15-fraction density gradient validated by proteomics, CD63-immunogold electron microscopy, and nanoparticle tracking analysis. Vesiculomics, comprising vesicular proteomics and small RNA-sequencing, was conducted on tissue EVs. TargetScan identified microRNA targets. Pathway network analyses prioritized genes for validation in primary human carotid artery smooth muscle cells and aortic valvular interstitial cells. Disease progression drove significant convergence (P<0.0001) of carotid artery plaque and calcified aortic valve proteomes (2318 proteins). Each tissue also retained a unique subset of differentially enriched proteins (381 in plaques; 226 in valves; q<0.05). Vesicular gene ontology terms increased 2.9-fold (P<0.0001) among proteins modulated by disease in both tissues. Proteomics identified 22 EV markers in tissue digest fractions. Networks of proteins and microRNA targets changed by disease progression in both artery and valve EVs revealed shared involvement in intracellular signaling and cell cycle regulation. Vesiculomics identified 773 proteins and 80 microRNAs differentially enriched by disease exclusively in artery or valve EVs (q<0.05); multiomics integration found tissue-specific EV cargoes associated with procalcific Notch and Wnt signaling in carotid arteries and aortic valves, respectively. Knockdown of tissue-specific EV-derived molecules FGFR2, PPP2CA, and ADAM17 in human carotid artery smooth muscle cells and WNT5A, APP, and APC in human aortic valvular interstitial cells significantly modulated calcification. The first comparative proteomics study of human carotid artery plaques and calcified aortic valves identifies unique drivers of atherosclerosis versus aortic valve stenosis and implicates EVs in advanced cardiovascular calcification. We delineate a vesiculomics strategy to isolate, purify, and study protein and RNA cargoes from EVs entrapped in fibrocalcific tissues. Integration of vesicular proteomics and transcriptomics by network approaches revealed novel roles for tissue EVs in modulating cardiovascular disease.

Human and porcine aortic valve endothelial and interstitial cell isolation and characterization.

Calcific aortic valve stenosis (AVS) is defined by pathological changes in the aortic valve (AV) and their predominant cell types: valvular interstitial (VICs) and endothelial cells (VECs). Understanding the cellular and molecular mechanisms of this disease is a prerequisite to identify potential pharmacological treatment strategies. In this study, we present a unique aortic valve cell isolation technique to acquire specific human and porcine cell populations and compared VICs and VECs of these species with each other for the first time. AV cells were isolated from tissue obtained from human patients undergoing surgical aortic valve replacement (SAVR) or from porcine hearts. Functional analysis and in vitro experiments revealed that endothelial-to-mesenchymal transition (EndMT) can be induced in hVECs, leading to a significant increase in mesenchymal markers. In vitro calcification experiments of VICs demonstrated pronounced expression of calcification markers and visible calcific deposits in Alizarin Red staining in both species after incubation with pro-calcific media. Cells isolated from patient-derived AVs showed mesenchymal and endothelial-specific gene signatures (VIC and VEC, respectively). For instance, von Willebrand factor (vWF) and platelet endothelial adhesion molecule-1 (PECAM1) were upregulated in VECs, while the myofibroblastic markers alpha-smooth muscle actin (α-SMA) and vimentin (VIM) were downregulated in VECs compared to VICs. Analysis of cell function by migration revealed that VECs are more migratory than VICs. Induction of EndMT in vitro in VECs displayed increased expression of EndMT markers and decreased expression of endothelial markers, confirming their mesenchymal transdifferentiation ability. In vitro calcification of VICs revealed upregulation of alkaline phosphatase (ALPL), a hallmark of calcification. In addition, other calcification-related genes such as osteocalcin (BGLAP) and runt-related factor 2 (RUNX2) were upregulated. Alizarin red staining of calcified cells provided a further layer of confirmation that the isolated cells were VICs with osteoblastic differentiation capacity. This study aims to take a first step towards standardizing a reproducible isolation technique for specific human and porcine VEC and VIC populations. A comparison of human and porcine aortic valve cells demonstrated that porcine cells may serve as an alternative cellular model system in settings where human tissue is difficult to obtain.

Functional differences in human aortic valve interstitial cells from patients with varying calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is characterized by progressive stiffening of aortic valve (AV) tissues, inducing stenosis and insufficiency. Bicuspid aortic valve (BAV) is a common congenital defect in which the AV has two leaflets rather than three, with BAV patients developing CAVD decades years earlier than in the general population. Current treatment for CAVD remains surgical replacement with its continued durability problems, as there are no pharmaceutical therapies or other alternative treatments available. Before such therapeutic approaches can be developed, a deeper understanding of CAVD disease mechanisms is clearly required. It is known that AV interstitial cells (AVICs) maintain the AV extracellular matrix and are typically quiescent in the normal state, transitioning into an activated, myofibroblast-like state during periods of growth or disease. One proposed mechanism of CAVD is the subsequent transition of AVICs into an osteoblast-like phenotype. A sensitive indicator of AVIC phenotypic state is enhanced basal contractility (tonus), so that AVICs from diseased AV will exhibit a higher basal tonus level. The goals of the present study were thus to assess the hypothesis that different human CAVD states lead to different biophysical AVIC states. To accomplish this, we characterized AVIC basal tonus behaviors from diseased human AV tissues embedded in 3D hydrogels. Established methods were utilized to track AVIC-induced gel displacements and shape changes after the application of Cytochalasin D (an actin polymerization inhibitor) to depolymerize the AVIC stress fibers. Results indicated that human diseased AVICs from the non-calcified region of TAVs were significantly more activated than AVICs from the corresponding calcified region. In addition, AVICs from the raphe region of BAVs were more activated than from the non-raphe region. Interestingly, we observed significantly greater basal tonus levels in females compared to males. Furthermore, the overall AVIC shape changes after Cytochalasin suggested that AVICs from TAVs and BAVs develop different stress fiber architectures. These findings are the first evidence of sex-specific differences in basal tonus state in human AVICs in varying disease states. Future studies are underway to quantify stress fiber mechanical behaviors to further elucidate CAVD disease mechanisms.

Morphology and Morphometry of Aortic Valve in Humans and Porcines – A Comparative Study

Background: The aortic valve is a semilunar valve with three leaflets that are in the left ventricle’s outflow tract and only permits unidirectional blood flow away from the ventricle. The valve leaflet can tolerate extremely high mechanical and hemodynamic forces resulting from the great complexity and complicated cellular and molecular functions. Aims: The aim of the study is to provide an add-on to the understanding of the morphological and morphometrical aspects of human and porcine aortic heart valves on comparative grounds so that they can be used as a guide in the construction of three-dimensional (3D)-printed mechanical valve. Materials and Methods: Twenty swine and twenty human hearts were compared and examined in this investigation. The porcine and human hearts weighing in between 300 g and 450 g were considered, and any variation from the normal and any mechanical damage rendered during procurement were all excluded from the study. Results: The length of the attached margin of the right coronary cusp, left coronary cusp, and noncoronary cusp of human and porcine were 45.15 mm, 36.47 mm, and 41.53 mm and 36.55 mm, 43.14 mm, and 36.10 mm, respectively. The length of the free margin and the height of the cusps from nadir to the sinotubular junction and the effective height of the cusp from nadir to the free margin of the cusps of human and porcine have been compared. Conclusion: The human and swine aortic valves share morphological and morphometrical characteristics, which can be used to create 3D valves and tissue engineering procedures with greater biocompatibility and biofunctionality.

IL-17A induces valvular endothelial inflammation and aggravates calcific aortic valve disease

Calcific aortic valve disease (CAVD) is an aging related disease characterized by inflammation and fibrocalcific remodeling. IL-17A is a key cytokine associated with pathophysiology of inflammatory and fibrotic disease. Previous studies showed accumulation of IL-17A-producing T helper lymphocytes in human calcified aortic valves and significantly elevated IL-17RA expression in calcified valves. However, the role of IL-17A signaling in the initiation and development of CAVD is still unclear. In this study, by analyzing public transcriptome databases, we found that IL-17A-IL-17RA signaling is activated in calcified valves. Gene expression analysis revealed significantly increased IL-17A, IL-17RA, and RUNX2 expression in calcified human aortic valves compared to in non-calcified valves, and the expression of IL-17A and IL-17RA were positively correlated with RUNX2 expression. A 5/6 nephrectomy was performed in Apoe−/− (Apoe knockout) mice to establish a CAVD mouse model. IL-17A-neutralizing antibodies significantly reduced valve calcium deposition and decreased expression of RUNX2 in aortic valves. Immunofluorescence staining of human aortic valves and qRT-PCR analysis of primary aortic valve cells revealed abundant expression of IL-17RA in valvular endothelial cells (VECs). RNA sequencing indicated that IL-17A promoted the activation of inflammatory signaling pathways in VECs. Furthermore, qRT-PCR and cytometric bead array analysis confirmed that IL-17A promoted the expression or secretion of inflammatory cytokines IL-6 and IL-1β, chemokines CXCL2 and CXCL8, and fibrosis-related gene COL16A1. Our findings indicate that elevated IL-17A in CAVD may promote valve inflammation, fibrosis, and calcification by inducing endothelial activation and inflammation. Targeting IL-17A-IL-17RA signaling may be a potential therapeutic strategy for CAVD.

EphrinB2 drives osteogenic fate of adult cardiac fibroblasts in a calcium influx dependent manner.

Cardiac calcification is a crucial but underrecognized pathological process, greatly increasing the risk of cardiovascular diseases. Little is known about how cardiac fibroblasts, as a central mediator, facilitate abnormal mineralization. Erythropoietin-producing hepatoma interactor B2 (EphrinB2), previously identified as an angiogenic regulator, is involved in fibroblast activation, while its role in the osteogenic differentiation of cardiac fibroblasts is unknown. Bioinformatics analysis was conducted to characterize the expression of the Ephrin family in human calcified aortic valves and calcific mouse hearts. The effects of EphrinB2 on cardiac fibroblasts to adopt osteogenic fate was determined by gain- and loss-of-function. EphrinB2 mRNA level was downregulated in calcified aortic valves and mouse hearts. Knockdown of EphrinB2 attenuated mineral deposits in adult cardiac fibroblasts, whereas overexpression of EphrinB2 promoted their osteogenic differentiation. RNA sequencing data implied that Ca2+-related S100/receptor for advanced glycation end products (RAGE) signaling may mediate EphrinB2-induced mineralization in cardiac fibroblasts. Moreover, L-type calcium channel blockers inhibited osteogenic differentiation of cardiac fibroblasts, implying a critical role in Ca2+ influx. In conclusion, our data illustrated an unrecognized role of EphrinB2, which functions as a novel osteogenic regulator in the heart through Ca2+ signaling and could be a potential therapeutic target in cardiovascular calcification.NEW & NOTEWORTHY In this study, we observed that adult cardiac fibroblasts but not neonatal cardiac fibroblasts exhibit the ability of osteogenic differentiation. EphrinB2 promoted osteogenic differentiation of cardiac fibroblasts through activating Ca2+-related S100/RAGE signaling. Inhibition of Ca2+ influx using L-type calcium channel blockers inhibited EphrinB2-mediated calcification of cardiac fibroblasts. Our data implied an unrecognized role of EphrinB2 in regulating cardiac calcification though Ca2+-related signaling, suggesting a potential therapeutic target of cardiovascular calcification.

Human antigen R regulates autophagic flux by stabilizing autophagy-associated mRNA in calcific aortic valve disease.

The incidence of calcific aortic valve disease (CAVD) has risen over the last decade and is expected to continue rising; however, pharmacological approaches have proven ineffective. In this study, we evaluated the role and underlying mechanisms of human antigen R (HuR)-mediated post-transcriptional regulation in CAVD. We found that HuR was significantly upregulated in human calcified aortic valves and primary aortic valvular interstitial cells (VICs) following osteogenic stimulation. Subsequent functional studies revealed that HuR silencing ameliorated calcification both in vitro and in vivo. For the first time, we demonstrated that HuR directly interacted with the transcript of phosphatidylinositol-5-phosphate 4-kinase, type II, alpha (PIP4K2A), which mediates phosphatidylinositol signalling, facilitates autophagy, and acts as an mRNA stabilizer. HuR positively modulated PIP4K2A expression at the post-transcriptional level and consequently influenced the AKT/mTOR/ATG13 pathway to regulate autophagy and CAVD progression. Our study provides new insights into the post-transcriptional regulatory role of HuR in modulating autophagy-positive factors to regulate the pathogenesis of CAVD. Our findings highlight the potential of HuR as an innovative therapeutic target in CAVD treatment.

Abstract 340: γ-carboxylated Growth Arrest Specific Protein 6 Differentially Induces Calcification In Human Aortic Valves

Introduction: Growth arrest specific protein 6 (GAS6) is a secreted γ-carboxylated vitamin K-dependent protein with roles in cell proliferation and migration. The role of GAS6 and post-translational γ-carboxylation in calcific aortic valve disease (CAVD) has yet to be evaluated. Hypothesis: Using proteomics, we identified GAS6 as a novel protein abundant in valve tissue and valvular interstitial cells (VICs), and assessed the hypothesis that γ-carboxylated GAS6 has a mechanistic role in the development of CAVD. Methods: Whole aortic valve and tissue extracellular vesicle (EV) label-free proteomics, valve histopathology, and valvular interstitial cell isolation was performed on human specimens collected from surgical aortic valve replacements for aortic stenosis at Brigham and Women’s Hospital. EVs were isolated from whole valves using ultracentrifugation and Optiprep density gradients. Mass-spectrometry will confirm γ-carboxylation. Results: GAS6 abundance was elevated in valvular areas of calcification compared to non-diseased regions (log2 FC=5, q=4.2E-10), and were enriched in GO terms associated with exosome and vesicular transport (N=18). Due to the role of EVs in ectopic cardiovascular calcification, tissue EVs were isolated from calcified aortic valves (N=9), and proteomics identified GAS6 in the vesicle-enriched fractions. Immunohistopathology of GAS6 in CAVD specimens identified co-localization of GAS6 with inflammation and calcification. Protein expression of GAS6 from VICs was significantly increased by media supplemented with organic phosphate (β-glycerophosphate) (N=5-8, p&lt;0.05), but markedly decreased by supplementation with inorganic phosphate (NaH 2 PO 4 ) (N=5-8, p&lt;0.05), despite both conditions generating Alizarin red positive calcification. GAS6 knockdown prevented calcification only in the setting of organic phosphate stimulation. Conclusions: GAS6 is a novel protein of pathological significance in CAVD. EV enrichment of GAS6 and unappreciated differential mechanistic roles of phosphate sources impacts valvular calcification, potentially through γ-carboxylation regulation. GAS6 has an unrecognized role in CAVD, which may have important implications for future disease pharmacotherapy.

Abstract 100: Multi-omics And Single Nucleus Transcriptomics Reveal Aberrant Adaptive Immune Responses In Human Bicuspid Aortic Valves

Introduction: Bicuspid aortic valve (BAV) is the most common cause of adult congenital heart disease. Patients with BAVs comprise &lt;2% of the population but represent ~50% of valve replacement surgeries and the pathogenic mechanisms underlying this disproportionate incidence of calcific aortic valve disease (CAVD) are unknown. We aimed to identify molecular contributors to accelerated disease in BAV-CAVD. Methods: Bulk multi-omics (miRNA-seq, RNA-seq, proteomics) was performed on 70 human aortic valves (11 non-diseased; fibrotic and calcific portions of 32 tricuspid aortic valves [TAVs] and 27 BAVs with CAVD). Single nuclei RNA-seq (snRNA-seq) was completed on 54,608 nuclei from 13 additional human valves (4 non-diseased; 5 TAVs and 4 BAVs with CAVD). Results: Calcific burden did not differ between diseased TAVs and BAVs (p=0.77), but BAV mineralization was significantly accelerated by 11.5±1.7 years vs. TAVs (p&lt;0.001). Latent factor-based integration of covariate-corrected multi-omics analyses (q&lt;0.05), CT-derived valve calcification, and 29 clinical parameters uncovered extensive involvement of adaptive immune responses where T cell/B cell inflammatory activation associated with autoimmune signals was enhanced in the early stages of BAV disease. Integrated multi-omics also revealed significantly elevated platelet activation potentially as a result of lifelong exposure to the high-shear BAV microenvironment, and implicated altered SLIT-ROBO signaling in BAV-CAVD progression. snRNA-seq identified 5 major cell types (27 states) in human aortic valves: valvular interstitial cells (65% of cells; 7 states), endothelial cells (10%; 4 states), macrophages (13%, 7 states), T cells (8%; 5 states), and B cells (3%; 4 states). CAVD drove differentiation of valve interstitial cells along distinct myofibrogenic ( ACTA2/CARMN/MYH11 -high) vs. osteogenic ( RUNX2/CDH11/POSTN -high) lineages, induction of endothelial-to-mesenchymal transition in valve endothelium and significant accumulation of specific macrophage and T-cell states (p&lt;0.05) in BAV-CAVD. Conclusions: Aberrant adaptive immune responses are critical drivers of BAV-CAVD, and our findings delineate a novel strategy for discovery of morphology-specific therapeutics.

TRPM4: New actor in osteogenic differentiation of human valvular interstitial cells

Transient receptor potential melastatin 4 (TRPM4) protein, a non-selective cation channel activated by calcium, is involved in cardiac fibroblasts remodeling. Its expression was reported in human valvular interstitial cells (VIC) with similar properties than fibroblasts, and its protein expression is increased on calcified aortic valves from patients suffering of aortic stenosis. VIC osteogenic differentiation is a key element in valvular calcification, which is, at term, responsible of aortic stenosis. The purpose of this study is to evaluate the implication of TRPM4 in human VIC osteogenic differentiation. VIC are isolated by enzymatic digestion from human aortic valves obtained from patients undergoing surgical aortic valve replacement. They are maintained 14 days in procalcifying media (2.7 mM calcium/2.5 mM phosphate), with or without 9-phenanthrol, a pharmacological TRPM4 inhibitor. Channel expression and functionality were followed respectively by PCR, Western Blot and electrophysiological recordings in the inside-out configuration of the patch clamp technique. Osteogenic differentiation of VIC was evaluated by measuring hydroxyapatite crystals by Alizarin red staining and by monitoring the protein expression of the osteogenic markers, BMP2 and RUNX2. A typical TRPM4 current was detected on VIC: its current-voltage relationship was linear with a conductance of 20.3 pS; the channel exhibited similar permeability for Na+ and K+; it was more activated in positive voltages and with increasing concentrations of internal Ca2+; the current was dose-dependently inhibited by 9-phenanthrol. TRPM4 mRNA and protein expression on VIC was confirmed by PCR and Western Blot. When maintained in procalcifying media, VIC developed hydroxyapatite crystals. Application of 9-phenanthrol (3.10-6 M) reduced mineralization by 30% without affecting cell viability. Moreover, it significantly reduced BMP2 and RUNX2 expression. Thereby, TRPM4 participates in osteogenic differentiation of human VIC in culture. Next studies of our unit are to evaluate whether TRPM4 repression by shRNA confirms those results.

Molecular Mechanisms Underlying the Progression of Aortic Valve Stenosis: Bioinformatic Analysis of Signal Pathways and Hub Genes.

The calcification of the aortic valve causes increased leaflet stiffness and leads to the development and progression of stenotic aortic valve disease. However, the molecular and cellular mechanisms underlying stenotic calcification remain poorly understood. Herein, we examined the gene expression associated with valve calcification and the progression of calcific aortic valve stenosis. We downloaded two publicly available gene expression profiles (GSE83453 and GSE51472) from NCBI-Gene Expression Omnibus database for the combined analysis of samples from human aortic stenosis and normal aortic valve tissue. After identifying the differentially expressed genes (DEGs) using the GEO2R online tool, we performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. We also analyzed the protein-protein interactions (PPIs) of the DEGs using the NetworkAnalyst online tool. We identified 4603 upregulated and 6272 downregulated DEGs, which were enriched in the positive regulation of cell adhesion, leukocyte-mediated immunity, response to hormones, cytokine signaling in the immune system, lymphocyte activation, and growth hormone receptor signaling. PPI network analysis identified 10 hub genes: VCAM1, FHL2, RUNX1, TNFSF10, PLAU, SPOCK1, CD74, SIPA1L2, TRIB1, and CXCL12. Through bioinformatic analysis, we identified potential biomarkers and therapeutic targets for aortic stenosis, providing a theoretical basis for future studies.

Electrospun Elastomeric Leaflet Substrates that Structurally and Mechanically Mimic Human Aortic Valve Leaflets

Creating leaflet substrates that accurately mimic the trilayered structure, anisotropic tensile characteristics, and elastomeric features of native human heart valve leaflets is essential to the success of heart valve tissue engineering. Earlier leaflet substrates have successfully replicated the mechanical properties of animal (porcine) heart valve leaflets. However, not one of them has been able to simultaneously replicate the trilayered structure and the highly anisotropic and elastomeric mechanical properties of human aortic heart valve leaflets. In this study, we employed a combination of polymers – polycaprolactone (PCL), poly(trimethylene carbonate-co-l-lactide) (PTMC-LA), and poly(trimethylene carbonate-co-caprolactone) (PTMC-CL) – in an electrospinning system to create elastomeric trilayer PCL/PTMC leaflet substrates that possess mechanical, flexural, and anisotropic properties similar to those of human native heart valve leaflets. We then compared these substrates with non-elastomeric trilayer PCL leaflet substrates to determine their effect on leaflet tissue engineering. Porcine valvular interstitial cells (PVICs) were cultured on these substrates for 1 month in static conditions to develop trilayer PCL and PCL/PTMC cell-cultured constructs. The PCL/PTMC substrates exhibited lower crystallinity and hydrophobicity than the PCL substrate, resulting in better cell adhesion and infiltration. The PCL/PTMC substrates demonstrated significantly greater cell proliferation, extracellular matrix production, and preferable gene and protein expression than the PCL substrates. Furthermore, in an osteogenic environment, the PCL/PTMC substrates showed superior resistance to calcification compared to the PCL substrates. The development of trilayer PCL/PTMC substrates with mechanical and structural characteristics resembling native tissue enhances leaflet tissue engineering.

A Rabbit Aortic Valve Stenosis Model Induced by Direct Balloon Injury

Animal models are emerging as an important tool to understand the pathologic mechanisms underlying aortic valve stenosis (AVS) because of the lack of access to reliable sources of diseased human aortic valves. Among the various animal models, AVS rabbit models are one of the most commonly used in large animal studies. However, traditional AVS rabbit models require a long-term period of dietary supplementation and genetic manipulation to induce significant stenosis in the aortic valve, limiting their use in experimental studies. To address these limitations, a new AVS rabbit model is proposed, in which stenosis is induced by a direct balloon injury to the aortic valve. The present protocol describes a successful technique for inducing AVS in New Zealand white (NZW) rabbits, with step-by-step procedures for the preparation, the surgical procedure, and the post-operative care. This simple and reproducible model offers a promising approach for studying the initiation and progression of AVS and provides a valuable tool for investigating the underlying pathological mechanisms of the disease.

BMAL1 Promotes Valvular Interstitial Cells' Osteogenic Differentiation through NF-κ B/AKT/MAPK Pathway.

Calcific aortic valve disease (CAVD) is most common in the aging population and is without effective medical treatments. Brain and muscle ARNT-like 1 (BMAL1) is related to calcification. It has unique tissue-specific characteristics and plays different roles in different tissues' calcification processes. The purpose of the present study is to explore the role of BMAL1 in CAVD. The protein levels of BMAL1 in normal and calcified human aortic valves and valvular interstitial cells (VICs) isolated from normal and calcified human aortic valves were checked. HVICs were cultured in osteogenic medium as an in vitro model, and BMAL1 expression and location were detected. TGF-β and RhoA/ROCK inhibitors and RhoA-siRNA were applied to detect the mechanism underlying the source of BMAL1 during HVICs' osteogenic differentiation. ChIP was applied to check whether BMAL1 could directly interact with the runx2 primer CPG region, and the expression of key proteins involved in the TNF signaling pathway and NF-κ B pathway was tested after silencing BMAL1. In this study, we found that BMAL1 expression was elevated in calcified human aortic valves and VICs isolated from calcified human aortic valves. Osteogenic medium could promote BMAL1 expression in HVICs and the knockdown of BMAL1 induced the inhibition of HVICs' osteogenic differentiation. Furthermore, the osteogenic medium promoting BMAL1 expression could be blocked by TGF-β and RhoA/ROCK inhibitors and RhoA-siRNA. Meanwhile, BMAL1 could not bind with the runx2 primer CPG region directly, but knockdown of BMAL1 led to decreased levels of P-AKT, P-IκBα, P-p65 and P-JNK. Osteogenic medium could promote BMAL1 expression in HVICs through the TGF-β/RhoA/ROCK pathway. BMAL1 could not act as a transcription factor, but functioned through the NF-κ B/AKT/MAPK pathway to regulate the osteogenic differentiation of HVICs.

Activation of the Hedgehog signaling pathway leads to fibrosis in aortic valves

BackgroundFibrosis is a pathological wound healing process characterized by excessive extracellular matrix deposition, which interferes with normal organ function and contributes to ~ 45% of human mortality. Fibrosis develops in response to chronic injury in nearly all organs, but the a cascade of events leading to fibrosis remains unclear. While hedgehog (Hh) signaling activation has been associated with fibrosis in the lung, kidney, and skin, it is unknown whether hedgehog signaling activation is the cause or the consequence of fibrosis. We hypothesize that activation of hedgehog signaling is sufficient to drive fibrosis in mouse models.ResultsIn this study, we provide direct evidence to show that activation of Hh signaling via expression of activated smoothened, SmoM2, is sufficient to induce fibrosis in the vasculature and aortic valves. We showed that activated SmoM2 -induced fibrosis is associated with abnormal function of aortic valves and heart. The relevance of this mouse model to human health is reflected in our findings that elevated GLI expression is detected in 6 out of 11 aortic valves from patients with fibrotic aortic valves.ConclusionsOur data show that activating hedgehog signaling is sufficient to drive fibrosis in mice, and this mouse model is relevant to human aortic valve stenosis.

Sam68 promotes osteogenic differentiation of aortic valvular interstitial cells by TNF-α/STAT3/autophagy axis.

Calcified aortic valve disease (CAVD) is a major non-rheumatic heart valve disease in the world, with a high mortality rate and without suitable pharmaceutical therapy due to its complex mechanisms. Src-associated in mitosis 68-KD (Sam68), an RNA binding protein, has been reported as a signaling adaptor in numerous signaling pathways (Huot in Mol Cell Biol,29(7), 1933-1943, 2009), particularly in inflammatory signaling pathways. The effects of Sam68 on the osteogenic differentiation process of hVICs and its regulation on signal transducer and activator of transcription 3 (STAT3) signaling pathway have been investigated in this study. Human aortic valve samples detection found that Sam68 expression was up-regulated in human calcific aortic valves. We used tumor necrosis factor α (TNF-α) as an activator for osteogenic differentiation in vitro and the result indicated that Sam68 was highly expressed after TNF-α stimulation. Overexpression of Sam68 promoted osteogenic differentiation of hVICs while Sam68 knockdown reversed this effect. Sam68 interaction with STAT3 was predicted by using String database and was verified in this study. Sam68 knockdown reduced phosphorylation of STAT3 activated by TNF-α and the downstream gene expression, which further influenced autophagy flux in hVICs. STAT3 knockdown alleviated the osteogenic differentiation and calcium deposition promoted by Sam68 overexpression. In conclusion, Sam68 interacts with STAT3 and participates in its phosphorylation to promote osteogenic differentiation of hVICs to induce valve calcification. Thus, Sam68 may be a new therapeutic target for CAVD. Regulatory of Sam68 in TNF-α/STAT3/Autophagy Axis in promoting osteogenesis of hVICs.