Sex-dependent expression of galectin-1, a cardioprotective ß-galactoside-binding lectin, in human calcific aortic stenosis.

We aimed to analyze sex-related differences in galectin-1 (Gal-1), a ß-galactoside-binding lectin, in aortic stenosis (AS) and its association with the inflammatory and fibrocalcific progression of AS. Gal-1 was determined in serum and aortic valves (AVs) from control and AS donors by western blot and immunohistochemistry. Differences were validated by ELISA and qPCR in AS samples. In vitro experiments were conducted in primary cultured valve interstitial cells (VICs). Serum Gal-1 was not different neither between control and AS nor between men and women. There was no association between circulating and valvular Gal-1 levels. The expression of Gal-1 in stenotic AVs was higher in men than women, even after adjusting for confounding factors, and was associated with inflammation, oxidative stress, extracellular matrix remodeling, fibrosis, and osteogenesis. Gal-1 (LGALS1) mRNA was enhanced within fibrocalcific areas of stenotic AVs, especially in men. Secretion of Gal-1 was up-regulated over a time course of 2, 4, and 8 days in men's calcifying VICs, only peaking at day 4 in women's VICs. In vitro, Gal-1 was associated with similar mechanisms to those in our clinical cohort. ß-estradiol significantly up-regulated the activity of an LGALS1 promoter vector and the secretion of Gal-1, only in women's VICs. Supplementation with rGal-1 prevented the effects elicited by calcific challenge including the metabolic shift to glycolysis. In conclusion, Gal-1 is up-regulated in stenotic AVs and VICs from men in association with inflammation, oxidative stress, matrix remodeling, and osteogenesis. Estrogens can regulate Gal-1 expression with potential implications in post-menopause women. Exogenous rGal-1 can diminish calcific phenotypes in both women and men.

Role of CD34 in calcification of human aortic valve interstitial cells from patients with aortic valve stenosis.

Various osteogenic factors are involved in ectopic human aortic valve calcification; however, the key cell species involved in calcification remains unclear. In a previous study, we reported that mesenchymal stem (CD73, 90, 105) and endothelial (VEGFR2) cell markers are positive in almost all human aortic valve interstitial cells (HAVICs) obtained from a patient with calcified aortic valve stenosis (CAVS). Further, CD34-negative HAVICs are highly sensitive to calcification stimulations. Here, we aimed to pathophysiologically clarify the role of CD34 in HAVICs obtained from individual patients with severe CAVS. A DNA microarray between CD34-positive and CD34-negative HAVICs, separated by fluorescence-activated cell sorting, indicated that tenascin X (TNX) mRNA expression significantly decreased in CD34-negative cells. Furthermore, the inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß significantly downregulated CD34 expression in HAVICs. TGF-ß, a key cytokine of endothelial-mesenchymal transition, did not affect HAVIC calcification. CD34 overexpression strongly inhibited TNF-α- and IL-1ß-induced calcification and maintained TNX mRNA expression. These results suggest one possibility that CD34 is an inhibitory regulator of valve calcification. Furthermore, TNF-α- and IL-1ß-induced CD34 downregulation in HAVICs contributes to HAVIC calcification by downregulating TNX protein expression.

Hyperglycemia-simulating environment attenuated experimentally induced calcification in cultured human aortic valve interstitial cells.

Objectives: The role of diabetes mellitus as a risk factor for the development of calcific aortic valve disease has not been fully clarified. Aortic valve interstitial cells (VICs) have been suggested to be crucial for calcification of the valve. Induced calcification in cultured VICs is a good in vitro model for aortic valve calcification. The purpose of this study was to investigate whether increased glucose levels increase experimentally induced calcification in cultured human VICs. Design: VICs were isolated from explanted calcified aortic valves after valve replacement. Osteogenic medium induced calcification of cultured VICs at different glucose levels (5, 15, and 25 mM). Calcium deposits were visualized using Alizarin Red staining and measured spectrophotometrically. Results: The higher the glucose concentration, the lower the level of calcification. High glucose (25 mM) reduced calcification by 52% compared with calcification at a physiological (5 mM) glucose concentration (correlation and regression analysis: r = -0.55, p = .025 with increased concentration of glucose). Conclusions: In vitro hyperglycemia-like conditions attenuated calcification in VICs. High glucose levels may trigger a series of events that secondarily stimulate calcification of VICs in vivo.

Isolated Valve Amyloid Deposition in Aortic Stenosis: Potential Clinical and Pathophysiological Relevance.

Amyloid deposition within stenotic aortic valves (AVs) also appears frequent in the absence of cardiac amyloidosis, but its clinical and pathophysiological relevance has not been investigated. We will elucidate the rate of isolated AV amyloid deposition and its potential clinical and pathophysiological significance in aortic stenosis (AS). In 130 patients without systemic and/or cardiac amyloidosis, we collected the explanted AVs during cardiac surgery: 57 patients with calcific AS and 73 patients with AV insufficiency (41 with AV sclerosis and 32 without, who were used as controls). Amyloid deposition was found in 21 AS valves (37%), 4 sclerotic AVs (10%), and none of the controls. Patients with and without isolated AV amyloid deposition had similar clinical and echocardiographic characteristics and survival rates. Isolated AV amyloid deposition was associated with higher degrees of AV fibrosis (p = 0.0082) and calcification (p < 0.0001). Immunohistochemistry analysis suggested serum amyloid A1 (SAA1), in addition to transthyretin (TTR), as the protein possibly involved in AV amyloid deposition. Circulating SAA1 levels were within the normal range in all groups, and no difference was observed in AS patients with and without AV amyloid deposition. In vitro, AV interstitial cells (VICs) were stimulated with interleukin (IL)-1ß which induced increased SAA1-mRNA both in the control VICs (+6.4 ± 0.5, p = 0.02) and the AS VICs (+7.6 ± 0.5, p = 0.008). In conclusion, isolated AV amyloid deposition is frequent in the context of AS, but it does not appear to have potential clinical relevance. Conversely, amyloid deposition within AV leaflets, probably promoted by local inflammation, could play a role in AS pathophysiology.

PALMD regulates aortic valve calcification via altered glycolysis and NF-κB-mediated inflammation.

Recent genome-wide association and transcriptome-wide association studies have identified an association between the PALMD locus, encoding palmdelphin, a protein involved in myoblast differentiation, and calcific aortic valve disease (CAVD). Nevertheless, the function and underlying mechanisms of PALMD in CAVD remain unclear. We herein investigated whether and how PALMD affects the pathogenesis of CAVD using clinical samples from CAVD patients and a human valve interstitial cell (hVIC) in vitro calcification model. We showed that PALMD was upregulated in calcified regions of human aortic valves and calcified hVICs. Furthermore, silencing of PALMD reduced hVIC in vitro calcification, osteogenic differentiation, and apoptosis, whereas overexpression of PALMD had the opposite effect. RNA-Seq of PALMD-depleted hVICs revealed that silencing of PALMD reduced glycolysis and nuclear factor-κB (NF-κB)-mediated inflammation in hVICs and attenuated tumor necrosis factor α-induced monocyte adhesion to hVICs. Having established the role of PALMD in hVIC glycolysis, we examined whether glycolysis itself could regulate hVIC osteogenic differentiation and inflammation. Intriguingly, the inhibition of PFKFB3-mediated glycolysis significantly attenuated osteogenic differentiation and inflammation of hVICs. However, silencing of PFKFB3 inhibited PALMD-induced hVIC inflammation, but not osteogenic differentiation. Finally, we showed that the overexpression of PALMD enhanced hVIC osteogenic differentiation and inflammation, as opposed to glycolysis, through the activation of NF-κB. The present study demonstrates that the genome-wide association- and transcriptome-wide association-identified CAVD risk gene PALMD may promote CAVD development through regulation of glycolysis and NF-κB-mediated inflammation. We propose that targeting PALMD-mediated glycolysis may represent a novel therapeutic strategy for treating CAVD.

Decreased expression of RPL15 and RPL18 exacerbated the calcification of valve interstitial cells during aortic valve calcification.

Calcific aortic valve disease (CAVD) is the most common valvular heart disease, with an increasing prevalence due to an aging population. The pathobiology of CAVD is a multifaceted and actively regulated process, but the detailed mechanisms have not been elucidated. The present study aims to identify the differentially expressed genes (DEGs) in calcified aortic valve tissues, and to analyze the correlation between DEGs and clinical features in CAVD patients. The DEGs were screened by microarray in normal and CAVD groups (n = 2 for each group), and confirmed by quantitative real-time polymerase chain reaction in normal (n = 12) and calcified aortic valve tissues (n = 34). A total of 1048 DEGs were identified in calcified aortic valve tissues, including 227 upregulated mRNAs and 821 downregulated mRNAs. Based on multiple bioinformatic analyses, three 60S ribosomal subunit components (RPL15, RPL18, and RPL18A), and two 40S ribosomal subunit components (RPS15 and RPS21) were identified as the top 5 hub genes in the protein-protein interaction network of DEGs. The expression of RPL15 and RPL18 was also found significantly decreased in calcified aortic valve tissues (both p < .01), and negatively correlated with the osteogenic differentiation marker OPN in CAVD patients (both p < .01). Moreover, inhibition of RPL15 or RPL18 exacerbated the calcification of valve interstitial cells under osteogenic induction conditions. The present study proved that decreased expression of RPL15 and RPL18 was closely associated with aortic valve calcification, which provided valuable clues to find therapeutic targets for CAVD.

Ultrastructural and Immunohistochemical Detection of Hydroxyapatite Nucleating Role by rRNA and Nuclear Chromatin Derivatives in Aortic Valve Calcification: In Vitro and In Vivo Pro-Calcific Animal Models and Actual Calcific Disease in Humans.

Calcification starts with hydroxyapatite (HA) crystallization on cell membranous components, as with aortic valve interstitial cells (AVICs), wherein a cell-membrane-derived substance containing acidic phospholipids (PPM/PPLs) acts as major crystal nucleator. Since nucleic acid removal is recommended to prevent calcification in valve biosubstitutes derived from decellularized valve scaffolds, the involvement of ribosomal RNA (rRNA) and nuclear chromatin (NC) was here explored in three distinct contexts: (i) bovine AVIC pro-calcific cultures; (ii) porcine aortic valve leaflets that had undergone accelerated calcification after xenogeneic subdermal implantation; and (iii) human aortic valve leaflets affected by calcific stenosis. Ultrastructurally, shared AVIC degenerative patterns included (i) the melting of ribosomes with PPM/PPLs, and the same for apparently well-featured NC; (ii) selective precipitation of silver particles on all three components after adapted von Kossa reactions; and (iii) labelling by anti-rRNA immunogold particles. Shared features were also provided by parallel light microscopy. In conclusion, the present results indicate that rRNA and NC contribute to AVIC mineralization in vitro and in vivo, with their anionic charges enhancing the HA nucleation capacity exerted by PPM/PPL substrates, supporting the concept that nucleic acid removal is needed for valve pre-implantation treatments, besides better elucidating the modality of pro-calcific cell death.

The role of neutrophil elastase in aortic valve calcification.

BACKGROUND: Calcific aortic valve disease (CAVD) is the most commonly valvular disease in the western countries initiated by inflammation and abnormal calcium deposition. Currently, there is no clinical drug for CAVD. Neutrophil elastase (NE) plays a causal role in inflammation and participates actively in cardiovascular diseases. However, the effect of NE on valve calcification remains unclear. So we next explore whether it is involved in valve calcification and the molecular mechanisms involved. METHODS: NE expression and activity in calcific aortic valve stenosis (CAVD) patients (n = 58) and healthy patients (n = 30) were measured by enzyme-linked immunosorbent assay (ELISA), western blot and immunohistochemistry (IHC). Porcine aortic valve interstitial cells (pVICs) were isolated and used in vitro expriments. The effects of NE on pVICs inflammation, apoptosis and calcification were detected by TUNEL assay, MTT assay, reverse transcription polymerase chain reaction (RT-PCR) and western blot. The effects of NE knockdown and NE activity inhibitor Alvelestat on pVICs inflammation, apoptosis and calcification under osteogenic medium induction were also detected by RT-PCR, western blot, alkaline phosphatase staining and alizarin red staining. Changes of Intracellular signaling pathways after NE treatment were measured by western blot. Apolipoprotein E-/- (APOE-/-) mice were employed in this study to establish the important role of Alvelestat in valve calcification. HE was used to detected the thickness of valve. IHC was used to detected the NE and α-SMA expression in APOE-/- mice. Echocardiography was employed to assess the heat function of APOE-/- mice. RESULTS: The level and activity of NE were evaluated in patients with CAVD and calcified valve tissues. NE promoted inflammation, apoptosis and phenotype transition in pVICs in the presence or absence of osteogenic medium. Under osteogenic medium induction, NE silencing or NE inhibitor Alvelestat both suppressed the osteogenic differentiation of pVICs. Mechanically, NE played its role in promoting osteogenic differentiation of pVICs by activating the NF-κB and AKT signaling pathway. Alvelestat alleviated valve thickening and decreased the expression of NE and α-SMA in western diet-induced APOE-/- mice. Alvelestat also reduced NE activity and partially improved the heart function of APOE-/-mice. CONCLUSIONS: Collectively, NE is highly involved in the pathogenesis of valve calcification. Targeting NE such as Alvelestat may be a potential treatment for CAVD.

Nfatc1 Promotes Interstitial Cell Formation During Cardiac Valve Development in Zebrafish.

RATIONALE: The transcription factor NFATC1 (nuclear factor of activated T-cell 1) has been implicated in cardiac valve formation in humans and mice, but we know little about the underlying mechanisms. To gain mechanistic understanding of cardiac valve formation at single-cell resolution and insights into the role of NFATC1 in this process, we used the zebrafish model as it offers unique attributes for live imaging and facile genetics. OBJECTIVE: To understand the role of Nfatc1 in cardiac valve formation. METHODS AND RESULTS: Using the zebrafish atrioventricular valve, we focus on the valve interstitial cells (VICs), which confer biomechanical strength to the cardiac valve leaflets. We find that initially atrioventricular endocardial cells migrate collectively into the cardiac jelly to form a bilayered structure; subsequently, the cells that led this migration invade the ECM (extracellular matrix) between the 2 endocardial cell monolayers, undergo endothelial-to-mesenchymal transition as marked by loss of intercellular adhesion, and differentiate into VICs. These cells proliferate and are joined by a few neural crest-derived cells. VIC expansion and a switch from a promigratory to an elastic ECM drive valve leaflet elongation. Functional analysis of Nfatc1 reveals its requirement during VIC development. Zebrafish nfatc1 mutants form significantly fewer VICs due to reduced proliferation and impaired recruitment of endocardial and neural crest cells during the early stages of VIC development. With high-speed microscopy and echocardiography, we show that reduced VIC formation correlates with valvular dysfunction and severe retrograde blood flow that persist into adulthood. Analysis of downstream effectors reveals that Nfatc1 promotes the expression of twist1b-a well-known regulator of epithelial-to-mesenchymal transition. CONCLUSIONS: Our study sheds light on the function of Nfatc1 in zebrafish cardiac valve development and reveals its role in VIC formation. It also further establishes the zebrafish as a powerful model to carry out longitudinal studies of valve formation and function.

MiR-138-5p targets RUNX2 to inhibit osteogenic differentiation of aortic valve interstitial cells via Wnt/ß-catenin signaling pathway.

BACKGROUND: Human aortic valve interstitial cells (hAVICs) are a key factor in the pathogenesis of calcific aortic valve disease (CAVD). This research examines the role and mechanism of microRNA miR-138-5p in osteogenic differentiation of hAVICs. METHODS: RT-qPCR analysis was applied for detecting miR-138-5p and RUNX2 expression in valve tissues of CAVD patients and controls. On completion of induction of osteogenic differentiation of hAVICs, and after overexpression or interference of miR-138-5p expression, the condition of osteogenic differentiation and calcification of hAVICs was confirmed using alkaline phosphatase staining and alizarin red staining. Subsequently, western blot was utilized to detect the expression of osteogenesis-related proteins OPN and ALP, and Wnt/ß-catenin signaling pathway-related proteins. Finally, the relationship between miR-138-5p and RUNX2 was validated by dual-luciferase reporter assay and Pearson's correlation test. RESULTS: Down-regulation of miR-138-5p was found in CAVD patients and during osteogenic differentiation of hAVICs. Overexpression of miR-138-5p contribute to the inhibition of osteoblast differentiation and calcium deposition in hAVICs, and of ALP and OPN protein expression. RUNX2 was a target gene of miR-138-5p, and it was negatively correlated with miR-138-5p in CAVD. Additionally, overexpression of RUNX2 could reverse the inhibitory effect of miR-138-5p on osteogenic differentiation of hAVICs. CONCLUSION: miR-138-5p can act as a positive regulator of osteogenic differentiation in CAVD patients to involve in inhibiting valve calcification, which is achieved through RUNX2 and Wnt/ß-catenin signaling pathway.

Calcium-Dependent Cytosolic Phospholipase A2α as Key Factor in Calcification of Subdermally Implanted Aortic Valve Leaflets.

Calcium-dependent cytosolic phospholipase A2α (cPLA2α) had been previously found to be overexpressed by aortic valve interstitial cells (AVICs) subjected to in vitro calcific induction. Here, cPLA2α expression was immunohistochemically assayed in porcine aortic valve leaflets (iAVLs) that had undergone accelerated calcification subsequent to 2- to 28-day-long implantation in rat subcutis. A time-dependent increase in cPLA2α-positive AVICs paralleled mineralization progression depending on dramatic cell membrane degeneration with the release of hydroxyapatite-nucleating acidic lipid material, as revealed by immunogold particles decorating organelle membranes in 2d-iAVLs, as well as membrane-derived lipid byproducts in 7d- to 28d-iAVLs. Additional positivity was detected for (i) pro-inflammatory IL-6, mostly exhibited by rat peri-implant cells surrounding 14d- and 28d-iAVLs; (ii) calcium-binding osteopontin, with time-dependent increase and no ossification occurrence; (iii) anti-calcific fetuin-A, mostly restricted to blood plasma within vessels irrorating the connective envelopes of 28d-iAVLs; (iv) early apoptosis marker annexin-V, limited to sporadic AVICs in all iAVLs. No positivity was found for either apoptosis executioner cleaved caspase-3 or autophagy marker MAP1. In conclusion, cPLA2α appears to be a factor characterizing AVL calcification concurrently with a distinct still uncoded cell death form also in an animal model, as well as a putative target for the prevention and treatment of calcific valve diseases.

Metformin alleviates the calcification of aortic valve interstitial cells through activating the PI3K/AKT pathway in an AMPK dependent way.

BACKGROUND: Calcific aortic valve disease (CAVD) is the most prevalent valvular disease worldwide. However, no effective treatment could delay or prevent the progression of the disease due to the poor understanding of its pathological mechanism. Many studies showed that metformin exerted beneficial effects on multiple cardiovascular diseases by mediating multiple proteins such as AMPK, NF-κB, and AKT. This study aims to verify whether metformin can inhibit aortic calcification through the PI3K/AKT signaling pathway. METHODS: We first analyzed four microarray datasets to screen differentially expressed genes (DEGs) and signaling pathways related to CAVD. Then aortic valve samples were used to verify selected genes and pathways through immunohistochemistry (IHC) and western blot (WB) assays. Aortic valve interstitial cells (AVICs) were isolated from non-calcific aortic valves and then cultured with phosphate medium (PM) with or without metformin to verify whether metformin can inhibit the osteogenic differentiation and calcification of AVICs. Finally, we used inhibitors and siRNA targeting AMPK, NF-κB, and AKT to study the mechanism of metformin. RESULTS: We screened 227 DEGs; NF-κB and PI3K/AKT signaling pathways were implicated in the pathological mechanism of CAVD. IHC and WB experiments showed decreased AMPK and AKT and increased Bax in calcific aortic valves. PM treatment significantly reduced AMPK and PI3K/AKT signaling pathways, promoted Bax/Bcl2 ratio, and induced AVICs calcification. Metformin treatment ameliorated AVICs calcification and apoptosis by activating the PI3K/AKT signaling pathway. AMPK activation and NF-κB inhibition could inhibit AVICs calcification induced by PM treatment; however, AMPK and AKT inhibition reversed the protective effect of metformin. CONCLUSIONS: This study, for the first time, demonstrates that metformin can inhibit AVICs in vitro calcification by activating the PI3K/AKT signaling pathway; this suggests that metformin may provide a potential target for the treatment of CAVD. And the PI3K/AKT signaling pathway emerges as an important regulatory axis in the pathological mechanism of CAVD.

Activation of the Interleukin-33/ST2 Pathway Exerts Deleterious Effects in Myxomatous Mitral Valve Disease.

Mitral valve disease (MVD) is a frequent cause of heart failure and death worldwide, but its etiopathogenesis is not fully understood. Interleukin (IL)-33 regulates inflammation and thrombosis in the vascular endothelium and may play a role in the atherosclerotic process, but its role in mitral valve has not been investigated. We aim to explore IL-33 as a possible inductor of myxomatous degeneration in human mitral valves. We enrolled 103 patients suffering from severe mitral regurgitation due to myxomatous degeneration undergoing mitral valve replacement. Immunohistochemistry of the resected leaflets showed IL-33 and ST2 expression in both valve interstitial cells (VICs) and valve endothelial cells (VECs). Positive correlations were found between the levels of IL-33 and molecules implicated in the development of myxomatous MVD, such as proteoglycans, extracellular matrix remodeling enzymes (matrix metalloproteinases and their tissue inhibitors), inflammatory and fibrotic markers. Stimulation of single cell cultures of VICs and VECs with recombinant human IL-33 induced the expression of activated VIC markers, endothelial-mesenchymal transition of VECs, proteoglycan synthesis, inflammatory molecules and extracellular matrix turnover. Our findings suggest that the IL-33/ST2 system may be involved in the development of myxomatous MVD by enhancing extracellular matrix remodeling.

Identification of CD34+/PGDFRα+ Valve Interstitial Cells (VICs) in Human Aortic Valves: Association of Their Abundance, Morphology and Spatial Organization with Early Calcific Remodeling.

Aortic valve interstitial cells (VICs) constitute a heterogeneous population involved in the maintenance of unique valvular architecture, ensuring proper hemodynamic function but also engaged in valve degeneration. Recently, cells similar to telocytes/interstitial Cajal-like cells described in various organs were found in heart valves. The aim of this study was to examine the density, distribution, and spatial organization of a VIC subset co-expressing CD34 and PDGFRα in normal aortic valves and to investigate if these cells are associated with the occurrence of early signs of valve calcific remodeling. We examined 28 human aortic valves obtained upon autopsy. General valve morphology and the early signs of degeneration were assessed histochemically. The studied VICs were identified by immunofluorescence (CD34, PDGFRα, vimentin), and their number in standardized parts and layers of the valves was evaluated. In order to show the complex three-dimensional structure of CD34+/PDGFRα+ VICs, whole-mount specimens were imaged by confocal microscopy, and subsequently rendered using the Imaris (Bitplane AG, Zürich, Switzerland) software. CD34+/PDGFRα+ VICs were found in all examined valves, showing significant differences in the number, distribution within valve tissue, spatial organization, and morphology (spherical/oval without projections; numerous short projections; long, branching, occasionally moniliform projections). Such a complex morphology was associated with the younger age of the subjects, and these VICs were more frequent in the spongiosa layer of the valve. Both the number and percentage of CD34+/PDGFRα+ VICs were inversely correlated with the age of the subjects. Valves with histochemical signs of early calcification contained a lower number of CD34+/PDGFRα+ cells. They were less numerous in proximal parts of the cusps, i.e., areas prone to calcification. The results suggest that normal aortic valves contain a subpopulation of CD34+/PDGFRα+ VICs, which might be involved in the maintenance of local microenvironment resisting to pathologic remodeling. Their reduced number in older age could limit the self-regenerative properties of the valve stroma.

COX-2 Is Downregulated in Human Stenotic Aortic Valves and Its Inhibition Promotes Dystrophic Calcification.

Calcific aortic valve disease (CAVD) is the result of maladaptive fibrocalcific processes leading to a progressive thickening and stiffening of aortic valve (AV) leaflets. CAVD is the most common cause of aortic stenosis (AS). At present, there is no effective pharmacotherapy in reducing CAVD progression; when CAVD becomes symptomatic it can only be treated with valve replacement. Inflammation has a key role in AV pathological remodeling; hence, anti-inflammatory therapy has been proposed as a strategy to prevent CAVD. Cyclooxygenase 2 (COX-2) is a key mediator of the inflammation and it is the target of widely used anti-inflammatory drugs. COX-2-inhibitor celecoxib was initially shown to reduce AV calcification in a murine model. However, in contrast to these findings, a recent retrospective clinical analysis found an association between AS and celecoxib use. In the present study, we investigated whether variations in COX-2 expression levels in human AVs may be linked to CAVD. We extracted total RNA from surgically explanted AVs from patients without CAVD or with CAVD. We found that COX-2 mRNA was higher in non-calcific AVs compared to calcific AVs (0.013 ± 0.002 vs. 0.006 ± 0.0004; p < 0.0001). Moreover, we isolated human aortic valve interstitial cells (AVICs) from AVs and found that COX-2 expression is decreased in AVICs from calcific valves compared to AVICs from non-calcific AVs. Furthermore, we observed that COX-2 inhibition with celecoxib induces AVICs trans-differentiation towards a myofibroblast phenotype, and increases the levels of TGF-ß-induced apoptosis, both processes able to promote the formation of calcific nodules. We conclude that reduced COX-2 expression is a characteristic of human AVICs prone to calcification and that COX-2 inhibition may promote aortic valve calcification. Our findings support the notion that celecoxib may facilitate CAVD progression.

MicroRNA-638 inhibits human aortic valve interstitial cell calcification by targeting Sp7.

Calcific aortic valve disease (CAVD) is a complex heart valve disease involving a wide range of pathological changes. Emerging evidence indicates that osteogenic differentiation of human aortic valve interstitial cells (hAVICs) plays a key role in valve calcification. In this study, we aimed to investigate the function of miR-638 in hAVICs osteogenesis. Both miRNA microarray assay and qRT-PCR results demonstrating miR-638 was obviously up-regulated in calcific aortic valves compared with non-calcific valves. We also proved that miR-638 was significantly up-regulated during hAVICs osteogenic differentiation. Overexpression of miR-638 suppressed osteogenic differentiation of hAVICs in vitro, whereas down-regulation of miR-638 enhance the process. Target prediction analysis and dual-luciferase reporter assay confirmed that Sp7 transcription factor (Sp7) was a direct target of miR-638. Furthermore, knockdown of Sp7 inhibited osteogenic differentiation of hAVICs, which is similar to the results observed in up-regulation miR-638. Our data indicated that miR-638 plays an inhibitory role in hAVICs osteogenic differentiation, which may act by targeting Sp7. MiR-638 may be a potential therapeutic target for CAVD.

The methylation of Notch1 promoter mediates the osteogenesis differentiation in human aortic valve interstitial cells through Wnt/ß-catenin signaling.

Aortic valve interstitial cells (AVICs) have the potential to undergo calcification, which has been regarded as a critical issue during the pathology of calcific aortic valve disease (CAVD). In the past decade, epigenetics, in particular, DNA methylation dysregulation, has been reported to play a vital role in the occurrence and development of CAVD. In the present study, the expression of Notch1, which can inhibit the osteogenesis differentiation of valve interstitial cells, was downregulated whereas the expression of methyltransferases was upregulated in CAVD tissues, suggesting the potential role of DNA methylation in Notch1 expression and CAVD progression. As revealed by DNA extraction and bisulfite sequencing polymerase chain reaction (PCR), the methylation level in Notch1 promoter was much higher in CAVD tissues and human AVICs on Day 14 of osteogenesis differentiation induction. The silence of Notch1 intercellular domain (NICD) promoted while the treatment of demethylation agent, 5-Aza-dC, inhibited the osteogenesis differentiation. Moreover, NICD overexpression significantly suppressed the transcriptional activity of ß-catenin on TCF4, and the expression of osteogenesis differentiation factors, indicating the involvement of Wnt/ß-catenin signaling in Notch1 modulating the osteogenesis differentiation in human AVICs (hAVICs). Taken together, Notch1 promoter methylation leads to a decreased Notch1 expression and subsequent decreased release of NICD in the nucleus of hAVICs, therefore promoting the activation of Wnt/ß-catenin signaling and the expression of osteogenesis differentiation factors, finally promoting the osteogenesis differentiation in hAVICs. DNA methylation might act as an important bridge to link epigenetic variation and CAVD progression.

Meis2 represses the osteoblastic transdifferentiation of aortic valve interstitial cells through the Notch1/Twist1 pathway.

AIM: Calcific aortic valve disease (CAVD) is the most common valvular disease worldwide. The osteoblastic transdifferentiation of aortic valve interstitial cells (VICs) is the essential process of CAVD, but the underlying mechanisms are poorly understood. Aortic VICs are generated from epithelial-to-mesenchymal transition (EMT) and migration of neural crest cells (NCCs).Meis2 has been associated with EMT and NCCs migration during development, but its role in CAVD is unknown. This study aims to elucidate the specific functions of Meis2 and its downstream targets in aortic valve calcification. MATERIAL AND METHODS: Levels of Meis2 were examined in calcified (n = 30) and normal (n = 30) human aortic valve tissues, respectively. Meis2 was inhibited in porcine aortic VICs in vitro, and the effect on osteoblastic transdifferentiation and its downstream pathway were studied. RESULTS: Meis2 gene and protein expression decreased significantly in calcified human aortic valve tissue compared with the normal ones. Inhibiting Meis2 by siRNAs reduced the gene and protein expression of Notch1 and Twist1, and induced the osteoblastic transdifferentiation of the porcine aortic VICs in vitro. CONCLUSIONS: The present study indicated that Meis2 repress the osteoblastic transdifferentiation of aortic VICs through the Notch1/Twist1 signaling pathway. The Results identify Meis2 as a potential intervention target for the prevention of CAVD.

LncRNA OIP5-AS1 inhibits osteoblast differentiation of valve interstitial cells via miR-137/TWIST11 axis.

OIP5-AS1, a highly abundant imprinted long non-coding RNA (lncRNA), has been implicated in calcific aortic valve disease (CAVD). However, the function and underlying mechanism of OIP5-AS1 in CAVD progression remains unknown. In this study, osteoblastic differentiation of valve interstitial cells (VICs) isolated from human calcific aortic valves was induced by osteogenic medium. The protein levels of osteogenic markers were determined by immunofluorescence and western blotting. OIP5-AS1, miR-137 and TWIST-related protein 1 (TWIST1) expressions were detected by quantitative real-time PCR (qRT-PCR). ALP activity was evaluated by spectrophotometry. Mineralized bone matrix formation was assessed by Alizarin Red S staining. The interaction between OIP5-AS1 and miR-137 was studied using luciferase reporter assay, RNA pull-down assay and RNA-binding protein immunoprecipitation (RIP) assay. Luciferase reporter assay was also used to identify the possible interaction between miR-137 and TWIST11. The results showed that downregulated expression of OIP5-AS1 was observed in human aortic VICs after osteogenic induction. In vitro experiments revealed that OIP5-AS1 acted as a negative regulator of osteogenic differentiation. Mechanistically, we further showed that OIP5-AS1 could relieve osteogenic differentiation of VICs via upregulating miR-137 target gene TWIST1. Our study provides novel mechanistic insights into the cross-talk between OIP5-AS1, miR-137, and TWIST11, shedding light on the therapy for CAVD.

Matrix Gla protein negatively regulates calcification of human aortic valve interstitial cells isolated from calcified aortic valves.

Calcified aortic valve stenosis (CAS) is a common heart valve disease in elderly people, and is mostly accompanied by ectopic valve calcification. We recently demonstrated that tumor necrosis factor-α (TNF-α) induces calcification of human aortic valve interstitial cells (HAVICs) obtained from CAS patients. In this study, we investigated the role of matrix Gla protein (MGP), a known calcification inhibitor that antagonizes bone morphogenetic protein 2 (BMP2) in TNF-α-induced calcification of HAVICs. HAVICs isolated from aortic valves were cultured, and calcification was significantly induced with 30 ng/mL TNF-α. Gene expression of the calcigenic marker, BMP2, was significantly increased in response to TNF-α, while the gene and protein expression of MGP was strongly decreased. To confirm the role of MGP, MGP-knockdown HAVICs and HAVICs overexpressing MGP were generated. In HAVICs, in which MGP expression was inhibited by small interfering RNA, calcification and BMP2 gene expression were induced following long-term culture for 32 days in the absence of TNF-α. In contrast, HAVICs overexpressing MGP had significantly decreased TNF-α-induced calcification. These results suggest that MGP acts as a negative regulator of HAVIC calcification, and as such, may be helpful in the development of new therapies for ectopic calcification of the aortic valve.