Valvular Interstitial Cells Calcification Research Articles

The role of noncoding RNAs during aortic valve stenosis

Abstract Background Aortic valve stenosis (AVS) is the most common valve disease worldwide. Thought to be a purely degenerative disease, it is now clear that shear stress/endothelial dysfunction, lipid deposition and inflammation lead to calcification and stenosis of the valve. There is evidence, that extracellular vesicles (EVs) are actively involved in calcification processes. Practically all cells, including endothelial cells, can generate EVs, which can be shed into the blood stream and into the interstitial space. EVs contain lipids, proteins and nucleic acids, including noncoding RNAs (ncRNAs). EVs can be taken up by acceptor cells and their cargo, especially the ncRNA content, can change the phenotype of these cells. NcRNAs have been shown to have protective and damaging properties in AVS, which can lead to disease progression. EVs are actively involved in atherosclerosis and vascular calcification, but their role during AVS formation remains largely unknown. Purpose We hypothesize, that EV-derived ncRNAs play a crucial role during calcification of the aortic valve through regulation of endothelial to mesenchymal transition (EndMT) and calcification of valvular interstitial cells. Methods and results In initial screening experiments, we investigated ncRNA (micro RNA, miRNA and long noncoding RNA, lncRNA) content in aortic valve tissue from explanted human aortic valves from patients undergoing surgical aortic valve replacement. There is a differential expression of miRNAs and lncRNAs in aortic valve tissue from patients with AVS and patients without AVS. We could also show a differential packaging of ncRNAs into EVs generated from patient aortic valve tissues. Furthermore, ncRNA expression in aortic valve tissue is altered in a “wire-injury” mouse model of AVS. We can demonstrate in vitro that EVs and their content can be transferred from valvular endothelial cells (VECs) to valvular interstitial cells (VICs) and vice versa. Additionally, we have established an isolation method of VECs and VICs from human samples. To identify ncRNAs involved in EndMT, we are investigating the effect of laminar and pulsatile flow on the expression of ncRNAs in vitro. We can demonstrate that different flow patterns lead to a vast change in ncRNA expression in primary VECs. Downstream effects of identified ncRNAs are currently under investigation in our in vitro calcification and EndMT models. Transfection of VICs with miRNA mimics and inhibitors and lentiviral transduction of our identified targets lead to an altered calcification potential of valve cells. Conclusion The analysis of the cell type specific expression of ncRNAs and the intercellular communication via EVs will greatly help our understanding of the pathomechanisms leading to valvular calcification. Pathway analysis will generate new targets that could be used to develop therapeutics to ameliorate disease progression. EV-based miRNA mimics and inhibitors could be used to treat valvular calcification. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft, TRR259; Else-Kröner-Fresenius

MicroRNA-34c Inhibits Osteogenic Differentiation and Valvular Interstitial Cell Calcification via STC1-Mediated JNK Pathway in Calcific Aortic Valve Disease.

Calcific aortic valve disease (CAVD), a common heart valve disease, is increasingly prevalent worldwide and causes high morbidity and mortality. Here, we aimed to investigate a possible role for miR-34c in the development of osteogenic differentiation during CAVD and to find out the underlying mechanisms. Valvular interstitial cells (VICs) were isolated from the clinical aortic valve tissue samples of CAVD patients and patients with acute aortic dissection and collected. Then, RT-qPCR was performed to determine miR-34c expression and western blot analysis was applied to confirm the relevant protein expression in these VICs. Dual luciferase reporter gene assay was applied to confirm the relation between miR-34c and STC1. Alkaline phosphatase (ALP) staining and alizarin red staining was performed to further confirm the degree of calcification in these samples. MiR-34c was lowly expressed and STC1 was highly expressed in the CAVD tissues. Furthermore, STC1 was the target of miR-34c and was negatively regulated by miR-34c. Overexpression of miR-34c in VICs was concomitant with suppression of both STC1 expression and phosphorylation level of c-Jun N-terminal kinase (JNK). In addition, significant decrease of bone morphogenetic protein-2 (BMP2) and osteocalcin, as well as the decrease of calcification degree were also observed in VICs with miR-34c overexpressed. Taken together, miR-34c could inhibit osteogenic differentiation and calcification of VICs by suppressing the STC1/JNK signaling pathway in CAVD, making miR-34c a novel therapeutic target for the treatment of CAVD.

Hydrogen sulfide inhibits aortic valve calcification in heart via regulating RUNX2 by NF-κB, a link between inflammation and mineralization

IntroductionHydrogen sulfide (H2S) was revealed to inhibit aortic valve calcification and inflammation was implicated in the pathogenesis of calcific aortic valve disease (CAVD). ObjectivesWe investigate whether H2S inhibits mineralization via abolishing inflammation. Methods and resultsExpression of pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) were increased in patients with CAVD and in calcified aortic valve of ApoE-/- mice. Administration of H22S releasing donor (4-methoxyphenyl piperidinylphosphinodithioc acid (AP72)) exhibited inhibition on both calcification and inflammation in aortic valve of apolipoprotein E knockout mice (ApoE-/-) mice is reflected by lowering IL-1β and TNF-α levels. Accordingly, AP72 prevented the accumulation of extracellular calcium deposition and decreased nuclear translocation of nuclear factor-κB (NF-κB) in human valvular interstitial cells (VIC). This was also accompanied by reduced cytokine response. Double-silencing of endogenous H2S producing enzymes, Cystathionine gamma-lyase (CSE) and Cystathionine beta-synthase (CBS) in VIC exerted enhanced mineralization and higher levels of IL-1β and TNF-α. Importantly, silencing NF-κB gene or its pharmacological inhibition prevented nuclear translocation of runt-related transcription factor 2 (Runx2) and subsequently the calcification of human VIC. Increased levels of NF-κB and Runx2 and their nuclear accumulation occurred in ApoE-/- mice with a high-fat diet. Administration of AP72 decreased the expression of NF-κB and prevented its nuclear translocation in VIC of ApoE-/- mice on a high-fat diet, and that was accompanied by a lowered pro-inflammatory cytokine level. Similarly, activation of Runx2 did not occur in VIC of ApoE-/- mice treated with H2S donor. Employing Stimulated Emission Depletion (STED) nanoscopy, a strong colocalization of NF-κB and Runx2 was detected during the progression of valvular calcification. ConclusionsHydrogen sulfide inhibits inflammation and calcification of aortic valve. Our study suggests that the regulation of Runx2 by hydrogen sulfide (CSE/CBS) occurs via NF-κB establishing a link between inflammation and mineralization in vascular calcification.

Proteoglycan 4 is Increased in Human Calcified Aortic Valves and Enhances Valvular Interstitial Cell Calcification.

Aortic valve stenosis (AVS), a consequence of increased fibrosis and calcification of the aortic valve leaflets, causes progressive narrowing of the aortic valve. Proteoglycans, structural components of the aortic valve, accumulate in regions with fibrosis and moderate calcification. Particularly, proteoglycan 4 (PRG4) has been identified in fibrotic parts of aortic valves. However, the role of PRG4 in the context of AVS and aortic valve calcification has not yet been determined. Here, transcriptomics, histology, and immunohistochemistry were performed in human aortic valves from patients undergoing aortic valve replacement. Human valve interstitial cells (VICs) were used for calcification experiments and RNA expression analysis. PRG4 was significantly upregulated in thickened and calcified regions of aortic valves compared with healthy regions. In addition, mRNA levels of PRG4 positively associated with mRNA for proteins involved in cardiovascular calcification. Treatment of VICs with recombinant human PRG4 enhanced phosphate-induced calcification and increased the mRNA expression of bone morphogenetic protein 2 and the runt-related transcription factor 2. In summary, PRG4 was upregulated in the development of AVS and promoted VIC osteogenic differentiation and calcification. These results suggest that an altered valve leaflet proteoglycan composition may play a role in the progression of AVS.

Semicarbazide-Sensitive Amine Oxidase Increases in Calcific Aortic Valve Stenosis and Contributes to Valvular Interstitial Cell Calcification.

Introduction Calcific aortic valve stenosis (CAVS) is a common disease associated with aging. Oxidative stress participates in the valve calcification process in CAVS. Semicarbazide-sensitive amine oxidase (SSAO), also referred to as vascular adhesion protein 1 (VAP-1), transforms primary amines into aldehydes, generating hydrogen peroxide and ammonia. SSAO is expressed in calcified aortic valves, but its role in valve calcification has remained largely unexplored. The aims of this study were to characterize the expression and the activity of SSAO during aortic valve calcification and to establish the effects of SSAO inhibition on human valvular interstitial cell (VIC) calcification. Methods Human aortic valves from n = 80 patients were used for mRNA extraction and expression analysis, Western blot, SSAO activity determination, immunohistochemistry, and the isolation of primary VIC cultures. Results SSAO mRNA, protein, and activity were increased with increasing calcification within human aortic valves and localized in the vicinity of the calcified zones. The valvular SSAO upregulation was consistent after stratification of the subjects according to cardiovascular and CAVS risk factors associated with increased oxidative stress: body mass index, diabetes, and smoking. SSAO mRNA levels were significantly associated with poly(ADP-ribose) polymerase 1 (PARP1) in calcified tissue. Calcification of VIC was inhibited in the presence of the specific SSAO inhibitor LJP1586. Conclusion The association of SSAO expression and activity with valvular calcification and oxidative stress as well as the decreased VIC calcification by SSAO inhibition points to SSAO as a possible marker and therapeutic target to be further explored in CAVS.

IL-21 promotes osteoblastic differentiation of human valvular interstitial cells through the JAK3/STAT3 pathway.

Objectives: This study amied to whether IL-21 promotes osteoblast transdifferentiation of cultured human Valvular interstitial cells (VICs).Methods: We first confirmed that IL-21 alters gene expression between CAVD aortic valve tissue and normal samples by immunohistochemistry, qPCR, and western blotting. VICs were cultured and treated with IL-21. Gene and protein expression levels of the osteoblastic markers ALP and Runx2, which can be blocked by specific JAK3 inhibitors and/or siRNA of STAT3, were measured.Results: IL-21 expression was upregulated in calcified aortic valves and promotes osteogenic differentiation of human VICs. IL-21 accelerated VIC calcification through the JAK3/STAT3 pathway.Conclusion: Our data suggest that IL-21 is a key factor in valve calcification and a promising candidate for targeted therapeutics for CAVD.

Hydrogen sulfide inhibits calcification of heart valves; implications for calcific aortic valve disease.

Background and PurposeCalcification of heart valves is a frequent pathological finding in chronic kidney disease and in elderly patients. Hydrogen sulfide (H2S) may exert anti‐calcific actions. Here we investigated H2S as an inhibitor of valvular calcification and to identify its targets in the pathogenesis.Experimental ApproachEffects of H2S on osteoblastic transdifferentiation of valvular interstitial cells (VIC) isolated from samples of human aortic valves were studied using immunohistochemistry and western blots. We also assessed H2S on valvular calcification in apolipoprotein E‐deficient (ApoE−/−) mice.Key ResultsIn human VIC, H2S from donor compounds (NaSH, Na2S, GYY4137, AP67, and AP72) inhibited mineralization/osteoblastic transdifferentiation, dose‐dependently in response to phosphate. Accumulation of calcium in the extracellular matrix and expression of osteocalcin and alkaline phosphatase was also inhibited. RUNX2 was not translocated to the nucleus and phosphate uptake was decreased. Pyrophosphate generation was increased via up‐regulating ENPP2 and ANK1. Lowering endogenous production of H2S by concomitant silencing of cystathionine γ‐lyase (CSE) and cystathionine β‐synthase (CBS) favoured VIC calcification. analysis of human specimens revealed higher Expression of CSE in aorta stenosis valves with calcification (AS) was higher than in valves of aortic insufficiency (AI). In contrast, tissue H2S generation was lower in AS valves compared to AI valves. Valvular calcification in ApoE−/− mice on a high‐fat diet was inhibited by H2S.Conclusions and ImplicationsThe endogenous CSE‐CBS/H2S system exerts anti‐calcification effects in heart valves providing a novel therapeutic approach to prevent hardening of valves.Linked ArticlesThis article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc

Standardization of Human Calcific Aortic Valve Disease in vitro Modeling Reveals Passage-Dependent Calcification.

Aortic valvular interstitial cells (VICs) isolated from patients undergoing valve replacement are commonly used as in vitro models of calcific aortic valve disease (CAVD). Standardization of VIC calcification, however, has not been implemented, which impairs comparison of results from different studies. We hypothesized that different culture methods impact the calcification phenotype of human VICs. We sought to identify the key parameters impacting calcification in primary human VICs to standardize CAVD in vitro research. Here we report that in calcification media containing organic phosphate, termed osteogenic media (OM), primary human VICs exhibited a passage-dependent decrease in calcification potential, which was not observed in calcification media containing inorganic phosphate, termed pro-calcifying media (PM). We used Alizarin red staining to compare the calcification potential of VICs cultured in OM and PM between the first and fourth passages after cell isolation from human CAVD tissues. Human VICs showed consistent Alizarin red stain when cultured with PM in a passage-independent manner. VICs cultured in OM did not exhibit consistent calcification potential between donors in early passages and consistently lacked positive Alizarin red stain in late passages. We performed whole cell, cytoplasmic and nuclear fractionation proteomics to identify factors regulating VIC passage-dependent calcification in OM. Proteomics cluster analysis identified tissue non-specific alkaline phosphatase (TNAP) as a regulator of passage-dependent calcification in OM. We verified an association of TNAP activity with calcification potential in VICs cultured in OM, but not in PM in which VICs calcified independent of TNAP activity. This study demonstrates that media culture conditions and cell passage impact the calcification potential of primary human VICs and should be taken into consideration in cell culture models of CAVD. Our results help standardize CAVD modeling as part of a greater effort to identify disease driving mechanisms and therapeutics for this unmet medical need.

Valvular interstitial cells down regulate matrix metalloproteinase 9 activity and expression in human monocyte-derived macrophages: Potential impact on aortic valve stenosis pathophysiology

Aortic valve stenosis (AVS) involves inflammation, excess deposition of collagen-rich extracellular matrix (ECM) and calcification. Recent studies have shown that M1 macrophages observed in stenosed valve promote calcification of valvular interstitial cells (VIC), the most prevalent cell type in aortic valve.

Implication of endothelin-1 in human aortic valve calcification

Aortic Stenosis (AS) is the most common acquired valvulopathy in the Western world. AS is characterized by the differenciation of valvular interstitial cells (VICs) into osteoblastic cells, leading to valvular calcification. The physiopathology of AS is not fully understood and there is, to date, no treatments to stop or slow the progression of AS. The purpose of our study was to investigate the role of endothelin-1 (ET-1) on VICs calcification. Primary human valvular interstitial cells (hVICs) were first cultured in pro-osteogenic medium or incubated with ET-1. hVICs were also co-cultured with human valvular endothelial cells (hVECs) obtained from patients with (pathological) or without (healthy) AS in Transwell™ inserts. hVICs calcification was quantified using o-cresolphthalein assay. Real-time quantitative PCR (qRT-PCR) and Western blot analysis were performed to determine the expression of ET-1 (EDN1), inflammation (IL1B, IL6) and bone markers (RUNX2, OPG, OPN). Immunochemistry was performed using ACTA2 antibody to evaluate the phenotype of VICs. hVICs showed an increase of calcification and a decrease of ACTA2 when cultured in pro-osteogenic medium or co-cultured with pathological hVECs. qRT-PCR revealed higher mRNA levels of EDN1, RUNX2, OPG, OPN, IL1B and IL6. At the same time, fibroblast marker ACTA2 was decreased. VIC fibroblastic phenotype was restored and calcification decreased when hVICs were co-cultured with healthy hVECs or incubated with ET-1 antagonist. The present study suggests that ET-1 play a role in the pathogenesis of AS, and therefore, pharmacological ET-1 antagonists could be used to attenuate or stop VIC calcification.

Modulation of a new pathway prevent valvular interstitial cells calcification, a potential innovative therapeutic target in aortic valve stenosis

Aortic valve stenosis (AVS) is the narrowing of the aortic valve (AV) opening caused by fibrosis and calcification. Despite this paradigm, no targeted medical therapy has proven to prevent, slow down or treat AVS. Using transcriptomic approach on human valvular interstitial cells (VIC) isolated from fibrocalcific and normal valves, our team has identified an enzyme X downregulated in fibro-calcific VIC. This enzyme X and its product Y have documented anti-calcification effects in other cell types than VIC. Our goal is to decipher the implication of this pathway on an in vitro model of human primary cultures of VIC in ostegenic conditions. Normal AV unsuitable for grafting and fibrocalcific AV from valve replacement (ATHERAO protocol) were collected. Levels of enzyme X in AV was assessing by histology and by Western Blot on tissue homogenate. VIC isolation was realized by enzymatic digestion of normal AV. Primary cultures were exposed to ostegenic media (OM) or control media with or without the product Y of the enzyme (1–1000 nM) during 7 days. Evaluation of calcification was realized by alizarin red staining, mRNA levels of calcification markers. We confirmed a differential expression of enzyme X on tissue by histology and by western blot (− 5.2 fold, P < 0.05) from fibro-calcific AV compared to those from control AV. In OM, enzyme X is decreased at the mRNA (− 5.2 fold, P < 0.05) and protein level (− 5.2 fold, P < 0.05) compared to control media. VIC treatments with product Y in presence of OM decrease the formation of calcium nodules in a dose dependent manner (− 5.2 fold, P < 0.05, vs OM) and mRNA levels for RUNX2 (− 2.4 fold, P < 0.05 vs OM), attenuating osteoblastic VIC differentiation. Our results demonstrate the involvement of this new pathway in an in vitro model of AVS by decreasing human VIC calcification and associated markers. Enzyme X is a potential innovative pharmacological therapeutic target in AVS.

Iron alters valvular interstitial cell function and is associated with calcification in aortic stenosis.

AimsAortic valve stenosis (AS) is the most common valvulopathy and is characterized by inflammation, extracellular matrix (ECM) remodelling and calcification, causing a narrowing of the valve and the consequential obstruction of the cardiac outflow. Although intraleaflet haemorrhage is associated with AS progression, the mechanisms involved are not known. The aims of this study were to identify valvular iron in relation to pathological changes associated with AS and the effects on valvular interstitial cells (VIC) in terms of iron uptake and iron-induced responses.Methods and resultsValvular iron accumulation was detected by Perls' staining on aortic valve sections and shown to increase with the extent of calcification. Furthermore, qRT–PCR analysis revealed that iron-containing valve regions exhibited increased expression of genes involved in ECM remodelling and calcification. In addition, we demonstrate that iron transporters are regulated by pathways with major impact on AS and that VIC can take up and accumulate iron, which resulted in increased proliferation and decreased elastin production.ConclusionIron, which may accumulate in the aortic valve by means of intraleaflet haemorrhages, can be taken up by VIC in a pro-inflammatory environment and actively contribute to VIC proliferation, ECM remodelling and calcification. These findings suggest a possible mechanism through which iron uptake by VIC may favour AS progression.

Simulation of early calcific aortic valve disease in a 3D platform: A role for myofibroblast differentiation

PurposeCalcific aortic valve disease (CAVD) is the most prevalent valve disease in the Western world. Recent difficulty in translating experimental results on statins to beneficial clinical effects warrants the need for understanding the role of valvular interstitial cells (VICs) in CAVD. In two-dimensional culture conditions, VICs undergo spontaneous activation similar to pathological differentiation, which intrinsically limits the use of in vitro models to study CAVD. Here, we hypothesized that a three-dimensional (3D) culture system based on naturally derived extracellular matrix polymers, mimicking the microenvironment of native valve tissue, could serve as a physiologically relevant platform to study the osteogenic differentiation of VICs. Principal resultsAortic VICs loaded into 3D hydrogel constructs maintained a quiescent phenotype, similar to healthy human valves. In contrast, osteogenic environment induced an initial myofibroblast differentiation (hallmarked by increased alpha smooth muscle actin [α-SMA] expression), followed by an osteoblastic differentiation, characterized by elevated Runx2 expression, and subsequent calcific nodule formation recapitulating CAVD conditions. Silencing of α-SMA under osteogenic conditions diminished VIC osteoblast-like differentiation and calcification, indicating that a VIC myofibroblast-like phenotype may precede osteogenic differentiation in CAVD. Major conclusionsUsing a 3D hydrogel model, we simulated events that occur during early CAVD in vivo and provided a platform to investigate mechanisms of CAVD. Differentiation of valvular interstitial cells to myofibroblasts was a key mechanistic step in the process of early mineralization. This novel approach can provide important insight into valve pathobiology and serve as a promising tool for drug screening.

High-mobility group box-1 protein induces osteogenic phenotype changes in aortic valve interstitial cells

ObjectivesCalcific aortic valve (AV) disease is known to be an inflammation-related process. High-mobility group box-1 (HMGB1) protein and Toll-like receptor 4 (TLR4) have been reported to participate in several inflammatory diseases. The purpose of the present study was to determine whether the HMGB1-TLR4 axis is involved in calcific AV disease, and to evaluate the effect of HMGB1, and its potential mechanisms, on the pro-osteogenic phenotype change of valvular interstitial cells (VICs). MethodsExpression of HMGB1 and TLR4 in human calcific AVs was evaluated using immunohistochemical staining and immunoblotting. Cultured VICs were used as an in vitro model. The VICs were stimulated with HMGB1 for analysis, with versus without TLR4 small interfering ribonucleic acid (siRNA), c-Jun N-terminal kinase mitogen-activated protein kinase (JNK MAPK), and nuclear factor kappa-B (NF-κB) inhibitors. ResultsEnhanced accumulation of HMGB1 and TLR4 was observed in calcific valves. Moreover, we found that HMGB1 induced high levels of pro-inflammatory cytokine production and promoted the osteoblastic differentiation and calcification of VICs. In addition, HMGB1 induced phosphorylation of JNK MAPK and NF-κB. However, these effects were markedly suppressed by siRNA silencing of TLR4. In addition, blockade of JNK MAPK and NF-κB phosphorylation prohibited HMGB1-induced production of pro-osteogenic factors, and mineralization of VICs. ConclusionsThe HMGB1 protein may promote osteoblastic differentiation and calcification of VICs, through the TLR4-JNK-NF-κB signaling pathway.

Using Na3PO4 to Enhance In vitro Animal Models of Aortic Valve Calcification

The pathogenesis of calcific aortic valvular disease (CAVD) involves an active inflammatory process of valvular interstitial cells (VICs) characterized by the activation of specific osteogenic signaling pathways and apoptosis. This process can be studied by analyzing certain molecular markers and gene expression pathways of spontaneous calcification. The purpose of our study is to investigate the role of sodium phosphate (Na3PO4) as a calcification promoter, with the aim of improving in vitro animal models for testing potential calcification inhibitors. VICs were extracted from 6 healthy 6-month-old fresh porcine hearts by serial collagenase digestion. Quantitative polymerase chain reaction (qPCR) was used to quantify trans-differentiation of genes of interest during spontaneous calcification of VICs. Spontaneous calcification of VICs was increased by adding Na3PO4 (3 mM, pH 7.4). The degree of calcification was estimated by Alizarin Red staining for calcium deposition, and Sirius Red staining for collagen. Colorimetric techniques were used to determine calcium and collagen deposition quantitatively. Additionally, the enzymatic activity of alkaline phosphatase (ALP) was measured by a kinetic assay. For statistical analysis we used SPSS and Microsoft Office Excel 2013. Porcine VICs calcify spontaneously with demonstrable calcium and collagen deposition. In this study we observed an increase of calcium and collagen deposition from day 0 to day 14 (calcium: 376%; P<0.001, collagen: 3553%; P<0.001). qPCR analysis of mRNA by day 14 showed the following results: α-actin, a marker of myoblast phenotype, was increased to 1.6-fold; P<0.001. Runx2, an osteoblast marker, rose to 1.3 fold; P<0.05, TGF-β, a promoter of osteogenesis, increased to 3.2-fold; P<0.001, and RhoA, a regulator of nodular formation in myoblasts, increased to 4.5-fold; P<0.001, compared to their levels at day 0. RANKL mRNA and calponin did not change significantly. Treatment of porcine VICs with Na3PO4 (3 mM, pH 7.4) led to a marked increase in calcium deposition by day 14 (522%; P<0.001), and a significant increase in ALP activity by day 7 (228%; P<0.05). There were no significant changes in ALP activity between the groups by day 14. This study has demonstrated the upregulation of some specific molecules during spontaneous calcification of aortic VICs with an active increase of calcium, collagen and ALP activity. In this in vitro model it was possible to increase spontaneous VICs calcification with Na3PO4 (3 mM, pH 7.4) to a level in which inhibitors of calcification could be tested to identify a novel potential therapeutic strategy against calcific aortic stenosis.

Denosumab could be a Potential Inhibitor of Valvular Interstitial Cells Calcification in vitro.

Denosumab is a fully human monoclonal antibody and novel antiresorptive agent that works by binding receptor activator of nuclear factor kappa-β ligand (RANKL) and inhibiting the signaling cascade that causes osteoclast maturation, activity, and survival. We aimed to elucidate the effect of Denosumab in the process of spontaneous and induced calcification in an in vitro porcine valvular interstitial cells (VICs) model. VICs were extracted from fresh porcine hearts by serial collagenase digestion. Spontaneous calcification of VICs was increased in vitro by adding Na3PO4 (3 mM, pH 7.4) and different concentrations (0.1, 1 and 10 ng/ml) of transforming growth factor beta (TGFß). The degree of calcification before and after treatment with Denosumab was estimated by Alizarin Red staining for calcium deposition, and Sirius Red staining for collagen. Colorimetric techniques were used to determine calcium and collagen deposition quantitatively. For statistical analysis we used SPSS and Microsoft Office Excel 2013. Porcine aortic VICs in vitro were induced to calcify by the addition of either 3 mM Na3PO4, showing a 5.2 fold increase by 14 days (P<0.001), or 3 mM Na3PO4 + 10 ng/ml of TGFβ, showing a 7 fold increase by Day 14 (P<0.001). Denosumab inhibited induced calcification by 3 mM Na3PO4 and 3 mM Na3PO4 with the addition of TGFß at either 0.1, 1 or 10 ng/ml to basal levels only at a concentration of 50 μg/ml (P<0.001). This study has proved that Denosumab could be a potential inhibitor of the calcification of VICs in vitro. A fuller understanding of the actions of Denosumab may identify a novel therapeutic strategy for clinical intervention against aortic valve calcification and aortic stenosis.

Oxidized Low-Density Lipoprotein Promotes Osteoblastic Differentiation of Valvular Interstitial Cells through RAGE/MAPK

Objectives: We have previously shown that oxidized low-density lipoprotein (oxLDL) promotes the osteogenic differentiation of valvular interstitial cells (VICs) by inducing endoplasmic reticulum (ER) stress. We also demonstrated the detrimental role of the receptor for advanced glycation end products (RAGE) activation and signaling in the development and progression of aortic valve (AV) calcification. Here, we test the hypothesis that oxLDL may induce the osteoblastic differentiation of VICs via RAGE. Methods: Cultured porcine aortic VICs were used in an in vitro model. The VICs were incubated with oxLDL for analysis, with and without RAGE siRNA. Results: We found that oxLDL markedly increased the expression of RAGE, induced high levels of proinflammatory cytokine production and promoted the osteoblastic differentiation and calcification of VICs. oxLDL also induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) MAPK. However, these effects were found to be markedly suppressed by siRNA silencing of RAGE. Conclusions: Our data provide evidence that RAGE mediates oxLDL-induced activation of p38 and JNK MAPK and the osteogenic differentiation of VICs.

Age-Related Changes in Aortic Valve Hemostatic Protein Regulation

Although valvular endothelial cells have unique responses compared with vascular endothelial cells, valvular regulation of hemostasis is not well-understood. Heart valves remodel throughout a person's lifetime, resulting in changes in extracellular matrix composition and tissue mechanical properties that may affect valvular endothelial cell hemostatic function. This work assessed valvular endothelial cell regulation of hemostasis in situ and in vitro as a function of specimen age. Porcine aortic valves were assigned to 1 of 3 age groups: Young (YNG) (6 weeks); Adult (ADT) (6 months); or Elderly (OLD) (2 years). Histological examination of valves showed that secreted thrombotic/antithrombotic proteins localize at the valve endothelium and tissue interior. Gene expression and immunostains for von Willebrand factor (VWF), tissue factor pathway inhibitor, and tissue plasminogen activator in YNG porcine aortic valve endothelial cells were higher than they were for OLD, whereas plasminogen activator inhibitor 1 levels in OLD were higher than those for YNG and ADT. Histamine-stimulated YNG porcine aortic valve endothelial cells released higher concentrations of VWF proteins than OLD, and the fractions of VWF-140 fragments was not different between age groups. A calcific aortic valve disease in vitro model using valvular interstitial cells confirmed that VWF in culture significantly increased valvular interstitial cell nodule formation and calcification. Hemostatic protein regulation in aortic valve tissues and in valvular endothelial cells changes with age. The presence of VWF and other potential hemostatic proteins increase valvular interstitial cell calcification in vitro. Therefore, the increased capacity of elderly valves to sequester the hemostatic proteins, together with age-associated loss of extracellular matrix organization, warrants investigation into potential role of these proteins in the formation of calcific nodules.

Abstract 303: CD44 Activation Protects Human Sclerotic-derived Valvular Interstitial Cells from Calcification

Introduction. The activation of Valvular Interstitial Cells (VIC) towards an osteoblast-like phenotype is a cellular hallmark of pathological progression towards Aortic Stenosis (AS). In recent years several clinical trials have failed to halt or revert the progression of this prevalent disease. The ability to prevent end-stage AS requires the understanding of the molecular events associated with the early phase of valve degeneration, a condition known as Aortic Sclerosis (ASc). In the last few years the transmembrane receptor CD44 has been studied as a putative molecule for cardiovascular drug therapy. We reported that the functional interaction between CD44 and one of its ligand, Osteopontin (OPN), protects vascular smooth muscle cells from calcification. More recently, we demonstrated that sclerotic tissues show increased expression of Bone Morphogenetic Protein 4 (BMP4) and BMP4 directly stimulates osteoblast-like transdifferentiation and calcification of VICs. Therefore we hypothesized a direct role of CD44 activation in protecting human Aortic Sclerosis-derived VICs from calcification. Methods. Human VICs from Control, ASc, and AS (n=5 each group) were isolated. Histological, cellular and molecular analysis, and in situ Proximity Ligation Assay were used to investigate the role of CD44 and OPN in VIC calcification. BMP4 treatments were used to promote VIC activation. Osteoblast-like transdifferentiation was analyzed using Alkaline Phosphatase (ALP) expression. Results. CD44 and OPN, as well as their functional binding, were increased in sclerotic and stenotic tissues compared to healthy controls in vitro and ex vivo. CD44-OPN binding prevented in vitro calcification induced by inorganic phosphate on human ASc-derived VICs. A neutralizing antibody against CD44, under BMP4 treatments, promoted calcium deposition along with increased expression of OPN and ALP. Conclusion. Our results generate an important insight into the molecular mechanism of VIC calcification. We proved that CD44-OPN direct interaction inhibits calcification of Aortic Sclerosis-derived VICs, suggesting that CD44 activation could have a protective role against VIC osteoblast-like transdifferentiation and calcification in the early stage of the disease.

Regulation of valvular interstitial cell phenotype and function by hyaluronic acid in 2-D and 3-D culture environments

Disruption of the extracellular matrix (ECM) is frequently found in calcific aortic valve disease (CAVD), yet the role of ECM components in valvular interstitial cell (VIC) function and dysfunction remains poorly understood. This study examines the contributions of exogenous and endogenous hyaluronic acid (HA), in both two-dimensional (2-D) and 3-D environments, in regulating the phenotype and calcification of VICs. VIC calcification was first assessed in a 2-D setting in which the cells were exposed to different molecular weights of exogenous HA presented in either an immobilized or soluble form. Delivery of HA suppressed nodule formation in a molecular weight-dependent manner, while blocking VIC recognition of HA via an antibody to CD44 abolished these nodule-suppressive effects and stimulated other hallmarks of valvular dysfunction. These 2-D results were then validated in a more physiologically-relevant setting, using an approach that allowed the characterization of VIC phenotype in response to HA alterations in the native 3-D environment. In this approach, leaflet organ cultures were analyzed following treatment with anti-CD44 or with hyaluronidase to specifically remove HA. Disruption of VIC–HA interactions upregulated markers of VIC disease and induced leaflet mineralization. Similarly, HA-deficient leaflets exhibited numerous hallmarks of CAVD, including increased VIC proliferation, apoptosis, increased expression of disease-related markers, and mineralization. These findings suggest that VIC–HA interactions are crucial in maintaining a healthy VIC phenotype. Identification ECM components that can regulate VIC phenotype and function has significant implications for understanding native valve disease, investigating possible treatments, and designing new biomaterials for valve tissue engineering.