Porcine Aortic Valve Interstitial Cells Research Articles

Single-cell RNA-sequencing analysis of aortic valve interstitial cells demonstrates the regulation of integrin signaling by nitric oxide.

Calcific aortic valve disease (CAVD) is an increasingly prevalent condition among the elderly population that is associated with significant morbidity and mortality. Insufficient understanding of the underlying disease mechanisms has hindered the development of pharmacologic therapies for CAVD. Recently, we described nitric oxide (NO) mediated S-nitrosylation as a novel mechanism for preventing the calcific process. We demonstrated that NO donor or an S-nitrosylating agent, S-nitrosoglutathione (GSNO), inhibits spontaneous calcification in porcine aortic valve interstitial cells (pAVICs) and this was supported by single-cell RNA sequencing (scRNAseq) that demonstrated NO donor and GSNO inhibited myofibroblast activation of pAVICs. Here, we investigated novel signaling pathways that are critical for the calcification of pAVICs that are altered by NO and GSNO by performing an in-depth analysis of the scRNA-seq dataset. Transcriptomic analysis revealed 1,247 differentially expressed genes in pAVICs after NO donor or GSNO treatment compared to untreated cells. Pathway-based analysis of the differentially expressed genes revealed an overrepresentation of the integrin signaling pathway, along with the Rho GTPase, Wnt, TGF-β, and p53 signaling pathways. We demonstrate that ITGA8 and VCL, two of the identified genes from the integrin signaling pathway, which are known to regulate cell-extracellular matrix (ECM) communication and focal adhesion, were upregulated in both in vitro and in vivo calcific conditions. Reduced expression of these genes after treatment with NO donor suggests that NO inhibits calcification by targeting myofibroblast adhesion and ECM remodeling. In addition, withdrawal of NO donor after 3 days of exposure revealed that NO-mediated transcriptional and translational regulation is a transient event and requires continuous NO exposure to inhibit calcification. Overall, our data suggest that NO and S-nitrosylation regulate the integrin signaling pathway to maintain healthy cell-ECM interaction and prevent CAVD.

Glycosaminoglycans affect endothelial to mesenchymal transformation, proliferation, and calcification in a 3D model of aortic valve disease.

Calcific nodules form in the fibrosa layer of the aortic valve in calcific aortic valve disease (CAVD). Glycosaminoglycans (GAGs), which are normally found in the valve spongiosa, are located local to calcific nodules. Previous work suggests that GAGs induce endothelial to mesenchymal transformation (EndMT), a phenomenon described by endothelial cells’ loss of the endothelial markers, gaining of migratory properties, and expression of mesenchymal markers such as alpha smooth muscle actin (α-SMA). EndMT is known to play roles in valvulogenesis and may provide a source of activated fibroblast with a potential role in CAVD progression. In this study, a 3D collagen hydrogel co-culture model of the aortic valve fibrosa was created to study the role of EndMT-derived activated valvular interstitial cell behavior in CAVD progression. Porcine aortic valve interstitial cells (PAVIC) and porcine aortic valve endothelial cells (PAVEC) were cultured within collagen I hydrogels containing the GAGs chondroitin sulfate (CS) or hyaluronic acid (HA). The model was used to study alkaline phosphatase (ALP) enzyme activity, cellular proliferation and matrix invasion, protein expression, and calcific nodule formation of the resident cell populations. CS and HA were found to alter ALP activity and increase cell proliferation. CS increased the formation of calcified nodules without the addition of osteogenic culture medium. This model has applications in the improvement of bioprosthetic valves by making replacements more micro-compositionally dynamic, as well as providing a platform for testing new pharmaceutical treatments of CAVD.

Circ-CCND1 regulates the CCND1/P53/P21 pathway through sponging miR-138-5p in valve interstitial cells to aggravate aortic valve calcification.

To discuss the effect and mechanism of circular-CCND1 (circ-CCND1) on the regulation of calcified aortic valve disease (CAVD). Differentially expressed circRNAs were screened through the GSE155119 data set and biological prediction. Subsequently, the miR-138-5p, CCND1, and circ-CCND1 expression were detected in the non-calcified and calcified aortic valve. Then Pearson correlation analysis was performed to analyze the correlation between the above expression, and dual luciferase and RNA-pull down assays for verifying the target relationship. Porcine aortic valve interstitial cells (AVICs) were isolated and transfected with pcDNA-circ-CCND1, miR-138-5p inhibitor, and miR-138-5p mimics. The alkaline phosphatase (ALP) activity was quantitatively analyzed by ALP staining, and alizarin-red staining was to check the calcium nodules formation. Finally, Western blot was applied to detect the expression of proteins associated with osteogenic differentiation (Runx2, Osterix, OPN) and CCND1/P53/P21 pathway proteins. Circ-CCND1 was highly expressed in calcific aortic valves. After inhibiting circ-CCND1 expression, a significant reduction was shown in ALP activity, the degree of ossification and the formation of calcium nodules in AVICs, and osteogenic differentiation-related protein expression and CCND1/P53/P21 pathway protein expression. By contrast, inhibition of miR-138-5p and circ-CCND1 together promoted the calcification of AVICs and expression of CCND1/P53/P21 pathway proteins. P53 inhibitor (PFT-α) could significantly reduce activation of CCND1/P53/P21 pathway protein expression by circ-CCND1 overexpression. However, P53 activator (Nutlin-3) significantly restored the suppression of the above pathway-related protein expression by downregulation of circ-CCND1. Circ-CCND1 sponges miR-138-5p to regulate CCND1 expression, thereby promoting the calcification of AVICs.

Berberine inhibits osteogenic differentiation of aortic valve interstitial cells induced by osteogenic induction medium

This research focused on the effect and mechanism of berberine on osteogenic differentiation of valve interstitial cells(VICs) induced by osteogenic induction medium, in order to provide new insights into the clinical treatment of calcified aortic valve disease. The expression of osteogenic and fibrotic makers in three cases of calcified valve tissues and one case of normal control was assayed by Western blot. After the porcine aortic VICs were isolated, the effects of different concentrations of berberine on their viability were examined by MTT assay for determining the optimal concentration range. VICs were cultured in osteogenic induction medium and treated with different concentrations of berberine. Western blot and q-PCR were conducted to detect the effects of berberine on the expression of osteogenic and fibrotic makers in VICs. The effects of berberine on osteogenic differentiation of VICs in the early and late stages were separately measured by ALP staining and alizarin red S staining. The effects of berberine on the phosphorylation of ERK1/2 at different time points were assayed by Western blot. And PD98059, an inhibitor of ERK1/2, was added for verification. The results suggested that related osteogenic and fibrotic makers were significantly up-regulated in calcified valve tissues as compared with those in the normal control. The up-regulated fibrosis and osteogenic makers of VICs under osteogenic conditions were reversed by berberine and the ALP activity and calcium deposition in VICs were also reduced obviously. The level of ERK1/2 phosphorylation was decreased. Similarly, the osteogenic and fibrotic makers of VICs induced by osteogenic induction medium were lowered by PD98059. This study has confirmed that berberine is able to inhibit the differentiation of VICs into myofibroblasts or osteoblast-like cells, which may be associated with the inhibition of ERK1/2 signaling pathway.

The role of neutrophil elastase in aortic valve calcification

BackgroundCalcific 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.MethodsNE 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.ResultsThe 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.ConclusionsCollectively, NE is highly involved in the pathogenesis of valve calcification. Targeting NE such as Alvelestat may be a potential treatment for CAVD.

Rac1 mediates cadherin-11 induced cellular pathogenic processes in aortic valve calcification

Background: Calcific aortic valve disease (CAVD), a major cause for surgical aortic valve replacement, currently lacks available pharmacological treatments. Cadherin-11 (Cad11), a promising therapeutic target, promotes aortic valve calcification in vivo, but direct Cad11 inhibition in clinical trials has been unsuccessful. Targeting of downstream Cad11 effectors instead may be clinically useful; however, the downstream effectors that mediate Cad11-induced aortic valve cellular pathogenesis have not been investigated. Approach and Results: Immunofluorescence of calcified human aortic valves revealed that GTP-Rac1 is highly upregulated in calcified leaflets and is 2.15 times more co-localized with Cad11 in calcified valves than GTP-RhoA. Using dominant negative mutants in porcine aortic valve interstitial cells (PAVICs), we show that Cad11 predominantly regulates Runx2 nuclear localization via Rac1. Rac1-GEF inhibition via NSC23766 effectively reduces calcification in ex vivo porcine aortic valve leaflets treated with osteogenic media by 2.8-fold and also prevents Cad11-induced cell migration, compaction, and calcification in PAVICs. GTP-Rac1 and Trio, a known Cad11 binding partner and Rac1-GEF, are significantly upregulated in Nfatc1Cre; R26-Cad11Tg/Tg (Cad11 OX) mice that conditionally overexpress Cad11 in the heart valves by 3.1-fold and 6.3-fold, respectively. Finally, we found that the Trio-specific Rac1-GEF inhibitor, ITX3, effectively prevents Cad11-induced calcification and Runx2 induction in osteogenic conditions. Conclusion: Here we show that Cad11 induces many cellular pathogenic processes via Rac1 and that Rac1 inhibition effectively prevents many Cad11-induced aortic disease phenotypes. These findings highlight the therapeutic potential of blocking Rac1-GEFs in CAVD.

Emodin alleviates aortic valvular calcification by inhibiting the AKT/FOXO1 pathway

BackgroundValvular calcification commonly occurs in elderly individuals, and is increasingly considered an important economic and health burden. However, no efficient drugs against valvular calcification are available. The present work aimed to examine emodin’s suppressive effect on high-calcium–dependent valve calcification and explore the underpinning mechanisms. MethodsExperiments were carried out in mice receiving vitamin D (Vit D) to induce valvular calcification. Results: Cell viability and apoptosis assays demonstrated celastrol suppressed proliferation and increased apoptosis in porcine aortic valve interstitial cells (PAVICs) at concentrations higher than 10 μM. Emodin (5 μM) attenuated the upregulation of osteogenic genes as well as calcium accumulation in PAVICs under high-calcium conditions. The elevations of calcium content in serum and valve, and calcium accumulation in valve and artery were suppressed by emodin in mice with valvular calcification after joint treatment with adenine and Vit D. In addition, p-AKT and p-FOXO1 were upregulated in PAVICs under high-calcium conditions, and this effect was reversed by emodin treatment. SC79, an AKT activator, reversed emodin’s suppressive effects on increased calcium content, calcium deposition and osteogenic gene expression in PAVICs induced by calcific medium. ConclusionsThese data demonstrated emodin alleviates high-calcium–associated valvular calcification via AKT/FOXO1 signaling suppression, providing new insights into therapeutic strategies for clinical valvular calcification.

Chondroitin Sulfate Promotes Interstitial Cell Activation and Calcification in an In Vitro Model of the Aortic Valve.

Calcific aortic valve disease (CAVD), has been characterized as a cascade of cellular changes leading to leaflet thickening and valvular calcification. In diseased aortic valves, glycosaminoglycans (GAGs) normally found in the valve spongiosa migrate to the collagen I-rich fibrosa layer near calcified nodules. Current treatments for CAVD are limited to valve replacement or drugs tailored to other cardiovascular diseases. Porcine aortic valve interstitial cells and porcine aortic valve endothelial cells were seeded into collagen I hydrogels of varying initial stiffness or initial stiffness-matched collagen I hydrogels containing the glycosaminoglycans chondroitin sulfate (CS), hyaluronic acid (HA), or dermatan sulfate (DS). Assays were performed after 2 weeks in culture to determine cell gene expression, protein expression, protein secretion, and calcification. Multiple regression analyses were performed to determine the importance of initial hydrogel stiffness, GAGs, and the presence of endothelial cells on calcification, both with and without osteogenic medium. High initial stiffness hydrogels and osteogenic medium promoted calcification, while for DS or HA the presence of endothelial cells prevented calcification. CS was found to increase the expression of pro-calcific genes, increase activated myofibroblast protein expression, induce the secretion of collagen I by activated interstitial cells, and increase calcified nodule formation. This study demonstrates a more complete model of aortic valve disease, including endothelial cells, interstitial cells, and a stiff and disease-like ECM. In vitro models of both healthy and diseased valves can be useful for understanding the mechanisms of CAVD pathogenesis and provide a model for testing novel therapeutics.

Carbon Nanotubes Substrates Alleviate Pro-Calcific Evolution in Porcine Valve Interstitial Cells.

The quest for surfaces able to interface cells and modulate their functionality has raised, in recent years, the development of biomaterials endowed with nanocues capable of mimicking the natural extracellular matrix (ECM), especially for tissue regeneration purposes. In this context, carbon nanotubes (CNTs) are optimal candidates, showing dimensions and a morphology comparable to fibril ECM constituents. Moreover, when immobilized onto surfaces, they demonstrated outstanding cytocompatibility and ease of chemical modification with ad hoc functionalities. In this study, we interface porcine aortic valve interstitial cells (pVICs) to multi-walled carbon nanotube (MWNT) carpets, investigating the impact of surface nano-morphology on cell properties. The results obtained indicate that CNTs significantly affect cell behavior in terms of cell morphology, cytoskeleton organization, and mechanical properties. We discovered that CNT carpets appear to maintain interfaced pVICs in a sort of “quiescent state”, hampering cell activation into a myofibroblasts-like phenotype morphology, a cellular evolution prodromal to Calcific Aortic Valve Disease (CAVD) and characterized by valve interstitial tissue stiffening. We found that this phenomenon is linked to CNTs’ ability to alter cell tensional homeostasis, interacting with cell plasma membranes, stabilizing focal adhesions and enabling a better strain distribution within cells. Our discovery contributes to shedding new light on the ECM contribution in modulating cell behavior and will open the door to new criteria for designing nanostructured scaffolds to drive cell functionality for tissue engineering applications.

Abstract P332: Microfluidic Model Of Late-stage Calcific Aortic Valve Disease Develops Calcium Phosphate Mineralizations

Introduction: Calcific aortic valve disease (CAVD) is an active pathological process leading to severe valve calcification. Late-stage CAVD is characterized by increased leaflet stiffness, disorganized collagen bundles and the deposition of glycosaminoglycans, such as chondroitin sulfate (CS), in the fibrosa layer. However, many details of the cellular pathological cascade remain unknown. Animal models such as mice, rabbits, and pigs are used in understanding human CAVD, but mice do not have similar anatomy, rabbits cannot spontaneously develop atherosclerotic lesions, and pigs require long, expensive and complex studies. Here we utilize microfluidic devices of the aortic valve fibrosa to model late-stage CAVD. Hypothesis: We assessed the hypothesis that microfluidic calcification will increase with increased shear rates and CS content. Methods: Valve-on-a-chip devices contained a hydrogel of 1.5 mg/mL collagen I-only healthy controls or 1.5 mg/mL collagen I with 1 mg/mL or 20 mg/mL CS. Porcine aortic valve interstitial cells (PAVIC) were embedded within and endothelial cells (PAVEC) were seeded onto the matrix. Steady shear stress at 1 dyne/cm 2 and 20 dyne/cm 2 were applied using a peristaltic pump for 14 days. Alizarin Red S (ARS), an assay to assess calcium deposition, was used to quantify calcific nodule formation. Scanning electron microscopy with energy dispersive x-ray (SEM/EDX) was used to further analyze sample mineralization. Results: Co-cultures in the presence of increasing shear stress and CS exhibit increased calcific nodule formation compared to static controls, both qualitatively and quantitatively (n≥3). SEM revealed the microstructure of calcified nodules and EDX confirmed calcium phosphate mineralization with physiologically-relevant calcium to phosphorous ratios (Ca/P= 0.88 - 1.4). Conclusions: These results show that in vitro calcification is driven by shear stress in the presence of PAVEC and CS. As seen in ex vivo studies of human calcification, these microfluidic-derived nodules are similarly composed of a range of naturally-occurring calcium phosphates. Given that CAVD has no targeted therapy, the creation of a physiologically relevant model of the aortic valve can provide a test bed for novel therapeutic interventions.

Endothelial to Mesenchymal Transformation-derived Activated Fibroblast Behavior in a 3D Culture Environment

Objective: In calcific aortic valve disease (CAVD), glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) and hyaluronic acid (HA) are present in the normally collagen-rich aortic valve fibrosa near calcified nodules. Previous work using a 3D in vitro model has shown that GAGs can also promote endothelial to mesenchymal transformation (EndMT). The role of mesenchymal transformation in the progression of CAVD remains unclear. The goal of this study is to investigate the role of GAGs and EndMT on valve cell behavior in a 3D, in vitro model of healthy and diseased valves. Methods: Three dimensional in-vitro cultures, representing healthy (collagen 1 hydrogels) or diseased (collagen 1 hydrogels + GAGs) valve tissue, were seeded with Porcine Aortic Valve Endothelial Cells (PAVEC) and Porcine Aortic Valve Interstitial Cells (PAVIC). Cell cultures were grown for 14 days in regular medium and then evaluated for calcified nodule formation, alkaline phosphatase activity, proliferation and protein expression relating to EndMT activity Results: Introduction of GAGs into the in vitro model resulted in decreased alkaline phosphatase activity. Hydrogels containing CS conditions induced the greatest calcification, as measured with Alzarin Red quantification, while gels containing HA conditions did not have calcification that was significantly different than controls. Proliferation results suggest high levels of cell division activity, for both PAVEC and PAVIC in CS conditions. Protein expression analysis indicated increased EndMT in hydrogels containing GAGs. Conclusions: 3D, in vitro models of the aortic valve simulated progression toward late stage CAVD. Further investigation of the downstream role of EndMT within the aortic valve microenvironment is critical to subsequent improvement in treatment options.

Late-stage Calcific Aortic Valve Disease Within an Aortic Valve-on-a-chip Model

Objective: Calcific aortic valve disease (CAVD) is a progressive disease ranging from aortic sclerosis to severe aortic stenosis. Current treatments include total valve replacement and drug interventions tailored to other cardiovascular diseases. In late-stage CAVD, glycosaminoglycans (GAGs), such as chondroitin sulfate (CS), deposit in the fibrosa layer. Here we utilize 3D microfluidic devices to study the effects of shear stress, CS, and endothelial cells on calcification in an aortic valve fibrosa model to understand CAVD progression. Methods: Valve-on-a-chip devices were fabricated utilizing a wafer mold, soft lithography, and corona discharge. Devices have a flow channel and 3D matrix reservoir of collagen-only healthy controls or with 1mg/mL or 20mg/mL CS. Porcine aortic valve interstitial cells (PAVIC) are embedded within and endothelial cells (PAVEC) seeded onto the matrix. Steady shear stress at 1dyne/cm2 and 20dyne/cm2 was applied using a peristaltic pump at 37°C and 5%CO2. Alizarin Red S (ARS) was used to assess calcific nodule formation after 14-day cultures. Results: PAVIC/PAVEC co-cultures with increasing shear stress and GAGs exhibit increased calcification compared to static controls (Figure 1). Quantitatively, ARS increased as shear stress increased, regardless of GAGs. In the presence of CS, analysis of nodules indicated that nodule size decreased with shear stress, however it increased the total average percent area stained. Valve-on-a-chip models with PAVIC-only show decreased calcium deposition when compared to co-cultures. Conclusions: In vitro calcification increases in the presence of shear stress, PAVEC, and increased concentrations of GAGs. Further studies will identify the chemical composition of nodules and to understand early-CAVD pathophysiology. Given that CAVD has no targeted therapies, the creation of a physiologically-relevant test-bed of the aortic valve will lead to contributions in new therapeutics.

Therapeutic inhibition of microRNA-34a ameliorates aortic valve calcification via modulation of Notch1-Runx2 signalling.

Calcific aortic valve stenosis (CAVS) is the most common valvular heart disease and is increased with elderly population. However, effective drug therapy has not been established yet. This study aimed to investigate the role of microRNAs (miRs) in the development of CAVS. We measured the expression of 10 miRs, which were reportedly involved in calcification by using human aortic valve tissue from patients who underwent aortic valve replacement with CAVS or aortic regurgitation (AR) and porcine aortic valve interstitial cells (AVICs) after treatment with osteogenic induction medium. We investigated whether a specific miR-inhibitor can suppress aortic valve calcification in wire injury CAVS mice model. Expression of miR-23a, miR-34a, miR-34c, miR-133a, miR-146a, and miR-155 was increased, and expression of miR-27a and miR-204 was decreased in valve tissues from CAVS compared with those from AR. Expression of Notch1 was decreased, and expression of Runt-related transcription factor 2 (Runx2) was increased in patients with CAVS compared with those with AR. We selected miR-34a among increased miRs in porcine AVICs after osteogenic treatment, which was consistent with results from patients with CAVS. MiR-34a increased calcium deposition in AVICs compared with miR-control. Notch1 expression was decreased, and Runx2 expression was increased in miR-34a transfected AVICs compared with that in miR-control. Conversely, inhibition of miR-34a significantly attenuated these calcification signals in AVICs compared with miR-control. RNA pull-down assay revealed that miR-34a directly targeted Notch1 expression by binding to Notch1 mRNA 3' untranslated region. In wire injury CAVS mice, locked nucleic acid miR-34a inhibitor suppressed aortic velocity, calcium deposition of aortic valves, and cardiac hypertrophy, which were involved in decreased Runx2 and increased Notch1 expressions. miR-34a plays an important role in the development of CAVS via Notch1-Runx2 signalling pathway. Inhibition of miR-34a may be the therapeutic target for CAVS.

Inhibition of acetylation of histones 3 and 4 attenuates aortic valve calcification

Aortic valve calcification develops in patients with chronic kidney disease who have calcium and phosphate metabolic disorders and poor prognoses. There is no effective treatment except valve replacement. However, metabolic disorders put patients at high risk for surgery. Increased acetylation of histones 3 and 4 is present in interstitial cells from human calcific aortic valves, but whether it is involved in aortic valve calcification has not been studied. In this study, we found that treating cultured porcine aortic valve interstitial cells with a high-calcium/high-phosphate medium induced calcium deposition, apoptosis, and expression of osteogenic marker genes, producing a phenotype resembling valve calcification in vivo. These phenotypic changes were attenuated by the histone acetyltransferase inhibitor C646. C646 treatment increased the levels of class I histone deacetylase members and decreased the acetylation of histones 3 and 4 induced by the high-calcium/high-phosphate treatment. Conversely, the histone deacetylase inhibitor suberoylanilide hydroxamic acid promoted valve interstitial cell calcification. In a mouse model of aortic valve calcification induced by adenine and vitamin D treatment, the levels of acetylated histones 3 and 4 were increased in the calcified aortic valves. Treatment of the models with C646 attenuated aortic valve calcification by restoring the levels of acetylated histones 3 and 4. These observations suggest that increased acetylation of histones 3 and 4 is part of the pathogenesis of aortic valve calcification associated with calcium and phosphate metabolic disorders. Targeting acetylated histones 3 and 4 may be a potential therapy for inoperable aortic valve calcification in chronic kidney disease patients.

Low-level overexpression of p53 promotes warfarin-induced calcification of porcine aortic valve interstitial cells by activating Slug gene transcription

The most frequently used oral anti-coagulant warfarin has been implicated in inducing calcification of aortic valve interstitial cells (AVICs), whereas the mechanism is not fully understood. The low-level activation of p53 is found to be involved in osteogenic transdifferentiation and calcification of AVICs. Whether p53 participates in warfarin-induced AVIC calcification remains unknown. In this study, we investigated the role of low-level p53 overexpression in warfarin-induced porcine AVIC (pAVIC) calcification. Immunostaining, quantitative PCR, and Western blotting revealed that p53 was expressed in human and pAVICs and that p53 expression was slightly increased in calcific human aortic valves compared with non-calcific valves. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining indicated that apoptosis slightly increased in calcific aortic valves than in non-calcific valves. Warfarin treatment led to a low-level increase of p53 mRNA and protein in both pAVICs and mouse aortic valves. Low-level overexpression of p53 in pAVICs via an adenovirus vector did not affect pAVIC apoptosis but promoted warfarin-induced calcium deposition and expression of osteogenic markers. shRNA-mediated p53 knockdown attenuated the pAVIC calcium deposition and osteogenic marker expression. Moreover, ChIP and luciferase assays showed that p53 was recruited to the slug promoter and activated slug expression in calcific pAVICs. Of note, overexpression of Slug increased osteogenic marker Runx2 expression, but not pAVIC calcium deposition, and Slug knockdown attenuated pAVIC calcification and p53-mediated pAVIC calcium deposition and expression of osteogenic markers. In conclusion, we found that p53 plays an important role in warfarin induced pAVIC calcification, and increased slug transcription by p53 is required for p53-mediated pAVIC calcification.

Exploiting novel valve interstitial cell lines to study calcific aortic valve disease.

Calcific aortic valve disease (CAVD) involves progressive valve leaflet thickening and severe calcification, impairing leaflet motion. The in vitro calcification of primary rat, human, porcine and bovine aortic valve interstitial cells (VICs) is commonly employed to investigate CAVD mechanisms. However, to date, no published studies have utilised cell lines to investigate this process. The present study has therefore generated and evaluated the calcification potential of immortalized cell lines derived from sheep and rat VICs. Immortalised sheep (SAVIC) and rat (RAVIC) cell lines were produced by transduction with a recombinant lentivirus encoding the Simian virus (SV40) large and small T antigens (sheep), or large T antigen only (rat), which expressed markers of VICs (vimentin and α-smooth muscle actin). Calcification was induced in the presence of calcium (Ca; 2.7 mM) in SAVICs (1.9 fold; P<0.001) and RAVICs (4.6 fold; P<0.01). Furthermore, a synergistic effect of calcium and phosphate was observed (2.7 mM Ca/2.0 mM Pi) on VIC calcification in the two cell lines (P<0.001). Analysis of SAVICs revealed significant increases in the mRNA expression of two key genes associated with vascular calcification in cells cultured under calcifying conditions, runt related transcription factor-2 (RUNX2;1.3 fold; P<0.05 in 4.5 mM Ca) and sodium-dependent phosphate transporter-1 (PiT1; 1.2 fold; P<0.05 in 5.4 mM Ca). A concomitant decrease in the expression of the calcification inhibitor matrix Gla protein (MGP) was noted at 3.6 mM Ca (1.3 fold; P<0.01). Assessment of RAVICs revealed alterations in Runx2, Pit1 and Mgp mRNA expression levels (P<0.01). Furthermore, a significant reduction in calcification was observed in SAVICs following treatment with established calcification inhibitors, pyrophosphate (1.8 fold; P<0.01) and etidronate (3.2 fold; P<0.01). Overall, the present study demonstrated that the use of immortalised sheep and rat VIC cell lines is a convenient and cost effective system to investigate CAVD in vitro, and will make a useful contribution to increasing current understanding of the pathophysiological process.

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

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

Bone Morphogenetic Protein Signaling Is Required for Aortic Valve Calcification.

Calcific aortic valve disease (CAVD) is the most prevalent type of heart valve disease, affecting ≈2% of the US population. CAVD is characterized by the presence of calcific nodules, resulting in aortic valve (AoV) stenosis; however, the underlying mechanisms driving disease remain unknown. Studies of human diseased AoV provide initial evidence that bone morphogenetic protein (BMP) signaling, essential for normal bone formation, is activated during CAVD. Mice deficient in Klotho, an FGF23 transmembrane coreceptor, exhibit premature aging and develop AoV calcific nodules as occurs in human CAVD. The role of BMP signaling in the development of CAVD was examined in porcine aortic valve interstitial cells (VICs) and Klotho(-/-) mice. We show that activation of BMP signaling, as indicated by pSmad1/5/8 expression, precedes and later localizes with AoV calcification in Klotho(-/-) mice. In addition, cellular and extracellular matrix changes resembling features of normal bone formation are accompanied by increased osteochondrogenic gene induction in calcified Klotho(-/-) AoV. Likewise, osteogenic media treatment of porcine VICs results in BMP pathway activation, increased osteochondrogenic gene induction, and formation of calcific nodules in vitro. We demonstrate that genetic inactivation of the BMP type IA receptor in Klotho(-/-) aortic VICs, as well as BMP pathway inhibition of osteogenic media-treated aortic VICs in vitro, results in the inhibition of AoV calcification. BMP signaling and osteochondrogenic gene induction are active in calcified Klotho(-/-) AoV in vivo and calcified porcine aortic VICs in vitro. Importantly, BMP signaling is required for the development of AoV calcification in vitro and in vivo.

Deficiency in the Anti‐Aging Gene Klotho Promotes Aortic Valve Fibrosis through AMPKα‐mediated Activation of RUNX2

BackgroundFibrotic aortic valve disease (FAVD) is an important cause of aortic stenosis, yet currently there is no effective treatment for FAVD due to its unknown etiology. The purpose of this study is to investigate whether deficiency in the anti‐aging Klotho gene (KL) promotes high‐fat‐diet‐induced FAVD and to explore the underlying molecular mechanism.Methods &amp; ResultsHeterozygous Klotho‐deficient (KL+/−) mice and WT littermates were fed with a high‐fat diet (HFD) or normal diet for 13 weeks, followed by treatment with the AMPKa activator (AICAR) for an additional 2 weeks. A HFD caused a greater increase in collagen levels in the aortic valves of KL+/− mice than of WT mice, indicating that Klotho deficiency promotes HFD‐induced aortic valve fibrosis (AVF). AMPKa activity (pAMPKα) was decreased while protein expression of collagen I and RUNX2 was increased in the aortic valves of KL+/− mice fed with a HFD. Treatment with AICAR markedly attenuated HFD‐induced AVF in KL+/− mice. AICAR not only abolished the downregulation of pAMPKα but also eliminated the upregulation of collagen I and RUNX2 in the aortic valves of KL+/− mice fed with HFD. In cultured porcine aortic valve interstitial cells, Klotho‐deficient serum plus cholesterol increased RUNX2 and collagen I protein expression, which were attenuated by activation of AMPKa by AICAR. Interestingly, silencing of RUNX2 abolished the stimulatory effect of Klotho deficiency on cholesterol‐induced upregulation of matrix proteins, including collagen I and osteocalcin.ConclusionKlotho gene deficiency promotes HFD‐induced fibrosis in aortic valves, likely through the AMPKa–RUNX2 pathway.Support or Funding Informationsupported by NIH R01 HL118558, DK093403, and HL105302.

Ascorbic acid promotes extracellular matrix deposition while preserving valve interstitial cell quiescence within 3D hydrogel scaffolds.

Current options for aortic valve replacements are non-viable and thus lack the ability to grow and remodel, which can be problematic for paediatric applications. Toward the development of living valve substitutes that can grow and remodel, porcine aortic valve interstitial cells (VICs) were isolated and encapsulated within proteolytically degradable and cell-adhesive poly(ethylene glycol) (PEG) hydrogels, in an effort to study their phenotypes and functions. The results showed that encapsulated VICs maintained high viability and proliferated within the hydrogels. The VICs actively remodelled the hydrogels via secretion of matrix metalloproteinase-2 (MMP-2) and deposition of new extracellular matrix (ECM) components, including collagens I and III. The soft hydrogels with compressive moduli of ~4.3 kPa quickly reverted VICs from an activated myofibroblastic phenotype to a quiescent, unactivated phenotype, evidenced by the loss of α-smooth muscle actin expression upon encapsulation. In an effort to promote VIC-mediated ECM production, ascorbic acid (AA) was supplemented in the medium to investigate its effects on VIC function and phenotype. AA treatment enhanced VIC spreading and proliferation, and inhibited apoptosis. AA treatment also promoted VIC-mediated ECM remodelling by increasing MMP-2 activity and depositing collagens I and III. AA treatment did not significantly influence the expression of α-smooth muscle actin (myofibroblast activation marker) and alkaline phosphatase (osteogenic differentiation marker). No calcification or nodule formation was observed within the cell-laden hydrogels, with or without AA treatment. These results suggest the potential of this system and the beneficial effect of AA in heart valve tissue engineering. Copyright © 2015 John Wiley & Sons, Ltd.