Treatment with XAV-939 prevents in vitro calcification of human valvular interstitial cells

Calcified aortic valve disease (CAVD), the most common valvular heart disease, lacks pharmaceutical treatment options because its pathogenesis remains unclear. This disease with a complex macroenvironment characterizes notable cellular heterogeneity. Therefore, a comprehensive understanding of cellular diversity and cell-to-cell communication are essential for elucidating the mechanisms driving CAVD progression and developing therapeutic targets. In this study, we used single-cell RNA sequencing (scRNA-seq) analysis to describe the comprehensive transcriptomic landscape and cell-to-cell interactions. The transitional valvular endothelial cells (tVECs), an intermediate state during the endothelial-to-mesenchymal transition (EndMT), could be a target to interfere with EndMT progression. Moreover, matrix valvular interstitial cells (mVICs) with high expression of midkine (MDK) interact with activated valvular interstitial cells (aVICs) and compliment-activated valvular interstitial cells (cVICs) through the MK pathway. Then, MDK inhibited calcification of VICs that calcification was validated by Alizarin Red S staining, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blotting assays in vitro. Therefore, we speculated that mVICs secreted MDK to prevent VICs’ calcification. Together, these findings delineate the aortic valve cells’ heterogeneity, underlining the importance of intercellular cross talk and MDK, which may offer a potential therapeutic strategy as a novel inhibitor of CAVD.

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

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a303 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Paolo Poggio ◽

Emanuela Branchetti ◽

Rachana Sainger ◽

Juan Grau ◽

Eric Lai ◽

...

Keyword(s):

Ex Vivo ◽

Early Stage ◽

Interstitial Cells ◽

Calcium Deposition ◽

Proximity Ligation Assay ◽

Valvular Interstitial Cells ◽

Direct Role ◽

Aortic Sclerosis

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.

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

Cardiology ◽

10.1159/000369126 ◽

2014 ◽

Vol 130 (1) ◽

pp. 55-61 ◽

Cited By ~ 14

Author(s):

Fei Li ◽

Zhihong Zhao ◽

Zhejun Cai ◽

Nianguo Dong ◽

Yi Liu

Keyword(s):

Osteogenic Differentiation ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Interstitial Cells ◽

Osteoblastic Differentiation ◽

Mitogen Activated Protein Kinase ◽

Low Density ◽

Valvular Interstitial Cells ◽

Oxidized Low Density Lipoprotein

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.

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