Calcification of aortic valvular interstitial cells induced by endothelin receptor blockers

Abstract

Calcified aortic stenosis (AS) is the most common acquired valvulopathy for which there is still no pharmacological treatment. Endothelin-1 (ET-1) is not only a powerful vasoconstrictor but also a pro-inflammatory and pro-fibrotic peptide whose role in AS remains unclear. The aim of this study was to characterize the role of ET-1 in the aortic valve calcification. Valvular endothelial cells (VECs), isolated from human aortic valves, were cultured in a cell perfusion system to assess ET-1 production in different fluid flow shear stress conditions. In addition, valvular interstitial cells (VICs) were cultured in a pro-calcifying culture medium (inorganic phosphate: 2 mM) with or without small-molecule and monoclonal antibody ET A and ET B receptor antagonists during 10 days. Calcium content was assessed using an o-cresolphtalein-based assay and red alizarin staining. VEC prepro-ET-1 and VIC osteogenic mRNA expression levels were evaluated by RTqPCR. Visualization of VIC ET A and ET B receptor was performed by immunohistochemistry. Turbulent shear stress, mimicking the flow conditions suffered by the valve at the aortic side that is the most prone to calcify, increased VEC prepro-ET-1 mRNA expression level and ET-1 release compared to laminar shear stress. Unexpectedly, calcium content and alizarin red staining of VICs were increased after blockade of ET B receptor and this effect was potentiated by concomitant blockade of ET A receptor. The mRNA expression of osteopontin, RUNX2, and BMP2 was similarly increased by ET B blockade. A perinuclear localization of ET A and ET B receptors in VICS was observed, suggesting a direct implication in the regulation of calcification genes. These results support a protective role of the endothelin system against the development of AS. Further studies are warranted to characterize the intracellular pathways and to confirm these results in ex vivo and in vivo models.