Implication of endothelin-1 in human aortic valve calcification

Abstract

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.