Telocytes-derived extracellular vesicles alleviate aortic valve calcification by carrying miR-30b.

Abstract

AimsCalcific aortic valve disease (CAVD) is frequent in the elderly. Telocytes (TCs) are implicated in intercellular communication by releasing extracellular vesicles (EVs). This study investigated the role of TC‐EVs in aortic valve calcification.Methods and resultsTCs were obtained and identified using enzymolysis method and flow cytometry. EVs were isolated from TCs using differential high‐speed centrifugation method and identified using transmission electron microscope, western blot, and qNano analysis. The mouse model of CAVD was established. The changes of aortic valve activity‐related indicators were analysed by ultrasound, and the expressions of TC markers CD34 and vimentin in mouse valve tissues were detected using RT‐qPCR and western blot. The model mice were injected with TC‐derived EVs. The expressions of Runx2, osteocalcin, and caspase‐3 were detected using RT‐qPCR and western blot. The calcification model of valvular interstitial cells (VICs) was established. TC‐EVs were co‐cultured with calcified VICs, and calcium deposition was detected using alizarin red S staining. miR‐30b expression in calcified valvular tissues and cells was detected after EV treatment. miR‐30b expression in TCs was knocked down and then EVs were extracted and co‐cultured with calcified VICs. The target of miR‐30b was predicted through bioinformatics website and verified using dual‐luciferase assay. The levels of Wnt/β‐catenin pathway‐related proteins were detected. ApoE−/− mice fed with a high‐fat diet showed decreased aortic valve orifice area, increased aortic transvalvular pressure difference and velocity, reduced left ventricular ejection fraction, decreased CD34 and vimentin, and increased caspase‐3, Runx2, and osteocalcin. The levels of apoptosis‐ and osteogenesis‐ related proteins were inhibited after EV treatment. TC‐EVs reduced calcium deposition and osteogenic proteins in calcified VICs. EVs could be absorbed by VICs. miR‐30b expression was promoted in calcified valvular tissues and cells after EV treatment. Knockdown of miR‐30b weakened the inhibitory effects of TC‐EVs on calcium deposition and osteogenic proteins. miR‐30b targeted Runx2. EV treatment inhibited the Wnt/β‐catenin pathway, and knockdown of miR‐30b in TCs attenuated the inhibitory effect of TC‐EVs on the Wnt/β‐catenin pathway.ConclusionTC‐EVs played a protective role in aortic valve calcification via the miR‐30b/Runx2/Wnt/β‐catenin axis.