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

Calcific 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.

TCs 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.

TC-EVs played a protective role in aortic valve calcification via the miR-30b/Runx2/Wnt/β-catenin axis.