M1 macrophage-derived extracellular vesicle containing tsRNA-5006c promotes osteogenic differentiation of aortic valve interstitial cells through regulating mitophagy

DiI-labeled M1-polarized macrophages were internalized by aortic valve interstitial cells AVICs, resulting in significantly increased calcium nodule formation and expression of osteogenesis-related genes in AVICs, including RUNX2, BMP2, osteopontin, and SPP1, compared with NC-EVs. Small RNA sequencing revealed that 17 tsRNAs were significantly up-regulated such as tsRNA-5006c, while 28 tsRNAs were significantly down-regulated in M1-EVs compared with NC-EVs. Intriguingly, tsRNA-5006c-deleted M1-EVs treatment significantly reduced calcium nodule formation and expression of osteogenesis-related genes in AVICs relative to control group. Moreover, target genes of tsRNA-5006c were mainly involved in autophagy-related signaling pathways. Hallmarks of mitophagy activation in AVICs including mitophagosome formation, TMRM fluorescence were significantly elevated in the M1-EVs group compared with NC-EVs group, whereas M1-EVs tsRNA-5006c inhibitor led to a significantly reduction in these indicators.Conclusively, tsRNA-5006c regulates AVIC osteogenic differentiation from the perspective of mitophagy.