M1 macrophage-derived extracellular vesicle containing tsRNA-5006c promotes osteogenic differentiation of aortic valve interstitial cells through regulating mitophagy
Hao Xia, Mingjian Gao, Jun Chen, Guanshen Huang, Xiuting Xiang, Yuyan Wang, Zhaohui Huang, Yongchun Li, Shuang Su, Zewei Zhao, Qingchun Zeng, Yunjun Ruan
Abstract
Background: Osteogenic differentiation of aortic valve interstitial cells (AVICs) plays a key role in the calcific aortic valve disease progression. Extracellular vesicles (EVs)-derived from M1-polarized macrophages (M1-EVs) orchestrated intercellular communication by delivering non-coding RNAs such as tRNA-derived small RNAs (tsRNAs) is crucial for cardiovascular disease. However, the role and mechanism of M1-EVs tsRNAs in osteogenic differentiation of AVICs remains largely unclear. Methods: M1-EVs and PBS treated-RAW 264.7 cell-derived EVs (NC-EVs) were incubated with AVICs and subjected to small RNA sequencing. Candidate tsRNA in M1-EVs was silenced to explore their effects on AVIC osteogenic differentiation and mitophagy. Results: DiI-labeled M1-EVs were internalized by 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, such as MAPK, Ras, Wnt, and Hippo signaling pathway. Hallmarks of mitophagy activation in AVICs including mitophagosome formation, TMRM fluorescence, expression of LC3-II, BINP3, and PGC1α, were significantly elevated in the M1-EVs group compared with NC-EVs group, whereas M1-EVs tsRNA-5006c inhibitor led to a significant reduction in these indicators. Conclusion: M1-EVs carried tsRNA-5006c regulates AVIC osteogenic differentiation from the perspective of mitophagy, and we provide a new target for the prevention and treatment of aortic valve calcification.