线粒体外膜蛋白Tom70介导VICs成骨分化在主动脉瓣钙化中的作用及机制研究

The pathogenesis of calcific aortic valve disease (CAVD) is complex, and osteogenesis of valve interstitial cells (VIC) is considered to be a key link leading to its occurrence. Studies have shown that mitochondrial homeostasis can promote osteogenic differentiation of VICs. The translocase of the outer membrane complex (Tom complex) is involved in maintaining mitochondrial homeostasis, however, the role of the Tom complex in CAVD is unclear. Our experiments found that Tom70 in human calcified valves was significantly lower. Aortic valve was thickening in untreated Tom70 KO mice under physiological conditions. Knocking down Tom70 can promote osteogenic differentiation of isolated VICs. This suggests that Tom70 may promote the development of CAVD through mediated osteogenic differentiation of VIC. This project intends to use Tie2-Gfp and VICs cell-specific Cre mice model and CRISPR-Cas9 technology to knock out Tom70 of VICs cells in vitro, to verify the role of Tom70 in CAVD, and to explore the mechanism Tom70-mediated osteogenic differentiation of VICs in the occurrence of CAVD. This study will reveal new mechanisms for the development of CAVD and provide new strategies for the prevention and treatment of clinical CAVD.

钙化性主动脉瓣疾病(CAVD)发病机制复杂，瓣膜间质细胞(VICs)成骨分化被认为是导致其发生发展的关键环节。研究表明，线粒体稳态失衡可以促进VICs成骨分化。线粒体转运酶复合物(Tom)参与维持了线粒体稳态，然而，Tom复合物在CAVD中的作用尚不清楚。我们前期工作发现，人体钙化瓣膜的Tom70表达明显下降，Tom70 KO小鼠正常喂养生理状态下的主动脉瓣膜明显增厚，细胞水平敲低原代VICs的Tom70可以促进其成骨分化。这提示Tom70可能通过介导VICs成骨分化促进CAVD的发生发展。本项目拟通过Tie2-Gfp和VICs细胞特异性Cre小鼠以及CRISPER-Cas9技术体外敲除VICs的Tom70，验证Tom70在CAVD中的作用，探索Tom70介导VICs成骨分化导致CAVD发生的分子机制。本研究将揭示CAVD发生的新机制，为临床CAVD的防治提供新策略。

钙化性主动脉瓣疾病（CAVD）是一种以主动脉瓣进行性纤维化、钙化重塑为特点的老年人群中常见的心脏瓣膜病。Tom70在CAVD中的作用尚未见报道。基于此，本研究中我们探讨了①Tom70在CAVD主动脉瓣组织及活化的AVICs中的表达模式；②体外干预Tom70的表达水平，观察对AVICs活化的影响；③小鼠Tom70基因敲除对主动脉瓣功能、心功能和CAVD发生发展的影响；④Tom70作用于CAVD的具体机制。我们发现在CAVD的人主动脉瓣组织和活化的人/大鼠AVICs中Tom70表达降低；Tom70表达降低促进大鼠AVICs活化和小鼠主动脉CAVD发生发展，而Tom70过表达抑制AVICs活化，即Tom70在AVICs活化进程中具有保护性作用；Tom70低表达导致AVICs自噬流受损，而Tom70过表达可恢复AVICs中受损的自噬流，即Tom70可维持AVICs自噬流通畅。自噬流受损促进AVICs活化，自噬流通畅抑制AVICs活化；Tom70低表达通过破坏自噬流进而促进AVICs活化，过表达Tom70能够维持自噬流通畅从而抑制AVICs的活化。敲低Tom70破坏自噬流，进而导致AVICs活化的机制为：敲低Tom70导致AVICs中EPA含量下降。即EPA介导了Tom70对AVICs自噬流和活化的调控。本研究为CAVD的机制探索提供了新的见解，证明了Tom70在AVICs活化过程中的保护作用，为自噬流和主动脉瓣病变的关系提供了新的证据，并将Tom70-EPA-自噬流相关联，为应用EPA这种小分子脂质化合物用于临床早期干预CAVD的进展提供了重要的证据，具有转化前景。