MAM介导的EPCs线粒体自噬在动脉粥样硬化内皮损伤修复中的作用及机制研究

The impairment of vascular endothelium is a general pathological process in most cardiovascular diseases. Endothelial progenitor cells (EPCs) play a major role in the maintenance of endothelial integrity and contribute to re-endothelialization of injured vessels. However, the poor viability and proliferation limit the repairment of EPCs in atherosclerotic environment. Therefore, improving the survival of EPCs is crucial to delaying or blocking the development of atherosclerosis. Recently, we have confirmed that CAMKKII-mediated autophagy promoted EPCs survival and proliferation in atherosclerotic environment, but the type of autophagy as well as the exact mechanism remained unclear. Interestingly, our preliminary experiment revealed that PINK1-PARK2-mediated mitophagy was inhibited in EPCs from atherosclerosis model mice. Furthermore, both the autophagic marker LC3II and calcium signaling protein CAMKKII localized less on mitochondria-associated endoplasmic reticulum membrane (MAM) of EPCs from atherosclerosis model mice. Combining with our previous study, we speculate that atherosclerotic environment leads EPCs bio-function and mitophagy impaired, enhancement of MAM-mediated calcium flux from endoplasmic reticulum to mitochondrion reverses EPCs mitophagy through activating CAMKKII that localized on MAM, which will be able to promote EPCs survival and bio-function as well as accelerate vascular endothelium repairment in atherosclerotic environment. As a consequence, we plan to employ proteomics, gene transfection, electrophysiology and other molecular biological techniques to explore the mechanism of CAMKKII-CAMKI-PINK1 signaling pathway in MAM-mediated mitophagy as well as the role of mitophagy in EPC function and endothelium repairment. This study will provide new theoretical basis and intervention targets for the prevention and treatment of atherosclerosis.

血管内皮损伤是众多心血管疾病的共同病理基础，内皮祖细胞（EPCs）是内皮修复的主要参与者。然而EPCs在动脉粥样硬化（AS）环境下生存能力低，增殖能力弱，限制了血管内皮的修复；改善其在应激环境下生存能力是目前亟待解决的问题。申请人新近报道了CAMKKII介导的细胞自噬能够促进EPCs生存、维持增殖，但何种自噬途径及其分子机制尚待明确。结合既往文献及预实验我们推测：在AS环境下EPCs生物学功能和线粒体自噬受损，而增强线粒体-内质网膜偶联结构（MAM）介导的内质网-线粒体单向跨膜钙流，激活CAMKKII通路，上调线粒体自噬水平，促进EPCs生存，提高AS环境下血管内皮修复。本项目拟运用蛋白组学、基因转染、电生理等分子生物学技术，明确MAM介导的单向跨膜钙流激活CAMKKII-CAMKI-PINK1线粒体自噬途径，提高EPCs生物学功能和促进血管内皮修复的新分子机制，为血管内皮再生提供新方向。

自噬是维持细胞生存的重要机制。无论在动脉粥样硬化（AS），还是缺氧的环境中，自噬的缺失将导致细胞功能的抑制，甚至死亡；然而其中的具体分子机制仍尚待研究。在AS的发生中，内皮细胞的损伤和功能异常是其中的始动环节，循环中内皮祖细胞（EPCs）是内皮修复的主要参与者。然而EPCs在AS环境下生存能力低，增殖能力弱，限制了血管内皮的修复。我们围绕自噬在AS和缺氧环境下细胞的生存机制开展了系列研究。我们采用ApoE敲除小鼠高脂饮食的方式构建了AS模型小鼠，发现EPCs线粒体功能降低，线粒体自噬流受到抑制，EPCs增殖能力显著降低。我们还发现他汀类药物能够激活线粒体钙释放，上调EPCs内钙浓度，进一步磷酸化钙调蛋白CAMK1，而CAMK1作为一种全新的PINK1蛋白激酶磷酸化PINK1，从而促进经典的PINK1-PARK2依赖的线粒体自噬，改善了EPCs在AS环境下的增殖、生存能力。结合PINK1敲除小鼠的在体研究进一步证实了CAMK1-PINK1介导的线粒体自噬是EPCs修复受损血管内皮的关键分子机制。此外，为进一步探索内质网线粒体膜耦联结构（MAM）在缺氧致心肌损伤中的机制，我们通过将C57B/L小鼠置于低压低氧舱中，建立了低压缺氧模型小鼠，6周后我们分离小鼠的心肌组织并行蛋白组学和代谢组学高通量分析，发现了MAM中关键蛋白分子PACS2减少显著（减少了3.16倍），线粒体自噬流、能量代谢均受到抑制。MAM上PACS2介导的内质网线粒体单向跨膜钙流是线粒体自噬及氧化磷酸化维持的关键分子机制。在心肌细胞特异性过表达的PACS2小鼠（Pacs2flox/flox/CreαMHC+/−）后，不仅维持了缺氧环境下的线粒体自噬和能量代谢，更保护了缺氧环境下心脏功能。我们上述围绕自噬的相关研究为血管损伤修复提供了新的靶点，为缺氧环境下心脏的保护提供了新机制；同时也揭示了MAM中PACS2的缺失是缺氧环境下致心肌损伤的关键分子机制，为心血管系统的保护提供了新的防治方向。