激活血管内皮细胞TMEM16A钙激活氯通道通过PPARγ/ABCA1抑制胆固醇负向调控其表达和通道活性产生抑制胆固醇及血管生成的抗动脉粥状硬化的机制

Atherosclerosis is the main cause of cardiovascular and cerebrovascular diseases. Hypercholesterolemia is the main cause that results in endothelial cell injury leading to the development of atherosclerosis. Identification of new biomarkers will be a promising therapeutic strategy for treating atherosclerosis. We found that the Ca2+-activated chloride channel TMEM16A was expressed in the endothelial cells and mediated the Ca2+-activated chloride currents. TMEM16A overexpression activated PPARγ, and inhibited endothelial cell proliferation, migration and angiogenesis. In addition, we found that cholesterol inhibited TMEM16A expression, and directly inhibited TMEM16A-mediated Ca2+-activated Cl- currents in endothelial cells. Furthermore, TMEM16A overexpression reduced the cellular cholesterol contents. The purpose of this project is to identify the mechanisms by which TMEM16A inhibits endothelial cell angiogenesis via activation of PPARγ, to investigate the mechanisms by which cholesterol inhibits TMEM16A expression via EZH2/DNMT1, and inhibits TMEM16A Ca2+-activated Cl- currents via the cholesterol-binding site on the channel in the endothelial cells, and to explore the mechanisms by which TMEM16A promotes cholesterol efflux in the endothelial cells via PPARγ/ABCA1, and to elucidate the anti-atherosclerotic mechanisms by which TMEM16A reduces cholesterol contents and inhibits angiogenesis in endothelial cells. Achieving these goals will provide the theoretical and experimental basis for the development of TMEM16A-targeted drugs for the treatment of atherosclerosis.

动脉粥状硬化是导致心脑疾病的主要原因，高胆固醇血症是导致内皮损伤诱发动脉粥状硬化的主要因素。发现新靶点有望成为治疗动脉粥状硬化的突破口。申请者发现TMEM16A钙激活氯通道表达在血管内皮细胞上，激活PPARγ，抑制内皮细胞增生、迁移和血管生成。申请者还发现胆固醇抑制TMEM16A蛋白表达，直接抑制TMEM16A钙激活氯电流；而过表达TMEM16A抑制细胞内胆固醇含量。本项目拟明确TMEM16A通过激活PPARγ抑制内皮细胞血管生成的作用机制，阐明胆固醇通过EZH2/DNMT1抑制TMEM16A表达及通过胆固醇结合位点抑制TMEM16A钙激活氯通道功能的作用；验证TMEM16A通过激活PPARγ/ABCA1促进胆固醇外排，进而揭示激活TMEM16A通过PPARγ抑制内皮细胞胆固醇和血管生成的抗动脉粥状硬化作用机制。研究成果为研发靶向TMEM16A治疗动脉粥状硬化的药物提供理论和实验基础。

引言：Ca2+激活的Cl-通道TMEM16A在内皮细胞中表达，并与许多疾病，诸如高血压、血脑屏障功能障碍和肺动脉高压等相关。TMEM16A钙激活氯通道参与了许多生理和病理进程，但目前仍不清楚TMEM16A是否调节内皮细胞的血管生成。胆固醇调节包括TMEM16A钙激活氯通道在内的许多离子通道，高胆固醇水平能促进内皮功能紊乱的发生发展。在内皮细胞中，胆固醇是否参与调节TMEM16A的表达和功能还有待研究。.目的：本研究旨在研究胆固醇是否调控TMEM16A在内皮细胞血管生成中的表达和功能。.方法：使用全细胞膜片钳技术记录人主动脉内皮细胞（HAECs）和转染了TMEM16A高表达质粒的HEK293细胞中的钙激活氯电流。Western Blot实验来检测人主动脉内皮细胞中TMEM16A和DNMT1（DNA甲基转移酶）的表达。CCK-8实验、伤口愈合实验和血管生成实验分别用来检测内皮细胞的增殖、迁移和血管生成。.结果：TMEM16A介导了人主动脉内皮细胞（HAEC）中的钙激活氯通道。在人主动脉内皮细胞（HAEC）中，胆固醇通过上调DNMT1来抑制TMEM16A的表达，并且胆固醇对TMEM16A表达的抑制作用可以被DNMT1抑制剂5-aza阻断。此外，在HEK293细胞中，胆固醇能直接抑制TMEM16A电流，IC50为0.1209 lM。同样，胆固醇也直接抑制了HAECs中TMEM16A的电流。TMEM16A基因敲低促进了了HAECs的细胞增殖、迁移和血管生成，TMEM16A基因过表达则产生相反的效果。.结论：这项研究揭示了胆固醇介导的TMEM16A抑制的新机制，胆固醇通过DNMT1介导的甲基化降低TMEM16A的表达，并直接抑制通道活性。抑制TMEM16A通道可促进内皮细胞的血管生成。