ClC-3氯通道对高血压状态下内皮祖细胞功能的调节作用及机制研究

Endothelial progenitor cells (EPC) play a significant role in neovascularization of ischemic tissues and in re-endothelialization of injured blood vessels. Our previous study has demonstrated that ClC-3 chloride channel is involved in vascular remodeling and endothelial dysfunction during hypertension. However, whether ClC-3 channel participates in the regulation of EPC function is still unclear. In our pilot study, we found that the expression of ClC-3 protein was significantly increased in Ang II-treated EPCs. Ang II treatment induced apparent EPC apoptosis, which was significantly inhibited by ClC-3 gene knockout. Furthermore, NADPH oxidase activity, as well as O2- level, was significantly lower in EPC from ClC-3 knockout mice than that from wild-type mice after Ang II administration. Therefore, the objective of this project is to determine how ClC-3 deficiency would influence the EPC number and function and the underlying mechanisms in both AngII-induced and DOCA-salt hypertension. Our proposed research may provide new insights regarding the effects of ClC-3 in cardiovascular diseases featuring EPC dysfunction and also provide a mechanistic basis for developing new target for drug production to combat EPC damage in hypertension.

内皮祖细胞（EPC）的数目和功能对维持血管内皮完整性发挥了重要作用。我们以往研究证明ClC-3氯通道参与了高血压血管重构及内皮功能不全的调节， 然而，ClC-3 是否参与了高血压状态下EPC的功能调节尚不清楚。我们的预实验结果证实 Ang II 能够使小鼠EPC中ClC-3的蛋白表达明显增加，ClC-3基因敲除能够显著抑制AngII 诱导的EPC凋亡，并使EPC中NADPH 氧化酶活性降低及O2-生成减少。在以上研究的基础上，本项目拟利用ClC-3 基因敲除小鼠，将离体细胞实验与整体实验相结合，从分子和离子通道水平研究ClC-3 对高血压 EPC氧化应激水平及EPC 数目和功能的调节作用及可能机制。 为进一步阐明心血管疾病中EPC功能受损的机制,并寻找防治EPC受损的新药新靶点提供实验依据。

我们以往的研究发现，随着血压的升高，ClC-3 容积调节性氯通道的表达和活性明显增加，且和血压的升高呈正相关，ClC-3 参与了高血压诱导的血管重构及内皮功能不全，提示ClC-3 在高血压的发生发展中起了重要的作用。内皮祖细胞（EPC））在维持血管内皮完整性和血管新生中发挥了重要作用，然而ClC-3是否参与了EPC的功能调控仍不清楚。在本项目中，我们采用ClC-3 基因敲除小鼠及腺病毒转染ClC-3 siRNA 和cDNA等技术，探讨该通道蛋白对EPC数目和功能的影响及其机制。结果发现，小鼠骨髓EPC中有内源性ClC-3表达，AngII 可使ClC-3表达升高，并诱导EPC凋亡，bcl-2/bax比值下降，线粒体膜电位降低，和PARP激活。ClC-3基因敲除则抑制了AngII 诱导的这些作用。进一步研究发现其机制与ClC-3影响了AngII诱导的NADPH氧化酶的激活及ROS生成有关，沉默ClC-3可降低AngII诱导的NADPH氧化酶胞膜亚基NOX2和p22phox的蛋白表达；而且，ClC-3缺失还可通过抑制p38MAPK信号途径下调p47phox的磷酸化而抑制胞浆亚基p47phox 和p67phox向胞膜的转位，从而减少NADPH氧化酶复合物的组装，使酶活性降低，超氧阴离子生成减少。高表达ClC-3则促进了这些作用，并增加AngII诱导的ROS生成和细胞老化。这些结果提示，在AngII诱导的高血压等氧化应激状态下，抑制ClC-3的异常升高可保护EPC的存活。进一步的研究发现，如果在正常状态下，敲除ClC-3基因却明显影响了EPC的功能，与对照组小鼠相比，ClC-3敲除小鼠的骨髓EPC的迁移能力，粘附能力及成管能力均明显下降；在体后肢缺血模型显示，ClC-3敲除小鼠的缺血后肢血流恢复下降，新生毛细血管数目减少。这一作用可能与ClC-3抑制了EPC中趋化因子受体CXCR4的表达及其下游信号JAK2的激活有关。 该项目的研究从多个方面探讨了ClC-3对EPC数目和功能的调节作用，为更深入认识ClC-3在维持血管内皮功能稳态中的重要性提供了依据。