RhoA在AngⅡ介导的内皮祖细胞成血管中的作用及其机制研究

Renin-angiotensin system has become a novel therapeutic target in neovascularization-related disease. In our previous work, it was noticed that angiotensin Ⅱ (Ang Ⅱ) enhanced endothelial progenitor cell (EPC) angiogenetic-related function, however, the underlying mechanisms are still uncertain. In preliminary study, we observed that Ang Ⅱ increased EPC RhoA activation which was shown to play a significant role in neovascularization by a recent study. Therefore, we propose that RhoA and its following signaling play a role in Ang Ⅱ-induced angiogenic effects of EPC. We are about to carry out the research concerning the role and mechanism of RhoA in angiogenic effects of EPC. First, we are going to investigate the effects of Ang Ⅱ on EPC angiogenetic-related function in vitro. The involvement of RhoA and downstream effectors of MAPK family members in these effects also will be explored by using SiRNA and small molecule inhibitors. Second, we are going to investigate the effects of Ang Ⅱ on EPC angiogenetic property by tracing the transplanted EPC in vivo. Meanwhile, RhoA gene expression silenced-EPC will be transplanted in ischemic hindlimb model of Ang Ⅱ infused-nude rat, and then neovascularization and the activation of RhoA and MAPK in ischemic hindlimb will be observed. It is anticipated that an important target point and theory basis for the treatment of neovascular-related diseases will be proposed if this research is accomplished.

肾素-血管紧张素系统已成为血管新生相关疾病治疗的新靶点。我们前期研究提示：血管紧张素Ⅱ（Ang Ⅱ）促进内皮祖细胞（EPC）成血管，然而其具体作用机制尚不明确。在预实验中，我们观察到Ang Ⅱ促进EPC RhoA活化，而近期研究表明RhoA与血管新生有关。据此，我们推测，RhoA及其下游信号通路参与调控Ang Ⅱ介导的EPC成血管。为此，本项目拟首先在体外研究Ang Ⅱ对EPC成血管影响，并采用SiRNA及小分子抑制剂调控RhoA及其下游MAPK信号通路，探讨RhoA及MAPK通路在其中的作用；其次，在体实验采用细胞标记及活体示踪，观察Ang Ⅱ对EPC成血管影响，并采用RhoA基因沉默的EPC移植肢端缺血裸鼠，联合Ang II微泵，观察缺血肢端血管新生情况，并进一步明确RhoA及MAPK通路在Ang Ⅱ介导EPC成血管中的作用。本项目完成将为血管新生相关疾病的治疗提供重要靶点与理论依据。

研究发现：Ang II参与血管新生。在前期工作中，我们发现：Ang II促进内皮祖细胞（EPC）成血管（PLoS ONE, 2012）。鉴于EPC在血管新生中起重要作用，我们推测：Ang II促进血管新生与其增强EPC成血管有关。在预实验中，我们观察到Ang II促进EPC RhoA活化，而RhoA在血管新生中发挥重要作用。为此，我们拟深入探讨RhoA在EPC成血管中的作用及其机制，主要内容包括 (1)体外实验观察Ang II对EPC迁移、黏附、体外血管形成等成血管相关功能影响；(2)进一步探讨RhoA/ROCK及其下游MAPK通路在其中的作用。研究发现：(1) Ang II通过促进EPCs迁移、黏附功能和体外血管形成能力，从而促进血管新生，Ang II不影响EPCs增殖能力。(2) RhoA/ROCK通路抑制剂C3 exoenzyme, GGTI-286和Y-27632可有效抑制Ang II对EPCs迁移、黏附功能和体外血管形成能力的影响。(3) Ang II增加RhoA的活性，该作用能被Rho的抑制剂C3 exoenzyme有效抑制。(4) Ang II促进JNK和p38的激活，该作用能被C3 exoenzyme, GGTI-286和Y-27632抑制。研究成果以第一作者共发表SCI收录论文2篇，另以第一作者发表核心期刊论文1篇。本项目的完成明确RhoA在EPC成血管中的作用及其机制，为治疗血管新生相关疾病提供新靶点。