2-ME调控HIF-1α信号通路抑制硬脑膜动静脉瘘血管生成的作用机制研究

Angiogenesis is the pathological basis of dural arteriovenous fistula (DAVF), leading to arteriovenous shunt in dura. Our previous studies revealed that hypoxia-inducible factor (HIF) -1α mediated angiogenesis caused by hypoxia plays a key role in DAVF formation, and thus it is a potential therapeutic target in DAVF. Meanwhile, our studies also show that 2-methoxyestradiol (2-ME) can remarkably suppress dural angiogenesis mediated by HIF-1α. However, detailed upstream signal pathway and regulatory network are still unclear. In this project, we have firstly used human umbilical vein endothelial cell (HUVEC) for screening candidate upstream factors regulated by 2-ME through RNA microarray. Then we will check each pathway by inhibiting or overexpressing candidate regulatory factors in vitro. Furthermore, we will examine the interaction of those molecules by co-immunoprecipitation, bimolecular fluorescence complementation (BiFC) and mutant tecniques. In the last stage, we will verify each signal pathway which can suppress HIF-1α regulated by 2-ME in vivo, through a modified DAVF rat model and local lentivirus intervention. Finally we will try to build regulatory network of 2-ME among HIF-1α and upstream factors, which can provide theoretical basis for further clinical trials.

硬脑膜动静脉瘘(DAVF)的病理基础是硬脑膜动静脉之间形成新生血管，导致动静脉短路。我们曾发现低氧诱导因子（HIF-1α）介导的血管生成对DAVF发病起关键作用，是极具前景的治疗靶标。2-甲氧雌二醇（2-ME）能通过抑制HIF-1α逆转大鼠DAVF形成，但分子机制尚不明确。本项目前期已构建人脐静脉内皮细胞（HUVEC）低氧模型，并已利用RNA芯片筛选2-ME抑制HIF-1α的信号通路，拟采用基因敲减（shRNA）或过表达的方法对其中的关键调控因子加以验证，以免疫共沉淀、双分子互补实验（BiFC）和构建突变体验证上游调控因子与HIF-1α的相互作用。再采用经我们创新改良的DAVF大鼠模型，颅内注射搭载关键调控因子RNA/shRNA的慢病毒，在体内验证2-ME抑制DAVF中HIF-1α激活的信号通路，据此构建2-ME抑制DAVF中HIF-1α的调控网络，为应用2-ME治疗DAVF提供理论基础。

硬脑膜动静脉瘘(DAVF)的病理基础是硬脑膜动静脉之间形成新生血管，导致动静脉短路。我们曾发现低氧诱导因子（HIF-1α）介导的血管生成对DAVF发病起关键作用，是极具前景的治疗靶标。2-甲氧雌二醇（2-ME）能通过抑制HIF-1α逆转大鼠DAVF形成，但分子机制尚不明确。本项目前期已构建人脐静脉内皮细胞（HUVEC）低氧模型，并已利用RNA芯片筛选2-ME抑制HIF-1α的信号通路，拟采用基因敲减或过表达的方法对其中的关键调控因子加以验证上游调控因子与HIF-1α的相互作用，发现ID-1和p53可能介导2-ME对于HIF-1α的调控作用。项目分为体内外两部分，体内研究中采用经我们创新改良的DAVF大鼠模型，颅内注射敲低/过表达ID-1和p53病毒载体，在体内验证2-ME抑制DAVF中HIF-1α激活的信号通路，并进一步抑制了下游血管生成因子的表达，降低脑内微血管密度，延缓疾病进展；后通过体外实验，进一步明确2-ME通过ID-1和p53调控HIF-1α的作用机制，以及对于内皮细胞功能的影响。据此我们构建2-ME抑制DAVF中HIF-1α表达的调控网络，为应用2-ME治疗DAVF提供理论基础。