支架蛋白PHACTR1通过诱导血管内皮细胞Akt活化促进血管新生的机制研究

Angiogenesis plays an important role in ischemic diseases. We analyzed GWAS studies published recently, and found that rs9349379 is strongly associated with ischemic diseases, including myocardial infarction and ischemic stroke. rs9349379 is located in the intron region of PHACTR1 (phosphatase and actin regulator 1) and is the binding site of MEF2 to regulate PHACTR1 expression. However, there have been no in vivo studies investigating the function of PHACTR1 in angiogenesis. Therefore, we generated PHACTR1 knockout mice. The mice display impaired retinal angiogenesis but show no other phenotypes. PHACTR1 knockout by siRNA in human umbilical vein endothelial cells suppresses tube formation, and inhibits VEGF-induced Akt activation, but not ERK1/2 or p38. Thus we hypothesize that PHACTR1 promotes angiogenesis by inducing Akt activation as a scaffold protein in endothelial cells. To prove our hypothesis, we will analyze retina angiogenesis of postnatal mice and perform matrigel plug assay in adult mice using PHACTR1 knockout mice. In vitro, we will discuss the molecular mechanism of PHACTR functions in angiogenesis using lung microvascular endothelial cells from PHACTR1 knockout mice. Accomplishment of these studies will provide new potential therapeutic targets for angiogenesis-related diseases.

血管新生在缺血性疾病中至关重要。分析已有GWAS研究发现，位于PHACTR1（磷酸酶及肌动蛋白调节因子1）内含子并调控其表达的rs9349379与心肌梗死、缺血性脑卒中等缺血性疾病密切相关。但尚无在体研究揭示PHACTR1在血管新生中的作用。因此，我们制备了PHACTR1敲除小鼠。该小鼠视网膜血管新生明显缺陷，但未见其它表型变化；人脐静脉内皮细胞中siRNA敲减PHACTR1抑制芽管形成及VEGF诱导的Akt活化，但未影响ERK1/2及p38活化。因此我们提出假设：支架蛋白PHACTR1通过促进内皮细胞中Akt活化介导血管新生。本项目将用PHACTR1敲除小鼠进行新生小鼠视网膜血管新生分析及成年小鼠基质胶塞实验，明确PHACTR1在幼体及成体血管新生中作用；进而分离PHACTR1敲除小鼠肺微血管内皮细胞，在细胞水平探讨PHACTR1调控血管新生的分子机制，为血管新生相关疾病提供新治疗靶点。

血管新生在缺血性疾病中至关重要。分析已有GWAS研究发现，位于PHACTR1（磷酸酶及肌动蛋白调节因子1）上的多个SNP位点与心肌梗死、缺血性脑卒中等缺血性疾病密切相关。但尚无在体研究揭示PHACTR1在血管新生中的作用。分析发现这些SNP位点均与PHACTR1表达相关，因此，我们建立了PHACTR1全身敲除小鼠。表达谱分析显示，PHACTR1高表达于血管内皮细胞。PHACTR1敲除明显抑制新生小鼠视网膜血管新生及成年小鼠后肢缺血诱导的血管新生。细胞实验显示，siRNA敲减PHACTR1诱导内皮细胞凋亡，抑制细胞成管，并抑制Akt活化。机制方面，内皮细胞PHACTR1定位于细胞核，RNAseq结果提示PHACTR1与转录调控相关，转录因子分析表明PHACTR1主要影响PPARγ转录因子活性。生化实验显示PHACTR1为PPARγ转录共抑制分子。综上，内皮细胞PHACTR1作为PPARγ转录共抑制分子促进血管新生，深入研究PHACTR1-PPARγ相互作用的分子基础将为缺血性疾病提供新的治疗靶点。