Mipu1促血管新生的机制研究：对VEGF-VASH1/SVBP负反馈通路的转录调节

Novel gene Mipu1 (myocardial ischemic preconditioning up-regulated protein 1), was cloned from rat myocardium insulted with ischemic preconditioning. We recently have verified that Mipu1 serves as an endogenous gene characterized with anti-apoptosis and promoting cell growth in cardiomyocytes. However, it is not clear whether Mipu1 contributes to the regulation of angiogenesis process in heart. VEGF-VASH1/SVBP, the only endothelium-derived negative feedback pathway for regulating angiogenesis. VASH1 (vasohibin 1) plays inhibitory effect on angiogenesis. Its secretion from endothelial cell depends on SVBP (small vasohibin binding protein) and its expression is induced by VEGF while suppressed by hypoxia stress.In previous works, we found that Mipu1 can promote HUVEC proliferation and inhibit hydrogen peroxide-induced apoptosis; Mipu1 promoted hypoxia-mediated HUVEC migration and tube formation; it also increase the expression of VEGF and reduce the expression and secretion of VASH1 and SVBP. And found that the promoter regions of VEGF, VASH1 and SVBP contain several Mipu1 binding core sequence. Therefore, we assume that Mipu1 would promote positively hypoxia-mediated angiogenesis process by tanscriptional regulating the negative feedback pathway VEGF-VASH1/SVBP at multiple sites. This project is designed to clarify the molecular mechanism by which Mipu1 regulate positively hypoxia-mediated angiogenesis process via regulating VEGF-VASH1/SVBP negative feedback pathway of angiogenesis, through a series of experiments on the key biological events (apoptosis, proliferation, migration and tube formation) of angiogenesis process by using ChIP, EMSA and immunoprecipitation at animal，celullar and molecular models. We expect to further expand the biological function of novel gene Mipu1 in the research field of myocardial protection and provide new clues and experimental basis for the study of feedback regulation mechanism of angiogenesis.

Mipu1是本室从缺血预适应心肌中克隆的新基因，证实其具有促心肌细胞生长和抗心肌细胞凋亡作用，但其是否调节血管新生，尚无报道。VEGF-VASH1/SVBP是唯一的内皮源性血管新生负反馈调节通路。申请者近期发现：①Mipu1促进内皮细胞增殖和抑制其凋亡、促进管型形成；②Mipu1抑制VASH1和SVBP表达与分泌、促进VEGF表达；③VEGF、VASH1和SVBP启动子区含多个Mipu1结合序列。因此，我们假设Mipu1可能在转录水平通过多靶点调节VEGF-VASH1/SVBP负反馈通路，发挥促血管新生作用。本项目以VEGF-VASH1/SVBP通路中关键分子的转录调节为切入点，从整体、细胞和分子水平，采用ChIP、EMSA和免疫沉淀等方法，深入探讨Mipu1促血管新生的分子机制。期望进一步拓展新基因Mipu1在心肌保护领域的生物学功能，并为血管新生反馈调节机制研究提供新的线索和实验依据。

Mipu1/ZNF667是本室从缺血预适应心肌中克隆的新基因，我们前期研究证实其具有促心肌细胞生长和抗心肌细胞凋亡作用，但其是否调节血管新生既往尚无报道。VEGF-VASH1/SVBP 是唯一的内皮源性血管新生负反馈调节通路。申请者在前期研究的基础上提出假设 Mipu1 可能在转录水平通过多靶点调节 VEGF-VASH1/SVBP 负反馈通路，发挥促血管新生作用。本项目以 VEGF-VASH1/SVBP 通路中关键分子的转录调节为切入点，从整体、细胞和分子水平，采用 ChIP、EMSA 和免疫沉淀等方法，深入探讨 了Mipu1/ZNF667促血管新生的分子机制。本研究结果显示急性心肌梗死时心脏组织血管内皮细胞Mipu1/ZNF667的表达上调，血管新生增加，而血管内皮细胞中VASH1的蛋白表达明显下调。人脐静脉内皮细胞Mipu1/ZNF667的表达上调可明显增加低氧诱导的管型的形成并促进细胞迁移，但下调VASH1的蛋白表达，而上调人脐静脉内皮细胞中VASH1的表达，可明显减少管型的形成并抑制细胞迁移。VASH1过表达可显著抑制Mipu1/ZNF667过表达所致细胞迁移和管型形成的增加。进一步的生物信息学和PCR分析结果显示Mipu1/ZNF667-VASH1-Wnt可能促进了血管新生过程。因此，ZNF667/Mipu1可能通过下调VASH1的表达，影响Wnt通路，进而促进急性心肌梗死时血管新生。此外，我们还发现Mipu1/ZNF667过表达可增加人脐静脉内皮细胞中MMP14、VEGFA表达；MMP14低表达可抑制Mipu1/ZNF667促人脐静脉内皮细胞管型形成和迁移的作用，提示Mipu1/ZNF667的促血管新生的作用也可能部分依赖于其上调MMP14基因的表达。同时，本项目进一步拓展了Mipu1/ZNF667在缺血心肌组织炎症反应中的作用，我们发现炎性小体信号中的关键成员PKR、NLRP3、IL-1β可能是Mipu1/ZNF667直接调控的分子靶，但其具体调控机制有待于进一步研究证实。以上研究结果为进一步拓展ZNF667/Mipu1 在心肌保护领域的生物学功能及血管新生反馈调节机制研究提供了新的线索和实验依据，并可能发现治疗心肌缺血的新靶点。