SENP1介导的GATA2去SUMO化修饰调控移植动脉硬化内皮激活的研究

The progress of graft arteriosclerosis (GA) arises from inflammatory cell response to graft endothelial cells (EC), resulting in the production of proinflammatory cytokines, which, in turn, drives vascular smooth muscle cells proliferation, leading to intimal expansion and luminal stenosis. However, the mechanism is little known for the induction of endothelial adhesion molecules as the result of EC activation, which is the key event of GA initiation. The SUMO (small ubiquitin-like modifier) proteases (SENPs) deconjugate SUMO modified proteins and thus are critical for maintaining the level of SUMOylated and de-SUMOylated substrates required for normal physiology. Our previous work has demonstrated SENP1 mediates the inflammatory signaling in EC and on the other hand directly promotes GATA1 activation and subsequent erythropoiesis by deSUMOylating GATA1. In vascular system, our preliminary results show that endothelial deficiency of SENP1 reduces endothelial adhesion molecule induction and consequent inflammatory cell infiltration as well as GA progression. Since the regulatory role of GATA transcription factor family on adhesion molecules has been well established while a significant body of evidence indicates GATAs are SUMO substrates, it suggests that SENP1 regulated SUMOylation could be a novel endogenous regulatory system that coordinates endothelium activation dependent GA by targeting GATA2 in EC. The proposed research will: Aim 1. Determine the role of endothelial SENP1 in allograft GA model in vivo. We will use WT and SENP1-ecKO mice to determine endothelial activation with endothelial adhesion molecule induction and GA progression in two mouse GA models established in the lab - minor histocompatibility antigen-driven allograft GA and IFN?-induced GA models, to understand the role of SENP1 in GA under different inflammatory settings. Aim 2. Illustrate the cellular and molecular mechanisms of SENP1 regulating endothelial adhesion molecules in GA. We will combine a variety of biochemical, molecular and cell biological approaches to elucidate the role of SENP1 regulated SUMOylation on GATA2 protein stability, DNA binding capability and cellular localization in vascular EC, and consequent effect on endothelial adhesion molecules induction by proinflammatory cytokines. Collectively, this study will define the role of SUMO system in endothelial activation dependent GA progression and lead to novel therapeutics for cardiovascular diseases.

移植动脉硬化（graft arteriosclerosis，GA)的发生源于血管内皮激活和随之发生的的内皮粘附因子诱导表达。然而其调控机制还不清楚。蛋白翻译后SUMO化修饰和去修饰由SUMO蛋白酶SENP调控，对维持正常的生理机能具有重要作用。我们之前的工作展现了SENP1对炎症信号的调控和针对GATA1的去SUMO化维持造血功能。在血管系统中，我们初步研究显示，SENP1在内皮的敲除抑制了促炎症因子引起的内皮细胞粘附因子表达及相应的GA发生。 而作为SUMO的靶蛋白，内皮GATA2和粘附因子表达的关联已明确。因此我们设想：SENP1调控的GATA2 SUMO化修饰是GA中内皮激活的内源性调控机制。本课题拟确立血管内皮SENP1在小鼠GA模型中的作用；阐明GA中SENP1调控内皮粘附因子表达的细胞和分子机制。这些研究将为了解GA的发病机理提供新视野，并为相关心血管病的诊疗提供新策略。

免疫介导的移植动脉硬化(graft arteriosclerosis, GA)为导致移植器官发生病变的主要原因。GA 的病理性过程始于以内皮粘附分子诱导表达为标志的内皮激活，导致移植物血管中新生内膜的生成，血管功能障碍及管腔的闭塞。然而，GA中内皮激活的相关机制仍未明确。本项目首先从临床样品入手，对正常心脏及移植排斥心脏中的冠状动脉进行分析比较，发现蛋白翻译后SUMO化修饰系统的主要蛋白酶SENP1，在血管内皮细胞中被特异性诱导高表达且伴随着内皮激活介导的血管重塑。而GATA2作为调控内皮激活的主要转录因子，在血管内皮中的表达形式也与SENP1相似。基于内皮SENP1与GA的相关性，我们构建了SENP1内皮条件性敲除小鼠。通过动脉移植诱导的GA模型，发现缺少内皮SENP1的移植血管与对照组相比具更好的血管功能，更少的白细胞募集及新生内膜的生成。而在GA早期阶段，内皮SENP1的缺失抑制了移植血管内皮粘附分子的表达，这一发现也在体外的细胞实验中进一步得到了证实，说明了SUMO化修饰对内皮激活的调节作用。另一方面，在移植血管中以及培养的内皮细胞中均明确了内皮GATA2的SUMO化修饰，并且此修饰受炎症诱导的SENP1直接调控，提示SENP1可能通过调节GATA2的SUMO化修饰而在GA的病理性过程中发挥作用。进一步研究表明，SUMO化修饰的GATA2降低了其蛋白稳定性及转录活性，从而减弱了内皮激活及白细胞的粘附和侵润。我们也发现，GATA2 SUMO化修饰位点突变体的重组表达更能促进内皮激活，而SUMO-GATA2融合蛋白的重组表达则显著降低了内皮激活，进一步明确了GATA2的SUMO化修饰在GA中的重要作用。因此，本研究证明了炎症介导的SENP1通过调节GATA2的SUMO化修饰及其调控的内皮激活而促进GA的发生，丰富了GA的发病机制，为GA的诊疗以及相关药物的开发提供了新的靶点和方案。.部分研究成果发表在 《Journal of the American College of Cardiology》、《International Journal of Molecular Medicine》、《中国细胞生物学学报》， 1篇论文正在《Nature Communications》初步接受，正在格式修改。培养博士后1名，硕士生2名。