CD109通过调控转录因子ERG表达调节心肌微血管内皮屏障功能的机制研究

The barrier function of microvasculature is crucial for homeostasis of cardiac microenvironment. Transcriptional factor ERG (ETS-Related-Gene) regulates the expression of junctional proteins of endothelial cells such as VE-cadherin and Claudin 5. Recently, we discovered that CD109 is specifically expressed in myocardial microvascular endothelial cells. In addition, CD109 regulates the expression of ERG in endothelial cells. Based on the previous reports and our preliminary results, we hypothesized that CD109 regulates GATA3, which binds to the -172 or -756 GATA binding sites on the ERG promoter and inhibits ERG expression in myocardial microvascular endothelial cells. Repression of ERG expression subsequently reduces the expression of VE-cadherin and Claudin 5, resulting in loss of endothelial cell barrier function. In this proposal, we will examine the gene expression profile in human myocardial microvascular endothelial cells from human cardiac tissue obtained by laser-capture micro-dissection. The molecular mechanisms of CD109 regulated ERG expression and hyperpermeability will be investigated in the models of cell culture, VEC-CD109 transgenic mice and CD109-/- mice. In addition, Hprt-targeted transgenic mice of ERG promoter with site mutations will be generated to validate the transcriptional binding elements. This study will explore molecular mechanisms underlying the role of CD109 in the barrier function of myocardial microvasculature, and provide new therapeutic target for related diseases.

微血管内皮屏障功能对于维持心脏微环境稳态起关键作用。转录因子ERG(ETS-Related-Gene)介导VE-cadherin、Claudin5等内皮屏障蛋白的表达。我们的研究发现，细胞表面抗原CD109特异性表达在心肌微血管内皮细胞中，并降低ERG水平。结合预实验结果，本课题提出假说：CD109通过减少转录因子GATA3与ERG启动子上-172和-756GATA位点的结合，抑制ERG基因表达，导致内皮屏障蛋白表达降低和屏障功能障碍。我们计划使用激光显微切割技术采集人心肌组织内皮细胞检测基因表达，在体外细胞模型、内皮特异性CD109过表达鼠和CD109基因敲除鼠中探查CD109调控ERG基因表达的分子机制，并通过构建和分析Hprt靶向ERG启动子转基因鼠精确验证调控机制。本课题对于深入理解CD109对心肌微血管内皮屏障的作用机制和为相关疾病提供干预靶点都具有重要意义。

项目背景：血管内皮屏障功能维持心脏微环境稳态，心肌微血管屏障障碍是冠心病、心肌炎、心肌纤维化等疾病发生、发展和转归中的关键病理过程。血管内皮细胞特异性转录因子ERG介导VE-cadherin、Claudin5等内皮屏障蛋白的表达。我们的前期研究发现，细胞表面抗原CD109特异性表达在心肌微血管内皮细胞中，并降低ERG水平。本课题计划研究CD109通过调控ERG基因表达调节心肌微血管内皮屏障蛋白水平的具体分子机制。..主要研究内容：在人心肌组织中的血管内皮细胞中确定CD109、ERG、VE-cadherin和Claudin5等基因的表达水平；在内皮细胞特异性CD109转基因小鼠和在CD109基因敲除小鼠心肌中检测ERG等基因的表达水平；通过血管内皮细胞体外培养模型中探查CD109、ERG和GATA家族转录因子的调控关系并进行机制验证。..重要结果和关键数据：1，冠心病病人心脏血管内皮细胞中CD109基因表达升高，ERG、VE-cadherin和Claudin5基因表达降低；2，血管内皮细胞特异性CD109过表达小鼠心肌组织中ERG、VE-cadherin和Claudin5表达降低；3，GATA1、GATA2、GATA3、GATA4、GATA6在血管内皮细胞中调控ERG基因表达；4，血管内皮细胞中ERG基因启动子上-172、-756 和-1015 GATA结合点介导其转录调控。. .科学意义：本课题深入探查CD109、ERG和GATA家族转录因子对血管内皮细胞屏障蛋白表达的调控机制，为相关疾病提供潜在的干预靶点和治疗策略。