血管性血友病因子(vWF) 在心肌微血管内皮细胞中转录调控机制的研究

Microvasculature phenotypes display remarkable heterogeneity in health and disease. Phenotypic heterogeneity is largely mediated at the level of gene transcription in endothelial cells （EC）. The majority of EC-specific genes are regulated by vascular bed-specific transcriptional modules, the von Willebrand factor (vWF) gene being a prototype of this gene class. In addition, recent study has discovered that Adamts13(-/-) mice exhibited significantly severe infarction after myocardical ischemia/reperfusion (I/R)，while vwf(-/-) mice exhibited significantly reduced infarction phenotype, suggesting a detrimental role for vWF in myocardial I/R injury. Investigation of how vWF transcription is regulated in myocardial microvasculature will help us understand the mechanisms of vascular bed-specific regulation and suggest a new venue to intervene myocardial I/R injury. We have discovered that distinct regions of the vWF promoter contain information for selective expression in a subset of ECs. By series of in vitro modeling and deletion studies of human vWF promoter expression in Hprt transgenic mice, we have defined the -843- -620 region that contains regulatory elements for vWF expression in myocardial microvascular ECs. We also identified two highly conserved sequences -723 TCCTACTA and -650 GT(CA)repeats by phylogenetic footprinting and have proved the regulatory function of STAT signaling on this region. Thus we are uniquely poised to evaluate mechanisms of vascular bed-specific gene regulation. The central hypothesis of this project is that vWF gene expression in myocardial microvascular endothelial cells is maintained by the extracellular microenvironment and blood flow shear stress through STAT binding site and GT repeats, and is regulated through the related signaling transduction pathways of these DNA elements during myocardial I/R injury. We will perform a series of DNA/protein binding experiments to identify transcription factors that bind to specific modules on the -843- -620 region and their roles in regulating vWF transcription in human myocardial endothelial cells. The function of the transcription modules will be examined in culture systems including application of blood flow shear stress and primary endothelial cell-cardiomyocyte co-culture. The identified transcriptional modules will be examined by targeted Hprt transgenic mice. In addition, these transcriptional modules will be examined by in vitro and in vivo models of myocardial I/R injury to determine the mechanisms regulating vWF transcription throughout this process. The study should establish the detailed mechanisms of myocardial microvascular-bed specific expression of vWF gene, and provide essential information for developing new therapeutic approaches for vWF related pathological conditions in cardiac microvasculature such as I/R.

微血管内皮细胞在各个组织和器官中存在结构，功能和基因表达异质性，相关机理研究尚处在起步阶段。血管性血友病因子(vWF)是典型的异质性表达基因。 最近的研究也发现vWF是心肌缺血再灌注（I/R）损伤的重要促损伤因素。申请人前期发表了vWF异质性表达的部分机制，并确定了控制vWF在心肌微血管内皮细胞表达的增强子片段。本课题结合既往的研究基础和预实验结果，假设：增强子上的-723 STAT结合序列和-650 GT重复序列可能通过介导血管床微环境和血流剪切力变化来调控vWF在心肌微血管内皮细胞中的表达，而心肌I/R则通过同一机制调控vWF表达。我们拟通过构建检测相同位置靶向转基因鼠等精确的研究手段以及体内和体外I/R模型从各层次深入探查vWF增强子片段上转录调控机制。本研究将揭示控制vWF血管床特异性表达的具体分子机制，为I/R损伤等vWF相关的微血管疾病防治提供新思路和新靶点。

血管性血友病因子（vWF）在全身血管内皮细胞中均有表达，但在各个血管床中有独特的表达规律和调控机理，参与局部组织缺血再灌注损伤等病理生理过程。我们在前期研究中发现一段223bp的血管性血友病因子启动子片段控制其在心肌微血管内皮细胞中的表达。本项目研究通过人类心肌组织检测，体外细胞实验和构建分析靶向转基因小鼠，发现STAT1, STAT3,CTCF等转录因子在正常和缺血再灌注条件下通过该启动子片段调控血管性血友病因子在心肌微血管内皮细胞中的转录水平，并确定了部分DNA调控点。此外，通过体内体外模型和临床数据分析，发现血液中血管性血友病因子水平可以作为心肌缺血的临床标志物。本研究部分揭示了心肌微血管床中血管性血友病因子的表达调控机制，为血管性血友病因子相关的微血管疾病防治提供了新思路和新靶点。