Genetic Models to Study Glial Regulation of Angiogenesis

描述(申请人提供)：血管生成，或通过血管内皮细胞增殖和发芽形成新的血管，对所有脊椎动物器官的发育都是必不可少的。这在由大脑、脊髓和视网膜组成的中枢神经系统(CNS)中尤其相关，在CNS中，血管生成由分泌各种生长因子和细胞外基质(ECM)蛋白的胶质细胞调控。然而，内皮细胞如何正确解释神经胶质来源的信号，在很大程度上仍然没有定论。在小鼠中使用Cre/Lox策略，我们发现了第一个调控发育中中枢神经系统血管生成的神经胶质-内皮细胞信号轴。该途径的组成部分包括胶质细胞中的αvbeta8整合素，ECM中与其结合的潜伏的转化生长因子β(TGFbeta)蛋白配体，以及内皮细胞中典型的TGFbeta受体(TGFbetaR2和Alk5)。对这一通路中的任何成分进行特定细胞类型的消融都会导致中枢神经系统特有的血管生成缺陷和过早死亡。有趣的是，其他研究小组报告说，胶质细胞中Wnt生长因子的基因缺失，或内皮细胞中Wnt信号效应因子β-catenin的基因缺失， 也会导致血管生成病理，在很大程度上与α-β整合素和转化生长因子β受体基因敲除小鼠的表型相似。这些结果，以及我们在本申请中提出的未发表的机制数据，有力地支持了整合素激活的TGFbetas和WNTs之间的串扰，在这个项目中，我们将分析发育中的大脑中的这些事件。特别是，将产生一套独特的实验工具，包括小鼠和斑马鱼遗传模型，以表征神经胶质细胞控制血管生成的信号机制和基因调控途径。我们将用WNTS和/或TGFbetas刺激的原代脑内皮细胞的信号实验来补充体内模型，并使用生化和基因表达谱来表征这些通路的组成部分是如何在功能上相互联系的。这些实验不仅将揭示WNTS和TGFbetas如何参与血管生成的神经胶质细胞调控的新的重要见解，而且可能确定血管相关神经疾病的新治疗机制。

DESCRIPTION (provided by applicant): Angiogenesis, or the formation of new blood vessels via vascular endothelial cell proliferation and sprouting, is essential for the development of all vertebrate organs. This is particularly relevant in the central nervous system (CNS), comprised of the brain, spinal cord and retina, where angiogenesis is regulated by glial cells that secrete various growth factors and extracellular matrix (ECM) proteins. How endothelial cells properly interpret glial-derived cues, however, remains largely uncharacterized. Using Cre/lox strategies in mice we have discovered the first glial-endothelial cell signaling axis that regulates angiogenesis in the developing CNS. Components of this pathway include alphavbeta8 integrin in glial cells, its ECM-bound latent transforming growth factor beta (TGFbeta) protein ligands in the ECM, and canonical TGFbeta receptors (TGFbetaR2 and Alk5) in endothelial cells. Cell type-specific ablation of any component in this pathway leads to CNS-specific angiogenesis defects and premature death. Interestingly, other groups have reported that genetic deletion of Wnt growth factors in glial cells, or the Wnt signaling effector beta-catenin in endothelial cells, also leads to angiogenesis pathologies that largely phenocopy those in alphavbeta8 integrin and TGFbeta receptor knockout mice. These results, as well as unpublished mechanistic data that we present in this application, strongly support cross talk between integrin-activated TGFbetas and Wnts, and in this project we will analyze these events in the developing brain. In particular, a unique set of experimental tools, including mouse and zebrafish genetic models, will be generated to characterize signaling mechanisms and gene regulatory pathways underlying glial control of angiogenesis. We will complement the in vivo models with signaling experiments in primary brain endothelial cells stimulated with Wnts and/or TGFbetas, and characterize how components of these pathways are functionally interconnected using biochemistry and gene expression profiling. These experiments will not only reveal new and important insights into how Wnts and TGFbetas are involved in glial regulation of angiogenesis, but may identify novel therapeutic mechanisms underlying vascular-related neurological disorders.