Rap1 in VEGF signaling in endothelial cells

描述（由申请人提供）：血管内皮生长因子（VEGF）是正常情况下（如伤口愈合）和病理情况（如肿瘤生长）中血管形成的关键因素。不同的内皮细胞（EC）对VEGF刺激的反应：细胞迁移、增殖和血管通透性增加部分是通过细胞表面受体VEGF受体2 （VEGFR2）介导的。小GTPase Rac1参与内皮细胞中vegf介导的每一个读数。我们的初步数据表明，小GTPase Rap1通过Rac1起作用，是内皮细胞中VEGF信号传导的关键调节因子。我们的中心假设是，通过VEGFR2破坏EC连接导致血管通透性升高的VEGF信号在一定程度上是通过激活的Rap1、Rac1和Rap1 GEF（鸟嘌呤核苷酸交换因子）C3G之间的相互作用传递的。Aim 1的研究将描述Rap1在体外和体内vegf诱导的ve -钙粘蛋白连接拆卸中的异构体特异性功能。两种ec特异性Rap1a和Rap1b-KO小鼠模型的基础和vegf诱导的血管通透性将被检查。为了深入了解在缺乏Rap1亚型的情况下导致细胞-细胞连接溶解的细胞过程是如何改变的机制，我们将在从Rap1缺失小鼠中分离的ECs中研究VEGF诱导的信号在ve -钙粘蛋白上趋同以及Rap1响应VEGF和Epac激活对RhoA的差异调节。为了确定Rap1活性是如何在Aim 2中响应VEGF刺激而被调节的，我们将研究两种gef （C3G和Epac）在VEGF诱导的Rap1激活中的作用。生物分子荧光互补（BiFC）将用于可视化vegf诱导的Rap1与C3G之间以及Rap1与Epac之间的相互作用。我们将在WT和Rap1a-或rap1b缺陷ec中研究沉默GEF表达对vegf诱导的通透性的影响，以及使用Rap1生物传感器对Rap1活性的影响。为了在体内确定C3G和Epac是否参与血管生成，我们将研究基于morpholino敲低每种GEF对体间血管形成的影响。Aim 3的研究将在体外和体内确定Rap1介导的VEGFR2下游的信号机制。我们的工作假设是，Rap1通过调节活性Rac1的定位来调节EC对VEGF的反应动力学，我们将在WT和Rap1缺失的EC中使用Rac1生物传感器，在VEGF刺激的WT EC中使用BiFC来检测Rap1和Rac1共定位，并通过分析Rac1依赖的信号传导和肌动蛋白细胞骨架动力学来测试Rap1。此外，两个Rac gef和一个Rac1效应物IQGAP1参与rap1依赖的Rac1定位和响应VEGF刺激的功能将被检查。我们期望这些目标将确定Rap1异构体调节内皮细胞中VEGF诱导的通透性的关键控制机制。通过这项研究获得的知识有望通过操纵特定的Rap1异构体活性成功地调节VEGF反应。

DESCRIPTION (provided by applicant): Vascular endothelial growth factor (VEGF) is the critical factor responsible for blood vessel formation in normal conditions, such as in wound healing, and in pathological conditions, such as in tumor growth. Disparate endothelial cell (EC) responses to VEGF stimulation: cell migration, proliferation and increased vascular permeability are mediated in part via cell surface receptor VEGF Receptor 2 (VEGFR2). Small GTPase Rac1 is involved in each of the VEGF-mediated readouts in ECs. Our preliminary data indicate that small GTPase Rap1, acting via Rac1, is a key regulator of VEGF signaling in ECs. Our central hypothesis is that VEGF signaling via VEGFR2 that disrupts EC junctions leading to elevated vascular permeability is in part relayed via interactions between activated Rap1, Rac1 and Rap1 GEF (Guanine nucleotide Exchange Factor) C3G. Studies in Aim 1 will delineate isoform-specific functions of Rap1 in VEGF-induced VE-cadherin junction disassembly in vitro and in vivo. Basal and VEGF-induced vascular permeability in two EC-specific Rap1a and Rap1b-KO mice models will be examined. To gain mechanistic insight into how cellular processes leading to cell-cell junction dissolution are altered in the absence of Rap1 isoforms, VEGF-induced signaling converging at VE-cadherin and differential regulation of RhoA by Rap1 in response to VEGF and Epac activation will be examined in ECs isolated from Rap1-deficient mice. To identify how Rap1 activity is regulated in response to VEGF stimulation in Aim 2, involvement of two GEFs, C3G and Epac, in VEGF-induced Rap1 activation will be examined. Biomolecular Fluorescence Complementation (BiFC) will be used to visualize VEGF-induced interaction between Rap1 and C3G and between Rap1 and Epac. The effect of silencing expression of either GEF on VEGF-induced permeability and, using Rap1 biosensor, on Rap1 activity, will be examined in WT and in Rap1a- or Rap1b-deficient ECs. For in vivo determination of C3G and Epac involvement in angiogenesis, the effect of morpholino-based knockdown of each GEF on intersomitic vessel formation will be examined. Studies in Aim 3 will identify the signaling mechanism downstream from VEGFR2 that is mediated by Rap1 in vitro and in vivo. Our working hypothesis that Rap1 regulates dynamics of EC responses to VEGF by regulating localization of active Rac1 will be tested using Rac1 biosensor in WT and Rap1-deficient ECs, BiFC in VEGF-stimulated WT ECs to detect Rap1 and Rac1 colocalization and by analysis of Rac1-dependent signaling and actin cytoskeleton dynamics. Furthermore, the involvement of two Rac GEFs and a Rac1 effector IQGAP1 in Rap1-dependent Rac1 localization and function in response to VEGF stimulation will be examined. We expect that these aims will identify key control mechanisms through which Rap1 isoforms regulate VEGF- induced permeability in ECs. Knowledge gained through this research is expected to enable successful modulation of VEGF responses by manipulating specific Rap1 isoform activity.