Rap1 in VEGF signaling in endothelial cells

Rap1 在内皮细胞 VEGF 信号转导中的作用

• 批准号: 8822909
• 负责人: Magdalena Chrzanowska
• 金额: $ 40.95万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-16 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8822909
• 关键词: Actins; Agonist; Binding; Biology; Biosensor; Birth; Blood Vessels; Cell Surface Receptors; Cell physiology; Cells; Chronic; Clinic; Cytoskeleton; Data; Development; Disease; Endothelial Cells; Fluorescence; Goals; Grant; Guanine Nucleotide Exchange Factors; Guanine Nucleotide-Releasing Factor 2; Guanine Nucleotides; Heart Injuries; In Vitro; Intercellular Junctions; Internet; Ischemia; Kinetics; Knowledge; Mediating; Monomeric GTP-Binding Proteins; Mus; Myocardial Infarction; Myocardial Ischemia; Neoplasm Metastasis; Permeability; Physiological; Play; Protein Isoforms; Proteins; Publishing; RAPGEF1 gene; Regulation; Research; Retinal Diseases; Role; Signal Transduction; Specificity; Testing; Tissues; Vascular Endothelial Growth Factor Receptor; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; Vascular Permeabilities; Work; Wound Healing; angiogenesis; antiangiogenesis therapy; base; blood vessel development; cadherin 5; cell motility; in vivo; insight; migration; mouse model; new therapeutic target; novel therapeutic intervention; prevent; repaired; response; therapeutic angiogenesis; tissue repair; tumor; tumor growth; tumor progression

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.

描述(申请人提供)：血管内皮生长因子(VEGF)是在正常情况下(如伤口愈合)和病理条件(如肿瘤生长)中负责血管形成的关键因素。内皮细胞(EC)对血管内皮生长因子刺激的不同反应：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血管内皮生长因子受体2(VEGFR2)介导的。在内皮细胞中，小的GTPase rac1参与了血管内皮生长因子介导的每一次读数。我们的初步数据表明，小GTPase Rap1通过rac1发挥作用，是内皮细胞中血管内皮生长因子信号转导的关键调节因子。我们的中心假设是，通过VEGFR2破坏EC连接导致血管通透性增加的VEGF信号在一定程度上是通过激活的Rap1、rac1和Rap1 Global(鸟嘌呤核苷酸交换因子)C3G之间的相互作用来传递的。目标1的研究将在体外和体内阐明Rap1在血管内皮生长因子诱导的VE-钙粘附素连接解离中的异构体特异性功能。在两个EC特异性的Rap1a和Rap1b-KO小鼠模型中，将检测基础和血管内皮生长因子诱导的血管通透性。为了从机制上深入了解在缺少Rap1亚型的情况下导致细胞连接溶解的细胞过程是如何改变的，我们将在从Rap1缺陷小鼠分离的内皮细胞中检测血管内皮生长因子诱导的信号会聚到VE-钙粘蛋白以及Rap1对RhoA响应血管内皮生长因子和EPAC激活的差异调控。为了确定在AIM 2中，Rap1活性是如何响应于血管内皮生长因子的刺激而被调节的，我们将研究两个GEF，C3G和EPAC，在血管内皮生长因子诱导的Rap1激活中的作用。生物分子荧光互补(BIFC)将用于可视化血管内皮生长因子诱导的RAP1和C3G之间以及RAP1和EPAC之间的相互作用。在WT和缺乏Rap1a或Rap1b的内皮细胞中，将检测任一环境基金沉默表达对血管内皮生长因子诱导的通透性以及使用Rap1生物传感器对Rap1活性的影响。为了在体内确定C3G和EPAC在血管生成中的作用，我们将研究基于吗啡诺的基因敲除对体间血管形成的影响。AIM 3的研究将在体外和体内确定由Rap1介导的VEGFR2下游的信号机制。我们的工作假设是，Rap1通过调节激活的rac1的定位来调节内皮细胞对血管内皮生长因子的反应，这一假设将通过在WT和Rap1缺乏的ECs中使用rac1生物传感器来检测Rap1和rac1共存的情况以及通过分析rac1依赖的信号和肌动蛋白细胞骨架动力学来验证。此外，还将研究两个RAC GEF和一个rac1效应器IQGAP1在RAP1依赖的rac1定位和功能中对血管内皮生长因子刺激的反应。我们期望这些目标将确定关键的控制机制，通过这些机制，Rap1亚型调节血管内皮生长因子诱导的内皮细胞的通透性。通过这项研究获得的知识有望通过操纵特定的Rap1亚型活性来成功地调节血管内皮生长因子的反应。