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: 8304895
• 负责人: Magdalena Chrzanowska
• 金额: $ 42.92万
• 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 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8304895
• 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; 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. PUBLIC HEALTH RELEVANCE: Formation of new blood vessels is important in physiological and pathological conditions. This project will help understand mechanisms underlying blood vessel formation to help create new therapies to either promote new blood vessels required for wound healing and tissue repair, such as after an ischemic heart injury, or prevent blood vessel formation, as in tumor progression.

描述（由申请人提供）：血管内皮生长因子（VEGF）是导致正常情况下血管形成的关键因素，例如在伤口愈合和病理状况（例如肿瘤生长中）。对VEGF刺激的不同内皮细胞（EC）反应：细胞迁移，增殖和血管通透性增加，部分通过细胞表面受体VEGF受体2（VEGFR2）的部分介导。小型GTPase Rac1参与EC中的每个VEGF介导的读数。我们的初步数据表明，通过Rac1作用的小GTPase RAP1是EC中VEGF信号传导的关键调节剂。我们的中心假设是，通过VEGFR2通过VEGF信号传导破坏EC连接，导致血管通透性升高的EC连接部分是通过激活的RAP1，RAC1和RAP1 GEF（鸟嘌呤核苷酸交换因子）C3G之间的相互作用的。 AIM 1中的研究将描绘Rap1在VEGF诱导的VE-cadherin结脱拆分的体外和体内的同工型特异性功能。将检查两种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。为了洞悉如何在没有RAP1同工型的情况下改变导致细胞 - 细胞连接溶解的细胞过程，VEGF诱导的VE-钙粘蛋白的信号传导以及RAP1对RAP1响应VEGF和EPAC激活的RHOA调节，将在RAP1缺乏的EC中进行检查。为了确定在AIM 2中如何调节RAP1活性，如何调节AIM 2的VEGF刺激，将检查两个GEF，C3G和EPAC的参与，将检查VEGF诱导的RAP1激活。生物分子荧光互补（BIFC）将用于可视化VEGF诱导的RAP1和C3G之间以及RAP1和EPAC之间的相互作用。将使GEF表达对VEGF诱导的渗透性的影响以及使用RAP1生物传感器对RAP1活性的影响，将在WT和RAP1A-或RAP1B缺陷EC中进行检查。为了在体内确定C3G和EPAC参与血管生成，将检查每个GEF基于形态的敲低对肌间血管形成的影响。 AIM 3中的研究将确定由Rap1体外和体内介导的VEGFR2下游的信号传导机制。我们将RAP1通过调节活性RAC1的定位来调节EC对VEGF的动态的工作假设将使用RAC1生物传感器在WT和RAP1缺陷型EC中进行测试，而在VEGF刺激的WT EC中，BIFC检测RAP1和RAC1共定位，并通过分析RAC1依赖性信号和依赖性信号和cate cytosskeletic cytosickeletic dynamisic。此外，将检查两个RAC GEF和Rac1效应子IQGAP1参与RAP1依赖性RAC1的本地化和响应VEGF刺激的功能。我们预计这些目标将确定RAP1同工型调节EC中VEGF诱导的渗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同工型活性来成功调节VEGF响应。 公共卫生相关性：新血管的形成在生理和病理状况中很重要。该项目将有助于了解血管形成的基础机制，以帮助创建新的疗法，以促进伤口愈合和组织修复所需的新血管，例如缺血性心脏损伤或防止血管形成，例如肿瘤进展。