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: 8458112
• 负责人: Magdalena Chrzanowska
• 金额: $ 39.83万
• 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/8458112
• 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.

描述（由申请人提供）：血管内皮生长因子（VEGF）是在正常条件下（如伤口愈合）和病理条件下（如肿瘤生长）负责血管形成的关键因子。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通过细胞表面受体VEGF受体2（VEGFR 2）介导。小GTdR Rac 1参与EC中VEGF介导的每个读数。我们的初步数据表明，通过Rac 1起作用的小GTCRap 1是EC中VEGF信号传导的关键调节因子。我们的中心假设是，VEGF信号通过VEGFR 2破坏EC连接，导致血管通透性升高，部分是通过激活的Rap 1，Rac 1和Rap 1 GEF（鸟嘌呤核苷酸交换因子）C3 G之间的相互作用中继。目的1的研究将描述Rap 1在VEGF诱导的VE-钙粘蛋白连接解体中的异构体特异性功能。将在两种EC特异性Rap 1a和Rap 1b-KO小鼠模型中检查基础和VEGF诱导的血管通透性。为了深入了解Rap 1亚型缺乏时导致细胞-细胞连接溶解的细胞过程是如何改变的，将在Rap 1缺陷小鼠分离的内皮细胞中研究VEGF诱导的信号转导在VE-钙粘蛋白处的会聚以及Rap 1对VEGF和Epac激活的RhoA的差异调节。为了确定Rap 1活性是如何调节的目标2，参与两个GEFs，C3 G和Epac，在VEGF诱导的Rap 1激活VEGF刺激。生物分子荧光互补（BiFC）将用于可视化Rap 1和C3 G之间以及Rap 1和Epac之间的VEGF诱导的相互作用。将在WT和Rap 1a或Rap 1b缺陷型EC中检查GEF沉默表达对VEGF诱导的渗透性的影响，以及使用Rap 1生物传感器对Rap 1活性的影响。为了在体内确定C3 G和Epac参与血管生成，将检查基于吗啉基的每种GEF敲低对体间血管形成的影响。目的3中的研究将在体外和体内鉴定由Rap 1介导的VEGFR 2下游的信号传导机制。我们的工作假设，Rap 1调节动态的EC响应VEGF的调节本地化的活性Rac 1将使用Rac 1生物传感器在WT和Rap 1缺陷的EC，BiFC在VEGF刺激的WT EC检测Rap 1和Rac 1共定位和Rac 1依赖的信号和肌动蛋白细胞骨架动力学的分析进行测试。此外，两个Rac GEF和Rac 1效应IQGAP 1在Rap 1依赖性Rac 1定位和功能响应VEGF刺激的参与将被检查。我们期望这些目标将确定Rap 1亚型调节内皮细胞中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Rap 1亚型活性成功调节VEGF反应。