Reactive Oxygen Species and Endothelial Migration

活性氧和内皮迁移

基本信息

批准号：
7322030
负责人：
Masuko Ushio-Fukai
金额：
$ 37.63万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-03-15 至 2011-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7322030
关键词：
Actins Adenoviruses Adherens Junction Adhesions Angiogenic Factor Binding Binding Proteins Binding Sites Biological Assay Blood capillaries Blood flow Cell Adhesion Molecules Cell-Cell Adhesion Cells Complementary DNA Complex Confocal Microscopy Coupling Data Development E-Cadherin Endothelial Cells Event Goals Growth Hindlimb Hydrogen Peroxide In Vitro Ischemia Laboratories Link Localized Mediating Modeling Molecular Monomeric GTP-Binding Proteins Morphogenesis Mus NAD(P)H oxidase Peptides Production Protein Overexpression Reactive Oxygen Species Recovery Recruitment Activity Regulation Research Personnel Role Scaffolding Protein Signal Pathway Signal Transduction Signaling Molecule Small Interfering RNA Source Specificity Stress Tyrosine Phosphorylation Vascular Endothelial Growth Factor Receptor-2 Vascular Endothelial Growth Factors Wild Type Mouse Work Wound Healing angiogenesis base cadherin 5 capillary cell motility in vivo migration mutant novel novel therapeutics postnatal programs receptor response response to injury scaffold wound yeast two hybrid system

项目摘要

Endothelial cell (EC) migration is a key event for endothelial wound repair and angiogenesis. VEGF is a potent stimulator for EC migration primarily through the VEGF type2 receptor (VEGFR2). In quiescent ECs, one of the initial responses to stimulate endothelial migration by VEGF is the loosening of stable cell-cell contacts which is regulated by tyrosine phosphorylation of VE-cadherin. We showed that VEGF stimulates Rac1-dependent gp91phox-based NAD(P)H oxidase and that reactive oxygen species (ROS) are involved in VEGFR2-mediated signaling linked to EC migration. Underlying molecular mechanisms are poorly understood. We recently identified IQGAP1 as a novel VEGFR2 binding protein. It functions as a scaffold protein that controls cell motility and morphogenesis by interacting with cytoskeletal and cell-cell adhesion proteins including active Rac1 and E-cadherin. We also found that: 1) IQGAP1 expression is increased in the newly-formed capillary ECs in a mouse hindlimb ischemia model of angiogenesis; 2) Overexpression of IQGAP1 increases basal Rac1 activity, ROS production and cell migration in ECs; 3) VEGF promotes recruitment of activated VEGFR2 and Rac1 to the IQGAP1-VE-cadherin complex at adherens junctions, which may promote ROS-dependent loss of cell-cell contacts and subsequent EC migration; 4) Wound assays reveal that IQGAP1 colocalizes with active VEGFR2 and gp91phox at the leading edge inactively migrating ECs. We thus hypothesize that IQGAP1 functions as a VEGFR2 binding scaffold protein to link ROS-dependent signaling with VEGF-mediated endothelial migration. Aim1 will examine whether IQGAP1 functionally interacts with VEGFR2 and Rac1 to couple VEGFR2 to ROS-dependent signaling linked to EC migration. Aim2 will examine whether IQGAP1 functions as a scaffold to recruit activated VEGFR2 and Rac1 to adherens junctions through binding to VE-cadherin, thereby facilitating ROS-dependent loss of cell-cell contacts, which initiates EC migration. Aim3 will examine the scaffolding role of IQGAP1 in targeting VEGFR2 and NAD(P)H oxidase to the leading edge during directed cell migration. Aim4 will assess the functional significance of IQGAP1 in vivo using mouse hindlimb ishcemia model. The long-term goal is to understand the molecular mechanisms by which ROS regulate EC migration in the context of angiogenesis and endothelial wound repair, which should facilitate the development of new therapeutic strategies.

内皮细胞（EC）迁移是内皮伤口修复和血管生成的关键事件。血管内皮生长因子是一种主要通过 VEGF 2 型受体 (VEGFR2) 有效刺激 EC 迁移。在静态 EC 中， VEGF 刺激内皮细胞迁移的最初反应之一是稳定细胞间的松动接触是由 VE-钙粘蛋白的酪氨酸磷酸化调节的。我们发现 VEGF 可以刺激 Rac1 依赖的基于 gp91phox 的 NAD(P)H 氧化酶和活性氧 (ROS) 参与 VEGFR2 介导的信号传导与 EC 迁移有关。潜在的分子机制很差明白了。我们最近发现 IQGAP1 是一种新型 VEGFR2 结合蛋白。它起到脚手架的作用通过与细胞骨架和细胞间粘附相互作用来控制细胞运动和形态发生的蛋白质蛋白质包括活性 Rac1 和 E-钙粘蛋白。我们还发现：1) IQGAP1 表达增加小鼠后肢缺血血管生成模型中新形成的毛细血管内皮细胞； 2) 过度表达 IQGAP1 增加 EC 中的基础 Rac1 活性、ROS 产生和细胞迁移； 3）VEGF促进将活化的 VEGFR2 和 Rac1 募集至粘附连接处的 IQGAP1-VE-钙粘蛋白复合物，这可能会促进 ROS 依赖性细胞间接触的丧失和随后的 EC 迁移； 4) 伤口分析表明 IQGAP1 与活性 VEGFR2 和 gp91phox 共定位于非活性前沿迁移 EC。因此，我们假设 IQGAP1 作为 VEGFR2 结合支架蛋白发挥作用，以连接 ROS 依赖性信号传导与 VEGF 介导的内皮迁移。 Aim1将检查IQGAP1是否与 VEGFR2 和 Rac1 功能性相互作用，将 VEGFR2 耦合到与 EC 相关的 ROS 依赖性信号传导迁移。 Aim2 将检查 IQGAP1 是否作为支架来招募激活的 VEGFR2 和 Rac1 通过与 VE-钙粘蛋白结合来粘附连接，从而促进 ROS 依赖性细胞损失联系人，这会启动 EC 迁移。 Aim3 将检查 IQGAP1 在靶向中的支架作用 VEGFR2 和 NAD(P)H 氧化酶在定向细胞迁移过程中处于前沿。 Aim4 将评估使用小鼠后肢缺血模型观察 IQGAP1 体内的功能意义。长期目标是了解 ROS 在血管生成背景下调节 EC 迁移的分子机制和内皮伤口修复，这应该有助于新治疗策略的开发。