VEGF INDUCED MODULATION OF ENDOTHELIAL STRUCTURE/FUNCT

VEGF 诱导的内皮结构/功能调节

• 批准号: 6177993
• 负责人: RICHARD C SCHAEFFER
• 金额: $ 27.98万
• 依托单位: SIDNEY KIMMEL CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177993
• 关键词: biological signal transduction; cell adhesion; cell cell interaction; cell proliferation; charge coupled device camera; cytoskeleton; diabetes mellitus; enzyme activity; immunofluorescence technique; immunoprecipitation; membrane permeability; mitogens; phosphatidylinositol 3 kinase; protein kinase; vascular endothelial growth factors; vascular endothelium; vascular endothelium permeability; western blottings

DESCRIPTION: Diabetes mellitus affects 1-5% of the world's population and is the major cause of new blindness. Diabetic blindness is frequently characterized by proliferative retinopathy, which is associated with increased vascular permeability and neovascularization. Vascular endothelial growth factor (VEGF), an endothelial cell (CE) specific mitogen, is a major contributor to this disease process. VEGF initiates cell proliferation by binding to VEGF receptors -1 and -2 and activating a phospholipase C- /protein kinase C (PLC /PKC) signal transduction cascade. However, the exact intracellular signals that modulate the chronic increases in vascular permeability induced by VEGF have not been determined. The long-term goals of this research program are to decipher the cellular and molecular mechanisms by which VEGF directly signals chronic alterations of endothelial structure leading to increased monolayer permeability to macromolecules. The specific aims of this experimental program are to test whether VEGF-induced chronic barrier dysfunction and EC growth are dependent on 1) the activation of the mitogen-activated protein kinase cascade, 2) the activation of protein kinase a and bII isoforms, 3) the activation of phosphatidylinositol 3-kinase and/or Rho A. This study will use the following methods: 1) a size-dependent solute (20-200 A) permeability assay developed in this laboratory to measure the "pore" size of the cell-cell junctional space in large and microvessel EC monolayers, 2) immunoprecipitation and Western blotting to examine the signal transduction events initiated by VEGF, 3) digital immunofluorescent microscopy using a -25 degrees C CCD camera that permits sensitive detection of compartmentalization of intracellular signaling, cytoskeletal and cell-cell adhesion molecules, and 4) an EC proliferation assay to measure the effects of inhibitors on VEGF's mitogenic properties. These studies will allow us to identify the VEGF-induced signal transduction pathways that cause chronic endothelial barrier dysfunction and suggest new targets for therapeutic interventions.

描述： 糖尿病影响着世界上 1-5% 的人口 是新发失明的主要原因。 糖尿病性失明很常见 以增殖性视网膜病变为特征，与 血管通透性增加和新血管形成。 血管 内皮生长因子（VEGF），一种内皮细胞（CE）特异性有丝分裂原， 是导致该疾病过程的主要因素。 VEGF启动细胞 通过结合 VEGF 受体 -1 和 -2 并激活 磷脂酶 C-/蛋白激酶 C (PLC /PKC) 信号转导级联。 然而，调节慢性增加的确切细胞内信号 VEGF 诱导的血管通透性的影响尚未确定。 这 该研究计划的长期目标是破译细胞和 VEGF 直接发出慢性变化信号的分子机制 内皮结构导致单层渗透性增加 大分子。 该实验计划的具体目的是测试 VEGF 诱导的慢性屏障功能障碍和 EC 生长是否相关 1) 丝裂原激活蛋白激酶级联的激活，2) 蛋白激酶 a 和 bII 亚型的激活，3) 磷脂酰肌醇 3-激酶和/或 Rho A。本研究将使用 以下方法：1) 尺寸依赖性溶质 (20-200 A) 渗透性测定 该实验室开发用于测量细胞间“孔隙”尺寸 大血管和微血管 EC 单层的连接空间，2) 免疫沉淀和蛋白质印迹检查信号转导 由 VEGF 引发的事件，3) 使用数字免疫荧光显微镜 -25°C CCD 摄像头，可灵敏检测 细胞内信号传导、细胞骨架和细胞间的区室化 粘附分子，以及 4) EC 增殖测定来测量效果 VEGF 促有丝分裂特性的抑制剂。 这些研究将使我们 确定 VEGF 诱导的信号转导途径，从而导致慢性 内皮屏障功能障碍并提出新的治疗靶点 干预措施。