VEGF INDUCED MODULATION OF ENDOTHELIAL STRUCTURE/FUNCT

描述：糖尿病影响世界1%-5%的人口， 是导致新的失明的主要原因。糖尿病性失明是常见的 以增殖性视网膜病变为特征，这与 增加血管通透性和新生血管。血管 内皮生长因子(VEGF)是一种内皮细胞(CE)特异性有丝分裂原， 是这一疾病过程的主要贡献者。血管内皮生长因子启动细胞 通过与血管内皮生长因子受体-1和-2结合并激活 磷脂酶C-/蛋白激酶C(PLC/PKC)信号转导通路。 然而，调节慢性增加的确切细胞内信号 血管内皮生长因子诱导的血管通透性尚未确定。这个 这项研究计划的长期目标是破译细胞和 血管内皮生长因子直接信号转导慢性血管内皮细胞病变的分子机制 内皮结构导致单层通透性增加 大分子。这个实验项目的具体目的是为了测试 血管内皮生长因子诱导的慢性屏障功能障碍与内皮细胞生长是否相关 1)丝裂原活化蛋白激酶级联反应的激活；2)丝裂原激活的蛋白激酶级联反应 蛋白激酶a和BII亚型的激活，3)蛋白激酶A和BII亚型的激活 磷脂酰肌醇3-激酶和/或Rho A。本研究将使用 以下方法：1)大小相关的溶质(20-200A)渗透率测定 这个实验室用来测量细胞的“孔”大小--细胞 大血管和微血管EC单层的连接间隙，2) 免疫沉淀和Western blotting检测信号转导 由血管内皮生长因子发起的事件，3)使用数字免疫荧光显微镜 -25摄氏度的CCD摄像机，可灵敏地检测 细胞内信号、细胞骨架和细胞-细胞的划分 黏附分子，以及4)EC增殖试验以测量其影响 抑制剂对血管内皮生长因子有丝分裂特性的影响。这些研究将使我们能够 确定血管内皮生长因子诱导的信号转导通路导致慢性 内皮屏障功能障碍和提出新的治疗靶点 干预措施。

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.