VEGF INDUCED MODULATION OF ENDOTHELIAL STRUCTURE/FUNCT

糖尿病影响世界人口的1-5%， 是新失明的主要原因。 糖尿病性失明通常 特征为增殖性视网膜病变， 增加血管渗透性和新血管形成。 血管 内皮生长因子（VEGF），一种内皮细胞（CE）特异性丝裂原， 是导致这种疾病的主要因素 VEGF启动细胞 通过与VEGF受体-1和-2结合并激活细胞增殖， 磷脂酶C- /蛋白激酶C（PLC /PKC）信号转导级联。 然而，调节慢性炎症的确切细胞内信号增加， 在VEGF诱导的血管通透性中的作用尚未确定。 的 这项研究计划的长期目标是破译细胞， VEGF直接信号慢性改变的分子机制 内皮结构导致单层渗透性增加， 大分子 这项实验计划的具体目标是测试 VEGF诱导的慢性屏障功能障碍和EC生长是否依赖于 1）促分裂原活化蛋白激酶级联的激活，2） 蛋白激酶a和bII亚型的活化，3）蛋白激酶a和bII亚型的活化， 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 在这个实验室开发的测量“孔”的大小细胞 大血管和微血管EC单层中的连接空间，2） 免疫沉淀和Western印迹检测信号转导 VEGF引发的事件，3）使用数字免疫荧光显微镜检查， -25 ℃ CCD摄像机，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，和4）EC增殖测定以测量作用 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， 内皮屏障功能障碍，并提出新的治疗目标 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.