Blood-retinal Barrier Changes in Retinopathy

描述(申请人提供)：本项目旨在阐明机制 糖尿病通过改变视网膜内皮细胞屏障功能和存活率 并明确血管内皮细胞生长因子(VEGF)在血管内皮细胞生长中的作用。 病理学。血管内皮生长因子及其受体VEGFR2的表达增加 糖尿病视网膜病变。矛盾的是，虽然血管内皮生长因子是一个强有力的生存因素， 视网膜毛细血管丢失伴随着糖尿病引起的 表情。此外，即使在正常成年人中有丰富的血管内皮生长因子和血管内皮生长因子R2 视网膜、血-视网膜屏障功能保持正常。因此，增加了 VEGF/VEGFR2的表达不能完全解释糖尿病血管病变 改装。初步数据显示，糖尿病和高血糖会导致 视网膜一氧化氮合酶(NOS)表达和活性增加， 一氧化氮(NO)、超氧化物(O2-)及其产物的形成， 过氧亚硝酸盐(ONOO-)。已知这些活性氧物种(ROS)会改变 细胞内信号传递功能。初步数据显示，血管内皮生长因子增加 激活和核过程中的细胞旁通透性 β-连环蛋白易位导致尿激酶型纤溶酶原激活 激活剂受体(UPAR)和紧密连接蛋白阻断素减少。 在稳定的单层中，β-连环蛋白通过连接血管促进细胞黏附 内皮钙粘附素与肌动蛋白细胞骨架结合，但与生长因子结合 刺激β-连环蛋白结合并激活转录因子Lef-1 (淋巴细胞增强因子-1)导致uPAR表达。β-连环蛋白 血管内皮生长因子激活涉及与磷脂酰肌醇(PI)-3的相互作用 通过以下途径促进胞浆内游离β-连环蛋白的积累 糖原合成酶激酶3抑制其降解。PI-3激酶促进 细胞迁移和存活，是蛋白质酪氨酸硝化的目标 ONOO-。NO可增强β-连环蛋白/Lef-1的结合。NO和O2-抑制作用 酪氨酸磷酸酶。我们的工作模型是，血管内皮生长因子的作用增加 内皮细胞的通透性和促进细胞存活通常是紧密的 β-连环蛋白与PI-3激酶的协同作用 信号通路。糖尿病患者血管内皮生长因子的通透性和生存功能 由于NO、O2-和ONOO-在改变 β-连环蛋白和PI-3激酶信号通路。这将通过以下方式进行测试 使用细胞和分子分析方法相结合的实验 为了检验以下假设：1)血管内皮生长因子促进细胞旁增加 β-连环蛋白和PI-3激酶介导的通透性 UPAR增加。2)高糖增加通透性，降低存活率 通过增加ROS的形成和改变血管内皮生长因子、β-连环蛋白和PI-3激酶 细胞内信号。3)糖尿病增加血管通透性及其原因 血管细胞通过增加ROS的形成而死亡。

DESCRIPTION (provided by applicant): This project seeks to elucidate mechanisms by which diabetes alters retinal endothelial cell barrier function and survival and to define the role of vascular endothelial cell growth factor (VEGF) in the pathology. Expression of VEGF and its receptor, VEGFR2, is increased in diabetic retinopathy. Paradoxically, while VEGF is a potent survival factor, retinal capillary dropout accompanies the diabetes-induced increases in its expression. Moreover, even though VEGF and VEGFR2 are abundant in normal adult retinas, blood-retinal barrier function remains normal. Thus, increased VEGF/VEGFR2 expression does not fully explain the diabetes-induced vascular alterations. Preliminary data show that diabetes and high glucose cause increases in retinal nitric oxide synthase (NOS) expression and activity and in formation of nitric oxide (NO), superoxide (O2-) and their product, peroxynitrite (ONOO-). These reactive oxygen species (ROS) are known to alter intracellular signaling functions. Preliminary data suggest that VEGF increases paracellular permeability by a process involving activation and nuclear translocation of beta-catenin, leading to activation of urokinase plasminogen activator receptor (uPAR) and decreases in the tight junction protein occludin. In stable monolayers, beta-catenin promotes cell adhesion by linking vascular endothelial cadherin to the actin cytoskeleton, but on growth factor stimulation beta-catenin binds and activates the transcription factor LEF-1 (lymphocyte enhancer factor-1) leading to uPAR expression. Beta-catenin activation by VEGF involves an interaction with phosphatidylinositol (PI)-3 kinase, which promotes cytosolic accumulation of free beta-catenin by inhibiting its degradation by glycogen synthase kinase 3. PI-3 kinase promotes cell migration and survival and is a target for protein tyrosine nitration by ONOO-. Beta-catenin/LEF-1 binding is enhanced by NO. Both NO and O2- inhibit tyrosine phosphatases. Our working model is that the effects of VEGF increasing endothelial cell permeability and promoting cell survival are normally tightly coupled by the coordinated function of the beta-catenin and PI-3 kinase signaling pathways. In diabetes the permeability and survival functions of VEGF are uncoupled due to the effects of NO, O2- and ONOO- in altering the beta-catenin and PI-3 kinase signaling pathways. This will be tested by experiments using a combination of cellular and molecular analytical approaches to test the following hypotheses: 1) VEGF promotes an increase in paracellular permeability by a process involving beta-catenin and PI-3 kinase mediated increases in uPAR. 2) High glucose increases permeability and reduces survival by increasing ROS formation and altering VEGF, beta-catenin and PI-3 kinase intracellular signaling. 3) Diabetes increases vascular permeability and causes vascular cell death by increasing ROS formation.