Study of the RPE barrier

血视网膜屏障（blood-retina barrier，BRB）的内外层由紧密连接形成 在相邻的内皮或视网膜色素上皮（RPE）细胞之间。细目 BRBs是年龄相关性黄斑变性、糖尿病性黄斑变性和视网膜病变的主要病理改变。 视网膜病、早产儿视网膜病和葡萄膜炎。虽然之前的研究已经得出了 更好地了解生理和生理条件下内部BRB的作用， 病理条件下，令人惊讶的是，很少有人知道的调控， 外BRB的病理生理学。由于外部BRB负责约85%的血液 循环到视网膜，缺血引起的病理变化是难以想象的 外BRB在视网膜脉络膜的整体病理中起不显著的作用， 血管疾病研究外部BRB击穿的机理，并测试 抑制外部BRB渗透性作为治疗策略的概念， 视网膜脉络膜血管疾病，我们已经准备了基因敲除系统， 小鼠RPE和M？ller胶质细胞，调节内外BRB功能的细胞 通过血管内皮生长因子（VEGF-A）。利用这些条件基因 敲除系统，我们已经破坏了VEGF及其受体（VEGFR 2）在小鼠 RPE，并产生了小鼠与VEGF破坏的M UNK ller细胞。 在具体目标1中，我们将检验我们的假设，即外部BRB崩溃是一种 是糖尿病/缺血诱导的整体视网膜“血管渗漏”的重要因素 通过测量总视网膜，通过RPE中的自分泌VEGF/VEGF-R2信号传导 血管渗漏、外BRB中显著断点的数量以及 RPE特异性VEGF和VEGFR 2敲除中的外部BRB特异性渗漏量 诱导缺血或糖尿病后的小鼠。在具体目标2中，我们将测试 抑制外BRB渗透性作为葡萄膜炎的治疗策略， 视网膜血管渗漏总量，视网膜脱离的数量和严重程度， 外BRB特异性渗漏量和炎症生物标志物的表达 在RPE特异性VEGFR 2敲除小鼠中诱导葡萄膜炎后。作为内部 BRB分解，我们也将测量葡萄膜M？ller细胞中相同的参数- 特异性VEGF敲除小鼠。在具体目标3中，我们将确定分子 糖尿病/缺血引起的外部BRB崩溃的机制，通过研究 控制紧密连接蛋白调节的生化途径。

The inner and outer blood-retina barriers (BRBs) are formed by tight junctions between adjacent endothelial or retinal pigment epithelial (RPE) cells. Breakdown of BRBs is a major pathological change in age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, and uveitis. While previous studies have yielded a better understanding about the roles of the inner BRB under physiological and pathological conditions, surprisingly little is known about the regulation and pathophysiology of the outer BRB. Since the outer BRB is responsible for ~85% of blood circulation to the retina, it is unimaginable that ischemia-induced pathological change in the outer BRB plays an insignificant role in the overall pathology of retinochoroidal vascular diseases. To investigate the mechanisms of outer BRB breakdown and to test the concept of inhibiting outer BRB permeability as a therapeutic strategy for retinochoroidal vascular diseases, we have prepared gene knockout systems for the mouse RPE and M¿ller glia, cells that regulate the function of both inner and outer BRBs through vascular endothelial growth factor (VEGF-A). Using these conditional gene knockout systems, we have disrupted VEGF and its receptor (VEGFR2) in the mouse RPE and have generated mice with VEGF disruption in the M¿ller cells. In Specific Aim 1, we will test our hypothesis that outer BRB breakdown is a significant contributor to diabetes/ischemia-induced overall retinal "vascular leakage" through autocrine VEGF/VEGF-R2 signaling in the RPE by measuring the total retinal vascular leakage, the number of significant breakpoints in the outer BRB, and the quantity of outer BRB-specific leakage in the RPE-specific VEGF and VEGFR2 knockout mice after inducing ischemia or diabetes. In Specific Aim 2, we will test the concept of inhibiting outer BRB permeability as a therapeutic strategy for uveitis by examining the total retinal vascular leakage, the number and severity of retinal detachment, the quantity of outer BRB-specific leakage, and the expression of inflammatory biomarkers in the RPE-specific VEGFR2 knockout mice after inducing uveitis. As a control for inner BRB breakdown, we will also measure the same parameters in uveitic M¿ller cell- specific VEGF knockout mice. In Specific Aim 3, we will determine the molecular mechanism of diabetes/ischemia-induced outer BRB breakdown by investigating the biochemical pathway governing the regulation of tight-junction proteins.