Study of the RPE barrier

描述（由申请人提供）：内部和外部血视网膜屏障（brb）是由相邻内皮细胞或视网膜色素上皮细胞（RPE）之间的紧密连接形成的。brb的破坏是年龄相关性黄斑变性、糖尿病视网膜病变、早产儿视网膜病变和葡萄膜炎的主要病理改变。虽然以前的研究已经对内部BRB在生理和病理条件下的作用有了更好的了解，但令人惊讶的是，对外部BRB的调节和病理生理知之甚少。由于外BRB负责约85%的视网膜血液循环，缺血引起的外BRB病理改变在视网膜脉络膜血管疾病的整体病理中起着微不足道的作用是不可想象的。为了研究外BRB分解的机制，并测试抑制外BRB通透性作为视网膜脉络膜血管疾病治疗策略的概念，我们为小鼠RPE和Muller胶质细胞制备了基因敲除系统，这些细胞通过血管内皮生长因子（VEGF-A）调节内外BRB的功能。使用这些条件基因敲除系统，我们在小鼠RPE中破坏了VEGF及其受体（VEGFR2），并在Muller细胞中产生了VEGF破坏的小鼠。在Specific Aim 1中，我们将通过测量RPE特异性VEGF和VEGFR2敲除小鼠在诱导缺血或糖尿病后的视网膜血管渗漏总量、外BRB显著断点数量和外BRB特异性渗漏数量，来验证我们的假设，即外BRB破坏是通过RPE中自分泌VEGF/VEGF- r2信号导致糖尿病/缺血诱导的视网膜整体“血管渗漏”的重要因素。在Specific Aim 2中，我们将通过检测葡萄膜炎诱导后rpe特异性VEGFR2敲除小鼠的视网膜血管渗漏总量、视网膜脱离的数量和严重程度、BRB特异性外渗漏的数量以及炎症生物标志物的表达，来测试抑制BRB外通透性作为葡萄膜炎治疗策略的概念。作为内部BRB分解的对照，我们还将在葡萄膜Muller细胞特异性VEGF敲除小鼠中测量相同的参数。在Specific Aim 3中，我们将通过研究紧密连接蛋白调控的生化途径来确定糖尿病/缺血诱导外BRB分解的分子机制。

DESCRIPTION (provided by applicant): 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 Muller 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 Muller 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 Muller 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. PUBLIC HEALTH RELEVANCE: This application is relevant to an important public health issue: the pathogenic mechanism of leading causes of blindness: diabetic retinopathy, retinopathy of prematurity, and uveitis. Our study will focus on the regulatory mechanism and the pathophysiology of blood-retina barriers.