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）由相邻内皮或视网膜色素上皮（RPE）细胞之间的紧密连接形成。BRB的分解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变化。虽然以前的研究已经取得了更好的了解生理和病理条件下的内部BRB的作用，令人惊讶的是，很少有人知道的外BRB的调节和病理生理。由于外部BRB负责~85%的视网膜血液循环，因此外部BRB中缺血诱导的病理变化在视网膜脉络膜血管疾病的总体病理学中起不重要的作用是不可想象的。为了研究外部BRB分解的机制并测试抑制外部BRB渗透性作为视网膜脉络膜血管疾病的治疗策略的概念，我们已经制备了小鼠RPE和Muller神经胶质细胞的基因敲除系统，这些细胞通过血管内皮生长因子（VEGF-A）调节内部和外部BRB的功能。使用这些条件基因敲除系统，我们已经破坏了小鼠RPE中的VEGF及其受体（VEGFR 2），并产生了Muller细胞中VEGF破坏的小鼠。 在具体目标1中，我们将通过测量总视网膜血管渗漏、外BRB中显著断裂点的数量、以及在诱导缺血或糖尿病后RPE特异性VEGF和VEGFR 2敲除小鼠中外部BRB特异性渗漏的量。在特定目标2中，我们将通过检查诱导葡萄膜炎后RPE特异性VEGFR 2敲除小鼠中的总视网膜血管渗漏、视网膜脱离的数量和严重程度、外部BRB特异性渗漏的量以及炎症生物标志物的表达来测试抑制外部BRB渗透性作为葡萄膜炎的治疗策略的概念。作为内部BRB分解的对照，我们还将在葡萄膜炎Muller细胞特异性VEGF敲除小鼠中测量相同的参数。在具体目标3中，我们将通过研究控制紧密连接蛋白调节的生化途径来确定糖尿病/缺血诱导的外部BRB破坏的分子机制。 公共卫生相关性：本申请涉及一个重要的公共卫生问题：致盲主要原因的致病机制：糖尿病视网膜病变、早产儿视网膜病变和葡萄膜炎。我们的研究将集中在血视网膜屏障的调节机制和病理生理学。

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.