Endothelial Transmigration Across the RPE Barrier

内皮细胞跨 RPE 屏障的迁移

• 批准号: 6736240
• 负责人: Mary Elizabeth Ruth Hartnett
• 金额: $ 14.6万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6736240
• 关键词: angiogenesis; angiogenesis factor; blood aqueous barrier; cell migration; contact inhibition; enzyme linked immunosorbent assay; fluorescent dye /probe; human tissue; immunocytochemistry; laboratory mouse; membrane permeability; mixed tissue /cell culture; northern blottings; organ culture; polymerase chain reaction; retinal pigment epithelium; statistics /biometry; vascular endothelial growth factors; vascular endothelium permeability; western blottings

DESCRIPTION (provided by applicant): Choroidal neovascularization (CNV) is the primary cause of severe vision loss in age-related macular degeneration (AMD), but little is known about the complex mechanisms leading to CNV growth into the neurosensory retina. Human histopathology of occult CNV in AMD has shown that contact between the endothelial cells (EC) of the choriocapillaris and the retinal pigment epithelium (RPE) leads to compromise of the RPE barrier, which precedes the growth of CNV into the neurosensory retina. We will test the hypothesis that RPE-EC contact causes a functional change in the RPE monolayer that permits the transmigration of EC across the RPE. Specifically, we will determine whether 1) EC contact with RPE causes compromised RPE barrier function and structure; 2) EC contact with RPE causes a major change in the ratio of angiogenic stimulators to inhibitors (i.e., VEGF: PEDF) in the RPE; and 3) RPE-EC contact specifically activates cell-associated RPE VEGF isoforms that enable transmigration of EC across the RPE monolayer. Methods will include measurement of transepithelial electrical resistance (TER), cell counts, viability tests, and staining for ZO-1, n-cadherin, and actin in human cell co-cultures of RPE and EC to evaluate RPE barrier structure and function; ELISA, Western blot, and Northern blot to quantitate VEGF:PEDF ratios under various culture conditions; staining for actin and barrier proteins, TER, permeability, and RT-PCR to quantitate soluble and cell-associated VEGF isoforms; and fluorescent labeling of RPE and EC to follow transmigration of EC. Approximately 90% of the legal blindness that occurs in patients with AMD is the result of CNV that originates from the choriocapillaris and grows beyond the natural boundaries of Bruch's membrane and the outer blood-retinal barrier of the RPE into the neurosensory retina. Increased knowledge of the mechanisms that cause this destructive phenomenon may lead to future means to prevent CNV in the neurosensory retina, and thus, reduce vision loss in AMD.

描述（由申请人提供）：脉络膜新生血管（CNV）是年龄相关性黄斑变性（AMD）中严重视力丧失的主要原因，但对于导致 CNV 生长到神经感觉视网膜的复杂机制知之甚少。 AMD 中隐匿性 CNV 的人类组织病理学表明，脉络膜毛细血管内皮细胞 (EC) 与视网膜色素上皮 (RPE) 之间的接触导致 RPE 屏障受损，从而导致 CNV 生长到神经感觉视网膜。 我们将检验以下假设：RPE-EC 接触会导致 RPE 单层功能发生变化，从而允许 EC 跨 RPE 迁移。具体来说，我们将确定 1) EC 与 RPE 接触是否会导致 RPE 屏障功能和结构受损； 2) EC与RPE的接触导致RPE中血管生成刺激剂与抑制剂(即VEGF:PEDF)的比例发生重大变化； 3) RPE-EC 接触特异性激活细胞相关的 RPE VEGF 同工型，使 EC 能够跨 RPE 单层迁移。方法包括测量跨上皮电阻 (TER)、细胞计数、活力测试以及 RPE 和 EC 人细胞共培养物中 ZO-1、n-钙粘蛋白和肌动蛋白的染色，以评估 RPE 屏障结构和功能； ELISA、Western blot 和 Northern blot 定量各种培养条件下的 VEGF:PEDF 比率；肌动蛋白和屏障蛋白、TER、渗透性和 RT-PCR 染色，以定量可溶性和细胞相关 VEGF 亚型；以及 RPE 和 EC 的荧光标记，以跟踪 EC 的迁移。 AMD 患者中大约 90% 的法定失明是 CNV 造成的，CNV 起源于脉络膜毛细血管，生长超出布鲁赫膜和 RPE 的外血视网膜屏障的自然边界，进入神经感觉视网膜。增加对导致这种破坏性现象的机制的了解可能会导致未来预防神经感觉视网膜 CNV 的方法，从而减少 AMD 的视力丧失。