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; 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）：脉络膜新生血管化（CNV）是年龄相关黄斑变性（AMD）严重视力丧失（AMD）的主要原因，但对导致CNV生长进入神经体验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）之间的接触会导致RPE屏障的妥协，这在CNV中的生长之前是CNV进入神经膜的。 我们将检验以下假设：RPE-EC接触会导致RPE单层的功能变化，该单层允许EC跨RPE进行移动。具体而言，我们将确定1）EC是否与RPE接触会导致RPE屏障功能和结构受损； 2）EC与RPE接触会导致RPE中血管生成刺激剂与抑制剂（即VEGF：PEDF）的比率发生了重大变化； 3）RPE-EC接触特异性激活了与细胞相关的RPE VEGF同工型，这些RPE VEGF同工型可以使EC跨RPE单层迁移。方法将包括测量transepithelial电阻（TER），细胞计数，活力测试以及ZO-1，N-钙粘着蛋白和肌动蛋白在RPE和EC的人类细胞共培养中的染色，以评估RPE屏障结构和功能； Elisa，Western印迹和北印迹，以在各种培养条件下定量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的VEGF同工型；以及RPE和EC的荧光标记，以遵循EC的迁移。 AMD患者发生的法律失明大约90％是CNV的结果，该CNV起源于脉络膜毛细血管，并且超出了Bruch膜的自然界限，而RPE的外部血液屏障则超出了神经体积视网膜。对导致这种破坏性现象的机制的知识越来越多，可能会导致未来的手段预防神经感觉视网膜中CNV，从而减少AMD的视力丧失。