Role of RPE-derived VEGF in Choroid Development and Stability

RPE 衍生的 VEGF 在脉络膜发育和稳定性中的作用

• 批准号: 7856199
• 负责人: Patricia Ann D'Amore
• 金额: $ 7.63万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7856199
• 关键词: Adult; Adverse effects; Age related macular degeneration; Architecture; Binding Sites; Biological Assay; Blood Vessels; Blood flow; Bruch' s basal membrane structure; Cells; Choroid; Choroidal Neovascularization; Coupled; Development; Diffuse; Electron Microscopy; Engineering; Excision; Eye; Fundus photography; Genes; Hypoxia; In Vitro; Individual; Knowledge; LacZ Genes; Light; Luciferases; Maintenance; Mediating; Modeling; Molecular; Mus; Mutagenesis; Nonexudative age-related macular degeneration; Organism; Pathogenesis; Pathologic; Pathologic Neovascularization; Pathology; Phenotype; Physiological; Pigment Epithelium; Play; Production; Protein Isoforms; RPE65 protein; Regulation; Reporter; Retinal; Role; Saints; Small Interfering RNA; Structure of retinal pigment epithelium; Testing; Time; Tissues; Transgenic Mice; Up-Regulation; VEGFA gene; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; age related; autocrine; base; choroidal circulation; chromatin immunoprecipitation; geographic atrophy; in vivo; insight; mouse model; novel; promoter; public health relevance; relating to nervous system; transcription factor

DESCRIPTION (provided by applicant): The studies outlined in this proposal are aimed at understanding the role of VEGF in retinal pigment epithelium (RPE) - choriocapillaris interactions. Significant evidence supports a role for the RPE in the development and maintenance of the choroidal vasculature, and interactions between RPE cells and the choroidal vasculature are central in age-related macular degeneration (AMD). We hypothesize that tightly regulated expression of VEGF by RPE is necessary for the integrity and maintenance of the adult choroidal vasculature. To test this hypothesis, we propose the following. (1) To examine the role of RPE-derived VEGF in the stability of the adult choriocapillaris. Mice engineered for the inducible deletion of VEGF in the RPE will be generated to determine if VEGF derived from RPE is necessary to the integrity of the choriocapillaris in the adult. In addition, we have observed that transgenic mice that express only VEGF188 display an age-dependent degeneration of the choriocapillaris and RPE, which has many of the features of dry/atrophic AMD. The phenotype of these mouse models will be characterized using light and electron microscopy, fundus photography and ERG. (2) To determine if VEGF has autocrine effects on RPE in vivo and in vitro. RPE cells express VEGFR2 in adult, but not during development. This fact, coupled with the continuous expression of VEGF by RPE suggests a developmentally regulated autocrine role for VEGF. A possible role for VEGF in RPE will be investigated both in vitro using siRNA and in vivo by RPE- specific deletion of VEGFR2. (3) To elucidate the molecular regulation of physiologic VEGF expression by RPE. The mechanisms of VEGF expression in adult RPE will be investigated by co-expressing candidate transcription factors along with VEGF promoter-luciferase constructs. The role of transcription factors identified in promoter-reporter assays will be determined in RPE by knockdown using siRNA. The involvement of particular promoter binding sites will be verified by mutagenesis studies and by chromatin immunoprecipitation studies. Results of these studies will not only provide important insights into the control of VEGF expression, but should also be useful in the development of novel anti-VEGF therapies that can target VEGF involved in pathologic angiogenesis while sparing physiological VEGF expression. PUBLIC HEALTH RELEVANCE: Interactions between the retinal pigment epithelium and choroidal circulation are vital to normal retinal function and to pathologies such as age-related macular degeneration (AMD). Vascular endothelial growth factor (VEGF) is likely central to both. Understanding the role of VEGF in the maintenance of the choriocapillaris and the pigment epithelium is important to understanding the pathogenesis of AMD. In light of VEGF's role in vascular maintenance, current therapies aimed at VEGF neutralization in the eye for wet AMD may have unwanted side effects. Knowledge of the mechanisms of VEGF regulation in RPE under normal conditions may provide an opportunity to develop novel anti-VEGF therapies to inhibit the VEGF that mediates pathologic choroidal neovascularization, while sparing physiological VEGF expression.

描述（由申请人提供）：该提案中概述的研究旨在了解VEGF在视网膜色素上皮（RPE） - 脉络膜毛细血管相互作用中的作用。大量证据支持RPE在脉络膜脉管系统的发展和维持中的作用，RPE细胞与脉络膜脉管系统之间的相互作用在与年龄相关的黄斑变性（AMD）中至关重要。我们假设RPE严格调节VEGF是成年脉络膜脉管系统的完整性和维持所必需的。为了检验这一假设，我们提出以下内容。 （1）检查了RPE衍生的VEGF在成年绒毛膜毛细血管稳定性中的作用。将生成针对RPE中VEGF诱导缺失的小鼠，以确定成年人中脉络膜毛细血管的完整性是否需要从RPE中得出的VEGF。此外，我们已经观察到，仅表达VEGF188的转基因小鼠显示出脉络膜毛细血管和RPE的年龄依赖性变性，这些变性具有干/萎缩性AMD的许多特征。这些小鼠模型的表型将使用光和电子显微镜，眼底摄影和ERG进行表征。 （2）确定VEGF是否对体内和体外RPE产生自分泌作用。 RPE细胞在成人中表达VEGFR2，但在发育过程中不表达VEGFR2。这一事实以及RPE的VEGF的连续表达表明，VEGF的发展自分泌作用。 VEGF在RPE中的可能作用将在体外使用siRNA和体内通过rpe-特定于VEGFR2进行研究。 （3）通过RPE阐明生理VEGF表达的分子调节。 VEGF表达在成人RPE中的机制将通过共表达候选转录因子以及VEGF启动子 - 葡萄酸酶构建体来研究。使用siRNA敲除在RPE中确定的转录因子的作用将在RPE中确定。特定启动子结合位点的参与将通过诱变研究和染色质免疫沉淀研究来验证。这些研究的结果不仅将提供对VEGF表达的控制的重要见解，而且还应该在开发新型的抗VEGF疗法的开发中有用，这些抗VEGF疗法可以靶向参与病理血管生成的VEGF，同时保留生理VEGF表达。公共卫生相关性：视网膜色素上皮和脉络膜循环之间的相互作用对于正常的视网膜功能以及与年龄相关的黄斑变性（AMD）至关重要。血管内皮生长因子（VEGF）可能对两者都是核心。了解VEGF在维持绒毛膜毛细血管和色素上皮方面的作用对于理解AMD的发病机理很重要。鉴于VEGF在血管维持中的作用，目前旨在在湿AMD眼中进行VEGF中和的疗法可能会产生不必要的副作用。在正常条件下，RPE中VEGF调节机制的知识可能为开发新型的抗VEGF疗法提供机会，以抑制介导病理脉络膜新生血管形成的VEGF，同时抑制生理VEGF的表达。