Role of RPE-derived VEGF in Choroid Development and Stability

描述(由申请人提供)：本提案中概述的研究旨在了解血管内皮生长因子在视网膜色素上皮(RPE)-脉络膜毛细血管相互作用中的作用。大量证据支持RPE在脉络膜血管系统的发育和维持中的作用，RPE细胞和脉络膜血管系统之间的相互作用在老年性黄斑变性(AMD)中是核心的。我们推测，RPE严密调控血管内皮生长因子的表达对于成人脉络膜血管的完整性和维持是必要的。为了检验这一假设，我们提出了以下建议。(1)探讨RPE来源的血管内皮生长因子在成人脉络膜毛细血管稳定性中的作用。为RPE中的血管内皮生长因子的可诱导缺失而设计的小鼠将被产生，以确定来自RPE的血管内皮生长因子是否对成人脉络膜毛细血管的完整性是必需的。此外，我们观察到，仅表达VEGF188的转基因小鼠表现出脉络膜毛细血管和RPE的年龄依赖性退化，这具有许多干性/萎缩性AMD的特征。这些小鼠模型的表型将使用光学和电子显微镜、眼底照相和ERG进行表征。(2)检测血管内皮细胞生长因子在体内和体外对视网膜色素上皮是否具有自分泌作用。RPE细胞在成体表达VEGFR2，但在发育过程中不表达。这一事实，再加上视网膜色素上皮细胞持续表达血管内皮生长因子，提示血管内皮生长因子具有发育调节的自分泌作用。血管内皮生长因子在RPE中的可能作用将在体外利用siRNA和在体内通过RPE特异性缺失VEGFR2来研究。(3)阐明RPE对生理性血管内皮生长因子表达的分子调控作用。通过将候选转录因子与血管内皮生长因子启动子-荧光素酶构建物共表达来研究血管内皮生长因子在成人视网膜色素上皮中的表达机制。在启动子-报告分析中确定的转录因子的作用将通过使用siRNA敲除RPE来确定。特定启动子结合位点的参与将通过突变研究和染色质免疫沉淀研究来验证。这些研究结果不仅将为控制血管内皮生长因子的表达提供重要的见解，而且也将有助于开发新型的抗血管内皮生长因子疗法，该疗法可以靶向参与病理性血管生成的血管生成，同时抑制生理性血管内皮生长因子的表达。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正常的视网膜功能和老年性黄斑变性(AMD)等病理疾病至关重要。血管内皮生长因子(VEGF)可能是两者的中心。了解血管内皮生长因子在维持脉络膜毛细血管和色素上皮中的作用对于了解AMD的发病机制具有重要意义。鉴于血管内皮生长因子在血管维持中的作用，目前针对湿性AMD眼部血管内皮生长因子中和的治疗方法可能会有不必要的副作用。了解正常条件下视网膜色素上皮中血管内皮生长因子的调节机制，可能为开发新型的抗血管内皮生长因子疗法提供机会，以抑制介导病理性脉络膜新生血管的血管内皮生长因子，同时避免生理性血管内皮生长因子的表达。

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.