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VEGF在视网膜色素上皮(RPE) -绒毛膜毛细血管相互作用中的作用。重要的证据支持RPE在脉络膜血管的发育和维持中的作用，RPE细胞和脉络膜血管之间的相互作用是年龄相关性黄斑变性（AMD）的核心。我们假设RPE严格调节VEGF的表达对于成人脉络膜血管的完整性和维持是必要的。为了验证这一假设，我们提出以下建议。(1)探讨rpe来源的VEGF在成人绒毛毛细血管稳定性中的作用。将产生用于诱导RPE中VEGF缺失的工程小鼠，以确定来自RPE的VEGF对于成人绒毛膜的完整性是否必要。此外，我们观察到，仅表达VEGF188的转基因小鼠显示出绒毛膜和RPE的年龄依赖性变性，这具有干性/萎缩性AMD的许多特征。这些小鼠模型的表型将使用光学和电子显微镜，眼底摄影和ERG进行表征。(2)测定VEGF在体内和体外对RPE是否有自分泌作用。成人RPE细胞表达VEGFR2，但在发育过程中不表达。这一事实，再加上RPE持续表达VEGF，表明VEGF具有发育调节的自分泌作用。VEGF在RPE中的可能作用将在体外使用siRNA和体内通过RPE特异性删除VEGFR2进行研究。(3)阐明RPE对生理性VEGF表达的分子调控。VEGF在成人RPE中的表达机制将通过联合表达候选转录因子和VEGF启动子-荧光素酶构建体来研究。在启动子报告子试验中鉴定的转录因子的作用将在RPE中通过使用siRNA敲低来确定。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为VEGF表达的控制提供重要的见解，而且还将有助于开发新的抗VEGF疗法，这些疗法可以靶向参与病理性血管生成的VEGF，同时保留生理性VEGF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视网膜功能和年龄相关性黄斑变性（AMD）等病理至关重要。血管内皮生长因子（VEGF）可能是两者的核心。了解VEGF在绒毛膜毛细血管和色素上皮维持中的作用，对了解AMD的发病机制具有重要意义。鉴于VEGF在血管维持中的作用，目前针对湿性AMD的眼部VEGF中和疗法可能会产生意想不到的副作用。了解正常情况下RPE中VEGF调节的机制可能为开发新的抗VEGF疗法提供机会，以抑制介导病理性脉络膜新生血管的VEGF，同时保留生理性VEGF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.