Role of RPE-derived VEGF in Choroid Development and Stability

描述（由申请方提供）：本提案中概述的研究旨在了解VEGF在视网膜色素上皮（RPE）-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，并且RPE细胞与脉络膜血管系统之间的相互作用在年龄相关性黄斑变性（AMD）中是中心的。我们推测RPE严格调控VEGF的表达对于成人脉络膜血管系统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源性VEGF在成人脉络膜毛细血管稳定性中的作用。将产生针对RPE中VEGF的可诱导缺失而工程化的小鼠，以确定源自RPE的VEGF对于成人中脉络膜毛细血管的完整性是否是必需的。此外，我们观察到仅表达VEGF 188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，其具有干性/萎缩性AMD的许多特征。将使用光学和电子显微镜、眼底照相和ERG表征这些小鼠模型的表型。(2)探讨VEGF在体外和体内对视网膜色素上皮细胞的自分泌作用。RPE细胞在成人中表达VEGFR 2，但在发育过程中不表达。这一事实，再加上VEGF的持续表达的RPE表明了一个发育调节的自分泌作用的VEGF。VEGF在RPE中的可能作用将在体外使用siRNA和在体内通过RPE特异性缺失VEGFR 2来研究。(3)阐明RPE对VEGF生理性表达的分子调控。将通过共表达候选转录因子沿着VEGF启动子-荧光素酶构建体来研究成人RPE中VEGF表达的机制。在启动子-报告基因测定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。这些研究的结果将不仅提供重要的见解VEGF表达的控制，但也应该是有用的，在新的抗VEGF治疗，可以靶向VEGF参与病理性血管生成，同时保留生理性VEGF表达的发展。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视网膜功能和诸如年龄相关性黄斑变性（AMD）的病理是至关重要的。血管内皮生长因子（VEGF）可能是两者的核心。了解VEGF在维持脉络膜毛细血管和色素上皮中的作用对了解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的眼睛中VEGF中和的疗法可能具有不希望的副作用。了解正常条件下RPE中VEGF调控机制可能为开发新型抗VEGF疗法提供机会，以抑制介导病理性脉络膜新生血管形成的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.