Regulating retinal microvascular morphogenesis

描述(申请人提供)：视网膜微血管形态发生是一个复杂和高度协调的过程，发生在胚胎发育期间，出生后并与几种视力损害疾病有关，包括早产儿视网膜病变(ROP)、老年性黄斑变性和糖尿病视网膜病变。首席研究员实验室最近开展的工作表明，基于等肌动蛋白的细胞骨架重构是发育和病理性血管生成过程中观察到的微血管迁移和增殖所不可或缺的，包括在视网膜微血管成熟过程中观察到的基于周细胞的重构。在一个集中的、跨学科的研究计划中，我们将利用一系列成熟的分子、细胞生物学和分子遗传学方法，利用体外和体内模型，旨在揭示调控(I)视网膜内皮细胞迁移驱动正常和病理性血管生成，以及(Ii)基于周细胞控制内皮细胞增殖和毛细血管收缩的分子机制和基于等肌动蛋白的信号级联反应。对视网膜微血管内皮细胞培养的定量分析将与旨在过度表达实验室发现的新型(-肌动蛋白特异性结合和微丝覆盖蛋白)(Cap73)的实验相结合。为了揭示基于异肌动蛋白控制发育和病理性血管生成的分子机制，我们将利用“两只小鼠”转基因方法，其中我们将特异性地诱导(Cap73)在出生后血管内皮细胞过度表达。这些联合结果揭示了内皮运动和血管生成受损的变化，将有助于指导旨在识别控制这些关键微血管事件的关键信号效应因子的cDNA表达阵列分析。此外，为了揭示调控周细胞收缩和视网膜内皮细胞生长的分子信号机制，我们将研究RhoGTPase家族成员在微血管形态发生过程中在信号转导血管细胞骨架重构和等肌动学中所起的作用。基于最近获得的初步数据和提出的实验方法，我们的研究计划将为调节正常发育过程中视网膜微血管形态发生的分子机制以及与糖尿病视网膜病变的病理性血管生成相关的分子机制提供重要的新见解。

DESCRIPTION (provided by applicant): Retinal microvascular morphogenesis is a complex and highly-coordinated process, which occurs during embryonic development, post-natally and in association with several visually-impairing diseases, including retinopathy of prematurity (ROP), age-related macular degeneration and diabetic retinopathy. Recent work carried out in the principal investigator's laboratory has revealed that isoactin- based cytoskeletal remodeling is integral to the microvascular migration and proliferation observed during developmental and pathologic angiogenesis, including the pericyte-based remodeling seen during retinal microvascular maturation. In a focused, inter-disciplinary research plan that will take advantage of in vitro and in vivo models using a spectrum of well-established molecular, cell biology-based and molecular genetic approaches, we aim to reveal the molecular mechanisms and the isoactin-based signaling cascades regulating (i) retinal endothelial migration driving normal and pathologic angiogenesis, and (ii) pericyte-based control of endothelial proliferation and capillary contractility. Quantitative analyses of retinal microvascular endothelial cell cultures will be performed in conjunction with experiments aimed at over-expressing the novel (-actin-specific binding and filament capping protein, (cap73, discovered in the lab. To reveal the molecular mechanisms driving isoactin-based control of developmental and pathologic angiogenesis, we will take advantage of a 'two-mouse' transgenic approach, where we will specifically induce (cap73 over-expression within the post-natal vascular endothelium. These combined results, revealing alterations in endothelial motility and impaired angiogenesis, will serve to guide cDNA expression array analyses aimed at identifying key signaling effectors controlling these pivotal microvascular events. Further, to reveal the molecular signaling mechanisms regulating pericyte contractility and retinal endothelial growth, we will characterize the role that RhoGTPase family members play in signaling vascular cytoskeletal remodeling and isoactin dynamics during microvascular morphogenesis. Based on the preliminary data recently obtained and the experimental approaches proposed, we anticipate that our research plan will provide important new insights into the molecular mechanisms regulating retinal microvascular morphogenesis during normal development and in association with the pathologic angiogenesis accompanying diabetic retinopathy.