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 beta-actin specific binding and filament capping protein, betacap73, 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 betacap73 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 Rho GTPase 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.
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