Experiments carried out in the investigator's laboratory have tested the general hypothesis that cell-cell and cell-matrix interactions modulate retinal microvascular growth and differentiation. Specifically, it addressed whether the endothelial-derived and matrix-associated growth regulators fibroblast growth factor (FGF-2) and transforming growth factor beta-1 (TGF-beta-1) influence retinal pericyte growth and contractile phenotype by signaling through molecular regulators of cell cycle progression and myogenic determination. Through the creation of in vitro and in vivo models, advantage will be taken of dominant-negative mutations in pericyte receptor tyrosine and serine/threonine kinases (RTKs, STKs), and knock-out mice harboring targeted disruptions in TGF-beta-1 and/or the cell cycle inhibitor p27 to directly establish the molecular mechanisms regulating the pericyte recruitment and differentiation during retinal microvascular remodeling. Using molecular, biochemical and cell biologic approaches, retroviral and plasmid-mediated gene delivery systems will be used to stably express high levels of truncation and point mutant RTK and STK in neonatal and adult pericyte cultures. Promoter-luciferase, in vitro kinase and receptor complementation analyses, and immunoprecipitation/Western blot will conclusively establish those downstream signaling components and cell cycle regulators required for promoting FGF and TGF-beta-1 mediated signal transduction. In addition, based on recent evidence accumulating in the applicant's laboratory which indicate that novel relationships may exist between the regulation of pericyte myogenic determination and cell cycle arrest, it is proposes to identify the molecular events controlling pericyte myogenic determination and contractile phenotype by characterizing the cis-regulatory and trans-activating factors responsible for regulating pericyte smooth muscle contractile protein expression, beginning with the vascular smooth muscle actin (VSMA) gene as a model. Promoter-luciferase, electrophoretic mobility shift and super-shift assays as well as DNA sequence-specific affinity chromatography will not only reveal the transcriptional regulators of pericyte contractile phenotype, but may also help to provide important new insights regarding apoptosis protection during myogenic commitment/differentiation. This knowledge will prove invaluable in order to unravel the molecular events controlling retinal microvascular morphogenesis during normal development or in association with retinal vasoproliferative disorders.
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