Whereas it is well established that microvascular cells play crucial roles in the etiology of diabetic retinopathy and retinopathy of prematurity, the molecular and cellular mechanisms regulating disease inception remain obscure. To approach the mechanisms regulating these retinal vasoproliferative disorders, antibody and recombinant DNA probes will be used in model systems. Results will reveal the role that cell-matrix, growth factor-matrix and cell-cell interactions play in retinal microvascular differentiation and pathology. Retinal microvascular endothelial cells and pericytes isolated from developing and post-natal retinas will be cultured alone and together. Rigid or mechanically deformable substrates, which have been synthesized by retinal vascular cells and previously shown by our group to differentially regulate vascular cell growth and contractile phenotype, will be assessed. Specifically, we will characterize the molecular mechanisms regulating retinal pericyte development and differentiation as well as the retinal microvascular endothelial cell response to injury. To this end, we will use the combined approaches of: (i) antibody localization using contractile protein isoform-specific and growth factor-specific antibodies prepared and characterized in the lab, (ii) quantitative immunoprecipitation and fluorographic analysis of biolabeled subcellular fractions derived from developing, differentiated and injured cell populations, (iii) Northern blot and transcription run-off analyses of injured endothelial cells, growth-arrested and growth-stimulated pericytes purified from neonatal and adult retinas and (iv) PCR, homology probing and antibody screening of lambda Zap II cDNA libraries derived from retinal pericytes using probes designed to reveal genetic elements regulating retinal microvascular myogenic determination. Results of these interdisciplinary studies will help to determine whether growth factor-matrix, matrix-cell and/or cell-cell interactions modulate the expression of cis DNA regulatory elements and trans-acting factors responsible for controlling retinal microvascular growth, differentiation and the unmanageable vasoproliferation associated with diabetic retinopathy.
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