The goals of this research include, first, evaluation of factors restrain proliferation and enhance differentiation of cultured retinal cells, including pericytes and endothelial cells of the retinal microvessels, the retinal pigment epithelium (RPE), and retinal glia. Factors to be evaluated include basement membrane macromolecules; interactions with other, neighboring cells; and a substance present in aqueous extracts of bovine retina that promotes morphologic differentiation of neuroglia. The interaction of disease states (diabetes and galactosemia, simulated by elevated levels of glucose and galactose in the culture medium) with the above factors will be evaluated. The types, and quantities, of extracellular matrix macro-molecules synthesized by retinal vascular, RPE, and retinal glial cells cultured on a basement membrane matrix under normal conditions and with elevated glucose or galactose, with or without an aldose reductase inhibitor, will be studied. The role of RPE and choroidal vascular cells in modifying one another's behavior will be studied by histologic, autoradiographic, and immunocytochemical methods in a model of human subretinal neovascularization produced in pigmented rats by mild krypton laster photo-coagulation. The """"""""glial modulating factor"""""""" from retina will be further purified, monoclonal antibodies prepared to it, and biochemical and immunological comparisons will be made with acidic fibroblast growth factors from retina and brain, and the """"""""glial maturation factor"""""""" of Lim, isolated from bovine brain. Immunocytochemical localization of this factor in the retina will be attempted. A second goal of this research is to determine factors that stimulate contraction of retinal pericytes, cells whose abnormality may be critical in diabetic retinopathy. This will be approached using vasoactive agents (norepinephrine, angiotensin II, and vasopressin), and variations in the O2 and CO2 levels in the culture medium, on pericytes grown on deformable matrices, and the determining whether these agents produce an elevation in cytoplasmic free Ca++ in cultured pericytes, using fluorescent Ca++-binding probes and microspectrofluorimetry. A third goal of this project is to initiate additional long-lived non-neoplastic human retinal cell lines from epiretinal membranes excised during vitrectomy surgery on infants with stage 5 retinopathy of prematurity. Cells obtained through these cultures will be defined immunocytochemically and used in the retinal cell culture experiments described above.
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