BLOOD-RETINAL BARRIER CHANGES IN RETINOPATHY

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Early breakdown of the blood-retinal barrier (BRB) is common in retinal diseases that eventually progress to proliferative retinopathy and has been suggested to be a contributing factor. The sites and sequence of BRB changes have been described for many diseases, but little is known about the specific causes or effects of permeability dysfunction. In order to identify stimuli for permeability changes and understand the mechanisms of their actions, we have devised cell culture models for the BRB. Our strategy is to look for cellular sources of permeability inducing substances in RCS dystrophic rats and then to test their effects and identify the specific stimuli using cell culture models and conditioned medium approaches. Our data suggest the hypothesis that during retinal degeneration, altered cellular interactions stimulate increased permeability of retinal pigment epithelial (RPE) and retinal microvascular endothelial (RMVE) cell layers, possibly by effects on composition of the extracellular matrix (ECM). Alternative hypotheses are that the genetic defect in dystrophic rats is the cause of RPE permeability increases, or that substances released by the degenerating photoreceptors stimulate increased permeability of both RPE and RMVE layers. The following specific experimental aims will test these hypotheses: 1) Determine the barrier properties of normal and dystrophic RPE cells and normal RMVE cells in culture and assess the effects of altered cell-cell interactions (RPE cells with microglia, RMVE cells with glial and RPE cells), the genetic defect in dystrophic RPE cells, and substances released from degenerating photoreceptors. These experiments will use a biochemical assay to measure permeability and high resolution immunolocalization approaches to determine effects on cell junctions, intracellular transport, integrin receptor-ligand interactions, and cytoskeletal organization. 2) Determine whether stimuli that induce increased RMVE cell permeability in vitro also induce alterations in RMVE cell ECM fibronectin and laminin composition, synthesis, gene expression, or turnover. These experiments will use immunoprobe, molecular, and substrate hydrolysis technologies. 3) ldentify stimuli With activity in modulating RPE and RMVE cell permeability by testing, known growth factors for activity in stimulating or inhibiting RPE and RMVE permeability in vitro. Identification of factors which induce or promote increased permeability in the BRB will highlight candidate mechanisms for therapeutic intervention in diseases such as diabetic retinopathy.