The proposed studies focus on the mechanisms that underly the functions of the blood-retinal barrier.
The specific aims are: nature and mode of formation of luminal and abluminal endothelial vesicles, the mechanism of receptor-mediated endocytosis in vascular endothelial cells, the role of plasma membrane-associated phosphatases in active transport as it relates to permeable vessels, and the role of vesicular transport in breakdown of the blood-retinal barrier. Also included are related studies of antigenic components in the blood vessel basal lamina and study of the cytoskeleton in endothelial cells. These various studies are aimed at gaining a better understanding of retinal vascular function in normal and abnormal conditions. Luminal and abluminal endothelial vesicles will be studied in rapidly frozen, freeze-substituted tissues to verify the morphology of the vesicles and to determine the ratio of free (endocytic) to attached vesicles. Two physiologically important molecules, heparin and insulin, will be used to examine the pathways of receptor mediated endocytosis. Animals will be perfused with gold-ligand conjugates and examined for evidence of endocytic transport. We also plan to compare the localization of Na+,K+ ATPase, alkaline phosphatase and 5' nucleotidase activities in normal blood vessels and in those of streptozotocin-diabetic rats and galactose-fed rats which exhibit a diabetic-like condition. Breakdown of the blood-retinal barrier at the level of retinal capillaries will be induced by perfusion of animals with heparatinase, an enzyme which degrades heparan sulfate, and which has been shown to cause capillary leakage. This system will be used to investigate the morphology of luminal vesicles in frozen tissue and Possible changes in the polarity of the phosphatases mentioned above. In related work, several antigens, including type IV collagen, laminin, fibronectin and heparan sulfate proteoglycan will be localized in the vascular basal lamina using post- embedding, immunogold methods. The localizations obtained for normal vessels will be compared with those found in streptozotocin- diabetic and galactose-fed rats both of which show thickening of the vascular basal lamina. Results will be quantified using morphometric analysis. Finally, the endothelial cytoskeleton will be examined in normal arteriolar endothelial cells and in those of spontaneously, hypertensive-stroke prone rats.
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