The objectives of this proposal are: 1) to analyze mechanisms involved in transport of plasma macromolecules across microvascular endothelium ii) to define the physicochemical basis of the dependence of the permselectivity of the endothelial barrier on albumin interaction with the glycocalyx and iii) delineate the pathologic molecular mechanism(s) for increased solute transvascular exchange with diabetes. Specifically, the project will center on the interaction of albumin and glycoalbumin with the endothelium both in vivo and in vitro under normal and diabetic conditions. The work in vitro will involve growth in culture of endothelial cells isolated from various vascular beds (hearts, retina, brain, lung, fat pad, and aorta). These cell monolayers will be used to assess the binding of albumin and glycoalbumin. In order to define at the molecular level now albumin (A) and glycoalbumin(GA) interact with the endothelium in vitro and in situ/vivo, we will i) quantitate the transport rate of A and Ga across the endothelium of various microvascular beds; ii) determine the location and transendothelial transport pathway of A and GA using immunocytochemical techniques: iii) identify and characterize endothelial plasmalemmal proteins, especially those proteins that are preferentially glycated with hyperglycemia or involved in the binding of GA and A to the endothelium. We believe these approaches represent a unique system to study at the cellular and molecular level albumin-endothelium interactions under normal and pathologic conditions. Although much more work is necessary to understand more clearly microvascular transport phenomena, this project in basic microvascular research has been extended to include aspects of pathological change in the endothelial barrier and transvascular exchange with diabetes. It is clear that structural and functional alterations of capiliaries are involved in the development of serious sequelae of diabetes including diabetic retinopathy, nephropathy and polyneuropathy. There is increasing evidence that poor metabolic control and the resultant hyperglycemia plays an important role in the development of diabetic microangiopathy. This project proposes to examine the effects of hyperglycemia on the molecular mechanisms of tissue-blood transport of macromolecules. The effects of glycation of albumin and endothelial plasmalemmal surface protein on transendothelial transport will be examined.
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