The attenuated layer of endothelial cells lining the blood vessels forms the critical barrier controlling the exchange of molecules from the blood to the interstitial fluid. The broad focus of this project is to define the cellular and molecular basis of the endothelial barriers and pathways that mediate capillary permeability. The ability of the endothelium to act both as a passive, serum-modified barrier to transvascular exchange and as a specific receptor-mediated translocator of molecules is dependent on interactions occurring not only at its luminal surface but also with transport pathways such as plasmalemmal vesicles. Specific endothelial proteins involved in the structure and function of transport pathways will be examined. Since albumin is the major plasma protein and has multiple cardiovascular functions, it will be used as a probe to define at both the cellular and molecular levels protein interactions with the endothelium. Albumin can increase the permselectivity of the endothelial barrier while conversely, facilitating as a carrier the transendothelial transport of select molecules such as fatty acids. The cellular and molecular mechanisms mediating albumin's effects on capillary permselectivity are uncertain. Our work shows that albumin specifically interacts with the endothelium and traverses it via plasmalemmal vesicles. Four albumin binding proteins (ABP) have been identified (SPARC, albondin, gp30 and gpl8); however, it is unclear which ones mediate the effects of albumin on the transport of itself and its bound ligands versus that of other molecules. Albondin and SPARC appear to share a common binding domain for native albumin whereas gp30 and gpl8 appear to be scavenger receptors for modified albumins that may play an important role in various vascular disease processes including atherosclerosis, diabetes and aging. A novel technique for isolating endothelial membrane from tissue will be used to identify and purify the endothelial ABP. Antibody and/or peptide probes will be used to determine specific protein function and the location of ABP within transendothelial transport pathways and barriers. Antibodies to albondin have been made and can inhibit albumin binding and transport by endothelium. They will be used for cloning and sequencing to determine the apparent common albumin binding domains of albondin and SPARC. These approaches afford a means to examine at the cellular and molecular level the interaction of blood molecules with the endothelium and how these interactions influence capillary permeability. Although this project focuses on normal functions, it is clear that structural and functional alterations of capillaries are involved in the development of atherosclerosis and various serious sequelae of diabetes such as diabetic retinopathy, nephropathy and polyneuropathy. Furthermore, information about endothelial transport may be useful in the future for devising approaches to increase drug delivery to select tissues.
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