The brain, of all organs of the body, most tightly regulates entry of blood-borne substances. The site of this blood brain barrier (BBB) resides in endothelial cells. In close contact with endothelial cells are astrocytes, which are postulated to influence the expression of BBB properties. Some of these properties, such as specific transport systems and receptors, have been identified but many of the biochemical mechanisms that control entry of blood-borne substances into the brain are not yet characterized. An important step toward elucidation of such mechanisms is characterization of macromolecular components of the endothelial membrane that play a role in the BBB. We have isolated a mouse monoclonal antibody (anti-EBA), which reacts with three protein components of the capillary-luminal surface of endothelial cells of brain, but not of most other organs. These protein components, endothelial barrier antigens or EBA, are shown to be an integral part of the BBB both by their immunocytochemical localization to endothelial cells and by their loss in experimental allergic encephalomyelitis where the integrity of the BBB is severely compromised.
The aim of this proposal, therefore, is to define the role of EBA in establishing BBB properties. A multi-faceted approach will utilize immunocytochemistry of EBA and other brain antigens, classical protein chemistry of EBA, CDNA cloning of EBA genes, and functional studies of several BBB transport systems and EBA in isolated microvessels and endothelial cell cultures. This proposal is designed to answer a number of fundamental questions: 1) What is the site of synthesis of EBA? In endothelial cells or elsewhere? 2) Do astrocytes influence expression of EBA in endothelial cells? 3) What are the structural properties of the three EBA proteins? Are they peripheral or integral membrane proteins? What are their primary structures? Do differences in posttranslational modifications account for differences in apparent molecular weight? 4) During BBB development in the rat brain does EBA expression correlate with specific """"""""turn-on"""""""" or """"""""turn-off"""""""" of other BBB properties? Do EBA or anti-EBA affect transport function? 5) Does EBA perform a structural or regulatory role in the EC membrane? The BBB is of great physiological importance in regulating the delicate balance of nutrients and minerals required for proper brain function. It also is of clinical importance. In the adult, its breakdown correlates with certain Z diseases such as multiple sclerosis and hypertension. In addition, the BBB is the most important barrier to access of therapeutic drugs into the brain. In 0 the immature central nervous system, it allows penetration of toxins such as lead, causing mental retardation. Changes in the functional properties of endothelial cells occur during development and may be at least partially responsible for the different vulnerabilities of adult and developing brain. Successful completion of this proposal to characterize EBA protein components of the BBB will significantly advance understanding of this crucial system.
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