The major aims of this proposal are to develop means for the isolation, culture and characterization of adult human pulmonary endothelial cells. The cells will be cultured by methods that avoid exposure to damaging proteolytic enzymes at eiher the isolation step or during serial subculture. Cells will be isolated from large vessels such as the pulmonary artery by scraping with a scalpel and from cells of the pulmonary microvasculature by perfusion with microcarriers of known diameter. The cells will be identified by light microscopy, electron microscopy, immunocytochemistry, and by assay of endothelial surface enzymes such as angiotensin converting enzyme and carboxypeptidase N. The techniques used for identification will also be used to monitor the continuing expression of endothelial characteristics after long-term, large-scale culture on microcarriers. A major portion of the work will be devoted to developing growth media and conditions for culturing human pulmonary endothelial cells that retain differentiated characteristics during extended periods in vitro. Once cultures become successful, studies will begin of mechanisms of injury to endothelial cells caused by proteolytic enzymes and by complement components. Assessment of injury will be by examination of the endothelial glycocalyx, by expression of endothelial surface receptors (such as the Fc component of IgG and the C3b component of complement), expression of surface enzymes and by measurement of endothelial heomstatic factors.
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