The objective of this research program is to study the growth and function of cultured human endothelial cells (EC) isolated from diverse vascular sites. Vascular tissue will be obtained from brain-dead, heart-beating cadavers, and large vessel and capillary EC will be isolated. In initial experiments priority will be given to a human iliac vein endothelial cell line derived from a young, healthy individual. The following goals will be pursued: 1. Cultures of adult human vascular EC will be cultivated and characterized with respect to EC marker functions, karyotypes, and culture lifespans. 2. Human EC proliferation in vitro will be investigated as effected by cooperative interactions between heparin, brain-derived endothelial mitogen, fibronectin, and collagen. The following approaches will be used: a) Purification and fractionation of components; b) Definition of EC response to components from a); c) Interaction of components under cell-free conditions; d) Influence of serum on the effects and interaction of components. 3. A human EC function, the expression of angiotensin I-converting enzyme (ACE) in vitro, will be investigated as it is affected by cooperative interactions between heparin, brain-derived endothelial mitogen, fibronectin, and collagen. The following approaches will be used: a) Definition of EC response (in terms of ACE activity) to culture components purified and fractionated in Aim #2; b) Development of specific polyclonal or monoclonal antibodies to quantitate ACE amounts, synthesis and degradation; c) Analyses of ACE expression in cloned and uncloned cultures within a single subcultivation growth cycle. 4. After sufficient progress has been made in achieving the above goals, possible effects of vascular site, donor age and sex, and in vitro cultivation on function and proliferation in culture human EC will be investigated.
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