The production of tPA and its secretion into the vasculature has been long assumed to be the responsibility of the vascular endothelium. In vitro, tPA is constitutively produced by endothelial cells from a variety of sources, large as well as microvessels from numerous normal and pathologic tissues. Thus, it has been concluded that all endothelial cells produce tPA in vivo regardless of the anatomic location. In contradiction to these concepts, studies described in this application have shown that in vivo expression of endothelial cell tPA is restricted to a distinct minority of endothelial cells. In the femoral artery and vein, aorta, carotid and other large vessels, tPA antigen is not found in the large vessel endothelium but only in some of the small vessels within the adventitial layer, the vasa vasora. Likewise, analysis of the brain and lung microvasculature show an even more limited presence of tPA. In the primate brain only 3% of the vessels are stained positive for tPA and 90% of those are in post-capillary venules between 7.5 and 30 Xm in diameter. tPA is never found associated with the capillary endothelium. In the lung, constitutive tPA expression occurs in the endothelium of the bronchial arteries but in few of the vessels of the pulmonary vasculature. The few pulmonary vessels that do show expression are between 7.5 and 30 Xm. Thus, tPA expression is limited by type (bronchial arteries and vasa vasorum) or size (7.5-30 Xm) of vessel. Under conditions promoting an acute inflammatory response, the number of vessels expressing tPA increases while remaining limited to small, non-capillary endothelial cells. The hypothesis to be tested is that this expression pattern represents a refinement of the concept of tissue and organ specific gene expression to include gene expression specific for a subpopulation of endothelial cells and it is the intent of the proposed studies to define the molecular basis for this specificity, i.e., the portion of the tPA gene and the protein factors required for cell specific constitutive and inflammation-induced cell tPA gene expression. Studies of the molecular regulation of tPA in cultured endothelial cells have proven difficult given the low expression levels, the continuous constitutive production of tPA, and the small increases observed with physiologic and pathologic stimuli. Therefore, experiments are proposed using established transgenic mouse lines containing sized tPA promoter fragment-lacZ reporter constructs. These studies will have broad application for understanding the basis for cell specific regulation of not only tPA but also other endothelial cell specific functions.
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