Type 1 plasminogen activator inhibitor (PAI-1) plays a major role in determining net fibrinolytic activity in vivo. PAI-1 antigen and activity levels are increased in a number of pathological states, including cardiovascular and thromboembolic disease, and sepsis, and in the acute phase (AP) response to trauma, infection, or surgery. The source of plasma PAI01 in the acute phase (AP) response to trauma, infection, or surgery. The source of plasma PAI-1 in the acute phase response is unknown and a matter of controversy; both liver and endothelial cells have been proposed. Very little is known about the regulation of human hepatic PAI-1 gene expression. The objectives of this project are to hypothesis that human hepatocytes are a major site of PAI-1 synthesis during the AP response, and to characterize the regulation of human hepatic PAI-1 gene expression by mediators of the AP response. The role of human hepatocytes in PAI-1 synthesis in vivo will be determined by in situ hybridization analysis of both normal liver and liver from patients with diseases associated with elevated plasma PAI-1. Because species differences in the acute phase response between rats and humans obviate the use of an animal model, the regulation of PAI-1 gene expression will be investigated in primary cultures of human hepatocytes and in the highly differentiated human hepatoma cell line, HepG2. The effects of mediators of the acute phase response, including glucocorticoid, and the cytokines interleukin-1, interleukin-6, tumor necrosis factor a, and transforming growth factor beta, alone and in combination, will be characterized. The molecular basis of this combinational regulation will be elucidated by investigating endogenous PAI-1 gene transcription and mRNA stability, and by identifying the cis- acting sequences and trans-acting proteins responsible for this regulation. We will directly assess the relationship between in vivo formation of DNA: protein complexes and changes in PAI-1 gene transcription in hepatocytes treated with AP mediators. The acute phase response is a homeostatic mechanism that protects the organism form tissue damage; however, the aberrant expression of hepatic PAI-1 may cause significant morbidity. A detailed molecular characterization of the cytokine and glucocorticoid regulation of PAI-1 gene expression in human hepatocytes will enhance our understanding of the acute phase response and of the role of PAI-1 in it, and may suggest important therapeutic manipulation of this homeostatic mechanism.
