Regulation of plasminogen activator inhibitor-1 : role of triglyceride-rich lipoproteins, fatty acids and fibrate compounds

Abstract: Impaired fibrinolytic function secondary to elevated plasma plasminogen activator inhibitor-1 (PAI-1) activity is associated with atherothrombotic disease, in particular coronary heart disease. Both environmental and genetic factors contribute to determine plasma PAI-1 levels and PAI-1 production in vivo. Triglyceride-rich lipoproteins have been suggested to be important regulators of PAI- I expression, as serum triglyceride levels and plasma PAI- I activity are positively correlated. In addition, studies of endothelial cells and hepatocytes in culture have consistently found a dose-dependent activation of PAI-1 synthesis and secretion by triglyceride-rich very low density lipoproteins (VLDLs). Furthermore, the relation between serum triglycerides and plasma PAI-1 levels appears to be dependent on a common functional 4G/5G polymorphism in the promoter region of the PAI-1 gene. The present research programme was set up to investigate the mechanisms by which VLDLs activate PAI-1 expression in human cultured endothelial cells and to study the importance of the triglyceride and fatty acid content of VLDL particles for this activation. We found that VLDL stimulates PAI-1 expression in endothelial cells through transcriptional activation, and a VLDL response element (VLDLRE) was identified in the PAI-1 promoter, adjacent to and downstream of the 4G/5G polymorphic site. The VLDL-inducible transcription factor competed for binding with both the common transcriptional activator and the 5G allele-specific repressor. Various preparations of LDL particles were used to study the importance of the triglyceride content of lipoprotein particles for activation of PAI- I gene expression. Triglyceride-enriched LDL, but not native LDL, was found to increase PAI-1 protein and mRNA levels, and to enhance the binding of the VLDL-inducible transcription factor. Furthermore, unsaturated fatty acids were found to stimulate PAI-1 transcriptional activity, and to increase PAI-1 mRNA levels and PAI-1 protein synthesis and secretion, and the effect was shown to be mediated through the previously identified VLDLRE. In addition, the effect of fibrate compounds, derivatives of fatty acids, on PAI- I expression was studied in endothelial cells. Low concentrations of clofibric acid and bezafibrate increased PAI-1 transcription and secretion, whereas Wy-14643 increased PAI-1 synthesis in a dose-dependent way. In contrast, both fenofibric acid and gemfibrozil markedly decreased PAI-1 transcription and secretion from endothelial cells. The mechanisms by which endothelial cells bind and process VLDL particles, eventually leading to increased PAI-1 production, are unknown. Therefore, the potential role of lipoprotein receptors in mediating VLDL-induced PAI-1 activation was investigated. We found that the stimulating effect of VLDL on PAI-1 expression could be completely prevented by addition of receptor-associated protein (RAP) and to a large exent inhibited by inclusion of specific anti-VLDL receptor IgG, indicating that the VLDL receptor is a strong candidate for mediating VLDL effects on PAI-1 synthesis and secretion. The importance of fibrinolytic function for the risk of stroke was investigated in a prospective study of healthy individuals living in northern Sweden. We found that the 4G-allele of the PAI-1 polymorphism was associated with an increased risk of fature stroke. This relation was present for total stroke and for ischaemic stroke, and confined to hypertriglyceridaemic individuals. Conversely, serum triglyceride level was a predictor of stroke only in 4G homozygotes. Multiple logistic regression analysis identified systolic blood pressure, serum triglycerides, diabetes and presence of hypertriglyceridaemia in carriers of the 4G-allele as independent predictors of ischaernic stroke. Taken together, these results suggest that triglyceride-rich lipoproteins are important activators of PAI-1 gene transcription. The transcriptional activation appears to be dependent on the triglyceride and fatty acid content of the lipoprotein particles and is mediated through a VLDL response element located adjacent to and downstream of the 4G/5G polymorphic site. The interaction between the VLDL-inducible factor and the transcription factors binding to the 4G/5G polymorphic site gives a possible explanation for the genotype-dependent relation between serum triglycerides and plasma PAI-1 activity observed in several studies. The results of the prospective study of stroke suggest that this genotype-by-triglyceride interaction is an important in vivo determinant of fibrinolytic function and a predictor of future atherothrombotic disease.