In Vivo Stimulation of Vascular Plasminogen Activator Inhibitor-1 Production by very Low-Density Lipoprotein Involves Transcription Factor Binding to a VLDL-Responsive Element

Abstract

High plasma levels of plasminogen activator inhibitor-1 (PAI-1) are associated with an increased risk of cardiovascular disease. There is also a close relation between high plasma levels of PAI-1 and hypertriglyceridemia. Cell culture studies have shown that very low density lipoprotein (VLDL) increases the production and secretion of PAI-1 in endothelial cells and hepatocytes, suggesting a possible mechanism for this association. To determine whether VLDL stimulates PAI-1 production in vascular cells also in vivo, Sprague-Dawley rats were injected intravenously with 6 mg/kg of VLDL (derived from human subjects with type IV hyperlipidemia). Previous studies have demonstrated that this results in an accumulation of human VLDL in the aorta and other arteries followed by increased nuclear factor-kappa B (NF-kappaB) activation. Endothelial, but not smooth muscle cells, showed a basal PAI-1 mRNA and protein expression as assessed by in situ hybridization and immunohistochemistry, respectively. Six to twenty-four hours after the VLDL injection, lipoprotein particle accumulation was seen in the aortic wall, which was accompanied by increasing PAI-1 mRNA and protein expression in endothelial and smooth muscle cells. Within the rat PAI-1 promoter we identified a sequence located at -589 to -571 with 74% homology with the recently described VLDL responsive element in the human PAI-1 promoter and located adjacent to a 4-guanosine motif presumably corresponding to the human 4G/5G polymorphism. Transient transfection studies showed that VLDL exerts its stimulatory effects on rat PAI-1 gene expression in vascular cells by interaction with promoter sequences located within bp -656 and -505. Electrophoretic mobility shift assays showed that VLDL increases the binding of as yet incompletely characterized factors to this response element. Taken together these observations support a direct influence of VLDL on vascular PAI-1 gene expression ill vivo. This stimulation is exerted on the level of PAI-1 gene transcription, and involves transcription factor binding to a VLDL responsive element adjacent to a 4G motif within the PAI-1 promoter.