Hypoxia Induces Transcription of the Plasminogen Activator Inhibitor-1 Gene Through Genistein-Sensitive Tyrosine Kinase Pathways in Vascular Endothelial Cells

Abstract

Abstract-A decline in oxygen concentration perturbs endothelial function, which promotes local thrombosis. In this study, we determined whether hypoxia in the range of that observed in pathophysiological hypoxic states stimulates plasminogen activator inhibitor-1 (PAI-1) production in bovine aortic endothelial cells. PAI-1 production, measured by ELISA, was increased by 4.7-fold (PϽ0.05 versus normoxic control, nϭ4) at 12 hours after hypoxic stimulation. Northern blot analysis showed the progressive time-dependent increase in the steady-state level of PAI-1 mRNA expression by hypoxia, which reached a 7.5-fold increase (PϽ0.05 versus control, nϭ4) at 12 hours. Deferoxamine, which has been known to bind heme protein and to reproduce the hypoxic response, induced PAI-1 production at both the mRNA and protein levels. The half-life of PAI-1 mRNA, as determined by a standard decay assay, was not affected by hypoxia, suggesting that induction of PAI-1 mRNA was regulated mainly at the transcriptional level. Transient transfection assays of the human PAI-1 promoter-luciferase construct indicates that a hypoxia-responsive region lies between Ϫ414 and Ϫ107 relative to the transcription start site, where no putative hypoxia response element is found. The hypoxia-mediated increase in PAI-1 mRNA levels was attenuated by the tyrosine kinase inhibitors genistein (50 mol/L) and herbimycin A (1 mol/L), whereas PD98059 (50 mol/L, MEK1 inhibitor), SB203580 (10 mol/L, p38 mitogen-activated protein kinase inhibitor), and calphostin C (1 mol/L, protein kinase C inhibitor) had no effect on the induction of PAI-1 expression by hypoxia and deferoxamine. Genistein but not daidzein blocked the production of hypoxia-and deferoxamine-induced PAI-1 protein. Thus, we conclude that hypoxia stimulates PAI-1 gene transcription and protein production through a signaling pathway involving genistein-sensitive tyrosine kinases in vascular endothelial cells. Key Words: hypoxia Ⅲ PAI-1 Ⅲ tyrosine kinase Ⅲ mitogen-activated protein kinase Ⅲ endothelial cells T here is growing evidence indicating that vascular endothelium plays an obligatory role in regulating fibrinolytic activity, such as vascular tone and platelet activity. 1 Endothelial dysfunction leads to the derangement of vascular endothelial anticoagulant properties and contributes to the pathophysiology of several cardiovascular disorders, including intravascular thrombosis, intimal growth, and plaque rupture. 2 Endothelial cells have been shown to produce many vasoactive substances that intervene in the fibrinolysis and coagulation processes. Of the many components regulating the balance between procoagulant and anticoagulant properties of the endothelium, pathways controlling plasminogen activator activity are particularly important. This activity is controlled primarily by plasminogen activator inhibitor-1 (PAI-1), which is synthesized by endothelial cells, 3 smooth muscle cells, 4,5 platelets, 6 monocytes, 7 and hepatocytes. 8

Role of Hypoxia-Inducible Transcription Factors 1α and 2α in the Regulation of Plasminogen Activator Inhibitor-1 Expression in a Human Trophoblast Cell Line

“…Cells were maintained at an oxygen concentration of 1−3% oxygen, a level similar to that which the placenta is exposed to before 10 weeks of pregnancy [32]. These results are consistent with previous studies demonstrating hypoxic induction of PAI-1 expression in trophoblasts and other cell types, and the demonstration of a HRE in the PAI-1 promoter [21][22][23]. We observed that hypoxic treatment induced a small (∼40%) but significant increase in PAI-1 levels in culture media from BeWo choriocarcinoma cells, whereas PAI-1 levels were extremely low in JAR choriocarcinoma cells under normoxic and hypoxic conditions (not shown).…”

“…Previous work showed that PAI-1 expression is increased under hypoxic conditions in several cell types including trophoblasts [21,22]. The PAI-1 gene, like several other hypoxiaresponsive genes, has a hypoxia response element (HRE) in its promoter through which hypoxia-inducible factor (HIF) induces transcription [23].…”

Gene program for cardiac cell survival induced by transient ischemia in conscious pigs

“…It is just this type of approach that could open new avenues to understand myocardial disease processes in terms of mechanisms not currently considered. Examples of these genes are PAI-1 and -2, which have been described in endothelial cells and tumors (12,15,45). Because one could argue that these genes were upregulated in the heart, but not in myocytes, e.g., vessels, fibroblasts, macrophage, we needed to confirm that the up-regulation was specific to myocytes.…”

Role of plasminogen activator-plasmin system in tumor angiogenesis

“…In contrast, when endothelial cells migrate, they upregulate uPA, uPAR and PAI-1 [55][56][57][58]. Hypoxia, a major stimulus for angiogenesis has been reported to increase uPAR and PAI-1 expression in endothelial cells [59]. A variety of angiogenic factors (cytokines and growth factors) control the expression of the plasminogen system.…”