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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1155.)
© 2000 American Heart Association, Inc.

Thrombosis

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

Tsuyoshi Uchiyama; Masahiko Kurabayashi; Yoshio Ohyama; Toshihiro Utsugi; Nobuhiro Akuzawa; Mahito Sato; Shouichi Tomono; Shoji Kawazu; Ryozo Nagai

From the Second Department of Internal Medicine, Gunma University School of Medicine (T. Uchiyama, M.K., Y.O., T. Utsugi, N.A., M.S., R.N.), and the School of Health Science, Faculty of Medicine, Gunma University (S.T.), Gunma; Saitama Medical Center, Saitama Medical School (S.K.), Saitama; and the Department of Cardiovascular Disease, Graduate School of Medicine, University of Tokyo (R.N.), Tokyo, Japan.

Correspondence to Masahiko Kurabayashi, MD, Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15, Showa-machi, Maebashi, 371-8511, Japan.

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 (P<0.05 versus normoxic control, n=4) 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 (P<0.05 versus control, n=4) 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 -414 and -107 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 µmol/L) and herbimycin A (1 µmol/L), whereas PD98059 (50 µmol/L, MEK1 inhibitor), SB203580 (10 µmol/L, p38 mitogen-activated protein kinase inhibitor), and calphostin C (1 µmol/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 • PAI-1 • tyrosine kinase • mitogen-activated protein kinase • endothelial cells

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