Activation of NF-kB by Reactive Oxygen Species Mediates Induction of PAI-1 Expression in Vascular Endothelial Cells

Abstract number: P1768

Swiatkowska¹ M, Szemraj² J, Cierniewski¹ CS, Liao³ JK

¹¹Department of Molecular and Medical Biophysics, Medical University in Lodz, Poland ¹¹Department of Molecular and Medical Biophysics, Medical University in Lodz, Poland ²²Department of Biochemistry, Medical University in Lodz, Poland ³³Cardiovascular Division, Brigham & Women's Hospital and Harvard Medical School, Boston, MA 02115, USA

Objective:  Plasminogen activator inhibitor (PAI)-1 is an important physiological regulator of vascular hemostasis. PAI-1 is an acute phase reactant, which is induced by many pro-inflammatory and pro-oxidant factors such as TNFa and TGF-b1. We hypothesize that reactive oxygen species mediates TNFa-induced PAI-1 expression.

Methods:  Human endothelial cells were treated with TNFa or hydrogen peroxide with or without antioxidants, and intracellular superoxide anion production and oxidative stress were determined by lucigenin chemiluminescence and 2',7'-dichlorofluroscein (DCF) fluorescence, respectively. PAI-1 mRNA expression was determined by real time PCR. PAI-1 gene transcription was determined by transfection of a PAI-1 promoter linked to a luciferase reporter. NF-kB activity was determined by electrophoretic mobility shift assay shift as well as its activity modified either by antisense oligonucleotides or transfection of endothelial cells with wild type or mutated IkBa.

Results:  Treatment of endothelial cells with TNFa increased the generation of superoxide anion and oxidative stress. This corresponded to increase in PAI-1 mRNA expression, which was blocked by the antioxidant, N-acetylcysteine (NAC) and by overexpression of a super-suppressor phosphorylation-resistant IkBa. Indeed, TNFa stimulated NF-kB activity, which was inhibited by NAC.

Conclusion:  These findings indicate that NF-kB is an important regulator of TNFa-induced PAI-1 expression, and suggest that reactive oxygen species mediates PAI-1 expression via activation of NF-kB.