A Reactive Oxygen-Generating System Activates Nuclear Factor-κB and Releases Tumor Necrosis Factor-α in Coronary Smooth Muscle Cells

Abstract

Background. Recently we reported that bacterial lipopolysaccharide (LPS) stimulates release of tumor necrosis factor α (TNF-α) from porcine coronary arteries and smooth muscle cells cultured from those vessels. It has also been reported that plasma levels of TNF-α are elevated after myocardial infarction. Since it is known that the production of reactive oxygen intermediates (ROI) occurs during ischemia and ROI are suggested activators of the nuclear regulatory factor κB (NF-κB), we tested the hypothesis that release of TNF-α from smooth muscle cells could also be stimulated with a ROI-generating system.Materials and Methods. Smooth muscle cells were isolated from porcine coronary arteries. Confluent cells in 48-well culture dishes were treated for 30 min with 0.003 units/ml xanthine oxidase (XO) and 2 mM hypoxanthine (HX) added to the culture medium. The medium was then removed and the cells were washed three times and fresh medium without HX-XO was added. Then, at 1, 3, and 6 h the medium was removed and analyzed for biologically active TNF-α. In other experiments, smooth muscle cells were treated with 20 μg/ml LPS for 6 h and aliquots of medium analyzed for TNF-α. Untreated cells served as controls. Data were analyzed by two-way ANOVA with repeated measures. Extracts of total cell protein were prepared and activation of NF-κB was determined by electrophoretic mobility shift assay.Results. Treatment of cells with HX-XO stimulated release of TNF-α, which rose to a maximum of 17.5 ± 1.7 units/mg cell protein at 6 h. This was significantly higher (P < 0.05) than release stimulated by LPS (10.2 ± 1.0 units/mg at 6 h) or TNF-α detected in the culture medium from untreated control cells (4.2 ± 0.9 units/mg protein at 6 h). Both HX/XO and LPS activated NF-κB.Conclusions. These results support the conclusion that coronary smooth muscle cells are a potential source of TNF-α during events that are associated with formation of ROI such as myocardial ischemia.