Activation of the nuclear regulatory factor, NF-κb, by reactive oxygen intermediates (ROI) and release of TNF-α from cultured vascular smooth muscle cells

Introduction: We previously reported that vascular tissue and vascular smooth muscle cells are a source of TNF-α (Crit Care Med 25: 519, 1997). Since it has been shown that elevated levels of this cytokine accompany ischemia-reperfusion injury we hypothesized that NF-κB, activated by ROI formed during reperfusion, is involved in the production TNF-α by smooth muscle cells. Methods: Smooth muscle cells were cultured from the aortas of Sprague-Dawiey rats. To generate ROI, cells were exposed to 2 mM hypoxanthine and 0.003 units/ml xanthine oxidase (Hx/XO) added to the culture medium. At 0, 15, 30, 60, 90, 180 min. after addition of Hx/XO, the medium was removed and whole cell extracts were prepared for the determination of NF-fdB by electrophoretic mobility shift assay. In other experiments, medium removed from cells was analysed for TNF-α by the L929 cell cytotoxicity assay. Values of TNF-a are reported as units/mg cell protein. Results: In the presence of Hx/XO, basal levels (0 time) of NF-κB were detected as faint bands on gels of cell extracts from 3 rats. At 15 min. the bands were unchanged. However, by 30 min. activation of NFncB was indicated by intense buds which remained elevated at 90 min. Specificity for NFncB was shown by the absence of competition by an excess of oUgonadeotide containing the nuclear factor, CREB, recognition sequence, and by the elimination of bands by a 100 fold excess cold oligonuclcotide containing the NFncB binding site. Similar results with NF-tcB were obtained in cells from an additional 2 rats in which experiments were extended to 360 nun. At 0 time TNF-a was (indetectable in the medium from cells treated with Hx/XO. However, at 60.180 and 360 min. TNF-α was significantly elevated to 20 ±1, 39 ±5 and 25 ± 3 units/mg cell protein, respectively (n 3). The addition of an ROI scavenging system, Superoxide dismutase/catalase, significantly reduced TNF-a at all time points. Coclusios: These data suggest that TNF-α could be produced locally in blood vessels during an ischemia-reperfusion event and are consistent with the idea that NF-κB, activated by ROI is involved in the regulation.