Inhibition of inducible nitric oxide synthase gene expression and nitric oxide synthesis in vascular smooth muscle cells by granulocyte-colony stimulating factor in vitro

Abstract

Clinical and experimental evidence suggests that granulocyte-colony stimulating factor (G-CSF) acts as an anti-inflammatory modulator with beneficial effects in severe inflammatory diseases, e.g., sepsis and septic shock. Excessive production of nitric oxide (NO) is regarded as a potent mediator of the vascular changes leading to systemic hypotension that occurs during sepsis. Therefore, the aim of the present study was to investigate the influence of G-CSF on inducible nitric oxide synthase (iNOS) gene expression and NO synthesis in vascular smooth muscle cells (VSMC). Qualitative and quantitative analyses of iNOS cDNA revealed that G-CSF significantly reduced interferon-γ/lipopolysaccharide (IFN-γ/LPS) dependent iNOS gene expression (P<0.05) following 6, 18, 24, and 48 h incubation periods. In addition, the co-application of G-CSF resulted in a decreased IFN-γ/LPS mediated iNOS protein generation as detected by immunoblotting methods after 24 and 48 h. Measurement of the stable NO metabolites showed a significant reduction of nitrite/nitrate concentrations following co-incubation of VSMC with G-CSF+IFN-γ/LPS (242.57±10.73 nmol NO2−/NO3−/mg cell protein, n=8) as compared to IFN-γ/LPS treatment (306.20±19.26 nmol NO2−/NO3−/mg cell protein, n=8, P<0.05) following a 24-h incubation protocol. This inhibitory effect of G-CSF was still present after a 48 h incubation period (G-CSF+IFN-γ/LPS: 319.56±6.26 nmol NO2−/NO3−/mg cell protein; IFN-γ/LPS: 489.20±27.15 nmol NO2−/NO3−/mg cell protein (P<0.05), n=8, respectively). The present findings suggest that inhibition of iNOS gene expression and NO generation in VSMC might be one of the protective anti-inflammatory effects of G-CSF during sepsis.