Differential Regulation of The Expression and Function of iNOS By Oxidative Stress Inducers In Cultured Vascular Smooth Muscle Cells

P Bingi, S Thakur, A Baydoun. University of Hertfordshire, Hatfield, Hertfordshire, UK

Introduction : The pathogenesis of atherosclerosis is complex and includes mechanisms and pathways associated with inducible nitric oxide synthase (iNOS). The role of iNOS in atherosclerosis is however controversial. Initially it was thought to have pro-atherogenic effects and contribute to the pathogenesis of the disease. Recent studies have however, shown that iNOS induced nitric oxide (NO) has cardio-protective properties which could be beneficial in retarding the progression of atherosclerotic lesions (Kanno et al., 2000; Okazaki et al., 2011). Previous studies in our research group have confirmed that iNOS is down regulated by oxidative stress (OS) inducers such as antimycin A (AA) and diethyl maleate (DEM) suggesting that part of the detrimental consequences to OS may be associated with the obliteration of iNOS and thus its cardio-protective effects. This is an attractive hypothesis that needs to be investigated but intriguingly hydrogen peroxide (H2O2), a common pro-oxidant, did not affect iNOS expression and function, indicating that the observations we made earlier may not simply be due to OS alone.

Methods : Rat aortic smooth muscle cells (RASMCs) in culture were treated with antimycin A (AA; 25-150µM), hydrogen peroxide (H2O2; 50-300µM) or diethyl maleate (DEM; 1-10µM) 30minutes prior to stimulation with bacterial lipopolysaccharide (LPS; 100 µg/ml) and interferon-gamma (IFN-γγ; 100 units/ml) for 24 h. Atorvastatin (10µM), Polyethylene glycol-superoxide dismutase (PEG-SOD, 500U/ml) and Polyethylene glycol-catalase (PEG-CAT, 500U/ml) were added 30minutes prior to the treatment with OS inducers. Nitric Oxide was quantified by the Griess assay, estimation of protein by bicinchoninic acid (BCA) assay, 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was employed to assess cell viability and variations in iNOS expression by western blotting. Generation of reactive oxygen species (ROS) was determined in the presence and absence of AA, H2O2 and DEM using 2, 7-dichlorofluorescein and MitoSOX Red by confocal laser scanning microscopy. Fluorescence and iNOS expression was measured semi-quantitatively using image-J software. Statistical analysis was carried out using one-way ANOVA followed by Bonferroni as a post-hoc test.

Results : Antimycin A and DEM resulted in concentration dependent inhibition of NO production and iNOS expression (p<0.001) whereas H2O2 was without any effect. Atorvastatin (10µM) significantly (p<0.001) and PEG-SOD (500U/ml) partially reversed the inhibition of NO production and iNOS expression. PEG-CAT was without effect. Antimycin A, H2O2 and DEM induced ROS production in a concentration and time dependent manner (p<0.001) but AA and DEM caused more generation of superoxide radicals (p<0.001) than did H2O2 which generated mostly hydroxyl radicals.

Conclusion : These findings suggest that iNOS expression and nitrite production may be down regulated partly by pro-oxidants generating superoxide but not hydroxyl radicals and this could explain the differential regulation of these processes by the different OS inducing agents.

References:

Kanno, S., Lee, P.C., Zhang, Y., et al, Circulation; 101(23):2742-8, 2000.

Okazaki, T., Otani, H., et al, Journal of molecular and cellular cardiology: 534-544, 2011.