Inflammatory cytokines present in smokers’ serum modulate cyclooxygenase-2 expression via NADPH-oxidase in endothelial cells

Background: Cigarette smoking modulates inflammation and endothelial dysfunction, events implicated in athero-thrombotic disease.1,2 Recently, endothelial dysfunction has been related to reactive oxygen species (ROS)3 and to prostanoids produced via cyclooxygenase-2 (COX-2)4. We hypothesized that inflammatory cytokines presents in smokers’ serum induce ROS generation in endothelium by activation of NADPH oxidase with consequent COX-2 expression. Methods: Serum levels of interleukin-1beta (IL-1) and tumor necrosis factor alpha (TNF) were measured in 18 male volunteers (9 smokers and 9 non smokers). Human bone marrow endothelial cells (HBMEC) were exposed to serum from smokers or non-smokers, and ROS production (dichlorofluorescein fluorescence), NADPH oxidase activation (translocation of p47phox), COX-2 protein (Western blot) and mRNA (quantitative RT-PCR) were evaluated. Results: Serum from smokers compared with non smokers showed higher levels of IL-1 (P<0.05) and TNF (P<0.0008) and greater ability to induce ROS generation (P<0.002), translocation of p47phox from cytoplasm to plasma membrane (P<0.01), and increase COX-2 mRNA and protein (P<0.002) in HBMEC. Serum-induced ROS production and COX-2 mRNA by HBMEC correlated positively with IL-1 (r=0.723, P<0.002 and r=0.707, P<0.002) and TNF ( =0.781, P<0.0002 and r=0.772, P<0.0003). Moreover, there was a significant positive correlation between ROS generation and COX-2 mRNA (r=0.822, P<0.006). Simultaneous immunoneutralization of IL and TNF prevented ROS formation and COX-2 expression induced by smoker’s serum. Inhibitors of NADPH oxidase and p47phox siRNA dramatically diminished smokers’ serum-mediated endothelial ROS production and COX-2 expression. Conclusion: Our results suggest an essential role of inflammatory cytokines in the modulation of endothelial dysfunction induced by smoking. References 1. J. A. Ambrose et R. S. Barua. The pathophysiology of cigarette smoking and cardiovascular disease: an update. J. Am. Coll. Cardiol., 43, 1731-1737, 2004. 2. P.W. Wilson. Smoking, smoking cessation, and risk of cardiovascular disease. Curr. Treat. Options Cardiovasc. Med., 8, 276-281, 2006. 3. S.S. Barbieri, L. Ruggiero, E. Tremoli, B.B. Weksler. Suppressing PTEN activity by tobacco smoke plus interleukin-1beta modulates dissociation of VE-cadherin/beta-catenin complexes in endothelium. Arterioscler Thromb Vasc Biol., 28, 732-738, 2008 4. S. S. Barbieri et B. B Weksler. Tobacco smoke cooperates with interleukin-1 to alter β-catenin trafficking in vascular endothelium resulting in increased permeability and induction of ciclooxigenase-2 expression in vitro and in vivo. FASEB J., 21, 1-13, 2007