Inflammatory cytokines present in smokers\u2019 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\u2019 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