Cigarette smoke and LDL cooperate in reducing nitric oxide bioavailability in endothelial cells via effects on both eNOS and NADPH oxidase

The ubiquitous free radical nitric oxide (NO) plays an important role in many biological processes, including the regulation of both vascular tone and inflammatory response; however, its role in the effects of cigarette smoke exposure on atherosclerosis remains unclear. Our aim was to study the mechanisms of NO regulation in endothelial cells in response to cigarette smoke exposure in vitro. Using human umbilical vein endothelial cells (HUVEC), we have demonstrated that combining non-toxic concentrations of cigarette smoke bubbled through PBS (smoke-bubbled PBS [sbPBS]) with native LDL (nLDL) significantly reduces the amount of bioavailable NO. The effect is comparable to that seen with oxidized LDL (oxLDL), but has not been seen with sbPBS or nLDL alone. Mechanistic investigations showed that the combination of sbPBS + nLDL did not reduce the amount of endothelial nitric oxide synthase (eNOS), but did inhibit its enzymatic activity. Concomitantly, both sbPBS + nLDL and oxLDL significantly increased the production of reactive oxygen species (ROS) in the form of superoxide anions (O2-) and peroxynitrite (ONOO−) in HUVEC. Selective inhibition of NADPH oxidase prevented this response. Incubation of sbPBS + nLDL revealed the formation of 7-ketocholesterol (7-KC) and 7-hydroxycholesterol, which are indicators for oxidative modification of LDL. This could explain the reported increase in circulatory levels of oxLDL in smokers.Our results suggest that reduction of functional NO in response to a combination of sbPBS + nLDL is secondary to both reduction of eNOS activity and stimulation of NADPH oxidase activity. Because sbPBS alone showed no effect on eNOS activity or ROS formation, nLDL should be included in cigarette-smoke-related mechanistic in vitro experiments on endothelial cells to be more reflective of the clinical situation.

► Non-toxic concentrations of cigarette smoke with LDL reduces the amount of NO.
► This effect is comparable to that seen with oxidized LDL (oxLDL).
► sbPBS + LDL and oxLDL increased the production of reactive oxygen species.
► sbPBS + LDL revealed formation of oxysterols are indicators for oxidation of LDL.
► We show cigarette-smoke-related clinical relevant mechanistic in vitro experiments.