title: Cigarette smoke and LDL cooperate in reducing nitric oxide bioavailability in endothelial cells via effects on both eNOS and NADPH oxidase.
abstract: 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 ((·)O(2)(-)) 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.
authors: Philip Morris International R&D, Philip Morris Research Laboratories GmbH, Cologne, Germany. yvonne.steffen@pmi.com
authors: Yvonne
authors: Y
authors: Steffen
authors: Gregory
authors: G
authors: Vuillaume
authors: Katrin
authors: K
authors: Stolle
authors: Karin
authors: K
authors: Roewer
authors: Michael
authors: M
authors: Lietz
authors: Jutta
authors: J
authors: Schueller
authors: Stefan
authors: S
authors: Lebrun
authors: Thomas
authors: T
authors: Wallerath
mesh_terms: D001682:Biological Availability; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008297:Male; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism / Q000493:pharmacokinetics*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects*; D030421:Peroxynitrous Acid / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics; D014028:Tobacco Smoke Pollution; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D008077:Lipoproteins, LDL; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014028:Tobacco Smoke Pollution; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019255:NADPH Oxidases