Background-The mechanisms involved in the dysfunction of both endothelium-dependent vasodilatation (EDV) and NO biosynthesis related to smoking are unclear. In this study, EDV was assessed in healthy smokers and nonsmokers in vivo and, using serum from the same individuals, was related to the NO biosynthetic pathway in vitro. Methods and Results-Flow-mediated EDV of the brachial artery was measured in 23 male patients (8 nonsmokers and 15 smokers). Serum was collected, added to confluent (Ϸ85%) monolayers of human umbilical vein endothelial cells (HUVECs), and incubated for 12 hours. Basal and substance P-stimulated NO production was measured. The HUVECs used for measuring basal NO production were lysed, and both endothelial NO synthase (eNOS) protein expression and eNOS activity were determined. EDV was lower in smokers compared with nonsmokers (PϽ0.001). HUVECs treated with serum from smokers compared with nonsmokers showed significantly lower basal (PϽ0.0001) and stimulated (PϽ0.02) NO production, higher eNOS expression (PϽ0.0001), but lower eNOS activity (PϽ0.004). There was a significant positive correlation between in vivo EDV and in vitro substance P-stimulated NO production (rhoϭ0.57, PϽ0.01) and between basal NO production and eNOS activity (rϭ0.54, PϽ0.008) and a negative correlation between basal NO production and eNOS protein expression (rϭϪ0.60, PϽ0.003). Conclusions-This is the first study to combine an in vivo model with a near-physiological in vitro model to demonstrate an association between decreased NO production and reduced EDV. Cigarette smoking was associated with reduced EDV, NO generation, and eNOS activity in the presence of increased eNOS protein expression.

Background-Our group has previously shown that human umbilical vein endothelial cells exposed to smokers' serum decreased nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in the presence of increased eNOS expression. In the present study, we examined whether these observations extended to human coronary artery endothelial cells (HCAECs). In addition, the role of reactive oxygen species in the observed alterations was examined. Methods and Results-HCAECs were incubated with serum from 10 nonsmokers and 15 smokers for 12 hours with or without the addition of either polyethylene glycol-superoxide dismutase (PEG-SOD, 300 U/mL), PEG-SODϩPEG-catalase (1000 U/mL), chelerythrine (3 mol/L), or tetrahydrobiopterin (20 mol/L). At the end of incubation, NO, eNOS protein, and eNOS activity were measured from the same culture. HCAECs incubated with smokers' serum alone showed significantly lower NO production (PϽ0.05) and eNOS activity (PϽ0.005) but higher eNOS expression (PϽ0.005) compared with nonsmokers. In smokers, addition of PEG-SOD, PEG-SODϩPEG-catalase, or tetrahydrobiopterin significantly (PϽ0.05) improved NO levels and eNOS activity. Interestingly, in the same smokers, a significant decrease in eNOS expression was only seen with the addition of PEG-SODϩPEG-catalase (PϽ0.05) and treatment with PEG-SOD alone insignificantly increased eNOS expression. Conclusions-The present study indicates that in vitro, HCAECs show similar changes in NO biosynthesis as human umbilical vein endothelial cells when exposed to smokers' serum and also confirms that oxidative stress plays a central role in smoking-mediated dysfunction of NO biosynthesis in endothelial cells. Furthermore, these data support other studies suggesting a role for hydrogen peroxide in the upregulation of eNOS.

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These results indicate light smoking may have similar detrimental effects on EDV and NO biosynthetic pathway as does heavy smoking. These data may have important implications concerning the amount of active cigarette exposure that imparts cardiovascular risk.