Chronic cigarette smoke exposure triggers a vicious cycle of leukocyte and endothelial-mediated oxidant stress that results in vascular dysfunction

Abstract

Although there is a strong association between cigarette smoking exposure (CSE) and vascular endothelial dysfunction (VED), the underlying mechanisms by which CSE triggers VED remain unclear. Therefore, studies were performed to define these mechanisms using a chronic mouse model of cigarette smoking (CS)-induced cardiovascular disease mirroring that in humans. C57BL/6 male mice were subjected to CSE for up to 48 wk. CSE impaired acetylcholine (ACh)-induced relaxation of aortic and mesenteric segments and triggered hypertension, with mean arterial blood pressure at 32 and 48 wk of exposure of 122 ± 6 and 135 ± 5 mmHg compared with 99 ± 4 and 102 ± 6 mmHg, respectively, in air-exposed mice. CSE led to monocyte activation with superoxide generation in blood exiting the pulmonary circulation. Macrophage infiltration with concomitant increase in NADPH oxidase subunits p22^phox and gp91^phox was seen in aortas of CS-exposed mice at 16 wk, with further increase out to 48 wk. Associated with this, increased superoxide production was detected that decreased with Nox inhibition. Tetrahydrobiopterin was progressively depleted in CS-exposed mice but not in air-exposed controls, resulting in endothelial nitric oxide synthase (eNOS) uncoupling and secondary superoxide generation. CSE led to a time-dependent decrease in eNOS and Akt expression and phosphorylation. Overall, CSE induces vascular monocyte infiltration with increased NADPH oxidase-mediated reactive oxygen species generation and depletes the eNOS cofactor tetrahydrobiopterin, uncoupling eNOS and triggering a vicious cycle of oxidative stress with VED and hypertension. Our study provides important insights toward understanding the process by which smoking contributes to the genesis of cardiovascular disease and identifies biomarkers predictive of disease.NEW & NOTEWORTHY In a chronic model of smoking-induced cardiovascular disease, we define underlying mechanisms of smoking-induced vascular endothelial dysfunction (VED). Smoking exposure triggered VED and hypertension and led to vascular macrophage infiltration with concomitant increase in superoxide and NADPH oxidase levels as early as 16 wk of exposure. This oxidative stress was accompanied by tetrahydrobiopterin depletion, resulting in endothelial nitric oxide synthase uncoupling with further superoxide generation triggering a vicious cycle of oxidative stress and VED.

Keywords: cigarette smoking; endothelial dysfunction; hypertension; inflammation; nitric oxide synthase uncoupling; reactive oxygen species; superoxide.

Fig. 1.

Blood pressure (BP) in control and smoking-exposed mice. BP was measured using a noninvasive tail-cuff method. Cigarette smoking exposure (CSE) significantly elevated systolic (A), diastolic (C), and mean arterial (B) BP and lowered pulse pressure (D) in 32- and 48-wk CS-exposed mice compared with air-exposed controls. Data are presented as means ± SE of 10 independent measurements in each group at each exposure time. *Significance from controls at P ≤ 0.05.

Fig. 2.

Relaxation response of aorta and mesenteric artery to acetylcholine and nitroprusside. Mice were exposed to cigarette smoke (smoking) or fresh air (control). Aortic and mesenteric artery rings were mounted in a wire myograph and constricted with phenylephrine (PE; 1 µM), and the concentration-response to acetylcholine and sodium nitroprusside was measured. Endothelial-dependent and independent relaxation in aortic rings from mice exposed to cigarette smoke for 32 wk is shown in A and B, respectively, while C and D show similar data following 48 wk of exposure. E and F: endothelial-dependent and independent relaxation, respectively, of mesenteric artery following 48 wk of exposure. Vessels of CS-exposed mice exhibited significant endothelial dysfunction compared with air-exposed controls. Data are presented as means ± SE of 6 independent experiments. *Significance from controls at P ≤ 0.05.

Fig. 3.

Superoxide radical generation in mouse aorta. Optimal cutting temperature-preserved aortic sections from 48-wk CS (smoking)- or air (control)-exposed mice were incubated with dihydroethidium (DHE) alone or together with 100 µM superoxide dismutase mimetic (SOD_m) Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), NADPH oxidase inhibitors diphenyleneiodonium (DPI; 100 µM) and 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870; 10 µM), or NOS inhibitor N^G-nitro-l-arginine methyl ester (l-NAME; 1 mM). Sections were visualized with confocal fluorescence microscopy. Red fluorescence arises from superoxide-mediated oxidation of DHE (A). B: quantitation of the fluorescence in A. Data represent means ± SE of 3 independent experiments. *Significance from the corresponding control at P ≤ 0.05. #Significance from the untreated smoking sections (DHE) at P ≤ 0.05.

Fig. 4.

Expression of NADPH oxidase protein subunits p22^phox, gp91^phox in mouse aorta with variable duration of cigarette smoke exposure. A: immunoblot of NADPH oxidase subunits in aortic homogenate from cigarette smoke (CS) or air (C)-exposed mice. B: quantitation of p22^phox, gp91^phox band intensities from a series of immunoblots as in A. GAPDH is shown as a loading control. Data show time-dependent increase of NADPH oxidase subunit expression following smoke exposure. Data are presented as means ± SE of 3 independent experiments. *Significance from controls at P ≤ 0.05.

Fig. 5.

Macrophage activation and expression of NADPH oxidase protein subunits p22^phox and gp91^phox in mouse aorta. A: hematoxylin-eosin staining of aorta following 48 wk of cigarette smoke (smoking) or air (control) exposure. B: optimal cutting temperature-preserved aortic sections from mice exposed to 48 wk of cigarette smoke or air were incubated with antibodies against p22^phox (red) and gp91^phox (green). Blue fluorescence shown in the merged image (B) is due to nuclear staining with DAPI. Graph to the right shows the quantitative analysis of the fluorescence intensity of gp91^phox and p22^phox. C: aortic sections from mice exposed to 48 wk of cigarette smoke or air were incubated with antibodies against gp91^phox (red) and monocyte/macrophage marker (MOMA-2; green). In the merged image, yellow shows colocalization of gp91^phox and MOMA-2. Graph to the right shows the quantitative analysis of the fluorescence intensity of gp91^phox and MOMA-2. Data are presented as means ± SE of 3 independent experiments. *Significance from control at P ≤ 0.05.

Fig. 6.

Measurement of endothelial nitric oxide synthase (eNOS), p-eNOS Ser¹¹⁷⁷, p-Akt, and GAPDH with variable duration of cigarette smoking exposure. A: immunoblotting for eNOS, p-eNOS Ser¹¹⁷⁷, and p-Akt in aortic homogenates from cigarette smoke (CS) or air-exposed (C) mice with exposure times of 16, 32, or 48 wk. B: quantitation of band intensities from a series of experiments as in A. C: aortic sections from mice exposed to 48 wk of cigarette smoking or air (control) with sections incubated with antibody against eNOS (green) and the nuclear stain DAPI (blue). Data are presented as means ± SE of 3 independent experiments. *Significance from controls at P ≤ 0.05.

Fig. 7.

HPLC quantitation of plasma tetrahydrobiopterin (BH₄) at different times following cigarette smoke exposure. Data are presented as means ± SE of 3 independent experiments. *Significance from air-exposed controls at P ≤ 0.05.

Fig. 8.

Superoxide radical generation in mononuclear leukocytes following cigarette smoke exposure. Measurements are shown from blood obtained at 16 wk of exposure to cigarette smoke or air (control) (A). Lower panels show higher magnification images of representative mononuclear cells from control and smoke-exposed mice. In these images, the fluorescence derived from dihydroethidium in red is overlaid with Hoechst stain of nuclei in blue. B: mean fluorescence intensities in mononuclear cells from control and cigarette smoke-exposed mice. Smoking induced clear activation with enhanced reactive oxygen species generation. Data are presented as means ± SE of 8 independent experiments. *Significance from air-exposed control cells at P ≤ 0.05.

Fig. 9.

Mechanisms of oxidant production that trigger vascular endothelial dysfunction and cardiovascular disease. With chronic smoking exposure, oxidants in cigarette smoke activate monocytic leukocytes, traversing the pulmonary circulation and triggering their vascular adhesion and uptake. In parallel to this, oxidants in cigarette smoke deplete tetrahydrobiopterin (BH₄) and antioxidants, leading to endothelial nitric oxide synthase (eNOS) uncoupling and oxidative modification and dysfunction with loss of nitric oxide (NO) synthesis with switch to superoxide (O₂^·−) generation, which leads to formation of the potent oxidant peroxynitrite (ONOO⁻). Both NADPH oxidase and eNOS-derived reactive oxygen species (ROS) formation can feedback on each other and themselves, resulting in a vicious cycle of ROS-induced ROS release and injury.