Short-term e-cigarette vapour exposure causes vascular oxidative stress and dysfunction: evidence for a close connection to brain damage and a key role of the phagocytic NADPH oxidase (NOX-2)

Abstract

Aims: Electronic (e)-cigarettes have been marketed as a 'healthy' alternative to traditional combustible cigarettes and as an effective method of smoking cessation. There are, however, a paucity of data to support these claims. In fact, e-cigarettes are implicated in endothelial dysfunction and oxidative stress in the vasculature and the lungs. The mechanisms underlying these side effects remain unclear. Here, we investigated the effects of e-cigarette vapour on vascular function in smokers and experimental animals to determine the underlying mechanisms.

Methods and results: Acute e-cigarette smoking produced a marked impairment of endothelial function in chronic smokers determined by flow-mediated dilation. In mice, e-cigarette vapour without nicotine had more detrimental effects on endothelial function, markers of oxidative stress, inflammation, and lipid peroxidation than vapour containing nicotine. These effects of e-cigarette vapour were largely absent in mice lacking phagocytic NADPH oxidase (NOX-2) or upon treatment with the endothelin receptor blocker macitentan or the FOXO3 activator bepridil. We also established that the e-cigarette product acrolein, a reactive aldehyde, recapitulated many of the NOX-2-dependent effects of e-cigarette vapour using in vitro blood vessel incubation.

Conclusions: E-cigarette vapour exposure increases vascular, cerebral, and pulmonary oxidative stress via a NOX-2-dependent mechanism. Our study identifies the toxic aldehyde acrolein as a key mediator of the observed adverse vascular consequences. Thus, e-cigarettes have the potential to induce marked adverse cardiovascular, pulmonary, and cerebrovascular consequences. Since e-cigarette use is increasing, particularly amongst youth, our data suggest that aggressive steps are warranted to limit their health risks.

Keywords: Oxidative stress; Behavioural risk factor; E-cigarette vapour; Endothelial dysfunction; Lifestyle drug.

Figure 1

Effects of short-term e-cigarette vapour exposure on vascular function and oxidative stress in wild-type mice—validation of human vascular function data. Endothelial function in healthy subjects was measured by FMD (A), FMC (B), and arterial stiffness by PTT/PWV (C) after single use of e-cigarette in a cross-over fashion. Data are mean ± SD from n = 20 healthy subjects. (D) E-cigarette vaping for 6 h/day for 1, 3, and 5 days led to a significant impairment of the endothelial function in wild-type mice. (E) E-cigarette vaping increased aortic reactive oxygen species (ROS) formation as assessed by DHE staining, which was inhibited by NOX-2 inhibition by GSK2795039 (compound of GlaxoSmithKline). Representative microscopy images are shown besides the densitometric quantification. (F) E-cigarette vaping significantly decreased endothelial •NO synthase (type 3) (eNOS) and DHFR protein expression and (G) increased HO-1 protein expression. Representative blots are shown in Supplementary material online, Figure S3D, all normalized to α-actinin. Data are presented as box (first and third quartiles, line = median) and whiskers (min, max) with jitter plot of single values from n = 20 healthy subjects (A–C) or n = 6–18 (E) animals/group or 3–7 (F), 3–7 (G), or mean ± SD from n = 8–38 (D) samples/group (pooled from 2–3 mice/sample). P-values as indicated except for (D) with *, P < 0.05 vs. unexposed controls.

Figure 2

Effects of short-term e-cigarette vapour exposure on vascular function and oxidative stress in Nox2^−/− (gp91phox^−/−) vs. wild-type mice. (A) E-cigarette vaping caused a stronger oxidative burst in whole blood of wild-type mice, whereas no significant change was observed in whole blood of Nox2^−/− mice. In addition, the overall stimulated signal was much lower in Nox2^−/− mice. (B) E-cigarette vaping caused endothelial dysfunction in wild-type mice, which was prevented by Nox2^−/−. (C) E-cigarette vaping increased systolic and diastolic blood pressure in wild-type but not in Nox2^−/− mice. (D, E) E-cigarette vaping paradoxically increased protein expression of eNOS while no changes were observed in the regulation of expression of DHFR and HO-1. Representative blots are shown below the densitometric quantification. (F) Bilirubin levels (product of HO-1 activity) in plasma were increased in wild-type but not in Nox2^−/− mice. Representative chromatograms are shown along with the quantification. Nox2 knockout prevented e-cigarette vaping-induced increase in aortic ROS/superoxide formation as assessed by lucigenin enhanced chemiluminescence (ECL) (G), high performance liquid chromatography (HPLC)-based quantification of DHE oxidation products (H) and DHE staining (I). Representative chromatograms and microscopy images are shown along with the quantifications. Data are presented as box (first and third quartiles, line = median) and whiskers (min, max) with jitter plot of single values from n = 5–10 (A), 6–22 (C), 4 (D, E) and at least 5 (F), 6–15 (G), 4 (H), or 4–18 (I) or mean ± SD from 6–38 (B) animals/group. P-values as indicated except for (B) with *, P < 0.05 vs. unexposed controls.

Figure 3

Effects of short-term e-cigarette vaping on vascular nitrotyrosine (3-NT), endothelin-1 (ET-1), inducible nitric oxide synthase, and NADPH oxidase subunit (NOX-2) expression. (A) E-cigarette vaping increased vascular 3-NT and ET-1 as well as (B) iNOS and NOX-2 expression. Representative microscopy images are shown below the densitometric quantification. Data are presented as box (first and third quartiles, line = median) and whiskers (min, max) with jitter plot of single values from n = 4–8 (A) and 5–8 (B) animals/group. P-values as indicated.

Figure 4

Effects of short-term e-cigarette vaping on cerebral oxidative stress. (A) E-cigarette vaping increased cerebral ROS formation (assessed by DHE) which was inhibited by the specific NOX-2 inhibitor GSK2795039. (B) Cerebral ROS formation (assessed by DHE) was also inhibited by the specific nNOS inhibitor ARL-17477 and genetic Nox2 deletion. (C) nNOS in the frontal cortex tended to be decreased in response to E-cigarette vaping. Representative blots are shown next to the densitometric quantification. (D) E-cigarette vaping increased cerebral superoxide formation (assessed by HPLC-based quantification of DHE oxidation products) which was inhibited by genetic Nox2 deletion. Representative chromatograms are shown along with the quantification. (E) E-cigarette vaping decreased mRNA expression of Foxo3 and nNOS and increased expression of Nox1 mRNA levels. (F) These genes were differentially or not significantly regulated in the frontal cortex of e-cigarette vapour-exposed Nox2^−/− mice. Data are presented as box (first and third quartiles, line = median) and whiskers (min, max) with jitter plot of single values from n = 4 (GSK-group) and at least 4 (Ctr and exposures) animals/group (A, B) and 4 samples/group (pooled from 3 mice/sample) (C), 6–8 (D), 9–12 (E), and 4–8 (F) animals/group. P-values as indicated.

Figure 5

E-cigarette liquid and vapour condensate contain toxic aldehydes as revealed by DNPH derivatization with HPLC and LC-MS analysis exerting toxic effects on cultured endothelial cells. (A) Quantification of identified aldehydes by HPLC analysis and known DNPH-aldehyde standards. Representative chromatograms are shown in Supplementary material online, Figure S10. (B) List of identified compounds (DNPH or aldehyde-DNPH) adducts in e-cigarette vapour condensate by LC-MS analysis with a representative chromatogram. The relative abundance of the aldehyde-DNPH adducts is provided in Supplementary material online, Table S2. (C) Protein expression of p67^phox and p47^phox was determined by western blot analysis in membranous and cytosolic protein-fractions from pellets of EA.hy926 cells upon incubation with toxic aldehyde mixtures of formaldehyde (0.5–5 µM) and acetaldehyde/butyraldehyde/acrolein (each 0.05–0.5 µM). The ratio of membraneous/cytosolic p67^phox and p47^phox reflects NOX-2 activation state. Representative western blots are shown besides the densitometric quantification. (D) Protein adducts of acrolein were determined by western and dot blot analysis in lung tissues of control or E-cigarette vapour-exposed mice. Representative original western and dot blots are shown besides the densitometric quantifications. (E) Vascular ROS formation by acrolein was determined using dihydroethidium (DHE, 1 µM)-dependent fluorescence microtopography in aortic cryo-sections. The contribution of NOX-2 to this ROS signal was assessed by comparison of aortic sections of wild-type vs. Nox2 deficient mice and pharmacological inhibition by GSK2795039. Representative microscopy images are shown below the densitometric quantification. Data are presented as box (first and third quartiles, line = median) and whiskers (min, max) with jitter plot of single values from n = 4–5 (C, D) and 4–16 (E) or mean ± SD from n = 3 (A, B) experiments or animals per group. P-values as indicated.

Figure 6

E-cigarette vaping induced cardiovascular complications are prevented by pharmacological ET-1 receptor blockade and FOXO-3 activation. ET-1 receptor blockade by macitentan and FOXO-3 activation by bepridil prevent e-cigarette vaping induced blood pressure increase (A), endothelial dysfunction (B), aortic superoxide formation by lucigenin ECL (C), and aortic ROS formation by DHE staining (D). Representative microscopy images are shown besides the densitometric quantification. Data are presented as box (first and third quartiles, line = median) and whiskers (min, max) with jitter plot of single values from at least n = 8 (A), 7 (C), and 4 (D) or mean ± SD from at least n = 8 (B) animals per group. Significance as indicated except for (B) with *, P < 0.05 vs. unexposed controls. (E) Central scheme: proposed mechanisms of E-cigarette mediated cerebro/cardiovascular complications from studies in mice. The described adverse effects are also operative in the disease development and progression of classical cerebro/cardiovascular disease such as stroke, atherosclerosis, hypertension, and coronary artery disease. Light blue colour items represent pharmacological/genetic interventions. NOX and Nox refers to protein and mRNA of NOX-2 isoform.