Acute exposure to e-cigarettes causes inflammation and pulmonary endothelial oxidative stress in nonsmoking, healthy young subjects

Abstract

The effects of e-cigarette (e-cig) aerosol inhalation by nonsmokers have not been examined to date. The present study was designed to evaluate the acute response to aerosol inhalation of non-nicotinized e-cigarettes in terms of oxidative stress and indices of endothelial activation in human pulmonary microvascular endothelial cells (HPMVEC). Ten smoking-naïve healthy subjects (mean age ± SD = 28.7 ± 5.5 yr) were subjected to an e-cig challenge, following which their serum was monitored for markers of inflammation [C-reactive protein (CRP) and soluble intercellular adhesion molecule (sICAM)] and nitric oxide metabolites (NOx). The oxidative stress and inflammation burden of the circulating serum on the vascular network was also assessed by measuring reactive oxygen species (ROS) production and induction of ICAM-1 expression on HPMVEC. Our results show that serum indices of oxidative stress and inflammation increased significantly (P < 0.05 as compared with baseline), reaching a peak at approximately 1-2 h post-e-cig aerosol inhalation and returning to baseline levels at 6 h. The circulatory burden of the serum (ICAM-1 and ROS) increased significantly at 2 h and returned to baseline values 6 h post-e-cig challenge. ROS production by HPMVEC was found to occur via activation of the NADPH oxidase 2 (NOX2) pathways. These findings suggest that even in the absence of nicotine, acute e-cig aerosol inhalation leads to a transient increase in oxidative stress and inflammation. This can adversely affect the vascular endothelial network by promoting oxidative stress and immune cell adhesion. Thus e-cig inhalation has the potential to drive the onset of vascular pathologies.

Keywords: e-cigarette; inflammation; oxidative stress; pulmonary endothelial activation; reactive oxygen species.

Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Fig. 1.

Schema of blood collection from smoking-naïve subjects at various time points pre- and post-non-nicotinized e-cig inhalation.

Fig. 2.

C-reactive protein (CRP) in serum with e-cigarette (e-cig) inhalation. A: profile of CRP in the serum of all subjects post e-cig vaping. Blood was collected before (−30 min) and postvaping (30–360 min) from 10 subjects (S1–S10). CRP was assayed using quantitative ELISA and quantified using reference curves of standards provided by the manufacturer (R&D Biosystems). B: quantification of CRP levels across all subjects postvaping. For each time point, the CRP levels for all 10 subjects were expressed as n-fold increase over the respective baseline (i.e., the respective prevaping levels). Data are expressed as means ± SD. *P < 0.05 as compared with baseline.

Fig. 3.

Stable end products of nitric oxide (NO) oxidation, i.e., nitrate-nitrite [nitric oxide metabolites (NOx)] in serum with e-cig inhalation. A: profile of serum NOx at various time points pre- and post-e-cigarette (e-cig) inhalation for subjects 1–10 (S1–S10). Dotted red line represents the average NO values for 6 regular e-cig smokers. Samples were assayed for NOx colorimetrically using a nitrite-nitrate Griess reaction assay, and NOx was quantified by standard curves. B: average fractional change in NOx pre- and post e-cig inhalation in all 10 subjects. For each subject, the NOx values were expressed as n-fold change over their respective baseline (i.e., the prevaping levels normalized to 1). *P < 0.005; #P < 0.05 as compared with baseline.

Fig. 4.

Soluble intercellular adhesion molecule (sICAM) in serum with e-cigarette (e-cig) inhalation. A: time course of ICAM-1 in the serum of all subjects post-e-cig vaping, as assayed using quantitative ELISA. ICAM-1 was quantified using reference curves of standards provided by the manufacturer (R&D Biosystems). B: average ICAM levels in blood post-e-cig inhalation across all 10 subjects (S1–S10). Data in the form of means ± SD of ICAM refer to samples obtained from 10 subjects for different time points. ICAM-1 levels are expressed as n-fold increase above baseline (i.e., pre-inhalation levels normalized to 1). *P < 0.05 relative to baseline.

Fig. 5.

Effect of serum on endothelial inflammation as monitored by intercellular adhesion molecule 1 (ICAM-1) expression on endothelial cells exposed to serum post e-cigarette (e-cig) inhalation. A: human pulmonary microvascular endothelial cells (HPMVEC) were exposed to media with 15% serum from subjects pre- and post-e-cig inhalation (120 and 360 min) and immunostained for ICAM (green). Blue, nuclear stained DAPI. Cells were imaged on Leica STED super resolution microscope at λex laser line 488 nm, λem 500–560 (maximum ∼530 nm). Inset (+120 min) is magnified below showing perinuclear staining. Scale bar, 25 μm. B: ICAM-1 expression on HPMVEC as obtained from quantification of fluorescence intensity of green signal, as observed in Fig. 4A. Images were quantified by integrating the fluorescence intensity and normalized to equal number of cells, as measured by the no. of nuclei (blue DAPI stain). Data are presented as a box and whisker plot of ICAM expression on HPMVEC upon serum treatment pre- (−30 min) and post-e-cig inhalation (120, 360 min) for 10 subjects. Within each box plot, the central horizontal line represents the median, whereas top and bottom lines represent the 1st and 3rd quartiles, respectively. X indicates the mean. Vertical lines (whiskers) represent the range of values. Outliers are beyond the end of each whisker and are displayed as dots. #P < 0.001, 120-min value as compared with values at −30 and +360 min.

Fig. 6.

Endothelial activation potential as monitored by reactive oxygen species (ROS) production by human pulmonary microvascular endothelial cells (HPMVEC) upon treatment with subjects’ serum post e-cigarette (e-cig) vaping. A: ROS production in HPMVEC exposed to media with 15% serum from subjects post-e-cig vaping. Cells labeled with ROS-sensitive dye CellROX Green were imaged at low and high magnification on the stage of a microscope at λex 488 nm, λem 500–560 (maximum ~530 nm). Images here are representative of ROS production by serum of 2 subjects (subjects 1 and 3). Inset is magnified below. Arrows show subcellular organelles. Scale bar, 10 μm. B: endothelial activation potential in serum from e-cig smokers. ROS was monitored in HPMVEC upon treatment with serum obtained from 6 regular e-cig smokers. Images here are representative of ROS production by serum of 2 subjects. Scale bar, 10 µm. Inset is magnified below. All images in A and B were acquired at the same settings using Metamorph Imaging Software. C: ROS production profile in individual subjects (S1–S10). Images acquired at ×20 magnification were used for quantification. Data represent means of three separate experiments. SD values are not displayed for ease of visualization. ImageJ software was used to integrate the fluorescence across an entire field. Background fluorescence was subtracted and data normalized to equal pixel area, as reported earlier (13, 54, 73). Red dotted line represents the average “baseline” values (210 ± 11.7) across a group of 6 regular e- cig smokers. D: quantification of ROS production by HPMVEC across all subjects post-e-cig vaping. For each subject, the fluorescence intensity (denoting ROS production) is expressed as n-fold increase over pre-e-cig vaping levels (baseline). Data across all 10 subjects were obtained and are expressed as means ± SD. *P < 0.001; #P < 0.01; **P < 0.05 relative to baseline.

Fig. 7.

Apocynin pretreatment reduces reactive oxygen species (ROS) production in response to exposure to serum from subjects post e-cigarette (e-cig) challenge. Top: human pulmonary microvascular endothelial cells (HPMVEC) were pretreated for 1 h with 300 μM apocynin in regular media before incubation with serum (from subjects 60 min post-e-cig challenge). Cells with (+Apo) and without apocynin (−Apo) were imaged for ROS under identical assay conditions. Inset is magnified to show ROS within the cellular regions. Bottom: fluorescence intensity (denoting ROS production) integrated across the entire microscopic field for all 10 subjects (in arbitrary fluorescence units) is expressed as means ± SD. #P < 0.001.

Fig. 8.

Schema showing proposed outline of the signaling events post-e-cigarette (e-cig) inhalation. Non-nicotinized e-cig inhalation as an initiator of vascular inflammation and oxidative stress. The detrimental chemical species that are inhaled from e-cig trigger an endothelial signaling pathway that drives inflammation and oxidative stress and also increases the inflammatory burden of the circulating blood.