Acute e-cig inhalation impacts vascular health: a study in smoking naïve subjects

Abstract

This study was designed to investigate the acute effects of nonnicotinized e-cigarette (e-cig) aerosol inhalation in nonsmokers both in terms of blood-based markers of inflammation and oxidative stress and evaluate their association with hemodynamic-metabolic MRI parameters quantifying peripheral vascular reactivity, cerebrovascular reactivity, and aortic stiffness. Thirty-one healthy nonsmokers were subjected to two blood draws and two identical MRI protocols, each one before and after a standardized e-cig vaping session. After vaping, the serum levels of C-reactive protein, soluble intercellular adhesion molecule, and the danger signal machinery high-mobility group box 1 (HMGB1) and its downstream effector and the NLR family pyrin domain containing 3 (NLRP3) inflammasome (as monitored by its adaptor protein ASC) increased significantly relative to the respective baseline (prevaping) values. Moreover, nitric oxide metabolites and reactive oxygen species production decreased and increased, respectively. These observations were paralleled by impaired peripheral vascular reactivity (with reduced flow-mediated dilation and attenuated hyperemic response after a cuff-occlusion test) and metabolic alterations expressed by decreased venous oxygen saturation, postvaping. The current results suggest propagation of inflammation signaling via activation of the danger signaling axis (HMGB1-NLRP3). The findings indicate that a single episode of vaping has adverse impacts on vascular inflammation and function.NEW & NOTWORTHY Endothelial cell signaling and blood biomarkers were found to correlate with functional vascular changes in a single episode e-cigarettes inhalation in healthy adults. This is indicative of the potential of e-cigarettes (even when inhaled acutely) to lead of vascular dysfunction.

Keywords: e-cig inhalation; inflammasome; inflammation; oxidative stress; vascular function.

Figure 1.

C-reactive protein (CRP) and soluble ICAM-1 (sICAM-1) in serum (colored online). A and B: profiles of CRP and soluble ICAM-1 (sICAM-1) extracted from serum of all subjects before (pre, blue arrow) and after (post, red arrow) e-cigarette (e-cig) vaping. Solid black arrow denotes the point in time of e-cig aerosol inhalation. CRP and ICAM-1 were assayed using quantitative ELISA and quantified using reference curves of standards provided by the manufacturer (see materials and methods). C and D: box and whiskers plots of CRP and sICAM-1 pre- and postvaping, plotted in log scale. Group average and median are indicated by white circle and horizontal bar, respectively; ***P < 0.0005.

Figure 2.

Damage-associated molecular patterns (DAMP) protein, high-mobility group box 1 (HMGB1), and ASC, the adaptor protein of the NLR family pyrin domain containing 3 (NLRP3) inflammasome in plasma (colored online). A and B: profiles of HMGB1 and ASC extracted from the plasma of all subjects before (pre, blue arrow) and after (post, red arrow) e-cigarette (e-cig) vaping. Solid black arrow denotes the point in time of e-cig aerosol inhalation. HMGB1 and ASC were assayed using quantitative ELISA and quantified using reference curves of standards provided by the manufacturer (see materials and methods). C and D: box and whiskers plots of HMGB1 and ASC pre- and postvaping, plotted in log scale. Group average and median are indicated by white circle and horizontal bar, respectively; ***P < 0.0005.

Figure 3.

Nitrate-nitrite (NOx) in serum and reactive oxygen species (ROS) production. A and B: profiles of NOx and ROS for all subjects before (pre, blue arrow) and after (post, red arrow) e-cig vaping. Solid black arrow denotes point in time of e-cigarette (e-cig) aerosol inhalation. Serum samples were assayed for NOx colorimetrically using a nitrite-nitrate Griess reaction assay and NOx was quantified by standard curves. ROS production was quantified via fluorescence intensity measurements (see materials and methods). B, inset: box and whiskers plots of ROS, plotted in log scale (***P < 0.0005). C and D: box and whiskers plots of NOx pre- and postvaping, plotted in log scale. Group average and median are indicated by white circle and horizontal bar, respectively; ***P < 0.0005. E: human pulmonary microvascular endothelial cells exposed to media with 10% serum extracted pre- and post-e-cig vaping. Results from 3 sample subjects (004, 023, and 043) are shown. The cells were labeled with CellROX Green fluorescent dye, sensitive to ROS production. Cells were imaged on the stage of a microscope at λex 488 nm, λem 500-560, max ∼530 nm. All images were acquired at the same settings on Nikon Axiophot microscope using Metamorph Imaging Software. AFU, arbitrary fluorescence units.

Figure 4.

Fig. 4. Associations between blood-based biomarkers (colored online): A: high-mobility group box 1 (HMGB1) and ASC levels in plasma extracted from the blood-draw executed before e-cigarette (e-cig) vaping challenge (“pre”). B: reactive oxygen species (ROS) production and ASC concentration quantified before e-cig vaping. C: nitrate-nitrite (NOx) and C-reactive protein (CRP) levels in serum quantified from the blood-draw executed after the e-cig vaping challenge and the second MRI protocol. Spearman’s correlation coefficients and the respective P values are reported in the box; 95% prediction and regression lines are indicated. Log transforms were used since the data were not normally distributed

Figure 5.

Correlation between blood-based markers and MRI metrics (colored online). Scatterplots of biomarkers plotted vs MRI metrics, evaluated before (pre) and after (post) e-cig vaping. A and C: reactive oxygen species (ROS) vs Pulsatility Index (PI) respectively, before and after e-cig vaping. B: intercellular adhesion molecules (sICAM) vs resistivity index (RI). D: C-reactive protein (CRP) vs luminal flow-mediated dilation (FMD). E: high mobility group box 1 (HMGB1) vs. time to peak velocity during hyperemia (TTP). F: CRP vs. peak hyperemic velocity (V_P). The Spearman’s correlation coefficients and respective P value are provided, along with 95% prediction intervals and regression lines.

Figure 6.

Heatmaps of associations between biomarkers and MRI metrics. A and B: colormaps showing Spearman’s correlation coefficients ρ of the various associations between biomarkers and MRI metrics acquired respectively before and after the e-cig vaping challenge. Comparison pairs are indicated in bold for P values <0.05 (see key for actual P values). Acronyms are listed in Tables 2-, and 3. Notice that the association between flow-mediated dilation (FMD) and hematocrit (Hct) refers to prevaping.