Chronic E-Cigarette Exposure Alters the Human Bronchial Epithelial Proteome

Abstract

Rationale: E-cigarettes vaporize propylene glycol/vegetable glycerin (PG/VG), nicotine, and flavorings. However, the long-term health effects of exposing lungs to vaped e-liquids are unknown.

Objectives: To determine the effects of chronic vaping on pulmonary epithelia.

Methods: We performed research bronchoscopies on healthy nonsmokers, cigarette smokers, and e-cigarette users (vapers) and obtained bronchial brush biopsies and lavage samples from these subjects for proteomic investigation. We further employed in vitro and murine exposure models to support our human findings.

Measurements and main results: Visual inspection by bronchoscopy revealed that vaper airways appeared friable and erythematous. Epithelial cells from biopsy samples revealed approximately 300 proteins that were differentially expressed in smoker and vaper airways, with only 78 proteins being commonly altered in both groups and 113 uniquely altered in vapers. For example, CYP1B1 (cytochrome P450 family 1 subfamily B member 1), MUC5AC (mucin 5 AC), and MUC4 levels were increased in vapers. Aerosolized PG/VG alone significantly increased MUC5AC protein in human airway epithelial cultures and in murine nasal epithelia in vivo. We also found that e-liquids rapidly entered cells and that PG/VG reduced membrane fluidity and impaired protein diffusion.

Conclusions: We conclude that chronic vaping exerts marked biological effects on the lung and that these effects may in part be mediated by the PG/VG base. These changes are likely not harmless and may have clinical implications for the development of chronic lung disease. Further studies will be required to determine the full extent of vaping on the lung.

Keywords: chronic obstructive pulmonary disease; mucin; tobacco; vaping.

Figure 1.

Vaping leads to unique changes in the airway proteome. (A) Heat map of differentially expressed proteins from bronchial brush biopsies from the various groups. Vapers cluster together and are different from nonsmokers and smokers. (B) Log₂ fold change in abundance versus significance for all identified proteins for smokers (red dots) and vapers (black dots) relative to nonsmokers. The dashed line indicates a significance (P) value of 0.05. (C) Venn diagram of increased (red) or decreased (blue) proteins in smokers and vapers relative to nonsmokers. NS = nonsmokers; S = smokers; V = vapers.

Figure 2.

Proteins commonly altered in vapers and smokers. Heat map of protein expression (log₂ fold change and P < 0.05) for proteins that are commonly altered in vapers and smokers. Data from bronchial brushings of nonsmokers (n = 8), smokers (n = 9), and vapers (n = 9).

Figure 3.

Acute PG/VG exposure increases intracellular MUC5AC levels in cultured human bronchial epithelia. (A) Typical hematoxylin and eosin and Alcian blue–periodic acid–Schiff staining of fixed bronchial cultures after acute air versus 36.8% PG/VG/nicotine exposures. (B) Representative confocal microscopy images of fixed cultures stained with Hoechst (nuclei; blue), MUC5AC (green), and cilia (α-tubulin; white). (C) Mean log₂ fold changes in MUC5AC fluorescence after exposure to various doses of PG/VG (shaded columns) or PG/VG + nicotine (solid columns). *P < 0.05, different air exposure (i.e., 0% PG/VG). Data shown as means ± SEM. AB/PAS = Alcian blue–periodic acid–Schiff; H&E = hematoxylin and eosin; MUC5AC = mucin 5AC; PG/VG = propylene glycol/vegetable glycerin.

Figure 4.

Acute PG/VG exposure increases upper airway MUC5AC and STIM1 expression in mice. (A) Left: Representative Western blot of MUC5AC from the nasal epithelia of wild-type mice after 3 hours of acute exposure to PG/VG or air (control). Right: Western blot probing for MUC5AC in wild-type and MUC5AC knockout mice as an antibody control. (B) Densitometric analysis of MUC5AC expression in PG/VG- and air-exposed mice (n = 6 per group). (C) Representative Western blot of STIM1 with GAPDH as the loading control in nasal epithelia of mice after 3 hours of exposure to PG/VG or air. (D) Densitometric analysis of STIM1 expression normalized to GAPDH in PG/VG-exposed mice compared with air (both n = 6). *P ≤ 0.05 in PG/VG compared with air. Data shown as means ± SD. The numbers on Western blots indicate protein from individual mice. KO = knockout; MUC5AC = mucin 5AC; PG/VG = propylene glycol/vegetable glycerin; STIM1 = stromal interaction protein 1; WT = wild type.

Figure 5.

PG/VG affects membrane fluidity and protein diffusion. (A) Confocal micrographs (x–z plane) of human bronchial epithelial cultures (HBECs) stained with calcein (green) and Pixie Dust e-liquid excited at 405 nm (red). Representative images of three independent experiments were taken before and 10 minutes after addition of e-liquid. The dashed yellow lines indicate the apical surface of the culture. (B) Representative x–y-plane confocal micrograph of HEK293T cells (gray differential interference contrast image) before and after 5 minutes of exposure to Pixie Dust e-liquid (purple). (C) Emission scan of merocyanine 540 (M540) in HBECs. Black circles, vehicle at 37°C; red squares, 3% PG/VG at 37°C; blue triangles, vehicle at 4°C. All n = 9 cultures from three separate donors. (D and E) Fluorescence recovery after photobleaching of (D) Orai1-YFP and (E) Ano1-GFP in HEK293T cells after vehicle or 3% PG/VG exposure. Cells transfected with Orai1-YFP or Ano1-GFP were exposed to PG/VG for 1 hour before FRAP was measured. Data shown as means ± SEM. All n = 12–16 cultures per group from three or four independent experiments. *P ≤  0.05. Ano1 = anoctamin 1; FRAP = fluorescence recovery after photobleaching; GFP = green fluorescent protein; Orai1 = calcium release-activated calcium modulator 1; PG/VG = propylene glycol/vegetable glycerin; RG = Ringer’s glucose; YFP = yellow fluorescent protein.