Chronic inhalation of e-cigarette vapor containing nicotine disrupts airway barrier function and induces systemic inflammation and multiorgan fibrosis in mice

Abstract

Electronic (e)-cigarettes theoretically may be safer than conventional tobacco. However, our prior studies demonstrated direct adverse effects of e-cigarette vapor (EV) on airway cells, including decreased viability and function. We hypothesize that repetitive, chronic inhalation of EV will diminish airway barrier function, leading to inflammatory protein release into circulation, creating a systemic inflammatory state, ultimately leading to distant organ injury and dysfunction. C57BL/6 and CD-1 mice underwent nose only EV exposure daily for 3-6 mo, followed by cardiorenal physiological testing. Primary human bronchial epithelial cells were grown at an air-liquid interface and exposed to EV for 15 min daily for 3-5 days before functional testing. Daily inhalation of EV increased circulating proinflammatory and profibrotic proteins in both C57BL/6 and CD-1 mice: the greatest increases observed were in angiopoietin-1 (31-fold) and EGF (25-fold). Proinflammatory responses were recapitulated by daily EV exposures in vitro of human airway epithelium, with EV epithelium secreting higher IL-8 in response to infection (227 vs. 37 pg/ml, respectively; P < 0.05). Chronic EV inhalation in vivo reduced renal filtration by 20% ( P = 0.017). Fibrosis, assessed by Masson's trichrome and Picrosirius red staining, was increased in EV kidneys (1.86-fold, C57BL/6; 3.2-fold, CD-1; P < 0.05), heart (2.75-fold, C57BL/6 mice; P < 0.05), and liver (1.77-fold in CD-1; P < 0.0001). Gene expression changes demonstrated profibrotic pathway activation. EV inhalation altered cardiovascular function, with decreased heart rate ( P < 0.01), and elevated blood pressure ( P = 0.016). These data demonstrate that chronic inhalation of EV may lead to increased inflammation, organ damage, and cardiorenal and hepatic disease.

Keywords: cardiorenal dysfunction; e-cigarette; electronic cigarette; fibrosis; nicotine; systemic inflammation.

Fig. 1.

Diagram of an electronic (e)-cigarette (A). For our in vitro model of firsthand e-cigarette vapor (EV) exposure (B), the e-cigarette hooked up to rubber tubing and a 3-way stopcock, such that negative pressure is applied to the mouthpiece via pulling back the plunger on a 60-ml syringe, generating fresh EV. The syringe is filled with 50 ml of EV each time, and the EV is subsequently exhaled through the side port of the 3-way stopcock onto primary human airway epithelial cells. MPO, myeloperoxidase; RAGE, receptor for advanced glycation end product; M-CSF, macrophage colony-stimulating factor; RBP4, retinol-binding protein 4: LIF, leukemia inhibitory factor; WISP-1, WNT1-inducible signaling pathway; MMP-3, matrix metalloprotease-3.

Fig. 2.

C57BL/6 (A) and CD-1 (B) mice exposed daily to EV for 3 and 6 mo, respectively, had modulated levels of inflammatory proteins in the serum, consistent with an altered systemic inflammatory state. Sera were evaluated by 111-cytokine antibody array (Proteome Profiler Mouse XL Array; R&D Systems), and graphed as a ratio of EV/Air for proteins that increased with EV exposure and Air/EV for proteins that decreased with EV exposure. A: changes in C57BL/6 serum protein levels caused by EV exposure are shown, with a 20% threshold in either direction, including large rises in angiopoietin-1 and EGF in EV mice, and much decreased Chitinase 3-like 1 and MMP-3 in EV mice (n = 3 per group). B: serum protein changes in CD-1 mice, including large increases in LIF (murine equivalent of IL-8) and EGF, and large decreases in MMP-3 and WISP-1 (n = 6 per group, pooled). *Protein changes occurred in both CD1 and C57BL/6 mice.

Fig. 3.

Mice that inhaled EV for an hour daily had inflammatory changes only at the protein level. A: lung parenchyma was stained with hemotoxylin and eosin (H&E) and Masson’s trichrome stains. One lung slice per mouse, including large, medium, and small airways, was evaluated by a blinded pathologist, and no pulmonary inflammation, emphysema or fibrosis was found in EV mice relative to Air controls (n = 6 per group). B: the airways of mice, as measured through bronchoalveolar lavage (BAL), had alterations in the inflammatory cytokine profile. BAL was pooled within EV and Air control groups (n = 6 within groups) and was evaluated by 111-cytokine antibody array (Proteome Profiler Mouse XL Array; R&D Systems) and graphed as a ratio of EV/Air. BAL from EV mice had decreased levels of LIX (murine version of IL-8; 519-fold lower or ~0.2% of Air levels) and VCAM-1 (99-fold lower or 1% of Air levels). EV BAL had increased levels of DPPIV (1.7-fold or 58% higher than Air levels).

Fig. 4.

Primary normal human bronchial epithelial cells (NHBEs) became leaky and proinflammatory with daily short 15 min EV exposures for 2–5 days. A: EV-treated NHBE cells tested for permeability with FITC-dextran had greater passage of small molecules, compared with controls exposed to Air only, on both days 2 and 5 (P < 0.01; means ± SE; wells were run in triplicate). B: EV-exposed NHBEs secreted more IL-8 than Air controls in response to bacterial infection (37 vs. 227 pg/ml, respectively; P < 0.05; wells were run in triplicate). C: protein quantification of Western blots of the tight junction protein zona occludins (ZO1) found 3.3-fold lower quantities in NHBEs after EV exposure, as compared with Air controls (P = 0.024; n = 3). Levels of loading control tubulin were similar across samples (P = 0.99).

Fig. 5.

Chronic EV inhalation diminished cardiorenal function and induced renal fibrosis in C57BL/6 mice exposed to EV for 3 mo. A: EV induced a 20% reduction in glomerular filtration rate (GFR) as compared with experimental controls (P = 0.017). B: representative ×20 Masson’s trichrome, ×20 Picrosirius red bright-field, and ×10 polarized light photomicrographs of renal tissue fibrosis. C: fibrosis was quantified in kidneys from EV and Air mice, by blinded grading of kidney sections. EV kidneys had 87% (1.86-fold increase) more collagen vs. experimental controls. When only the 10 sections with the highest levels of collagen staining were compared, EV kidneys still had 1.88-fold higher levels of fibrosis, compared with controls. D: Picrosirius red staining also demonstrated higher collagen content in EV-exposed mice, relative to Air controls (1.62-fold increase, P = 0.034). Means ± SE are shown; n = 5–6 per group; *P < 0.05.

Fig. 6.

Induction of kidney fibrosis also occurred in CD-1 mice exposed to EV for 6 mo. A: kidney parenchyma stained with Masson’s trichrome and Picrosirius red stains. B: in CD-1 mice, daily EV inhalation for 6 mo led to a 3.2-fold increase in renal fibrosis, assessed by Masson’s trichrome stain, relative to Air controls (means ± SE are shown; P = 0.022). C: Picrosirius red staining also demonstrated 2.14-fold higher collagen content in EV-exposed mice, relative to Air controls (means ± SE are shown; P < 0.01). D–K: to assess for the origin of fibrosis, genes associated with fibrosis and extracellular matrix pathways were evaluated after only 4 wk of EV or Air exposure. Lower expression of the antifibrotic miRNA miR-29b-3p (D) and higher expression of collagen-1 within CD-1 renal parenchyma (E), suggest that fibrosis begins early in the course of daily EV inhalation. The expression of additional profibrotic factors, Col3a1 (F), Col4a1 (G), Itgb1 (I), and Fbn1 (J), were all significantly increased in renal tissues from e-cig-exposed animals (P < 0.05). Extracellular matrix remodeling factor Mmp2 trended up but not significantly (H). However, the fibrosis component Eln was not significantly different (K). *P < 0.05; n = 5–6 for all groups.

Fig. 7.

Chronic inhalation of EV induced cardiac fibrosis and altered cardiovascular function. A: Masson’s trichrome stain of fixed cardiac ventricular tissue from CD-1 mice exposed to EV daily for 6 mo. B: quantitative analysis of EV relative to control determined that EV hearts had 2.75-fold greater level of collagen staining in ventricular tissue compared with controls (***P < 0.001). C and D: when tissues were harvested after only 4 wk of EV exposure, cardiac tissues were found to have higher expression of collagen-3 mRNA (C) but normal expression levels of collagen-1 mRNA (D) (**P < 0.05). For A–D, n = 6 per group. E: in C57BL/6 mice, EV daily for 3 mo led to decreased heart rates (HR), as compared with Air controls (*P < 0.01). F: heart rates (HRs) were more variable in EV exposed mice, as indicated by greater SD within beat-to-beat measurements of each mouse. G: systolic blood pressure was increased in e-cigarette-exposed mice (P = 0.016). H: diastolic blood pressure trended up in EV mice (*P = 0.050). For E–H, n = 19 for EV and n = 20 for Air controls.

Fig. 8.

Chronic inhalation of EV led to hepatic fibrosis in CD-1 mice exposed to EV for 6 mo. A: representative Masson’s trichrome photomicrographs of fixed hepatic tissue. B: quantitative analysis of EV relative to control determined that EV livers exposed to EV daily for 6 mo had 1.9-fold higher collagen deposition, relative to Air controls. Means ± SE are shown; n = 6 per group. ****P < 0.0001.