The Impact of Electronic Cigarettes on Airway Epithelial Barrier Integrity in Preclinical Mouse Model

Abstract

The increasing use of electronic cigarettes (e-cigs) among adolescents poses significant public health risks. This study investigates the impact of e-cigs on the airway epithelial barrier, focusing on apical junctional complexes (AJCs), including tight junctions (TJs) and adherens junctions (AJs). We hypothesized that e-cigs disrupt AJCs in a mouse model, leading to increased airway barrier permeability. C57BL/6 mice were exposed to 36 mg/mL e-cig aerosols (3 puffs per minute) for one hour daily over four days. Bronchoalveolar lavage (BAL) fluid analysis, lung inflammation assessment, immunohistochemistry (IHC) staining, Western blotting (WB), and permeability assays were performed to evaluate the structure and function of the airway barrier. E-cig-exposed mice showed weight loss and elevated serum cotinine levels. BAL fluid analysis revealed elevated white blood cells. Histological analysis confirmed lung inflammation, while IHC and WB showed significant AJC disruption. Notably, claudin-2 levels were elevated in e-cig-exposed mice compared to controls. Claudin-2, known for its role in promoting permeability in “leaky” epithelia, increased alongside decreases in other TJ components, signifying structural barrier impairment. After e-cig exposure, instilling FITC-dextran into the airway increased serum FITC-dextran levels, indicating enhanced barrier permeability. E-cig aerosol exposure disrupts airway epithelial barrier structure and function, primarily through the disassembly of TJs and AJs. These findings suggest potential pathways for further clinical investigation into the health risks of e-cig use.

Graphical Abstract

INTRODUCTION

Since their introduction to the US market in 2007, electronic cigarettes (e-cigs) have surged in popularity. These battery-powered devices aerosolize liquid mixtures containing propylene glycol (PG), vegetable glycerin (VG), nicotine, and flavorings. The landscape has witnessed a significant expansion in both the variety of e-cig devices and the formulations of e-liquids. A myriad of flavors, ranging from menthol, fruits, and spices have been advertised, with a particular focus on attracting the youth market.

In 2021, the number of adult cigarette smokers in the United States was reported to be 28.3 million. Concerningly, the U.S. Centers for Disease Control and Prevention (CDC) and the U.S. Food and Drug Administration (FDA) found that by 2023, 2.8 million American youth admitted to current (past-30 days) usage of e-cigs, with 12.6% of high school students and 6.6% of middle school students being users. Recent case series have highlighted the complications associated with vaping among children and adolescents admitted to healthcare facilities (1). The act of inhaling an e-cig initiates the heating of e-cig liquid, producing a mixture of potentially hazardous liquid droplets and airborne gases that are directly drawn into the user’s lungs. Despite the acknowledgment of youth e-cig usage as an epidemic in the U.S. and the committed efforts of the CDC and FDA to protect youth from this preventable health risk, the toxicity of e-cigs remains inadequately documented.

Airway epithelial cells maintain the integrity of the respiratory system while serving as the frontline defense against inhaled viruses, particles, and toxins (2, 3). Exposure to conventional and e-cig smoke has been shown to disrupt airway epithelial integrity, leading to increased permeability and decreased transepithelial electrical resistance (TEER) (4, 5). A few studies specifically investigated the impact of e-cig vapor, which contains nicotine, on airway permeability in vitro (4, 5). However, the impact on the structure and function of apical junctional complexes (AJCs) in animal models remains understudied. AJCs, consisting of tight junctions (TJs) and adherens junctions (AJs), play a crucial role in maintaining the epithelial barrier (6).

Numerous proteins, including those from the zonula occludens (ZO) family, occludin, α-catenin and β-catenin, cadherin family, claudin family, tricellulin, and the junctional adhesion molecules comprise the apical TJ and AJ (7, 8). Dysfunction in AJCs may contribute to airway inflammation by facilitating the “outside/in” translocation of inhaled pathogens, allergens, and particles (2, 3, 6). The impact of PG, VG, nicotine, and flavoring components in e-cig liquid on airway epithelial barrier function and structure has been documented by our lab using a well-established in vitro model (5). However, the alterations in the structure and composition of the epithelial AJC during pulmonary inflammation in vivo remain insufficiently documented.

This study aims to establish a murine model to investigate the effects of e-cig exposure on the structure and function of airway epithelial TJs in vivo. Such research is critical for understanding the potential risks associated with e-cig usage, especially in conjunction with viral infections, and for developing strategies to mitigate these risks.

MATERIALS AND METHODS

Antibodies and reagents

The primary antibodies included occludin (33–1500, Invitrogen), E-cadherin (610181, BD Biosciences), β-catenin (ab32572, Abcam), claudin-2 (ab53032, Abcam for Immunohistochemistry (IHC) and 32–5600, Invitrogen for Western blot) (Table 1- online Supplemental Data). Secondary antibodies, Alexa Fluor™ 488 anti-mouse IgG (A21202), Alexa Fluor™ 568 anti-rabbit IgG (A10042), and HRP-conjugated goat anti-mouse IgG (31430), were obtained from Invitrogen for immunohistochemistry staining (1:500) and Western blotting (1:5000). E-cig nicotine in a 50:50 PG:VG ratio from Air Factory E-Liquid, Irvine, CA, was purchased from local vape shops.

Animals

Female C57BL/6 mice, aged 8–12 weeks and weighing between 16 and 23 grams with an average of 20 grams, were procured from Jackson Laboratories (Bar Harbor, ME) and housed under standard conditions with a 12-hour light–12-hour dark cycle, provided with ad libitum access to water and a rodent diet. Mice were exposed to either e-cig aerosols or HEPA-filtered air, weighed daily, and euthanized at the specified time post-exposure. Experiments were repeated at least three times, with 5–10 mice per group.

Ethics approval and consent to participate

All procedures involving animals in this study were conducted in compliance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Approval for the study was granted by the Institutional Animal Care and Use Committee (IACUC) at the Lerner Research Institute, Cleveland Clinic Foundation, under protocol 0000–2030. This facility is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) under accreditation number 000383, in compliance with federal law and NIH regulations.

Exposure to e-cig in vivo

Mice were exposed to aerosolized e-cig chemicals using an inExpose whole-body mouse exposure chamber attached to a 3rd generation e-cig extension (Joyetech eVIC VTC mini, SCIREQ Inc. Montreal, Canada). The e-cig device was activated through a stroke controller under the regulation of SCIREQ’s flexiware software (version 6.1). This software was set to simulate a puff profile based on realistic topography data from e-cig users. Exposure of wild-type C57BL/6 mice occurred within a fume hood, following a puff profile of 3 puffs/min for 60 minutes with a puff volume of 70 mL. One exposure session occurred 4 days a week for a total of 4 exposures. Before each exposure, the flow rates for the two active air pumps were calibrated using a rotameter provided by SCIREQ. E-cig aerosols were generated and passed through a condensing chamber, then entered the mixing chamber via pump 4 at a flow rate of 2.0 L/min. The vape was diluted with room-temperature air before reaching the whole-body exposure chamber. Additionally, pump 1 removed the air and e-cig aerosol mixture from inside the exposure chamber into the biosafety cabinet. Post-exposure, the mice remained in the chamber for 5 to 10 minutes until the aerosol levels decreased, after which they were returned to their cages for observation. To maintain uniform environmental conditions, mice in the air group were housed in the same room, and dividers were used to keep the mice separated.

FITC-dextran permeability assay in vivo

Fluorescein isothiocyanate (FITC)-conjugated dextran 4 kD (46944, Sigma) was used as previously described (9, 10). FITC-dextran was dissolved in sterile phosphate buffered saline (PBS) at a 5 mg/ml concentration and intranasally administered at 10 μg/g body weight. After 1 hour, mice were euthanized, and blood was collected via cardiac puncture in BD Microtainer serum separators, centrifuged, and blood serum was collected. The FITC-dextran standard curve was made by serial diluting the 10 mg/ml stock solution in PBS. Using a FlexStation 3 (Molecular Devices, San Jose, CA), fluorescence intensity was measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The fluorescence intensity of an untreated serum sample that did not receive FITC-dextran was subtracted from the fluorescence values of all experimental samples.

Immunohistochemistry labeling and confocal microscopy

Mouse lung tissues underwent IHC labeling, which was performed as previously described (8, 9). Briefly, the lungs were perfused by injecting PBS into the right ventricle, inflated with zinc-buffered formalin (5701ZF, Richard-Allan Scientific LLC, Kalamazoo, MI), harvested, and fixed in zinc-buffered formalin. The tissue was embedded in paraffin and sectioned into 5 μm thick slices, followed by deparaffinization and rehydration, progressing through xylene, flex 100, flex 95, and flex 70 at room temperature (RT). The antigen retrieval process was conducted with two different buffers along with different primary antibodies: for occludin, β-catenin, claudin-2 (10 mM Tris-1 mM EDTA, 0.05% Tween-20, pH 9); for E-cadherin (10 mM trisodium citrate, 0.05% Tween-20, pH 6.0). Tissue sections were blocked with 10% normal donkey serum with 0.5% Triton X-100 for 1 hour at RT. The sections were incubated overnight with primary antibodies at 4°C in a humid chamber and subsequently incubated with Alexa Fluor™-conjugated secondary antibodies for 1 hour at RT. The nuclei were stained with DAPI (Sigma-Aldrich, St. Louis, MO) and mounted with Vectashield anti-fade mounting medium (H-1000, Vector Laboratories Inc., Newark, CA). Samples were imaged with 40X oil objective using an upright fluorescent or confocal microscope (Leica Microsystems, Wetslar, Germany). The images were processed using Adobe Photoshop.

Western Blot analysis

Mouse lung tissues were homogenized using RIPA buffer, and Western blot analysis performed as previously described (5, 10). Enhanced chemiluminescence (Thermo Scientific, cat#32106, Waltham, MA) was used to visualize the membranes, and the ChemiDocTM imager system (Bio-Rad Laboratories, RRID:SCR_008426, Hercules, CA) was used to take images of the membranes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a lane protein-loading control. Super Signal West Pico Plus chemiluminescence solutions (Thermo-Fisher). Densitometry was performed using ImageJ software (RRID:SCR_003070) for each band of the focus protein with bands normalized to the GAPDH band.

ELISA

The plasma cotinine levels were measured using a commercially available ELISA kit (C0096D, Calbiotech). The standard, quality control, and samples were processed according to the manufacturer’s instructions.

Statistical analysis

Data analysis was carried out using both Microsoft Excel and Prism software (GraphPad Prism, RRID:SCR_002798). Data are shown as the mean ± standard deviation (SD) representing outcomes from three experiments. Statistical analysis for comparing multiple groups involved using one-way ANOVA when examining more than two groups, followed by the Tukey post hoc test for all experimental groups. A t-test was used to assess the difference between the two groups. Statistical difference was considered when p<0.05 and p values were displayed as *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001), while no significant difference was indicated as ns.

RESULTS

Impact of e-cig exposure on mice weight and blood cotinine.

Wild-type female C57BL/6 mice were exposed to aerosolized nicotine (36 mg/mL in 50:50 PG:VG) using the SCIREQ InExpose system, delivering 3 puffs/min with a puff volume of 70 mL, for 60 minutes per session, once daily for 4 days. The control group was exposed to HEPA-filtered air. We observed that body weight remained relatively stable on days 1–3 of exposure in both the HEPA-filtered air and aerosolized e-cig groups. However, by day 4, mice exposed to e-cig aerosol showed a small but significant decrease in body weight (8.00 ± 1.37%) compared to the control group, which exhibited a weight gain of (1.14 ± 1.37%) (Fig. 1A). Exposure to e-cig aerosol led to elevated serum cotinine levels, consistent with ranges observed in human e-cig users and other mouse studies (11, 12) (Fig. 1B).

Fig. 1: Acute exposure of mice to e-cig aerosol induces weight loss.

C57BL/6 mice were exposed to e-cig aerosol at 36 mg/mL using the SCIREQ inExpose system, delivering 3 puffs/min with a puff volume of 70 mL for 60 minutes per session, once a day for 4 days. (A) Weight reduction was noted on day 4 in mice exposed to e-cig aerosol in comparison to controls. Data are shown as mean ± SD, 35 mice per group, ****p = 0.000001 (B) Elevated cotinine levels were observed in the blood serum of mice exposed to e-cig aerosol in comparison to control mice. Data are shown as mean ± SD, 6 mice per group, ****p < 0.0001, as determined by using unpaired t tests with two-stage step-up for grouped analyses (Benjamini, Krieger, and Yekutieli) followed by Shapiro-Wilk test.

Impact of e-cig exposure on airway inflammation

Elevated white blood cell (WBC) counts in bronchoalveolar lavage (BAL) fluid serve as crucial indicators of airway inflammation. Our findings revealed an increase in WBC count in BAL fluid from animals exposed to e-cig aerosols, suggesting inflammation and heightened airway permeability (Fig. 2A). However, there was no notable difference in BAL cell differentiation between control mice and those exposed to e-cigs, where an average of 87% of cells were identified as macrophages (Fig. 2B). Histological examination with H&E staining further confirmed lung inflammation in e-cig-exposed mice, characterized by enhanced peribronchial infiltration of immune cells (Fig. 2C). Previously, both our research group and others have employed semi-quantitative and multi-parametric approaches to assess pulmonary inflammation in mice (9). Utilizing a histopathology scoring system, we observed a significantly augmented inflammatory response in e-cig-exposed mice compared to those exposed to HEPA-filtered air. Inflammation levels were notably higher in e-cig-exposed mice (6.765 ± 0.338) compared to HEPA-filtered air-exposed mice (3.250 ± 0.338) (Fig. 2D).

Fig. 2: E-cig aerosol exposure increases airway and lung inflammation.

C57BL/6 mice were exposed to e-cig aerosol at a concentration of 36 mg/mL using the SCIREQ inExpose system. The exposure regimen consisted of 3 puffs/min, each with a volume of 70 mL, administered for 60 minutes per session, once daily for 4 consecutive days. (A) BAL fluid analysis demonstrated a significant increase in white blood cell (WBC) counts in mice exposed to e-cig aerosols compared to control mice. Data are shown as mean ± SD, 7–8 mice per group, **p = 0.0078 (B) WBC counts showed no notable differences in differentiation between control and e-cig mice. (C) Histological examination with H&E staining revealed lung inflammation in e-cig-exposed mice, characterized by enhanced peribronchial infiltration of immune cells. (D) Inflammation levels quantified through histopathology scoring, were notably higher in e-cig-exposed mice compared to HEPA-filtered air-exposed mice. Data are shown as mean ± SD, 17–20 mice per group, ****p < 0.0001. Scale bar of 200 μm. Statistical analyses were completed using unpaired t tests with two-stage step-up for grouped analyses (Benjamini, Krieger, and Yekutieli) followed by Shapiro-Wilk test.

Impact of e-cig exposure on tight junction structure, and expression

Through immunofluorescence labeling and confocal microscopy on lung tissue sections collected on day 4 post-e-cig exposure, we examined the localization of key TJ and AJ proteins (Fig. 3A). In control mice, a well-defined AJC structure was observed in the epithelium lining of the bronchiolar lumen. However, exposure to e-cig aerosol resulted in a noticeable disruption of epithelial AJ integrity. We observed a reduction in the intensity of both TJs and AJs. Interestingly, occludin, which is located more apically than AJs, was more severely impacted by e-cig aerosol exposure. Intriguingly, claudin-2 levels were elevated in e-cig-exposed mice compared to controls. Importantly, cell sloughing was not observed, as evidenced by intact nuclear counterstaining using DAPI. Our investigation utilized a quantitative immunoblotting approach to assess the effect of e-cig exposure on the expression of TJ proteins, and AJ proteins. In lung homogenates of mice exposed to e-cigs, we observed a significant decrease in the protein expression of occludin (Fig. 3B-C). However, a minor decrease was noted in β-catenin expression, and no changes were observed in E-cadherin expression at the protein level (Fig. 3B, and D-E). Additionally, the protein expression of claudin-2 significantly increased (Fig. 3B, and F). These findings suggest that e-cig exposure induces alterations in the structure and composition of epithelial TJs, correlating with the observed disruption of the airway barrier.

Fig. 3: Impact of e-cig aerosol exposure on apical junctional complexes structure.

(A) Lung tissue sections collected on day 4, post-exposure, were examined for the localization of key AJC proteins including occludin, E-cadherin, β-catenin, and claudin-2. In control mice, a well-defined AJC structure was observed in the bronchiolar epithelium, while exposure to e-cig aerosol led to a noticeable disruption of epithelial TJ and AJ integrity. Scale bar of 50 μm. (B) In lung homogenates, were subjected to Western blot analysis. (C-F) Densitometry analysis confirmed the significant reduction in occludin, no significant changes in E-cadherin or β-catenin, and a significant increase in claudin-2 expression in e-cig-exposed mice. The data is represented as mean ± SD, 3 mice per group, not significant (ns), *p =0.0204, **p = 0.0031, as determined by paired t tests followed by the Shapiro-Wilk test.

Impact of e-cig exposure on airway barrier function

To quantify the inside-out increase in permeability, we collected BAL fluid and measured protein levels in mice exposed to air and e-cig. We observed a significant increase in protein concentrations in e-cig-exposed mice (Fig. 4A). Additionally, we intranasally inoculated mice on day 4 of e-cig aerosol exposure. The serum level of FITC-dextran was significantly higher in e-cig aerosol-exposed animals compared to controls (Fig. 4B). These findings demonstrate enhanced bi-directional pulmonary barrier permeability to large proteins post e-cig exposure.

Fig. 4: Exposure of mice to e-cig aerosol induces leaky airway.

Mice were exposed to e-cig aerosol at a concentration of 36 mg/mL, 3 puffs/min, each with a volume of 70 mL, administered for 60 minutes per session. (A) BAL fluid was collected, and protein levels were measured. Data are shown as mean ± SD, 9–10 mice per group, **p = 0.0083 (B) Mice were intranasally inoculated with 5 μg/g FITC-dextran in PBS or vehicle on day 4, and blood was collected 1 hour later. The serum level of FITC-dextran was significantly higher in e-cig aerosol-exposed animals compared to controls. A significant increase in protein concentrations was observed in e-cig-exposed mice. Data are normalized to the average of control group. Data are shown as mean ± SD., with 7 mice per group, **p = 0.009. Statistical analyses were completed using unpaired t tests followed by Shapiro-Wilk test.

DISCUSSION AND CONCLUSION

Our study elucidates the deleterious effects of e-cig aerosol exposure on airway epithelial barrier integrity using a preclinical mouse model and provides significant insights into the adverse effects of e-cigs aerosol exposure on the airway barrier function. These findings reinforce concerns raised by previous research and align with our previous in vitro study, demonstrating that e-cig exposure compromises the epithelial barrier, leading to increased permeability due to the disassembly of AJCs (5). While prior studies have shown in vitro barrier disruption by e-cigs, our study is the first to demonstrate these effects in vivo. We found that e-cig exposure not only disrupts epithelial barriers but might increase endothelial permeability, suggesting a broader impact on vascular health. This in vivo evidence highlights the potential cardiovascular risks of e-cigs, extending beyond airway damage to include possible endothelial dysfunction. These findings underscore the importance of further research into the long-term effects of e-cigarette use on vascular integrity.

One of the key findings of our study is the significant weight loss observed in mice following acute e-cig exposure (Fig. 1). Similarly, Shao et al. showed significant weight reduction in ApoE−/− mice exposed to chronic intermittent e-cig aerosol (13). Neonatal mice also exhibited lower total body weights after inhalation of e-cig emissions, indicating that the adverse effects of e-cig exposure are especially impactful in neonatal development (14). Additionally, our study demonstrates marked airway inflammation in e-cig-exposed mice, characterized by increased white blood cell counts in bronchoalveolar lavage (BAL) fluid and histological evidence of peribronchial infiltration of immune cells (Fig. 2). These results are in agreement with earlier reports of e-cig-induced pulmonary inflammation (15). In our study, despite increased total WBCs in BALF, specific cell proportions remained unchanged, likely reflecting a generalized inflammatory response to e-cigarette exposure. This may indicate an early immune reaction with leukocyte recruitment or systemic inflammation elevating blood leukocytes. Further studies are needed to confirm these mechanisms.

Moreover, our research highlights the impact of e-cig exposure on the structural integrity of tight junctions (TJs) and adherens junctions (AJs) within the airway epithelium. Claudin-2 is recognized for enhancing permeability in “leaky” epithelial barriers. The observed reduction in the expression of key TJ proteins such as occludin, alongside increased levels of claudin-2, indicates a compromised epithelial barrier (Fig. 3). This disruption was further evidenced by increased serum FITC-dextran levels and higher protein concentrations in BAL fluid (Fig. 4). These changes suggest enhanced bi-directional permeability and impaired barrier function, which could predispose the airways to pathogen invasion and systemic inflammation. Our findings are supported by previous studies as well. For instance, another study by Crotty Alexander et al. found that e-cig exposure via aerosol caused increased permeability and decreased TJ protein expression in normal human bronchial epithelial (NHBE) cells (16). However, the FITC-dextran assay may reflect alveolar rather than airway-specific permeability due to its reach to the alveolar-capillary barrier. While we aimed to assess airway barrier function, our method includes contributions from alveolar and endothelial barriers. Future studies will incorporate bronchial lavage or airway-specific analysis for greater precision. Similarly, as the inside-out assay mainly measures alveolar and endothelial permeability, we will refine our approach to better target airway-specific permeability.

Overall, our study provides comprehensive evidence that e-cig aerosol exposure significantly disrupts airway epithelial barrier integrity through structural disassembly of TJs and AJs, leading to increased permeability and inflammation. This understanding is crucial for the development of effective strategies for mitigating these health risks. Given the increasing prevalence of e-cig use among youth and the potential for long-term respiratory health consequences, this research is of utmost importance. Our study has several limitations. First, it is restricted to short-term exposure to e-cig liquid. It would be beneficial to investigate the impact of e-cig on the airway barrier in both subacute and chronic models. Second, the results may not be generalizable to other brands, e-cig models, or flavors. It is essential to investigate the effects of vaping e-cigs with or without nicotine in comparison to smoking conventional cigarettes. Additionally, this study does not examine the impact of different flavors on the outcomes of e-cig exposure. In this study, to minimize variability and enhance statistical rigor, we used female mice, as sex-specific differences were beyond this study’s scope. However, we recognize the importance of including both sexes and plan to address this in future research. Additionally, we acknowledge that using whole lung tissue for immunoblots is a limitation of our study. This approach does not allow us to distinguish cell-type-specific protein expression or regional variations within the lung, which may influence the interpretation of the results. Future studies will aim to use more refined techniques, such as cell-specific protein isolation or regional sampling, to provide a more detailed analysis of the observed effects.

Future research should focus on identifying specific molecular pathways involved in e-cig-induced barrier dysfunction and exploring therapeutic interventions to mitigate these adverse effects. It would be interesting to determine whether the observed impact of e-cigs in our study is a direct effect of e-cigs or a consequence of released inflammatory cytokines and chemokines in response to e-cig exposure. Additionally, studying the recovery of barrier structure and function after cessation of e-cig exposure is critical.

Supplementary Material

All supplemental material is available at https://doi.org/10.6084/m9.figshare.28278806.

NEW & NOTEWORTHY:

The rising use of e-cigs among youth has become a significant public health concern. This study, using a mouse model, demonstrates that exposure to e-cig aerosol leads to airway inflammation, structural damage to the airway epithelial barrier, and increased epithelial barrier permeability.

AVAILABILITY OF DATA AND MATERIALS

Data will be made available upon reasonable request.