Inhalation of nicotine-containing electronic cigarette vapor exacerbates the features of COPD in βENaC-overexpressing mice

: Background: Chronic obstructive pulmonary disease (COPD) is currently listed as the 3 rd leading cause of death in the United States. COPD is marked by limitations in expiratory airflow, emphysematous destruction of the lungs, bronchitis, and chronic inflammation of the lung tissue. With the emerging prevalence in usage of electronic nicotine delivery systems (ENDS), the impact of ENDS on the development of lung diseases such as COPD requires further attention. Previous studies have shown that  -epithelial Na + channel (  ENaC)- overexpressing mice have multiple emphysematous phenotypes, such as mucus accumulation and chronic inflammation. This study was aimed to elucidate the effects of e-cigarettes, a type of ENDS, on the development of COPD. Methods: We hypothesized that acute repetitive usage of ENDS will exacerbate the features of COPD including mucus production, inflammation, and fibrosis, ultimately leading to lung injury. Twenty  ENaC mice (10 mice per group) underwent whole body ENDS or sham air exposure for 2 hours daily over 10 days. Bronchoalveolar lavage (BAL) fluid and lung tissues were collected to assess for inflammation, fibrosis, mucus accumulation, and alveolar damage. Results and Discussion: Our data showed that ENDS exposure significantly increased the alveolar size as compared with the control. We also found that level of inflammatory cytokines, such as CCL2, IL-13, and IL-10, were elevated in animals exposed to ENDS compared with control. Moreover, we observed ENDS exposure caused mucus accumulation, alveolar destruction, and fibrosis in the bronchioles of the  ENaC mice. Additionally, ENDS exposure promoted apoptosis in pulmonary endothelial cell and epithelial cells. Conclusion: Our data suggest that nicotine-containing e-cigarettes exacerbates features of COPD involving abnormal lung inflammation, mucus accumulation and


Background
Chronic obstructive pulmonary disease (COPD) is a primary leading cause of death with limited treatment options, accounting for more than 300 million deaths worldwide annually [1]. The phenotypes of COPD are characterized by limitations in expiratory airflow, emphysematous destruction of the lungs, chronic bronchitis, and inflammation of the lung tissue. Patients with COPD experience a variety of symptoms including shortness of breath, wheezing, and/or a chronic cough [2]. Cigarette smoking is a major risk factor for COPD. While cigarette smoking use in the U.S has decreased over the past 50 years, there has been a dramatic increase in the usage of electronic nicotine delivery systems (ENDS) as the substitutes [1]. As ENDS are perceived as a safer option compared to traditional cigarettes, they are often used to assist in cigarette smoking cessation. The risk of cigarette smoking and its contribution to chronic lung diseases has been well characterized, however, the health effects of ENDS are poorly known. [3]. Therefore, a thorough investigation into the effects of e-cigarette vapor on chronic lung conditions is essential.
ENDS include vape pens, hookah pens, electronic cigarettes (e-cigarettes or e-cigs), which transport noncombustible vaporized nicotine products to the lungs [4]. The common components of e-cigarette liquid include nicotine glycerin, propylene glycol, flavorings, and other ingredients. When heated, the nicotine-containing e-cigarette liquid generates vapor, which will be inhaled into the lungs. Vaporization of e-liquid generates toxic compounds similar to cigarette smoke. Reactive aldehydes, such as formaldehyde and acrolein, are common products from the thermal decomposition of propylene glycol and glycerol, the major vehicle components of e-liquids [5]. Notably, reactive aldehydes generated from ENDS have been noted in much greater concentrations than recommended occupational safety standards [6]. Accumulating data suggest that e-cigarette vapor induces oxidative stress and inflammation in vitro [7,8] and in vivo [9,10].
For example, e-cigarette liquid alone caused morphology changes in lung epithelial cells, by initiating a stress phenotype and inflammatory response [9]. As a major component of ENDS, nicotine, the addictive component in cigarettes and e-cigarettes, inhibits mucus hydration [11] and induces pro-inflammatory dendritic cell responses [12]. Similar results were evidenced in mice where e-cigarette vapor induced airway responses similar to cigarette smoke, including airway inflammation, tissue remodeling, and enhanced mucin expression following four months of exposure [10]. These findings suggest the ability of nicotine-containing e-cigarettes could induce physiological changes akin to human COPD.
As the primary risk factor for COPD, cigarette smoking is widely used to study the pathogenesis of COPD in various animal models [13]. Despite the recent popularity of ENDS, there are limited investigations aimed at elucidating the effects of e-cigarettes on the development and progression of COPD [4]. Therefore, there is an unmet need to study the effects of ENDS, ecigarettes in particular, on COPD progression utilizing the proper animal model with pre-existing phenotypes of COPD. In this study, we utilized the ENaC mouse, a transgenic line that exhibits defective airway mucus clearance due to the overexpression of the epithelial sodium channel non-voltage gated 1, beta subunit (Scnn1b) [14][15][16]. ENaC mice have been used in the investigation of respiratory diseases including COPD and cystic fibrosis [17,18]. As this strain of mice has symptoms of emphysema and muco-obstruction [17], we postulated that ENDS could exacerbate the phenotypic response of COPD following acute e-cigarette vapor exposure. The specific goals of this study were to evaluate the effects of nicotine-containing e-cigarette vapor in the development and progression of a COPD phenotype including 1) inflammatory response in the lung tissues, 2) mucus accumulation within the bronchioles, 3) small airway fibrosis, and 4) the destruction and degradation of alveolar structure.

Animals and genotyping
Male and female ENaC were purchased from The Jackson Laboratory (congenic C57BL/6J background, Stock No. 006438, Bar Harbor, ME). Mice were housed in an environmentally controlled animal facility and maintained on a 12 h light/dark cycle with free access to water and standard chow diet throughout treatment. All animal procedures described were approved by the

ENDS exposure to ENaC mice
At 12-weeks of age, mice (n=10, five male and five female per group) were randomized into either ENDS exposure group or the control group (sham air). Mice in the ENDS group were exposed to e-cigarette vapor for 1-hour twice daily at a frequency of 5 times per week for a total duration of 10 days as shown in

Bronchoalveolar lavage fluid and cytokine measurements
After the mice were euthanized, tracheostomy was performed and lungs were immediately flushed with 1 mL ice-cold phosphate buffered saline (PBS) twice for collection of BAL fluid. Blood was collected via the abdominal aorta, allowed to sit for 30 min to promote coagulation and centrifuged at 5,000 rpm for 10 min for isolation of serum. Total cells from the BAL were centrifuged and counted using Countess™ II Automated Cell Counter (Invitrogen, Carlsbad, CA) as previously described [16]. Differential immune cell count (5,000 cells/slide) was performed on

Morphometric assessment
In order to determine the effects of ENDS exposure on the development of emphysema, we did a morphometric assessment according to the previous protocol [17,19]. Briefly, the mouse lungs were inflated with 1% low melting-point agarose to 25 cm of fixative pressure. After 48 hours fixation, lungs were paraffin-embedded, sectioned and stained with hematoxylin and eosin (H&E).
Mean linear intercepts were determined and calculated [20].
Immunohistochemical and histology analysis.
The mouse lungs were inflated with 1% low melting-point agarose to 25 cm of fixative pressure and then fixed with 4% neutral buffered formalin for 48 hours. Later, these tissue samples were embedded in paraffin, and sectioned into 4 µm sections using a rotary microtome. For immunohistochemical analysis, tissue sections were dewaxed with xylene and rehydrated with graded concentrations of ethanol, followed by antigen retrieval in 10 mM citric acid solution (pH = 6) for 20 min using a pressure cooker. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide. After blocking with 5% BSA for 30 min, the slides were incubated with the primary antibodies against the following proteins at 4°C overnight: α smooth muscle actin (α-SMA) 1:500 (Ab5694; Abcam); Von Willebrand Factor (vWF) 1:300 (A008229; Agilent). After washing, the sections were incubated with the appropriate HRP polymers and developed with 3-3′ diaminobenzidine solution (DAB substrate kit; Vector Laboratories). After counterstaining with hematoxylin, the slides were dehydrated and mounted with a mounting medium.

Data analysis.
In the case of only two groups, two-tail t-tests were performed to assess the differences. All analyses were carried out using Prism7.0 software (GraphPad Software Inc., San Diego, CA).
Values are represented as mean ± SEM. Levels of significance designated as p < 0.05 unless otherwise indicated.

Results
ENaC mice characteristics.
Results of genotyping were used to dictate the use of animals in the treatment procedure as shown in Fig. 1A. Elevated levels of immune cells, specifically neutrophils, have been associated with COPD progression and exacerbation [21,22]. Previous studies have demonstrated that ENaC mice have a higher mucin expression and persistent increases in neutrophils and macrophages [16,17]. These phenotypes indicate an active immune status in the lungs of these animals before e-cigarette exposure, which could be a model to explore the effects of ENDs on the development of COPD.

ENDS exposure induces airspace enlargement and mucus accumulation in the lungs.
COPD is distinguished by airway remodeling, mucus accumulation, goblet cell metaplasia, and inflammation [23]. We first measured the airspace enlargement, which are the characteristics of COPD, in ENaC mice exposed to air or ENDs. As shown in Fig. 1B and 1C, ENDs exposure significantly induced the airspace enlargement compared with the sham air control in EnaC mice.
These data suggest acute ENDS exposure impairs the alveolar structure in EnaC mice.
Because ENaC mice exhibit mucus accumulation due to dehydration in the airway epithelium [16], these strain of mice are known as a model of muco-obstructive lung disease for the investigation of COPD [15]. Airway mucus content was evaluated histologically using PAS staining [16]. As mucus accumulation is a feature of COPD, we therefore asked if ENDS exposure could further induce airway mucus accumulation and progress the development of COPD. As shown in Fig. 1D, ENDS exposure increased the deposition of mucus in the bronchioles compared with control. Goblet cells are the primary cell type for mucus production, and goblet cell metaplasia commonly occurs in association with exposure to cigarette smoke in animals models [24].
Although goblet cells are not prevalent in all the animal models, a previous study found goblet cell metaplasia as well as epithelial cell hypertrophy exist in the ENaC mice [17]. Goblet cell metaplasia is marked by the accumulation of goblet cells in the epithelium lining of the bronchiole and responsible for tissue regeneration. Because goblet cells are the cells primarily responsible for mucus production [25], our data suggest the ENDS exposure causes mucus overproduction by activating goblet cell metaplasia in the lung.

ENDs exposure causes immune cells infiltration and inflammation in the lungs
The dysregulation of immune cells, especially the elevated presence of neutrophils is known to cause the goblet cell metaplasia [26]. In order to profile the involvement of different immune cells, we then performed the Diff-Quik staining on the cells from the BAL. As shown in Fig. 2A and 2B, we found ENDS exposure causes increased infiltration of immune cells including macrophages and neutrophils in ENaC mice as compared with the control.
To elucidate the effects of ENDS exposure on airway inflammation, we performed mice cytokine arrays using BAL supernatant from the experimental mice. As shown in Fig. 2C,  (Fig. 2E-G). CCL-2, IL-10, and IL-13 have been indicated in contributing to macrophage infiltration, M2 macrophage polarization, and pulmonary fibrosis [27][28][29]. Thus, these results indicate ENDS exposure contribute to immune cell infiltration such as macrophages and neutrophils as well as inflammatory cytokines productions within the airway in the ENaC mice.
ENDs exposure promotes exacerbation of small airway fibrosis.
Small airway fibrosis is considered an early feature of COPD. High-resolution computerized tomography (HRCT) suggest that small airway remodeling is the earliest feature of COPD and could be used to determine the trajectory of disease progression [30]. Recent study showed that e-cigarette vapor exposure promotes fibroblast differentiation to myofibroblasts, which leads synthesis of abnormal amounts of extracellular matrix (ECM) proteins and fibrosis [31][32][33]. Other evidence points out that chronic nicotine-containing e-cigarette vapor exposure causes multiorgan fibrosis in mice [34]. Airway remodeling involves multiple physiological changes in the lung such as thickening of the airway wall, goblet cell hyperplasia, mucous gland hypertrophy, the luminal obstruction caused by inflammatory exudates [35]. Moreover, destruction and fibrosis of the alveolar wall with the deposition of ECM uniquely occurs in patients with COPD rather than in patients with other respiratory conditions, such as asthma [36,37]. Therefore, we examined the effects of ENDS on the progression of fibrosis in this animal model. We assessed the deposition of collagen, a major constituent in ECM within lung tissue following exposure to ENDS. Picro-Sirius red (Fig. 3A) was used to stain for all types of collagen in the lung tissue of ENaC mice with and without ENDS exposure. As shown Fig. 3A, 10-day exposure to ENDS elevated collagen deposition in ENaC mice within the airway and lung parenchyma (Fig. 3A bottom panel) compared with the control (Fig. 3A top panel). Quantitative analysis of Picro-Sirius red staining was shown in Fig. 3B. These results were confirmed by Masson's Trichrome staining (Fig. 3C).
In addition, small airway destruction was also observed in mice upon ENDS exposure (Fig. 3A, bottom panel) compared with the control (Fig. 3A, top panel).
Myofibroblasts, the activated form of fibroblasts, contribute to ECM deposition and therefore are responsible for small airway fibrosis within the lung [38]. As myofibroblasts specially express the protein α-SMA, IHC staining of α-SMA was used to evaluate the presence of fibrotic tissue in the lung interstitium. As shown in the top panel of Fig. 3E, ENDS exposure elevated the expression α-SMA in the subpleural and interspersed in the parenchyma of ENaC mice compared with the control (Fig. 3C, bottom panel). These results are supported by a previous study showed that chronic exposure to nicotine-containing ENDS caused fibrosis in kidney and heart tissue [34]. These data suggest that exposure to nicotine-containing ENDS induces ECM deposition, and therefore fibrosis within the lung tissue, which is responsible for lung tissue remodeling by the activation of myofibroblasts.

ENDS exposure causes lung vascular injury by inducing lung endothelial cell apoptosis
Vascular cell dysfunction and death are the primary phenotypes in cigarette smoking-related lung diseases [39]. Newly emerging evidence suggests ENDS exposure induces endothelial inflammation [40] and oxidative stress [41]. Therefore, we examined the effect of ENDS on vascular injury in the lungs. Pulmonary endothelial cell apoptosis has been known to promote the progression of emphysema [42]. Thus, we hypothesized that ENDS exposure would lead to the apoptosis of pulmonary endothelial cells. Using TUNEL staining, we were able to detect apoptosis in the fixed lung tissues. Consistent with the pattern from the positive control, we found ENDS exposure leads to the elevation of apoptosis in pulmonary endothelial cells (Fig. 4A bottom, black dots indicated by green arrows) whereas the apoptosis was barely seen in the control (Fig. 4A Top). Quantitative analysis of TUNEL staining from βENaC mice without and with e-cigarette vapor exposure was shown in Fig. 4B.
In order to confirm the death of endothelial cells, we also performed immunohistochemical staining of von Willebrand Factor (vWF), a widely-used biomarker for vascular endothelial cells in the lungs of mice without or with ENDS exposure. We observed that ENDS exposure significantly diminished the amount of vWF positive endothelial cells compared with the control (Fig. 4C and   4D).
Based on the results obtained in vivo, we further employed the HAEC cells to test the effects of ENDS in vitro. As shown in Fig. 5F, exposure to nicotine-containing e-cigarette liquids ( Fig. 5F bottom right, 57%), significantly decreased the cell viability compared with the control ( Fig. 5F top left, 82%). Interestingly, a significant toxic effect of e-cigarette liquid exposure without nicotine ( Fig. 5F bottom left, 75%) was also observed as shown in Fig. 5F bottom left and 5E.
Thus, these data suggest that ENDS exposure, particularly nicotine-containing e-cigarette liquid, causes lung vascular injury by inducing lung endothelial cell apoptosis.

ENDS exposure causes apoptosis in lung epithelial cells
COPD consists of chronic bronchitis and emphysema, in which alveolar destruction leads to impaired air exchange [43]. Previous study showed that ENaC mice spontaneously develop an emphysematous phenotype, and chronic e-cigarette exposure causes alveolar destruction in a nicotine-dependent manner [10]. We, therefore, tested whether nicotine-containing e-cigarette vapor exposure could cause alveolar destruction. As expected, we detected apoptosis in the fixed lung tissues by using TUNEL staining. Consistent with the pattern shown in endothelial cells, we found ENDS exposure leads to the elevation of apoptosis in airway epithelial cells (Fig. 5A bottom, black dots indicated by green arrows) compared to the control (Fig. 5A top). Quantitative analysis of TUNEL staining from βENaC mice without and with e-cigarette vapor exposure was shown in Fig. 5B. We then validated the data in vitro by culture of airway epithelial cells (16HBE) with ecigarette liquid. As shown in Fig. 5C and 5D, e-cigarette liquid with nicotine and without nicotine significantly leads the cell death compared with the control. Altogether, our results suggested that ENDS exposure leads to epithelium damage and remodeling by causing apoptosis in lung epithelium.

Discussion
Cigarette smoking is considered the major risk factor for the development of lung diseases such as COPD and lung cancer in the developed world. The toxic effects of smoking are attributed to the notorious mixture of oxidants, irritants, and carcinogens. Based on the assumption that ENDS vapor containing fewer toxins than conventional cigarette smoke, they were initially considered to be safer [44,45]. While ENDS have a less detrimental effect in terms of environmental pollutants [46] and production of carcinogens [47], the health effects of ENDS usage on chronic lung conditions, such as COPD, and associated public health recommendations remain scant.
Recent data classified ENDS aerosol is unsafe, as it contains heavy metals such as nickel and chromium, volatile organic, and carcinogen compounds such as acrolein and crotonaldehyde [45,48]. Results from cell culture and animal studies suggest nicotine is the driving force of emphysema, lung tissue inflammation, and airway surface dehydration [10]. However, differences in heating elements, humectants, and flavorings have also been shown to produce different physiological responses in terms of reactive oxygen species (ROS) production [9]. In addition, ENDS users often combine e-cigarette liquid with tetrahydrocannabinol (THC) and cannabidiol (CBD) for vaping, potentially worsening the effects [49]. By February of 2020, the Centers for Disease Control and Prevention reported approximately 3,000 hospitalizations associated with ecigarette or vaping, product use-associated lung injury (EVALI) [50]. Given the clinical health issues of ENDS in lung pathobiology, our current study was to investigate the effects of ENDS on pro-inflammatory and pro-fibrotic phenotypes in vivo and in vitro.
In this present study, an animal model with pre-existing phenotypes of COPD was used to determine the effect of ENDS on the progression and exacerbation of COPD. Following a 10day acute exposure to nicotine-containing e-cigarette vapor, significant mucus accumulation was noted within the bronchioles of ENaC mice as evidenced by PAS staining. Given the phenotypes of this transgenic line, mucus accumulation was expected and was exacerbated by ENDS vapor exposure. Nicotine is not only the addictive component of cigarettes, but is also a key factor in the initiation and progression of COPD and is known to hinder the hydration of mucus [12].
Cigarette smoke generated from high-dose nicotine cigarettes induced more emphysematous changes than low-dose nicotine cigarettes in elastase-treated rats [54]. Similarly, an in vitro study showed that nicotine induced mucus production in bronchial cells [55]. Consistent with these data, our results indicated that mucus accumulation occurs in the lungs of βENaC mice following exposure to nicotine-containing ENDS vapor (Fig. 1D). Reactive aldehydes, such as formaldehyde and acrolein, are common products from the thermal decomposition of propylene glycol and glycerol, the major vehicle components of e-liquids [5]. In addition, popular sweet and creamy flavors used in e-liquids containing dicarbonyls such as diacetyl and 2,3-pentanedione could also cause significant pulmonary epithelial apoptosis and inflammation in animal models [51,52]. These reactive carbonyls generated from flavors have been noted in much greater concentrations than occupational safety standards [6]. Inhalation of high levels of volatile butter flavoring (2,3-butanedione, an alpha-diketone) could cause bronchiolitis obliterans [53]. Although flavored ENDS aerosols have lower levels of toxins than tobacco smoke, inhalation of ENDS aerosols will cause the accumulation of carbonyls in the circulation [54], which may be detrimental to the endothelium. Our results support the role of nicotine in the progression of muco-obstructive conditions. However, further studies are needed to compare the pulmonary consequences of inhaled nicotine with different flavorings and the potential for dose dependent responses.
As expected, ENDS vapor exposure increases immune cell infiltration and proinflammatory cytokines production within BAL fluid. Most notably, CCL2, also known as monocyte chemotactic/chemoattractant protein 1 (MCP1), is a soluble factor in driving monocytic infiltration of tissues during inflammatory processes [55]. In a previous study, CCL2-producing macrophages are highly activated in patients with COPD, which is associated with increased monocyte migration [55]. Additionally, significant increases in IL-10, a regulatory anti-inflammatory cytokine, was also observed in response to ENDS exposure. Elevation of IL-10 was shown to be present with other pro-inflammatory cytokines, although the presence of IL-10 may negatively regulate these pro-inflammatory cytokines [50]. In addition, IL-10 is known to coincide with the switching of macrophages towards the M2-like phenotype in patients with COPD [27]. Similar to IL-10, IL-13 was also observed increased upon ENDS exposure, another cytokine known to induce M2macrophage polarization [28]. Given that the attenuation of M2-macrophages slows the progression of pulmonary fibrosis [29], these data suggested M2-macrophages play an important role during the development of COPD in our animal model.
We premised that ENDS exposure might cause the elevation of inflammatory markers indicates inflammation and pro-fibrotic response. As seen in Fig. 3A and C, ENDS vapor exposure caused collagen deposition within the lung tissues. In addition, we also found e-cigarette vapor containing nicotine caused a significant increase in the expression of -SMA (Fig. 3E), suggesting activation of fibroblasts and the fibrotic phenotype in our animal model. The detailed mechanism in which ENDS contributes to lung tissue damage is worth further investigation.
While the majority of research focus is on the epithelial lining within the airway, recent evidence suggests endothelial dysfunction plays an essential role in the oxidative stress response to e-cigarette vapor [41]. Here, we validated the effects of ENDS exposure on the cell viability of endothelial cells (HAEC) and epithelial cells (16HBE) via flow cytometry. Cell viability was significantly decreased following treatment with e-cigarette liquid containing nicotine ( Fig. 4F and   5C). Interestingly, e-cigarette liquid without nicotine did not have the same effect on cell death and viability remained comparable to control, suggesting nicotine plays a significant role in apoptosis of endothelial and epithelial cells responsive to inflammation and oxidative stress. We also found e-cigarette liquid without nicotine affect the cell viability, indicating other components of e-cigarette could be toxic to endothelial cells as well. A recent study found that chronic ENDS exposure without nicotine adversely affect the vascular endothelial network by promoting oxidative stress and immune cell adhesion [41]. Further investigation is needed to elucidate the impact of chronic ENDS exposure without nicotine on the endothelium within the lung.

Conclusion
Taken together, our results demonstrate nicotine-containing ENDS, in the form of e-cigarette vapor, causes the exacerbation of features of COPD in βENaC-overexpressing mice. Specifically, acute exposure to nicotine-containing e-cigarette vapor results in mucus accumulation, overproduction of inflammatory cytokines, deposition of ECM, and fibrosis. Last but not the least, e-cigarette exposure induces cell death in epithelium and endothelium in the lung of βENaCoverexpressing mice.

Ethics approval:
All animal procedures described were approved by the Institutional Animal Care and Use Committee (IACUC) at Georgia State University.

Consent for publication
All the authors have approved to the material to be submitted to the journal of inflammation.

Availability of data and materials
All data are available from the corresponding author on request.    The cell viabilities were quantified as the Annexin V/ PI double-negative cell percentages of total acquired cells. Columns and error bars represent means ± SD, n=4 * p<0.05; **p<0.01; ***p<0.0001.

Figure 6. Potential molecular mechanism of ENDS on inflammation, fibrosis, and apoptosis
The exposure of ENDS causes inflammation, fibrosis, and apoptosis of endothelial and epithelial in the lung. The acute inflammation leads to the production of IL-10 and IL-13, infiltration of macrophages, and mucus accumulation. Both cytokines are involved in the infiltration of immune cells and contribute to the inflammatory process. The elevation of macrophages in the lung was associated with fibrosis, collagen deposition, and myofibroblast differentiation. The acute ENDS exposure also stimulates the apoptosis in the endothelial and epithelial cells, which contribute to the destruction and degradation of lung alveoli.