Mainstream Cigarette Smoke Impacts the Mouse Vocal Fold Epithelium and Mucus Barrier

Abstract

Objectives/Hypothesis Cigarette smoke (CS) is a primary risk factor for the development of numerous benign and malignant laryngeal diseases. The epithelium and mucus lining the vocal folds (VF) are the first barriers against CS. The primary objective of this study was to investigate epithelial and mucus barrier changes in the mouse laryngeal mucosa upon exposure to subacute CS. The secondary objective was to compare mucus barrier changes in mice and human smokers and nonsmokers. Study DesignAnimal model. Methods Mice were exposed to CS for 4 weeks for 4 hours (N = 12, high dose [HD]) or 1 hour (N = 12, low dose [LD]) per day. Air‐exposed mice were used as a control group (N = 10). Larynges were harvested and VF epithelial barrier integrity was evaluated including cellular proliferation and expression of cell junctions. We also investigated mucus production by examining mucus cell area and mucin expression in mice and human smokers and nonsmokers. Results HD CS increased VF epithelial cellular proliferation but did not alter the expression of cell junctions. HD CS also induced hypertrophy of the mucus‐producing submucosal glands. However, only LD CS increased MUC5AC gene expression. MUC5AC staining appeared elevated in laryngeal specimens from smokers, but this was not significant as compared to nonsmokers. Conclusions These findings help us identify potential adaptive mechanisms to CS exposure as well as set the foundation for further study of key aspects of epithelial and mucus barrier integrity that may be implicated in laryngeal disease development following prolonged smoking. Level of Evidence NA Laryngoscope, 131:2530–2539, 2021

Expanded Basal Compartment and Disrupted Barrier in Vocal Fold Epithelium Infected with Mouse Papillomavirus MmuPV1

“…In uninfected laryngeal tissues, the proliferation index (% Ki67 + cells) was about 1%. This is consistent with the literature reporting very few proliferating cells in adult mouse vocal folds [ 55 , 57 , 73 ]. Proliferation index increased to a median of 3.4% in diseased murine laryngeal tissues, and Ki67-expressing cells were found above the basal layer of the epithelium.…”

“…Vocal fold epithelium is thinner in mice than in humans, comprising 2–4 cell layers rather than 5–10 [ 20 , 54 ]. Likely because of this difference, there is one layer of proliferating basal cells rather than two or three [ 54 , 55 , 56 , 57 ]. The same markers of basal cells (p63), proliferating cells (Ki67), differentiated cells (cytokeratin 13 (K13)), cell–cell junctions (E-cadherin, β-catenin, zonula occludens 1 (ZO-1)), and basement membrane (laminin, collagen IV) are present in the vocal fold epithelium of humans [ 20 , 21 , 22 , 34 , 58 , 59 , 60 ] and mice [ 34 , 47 , 54 , 55 , 56 , 57 , 61 ].…”

Modulation of mouse laryngeal inflammatory and immune cell responses by low and high doses of mainstream cigarette smoke

“…Mainstream CS from Kentucky 3R4F reference cigarettes was generated using inExpose smoking equipment (SCIREQ, Montreal, QB, Canada), consistent with our previous studies [12][13][14] . Exposure conditions were per the Federal Trade Commission (FTC)/International Standard Organization (ISO) standards (ISO 1991).…”

“…HD mice were exposed to 4 h of CS per day in 1-1.5 h increments, with room air breaks. Earlier studies in the airways and speci cally in the larynx, have shown that 1-4 h/day of CSE is su cient to induce histopathological changes 12,13,77,78 . LD and HD CS exposures were performed for 5 days/week, M-F for a total of 4 weeks as per the subacute inhalation toxicity guidelines regulated by the Organization for Economic Co-operation and Development (OECD) 79 .…”

“…We have previously characterized tissue remodeling in the laryngeal mucosa after CS exposure (CSE) [12][13][14] . Morphological alterations to the laryngeal epithelium, as driven by changes to cell proliferation, cell death, epithelial barrier integrity, subglandular hypertrophy, and mucin dynamics were observed [12][13][14] , consistent with previous reports [15][16][17] . The mode of action by which CS induce these changes is complex and not well elucidated.…”