Electronic Cigarette Aerosol Exposure Induces Airway Remodeling in 3D Human Tracheobronchial Epithelial Tissues: From Goblet Cell Hyperplasia to Squamous Metaplasia

Electronic cigarette (EC) aerosols are increasingly recognized for their disruptive effects on airway physiology, yet the temporal progression of epithelial remodeling remains poorly characterized. While squamous metaplasia, mucin hypersecretion, and ciliary dysfunction are hallmark features of smoking-related airway disease, analogous data for EC exposure are limited. To address this gap, we employed a 3D human tracheobronchial epithelial tissue (hTET) model derived from airway basal stem cells and exposed it daily to NJOY ACE "Classic Tobacco" aerosols (1 or 5 puffs) or clean air for 1, 2, or 3 weeks. Remodeling was assessed via immunolabeling and Western blot analysis. MUC5AC expression revealed a transient hypersecretory response, peaking at week 2 and declining by week 3. Multiparametric analysis demonstrated progressive squamous remodeling: early ciliary loss and involucrin upregulation emerged by week 1, with severity increasing in a puff-dependent manner. By week 2, tissues exhibited marked ciliary depletion, altered epithelial thickness, horizontal nuclear reorientation, and elevated involucrin. At week 3, both exposure groups developed a squamous metaplasia phenotype, characterized by involucrin-positive squamous cells replacing ciliated cells. Quantitative analysis confirmed sustained involucrin elevation, with the 1-puff group reaching levels comparable to the 5-puff group by week 3. These findings delineate a sequential pathological trajectory initiated by EC aerosols, progressing from transient goblet cell activation to squamous metaplasia. This human-relevant model underscores the potential for repeated low-level EC exposure to induce airway epithelial remodeling and pathology.