A76-03 E-cigarette Aerosols Induce Airway Epithelial Barrier Dysfunction Via Matrix Metalloproteinase Activation

Abstract Rationale The rise in electronic cigarette (e-cigarette) use among adolescents has emerged as a pressing public health issue. Harmful compounds found in e-cigarette aerosols can damage the airway epithelial barrier, contributing to lung injury and increased permeability. Barrier integrity depends on tight junctions (TJs) and adherens junctions (AJs), which maintain epithelial cohesion through interactions with other junctional proteins and the actin cytoskeleton. We investigated how matrix metalloproteinases (MMPs), enzymes upregulated during airway epithelial dysfunction, destabilize TJs and AJs, thereby compromising junctional networks and barrier integrity. Specifically, we examined whether exposure to nicotine-containing and flavored e-cigarette aerosols increases MMP activity, leading to airway epithelial barrier dysfunction. Methods Human bronchial epithelial (16HBE14o-) cells and well-differentiated normal human bronchial epithelial (NHBE) cells were exposed to nicotine-containing or nicotine-containing mint-flavored e-cigarette aerosols. Exposures occurred at 0, 8, and 24 hours, with each session lasting 15 minutes, using an ASL 5000 breathing simulator (IngMar Medical, Pittsburgh, PA), which generates realistic breathing patterns. To assess the role of MMPs, cells were co-treated with fisetin, a flavonoid inhibitor of MMPs. Immunofluorescence imaging was used to visualize TJ proteins (ZO-1, Occludin), AJ proteins (E-cadherin, β-catenin), and the actin cytoskeleton to assess protein localization and structural integrity. Epithelial permeability was evaluated using a FITC-Dextran assay and transepithelial electrical resistance (TEER). Cell lysates collected 27 hours post-exposure were analyzed by western blotting for TJ and AJ proteins, as well as cortactin, an actin-binding protein. Densitometric analysis quantified relative protein expression compared to air-exposed controls. Results Exposure to both nicotine-containing and mint-flavored aerosols disrupted TJs and AJs at cell interfaces. MMP inhibition by fisetin prevented these morphological changes. FITC-Dextran assays and TEER demonstrated increased epithelial permeability following nicotine-aerosol exposure, an effect significantly reduced by MMP inhibition. However, western blot analysis showed no significant changes in junctional proteins, likely because these structural alterations occur primarily through protein relocalization and cytoskeletal remodeling rather than changes in total protein levels. Conclusions Acute exposure to nicotine-containing and flavored e-cigarette aerosols induces both structural and functional damage to the airway epithelial barrier. These effects appear to be mediated, at least in part, by MMP activation, which disrupts junctional organization and impairs barrier function. E-cigarettes, particularly products containing nicotine and flavoring agents, pose significant risks to airway health. Further studies are warranted to evaluate the long-term effects of chronic exposure, especially among youth and other vulnerable populations. This abstract is funded by: National Institutes of Health (NIH) grant R01-HL-148057 (F. Rezaee), Research Program Committees, Cleveland Clinic 4159 (F. Rezaee)