Abstract Rationale The increasing use of electronic cigarettes (e-cigarettes), particularly among adolescents, represents an emerging public health concern due to the presence of toxic chemicals in their aerosols. We previously showed that exposure to e-cigarette aerosols increases epithelial permeability and disrupts apical junctional complex (AJC) structures in vivo. E-cadherin, a key protein for cell-cell adhesion, is cleaved under stress to generate soluble E-cadherin (sE-cad), a marker of epithelial disruption elevated in inflammation, infection, and malignancy. Matrix metalloproteinases (MMPs) are proteolytic enzymes that mediate E-cadherin cleavage and are upregulated during inflammation and injury. We hypothesized that inhibiting MMP activity would prevent sE-cad shedding induced by e-cigarette exposure, thereby preserving epithelial junction integrity. Methods Human bronchial epithelial (16HBE14o-) and primary normal human bronchial epithelial (NHBE) cells were exposed to 18 mg/mL unflavored nicotine aerosol using an ASL 5000 breathing simulator programmed to replicate normal adolescent breathing. Cells were exposed for 15-minute intervals at 0, 8, and 24 hours, with or without pretreatment with 20 µM fisetin, a known flavonoid MMP inhibitor. Barrier integrity was evaluated via transepithelial electrical resistance (TEER). Protein expression and activity was assessed by western blotting and gelatin zymography. In parallel, C57BL/6 mice (8-12 weeks old) were exposed to e-cigarette aerosols (36 mg/mL) using an e-cigarette extension system (Joyetech, SCIREQ Inc., Montreal, Canada). Results 16HBE and NHBE cells exposed to e-cigarette aerosols exhibited decreased TEER indicating increased airway epithelial barrier permeability, which was partially restored with MMP inhibitor treatment. Western blot analysis of bronchoalveolar lavage (BAL) fluid from e-cigarette-exposed mice after four days revealed a significant increase in sE-cad compared to air-exposed controls. Similarly, elevated sE-cad levels were observed in 16HBE and NHBE culture supernatants exposed to e-cigarette aerosols. E-cigarette exposure also increased MMP-2, MMP-9, and MMP-12 expression and secretion in BAL fluid and cell culture supernatants. In vitro, pretreatment with fisetin significantly reduced MMP activity and sE-cad release, indicating that MMP inhibition mitigates E-cadherin cleavage and preserves epithelial barrier integrity. Conclusions E-cigarette aerosol exposure compromises airway epithelial barrier function through MMP-mediated E-cadherin cleavage, leading to increased sE-cad shedding. Upregulation of MMPs provides a mechanistic basis for epithelial disruption. Targeting MMP-mediated pathways offers a promising treatment strategy to protect against e-cigarette-induced airway injury and to maintain epithelial barrier integrity. This abstract is funded by: National Institutes of Health (NIH) grant R01-HL-148057 (F. Rezaee), Research Program Committees, Cleveland Clinic 4159 (F. Rezaee)
Beaumont et al. (Fri,) studied this question.