Centner, A. M.; Khalili, L.; Ukhanov, V.; Park, G.; Kadyan, S.; Hwang, H. S.; Nagpal, R.; Salazar, G.

Rationale: The role of sex in the effects of vaping and individual aerosolized e-liquid constituents on atherosclerosis, vascular aging, and gut microbiome remodeling remains poorly characterized. Objective: To determine the contribution of e-cigarette aerosol components to vascular senescence, atherosclerosis, and gut microbiome dysbiosis in ApoE-/- mice and vascular smooth muscle cell (VSMC) viability and senescence. Methods: Male and female ApoE-/- mice were exposed to e-liquid constituents (vehicle, vehicle plus nicotine, and vehicle plus nicotine plus menthol) for 48 minutes per day, 5 days per week for 16 weeks, with vascular pathology assessed in vivo. VSMCs isolated from aortas of wild-type and ApoE-/- male and female mice were exposed to aerosolized e-liquids and evaluated for cellular senescence. Results: Exposure to all tested e-liquid formulations, including vehicle, nicotine-containing, and menthol-containing aerosols, increased atherosclerosis in both male and female mice, with the most robust effects observed in the nicotine-containing formulation and in the descending aorta. Females exhibited greater sensitivity to e-liquid exposure, with increased plaque accumulation in both the aortic arch and descending aorta, while the addition of menthol was associated with reduced plaque burden compared with nicotine alone in both sexes. Novel findings show that e-liquid exposure also altered gut microbial composition in a sex- and exposure-dependent manner, with nicotine causing the greatest dysbiosis and menthol exerting modulatory, but not restorative, effects. Notably, Alloprevotella emerged as a key discriminating genus associated with reduced plaque burden, supporting a potential link between gut microbial remodeling, inflammatory regulation, and atherosclerosis. Conclusions: These findings demonstrate that individual e-liquid aerosol components increase atherosclerosis and alter the gut microbiome in a sex-specific manner, with nicotine producing the most pronounced pro-atherogenic effects and the addition of menthol reducing these effects, without eliminating overall atherosclerotic risk.