BACKGROUND:Electronic cigarettes (e-cigarettes) have experienced a tremendous increase in use. Unlike cigarette smoking, the effects of e-cigarettes and their constituents on mediating vascular health remain understudied. However, given their increasing popularity, it is imperative to evaluate the health risks of e-cigarettes, including the effects of their ingredients, especially nicotine and flavorings. OBJECTIVES:The purpose of this study was to investigate the effects of flavored e-cigarette liquids (e-liquids) and serum isolated from e-cigarette users on endothelial health and endothelial cell-dependent macrophage activation. METHODS:Human-induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and a high-throughput screening approach were used to assess endothelial integrity following exposure to 6 different e-liquids with varying nicotine concentrations and to serum from e-cigarette users. RESULTS:The cytotoxicity of the e-liquids varied considerably, with the cinnamon-flavored product being most potent and leading to significantly decreased cell viability, increased reactive oxygen species (ROS) levels, caspase 3/7 activity, and low-density lipoprotein uptake, activation of oxidative stress-related pathway, and impaired tube formation and migration, confirming endothelial dysfunction. Upon exposure of ECs to e-liquid, conditioned media induced macrophage polarization into a pro-inflammatory state, eliciting the production of interleukin-1β and -6, leading to increased ROS. After exposure of human iPSC-ECs to serum of e-cigarette users, increased ROS linked to endothelial dysfunction was observed, as indicated by impaired pro-angiogenic properties. There was also an observed increase in inflammatory cytokine expression in the serum of e-cigarette users. CONCLUSIONS:Acute exposure to flavored e-liquids or e-cigarette use exacerbates endothelial dysfunction, which often precedes cardiovascular diseases.