PROJECT SUMMARY This application is being submitted in response to NOT-HL-19-724. The use of e-cigarettes has been rapidly increasing among young adults in the US but the health effects of prolonged e-cigarette use are unknown. The toxicity of e-cigarette was recently highlighted by the large number of recent cases of acute lung injury associated with e-cigarette use or vaping (EVALI), resulting in a total of 2,807 hospitalized EVALI cases or deaths. Whether these cases are linked to the vaping of illicit compounds-containing e-liquids or the widely used nicotine- containing e-liquids remain to be determined. Importantly, characterization of EVALI patients who had to be rehospitalized or died after initial discharge revealed the presence of one or more comorbities including cardiac disease, respiratory diseases and diabetes which suggests certain cohort of e-cigarette users are at increased risk of EVALI. In this Supplement to the Parent R00 HL130416, we propose to leverage patient-derived human induced pluripotent stem cell (iPSC) platform towards determing the pathogenesis of EVALI as a consequence of e-cigarette or its constituents and how the presence of susceptibility factors may worsen the outcome. We hypothesize diseased iPSC-derived cells will exhibit increased cytotoxicity compared to healthy cells when exposed to e-cigarette and its constituents. Aim 1 will be based on the differentiation of iPSCs representing different risk factors (healthy, diabetes alone, diabetes with cardiomyopathy) into endothelial cells and alveolar epithelial cells. Upon exposure of these cells to e-cigarette or vitamin E acetate and its toxic derivative ketene, phenotypic characterization of cellular health will be performed to determine which of these constituents induces the most toxicity. In Aim 2, we will investigate the mechanism of e-cigarette-induced endothelial dysfunction and focus on the p62/NRF2/KEAP1 anti-oxidative stress pathway since we observed that only healthy cells had sustained levels of p62 upon exposure. We will also perform proteomic profiling of exosomes isolated from healthy and diseased iPSC-ECs upon exposure in order to identify novel biomarkers that can potentially predict the development of EVALI. In summary, this Administrative Supplement proposal will create novel opportunities to establish a human-based iPSC repository representing different risk factors that can be used to evaluate health outcomes from vaping. The combination of biochemical methodologies coupled with proteomics will also allow a deeper mechanistic understanding behind the increased susceptibility of certain patients against EVALI and provide biomarkers that may be predictive of EVALI development.