We developed a novel young‐adult murine model of subacute VEA exposure to investigate the temporal relationship between recreational use and lung injury onset and to examine sex‐stratified pathobiological responses. After 10 days, VEA‐exposed mice exhibited acute features of pathologic lung injury, inflammation, and higher systemic plasma sRAGE levels. CCSP expression was reduced in large airways, most prominently in males. Notably, early changes in alveolar barrier integrity were evident as soon as 3 days post‐exposure, with increased BAL neutrophils, total protein, and plasma sRAGE despite the absence of overt histopathology. Sex‐stratified analyses showed greater histologic lung injury and immune dysregulation in males, with elevated Il1b, Il6, and Kc expression, while females had higher sRAGE levels despite milder pathology. Notably, all mice maintained stable vital signs consistent with normative murine values under basal conditions (Dutta & Sengupta, 2016), even with the pronounced histologic changes observed after 10 days of repeated VEA exposure. This finding suggests an indolent disease trajectory, with clinical manifestations likely emerging only at later stages.

Despite comparable aerochamber VEA concentrations and anatomic size, male mice exhibited markedly higher BALF VEA levels over time, with significant elevations relative to females by day 10. These findings align with prior in vivo studies demonstrating the pulmonary toxicity of aerosolized VEA in short‐term (6–28 days), intermittent (1–5 h/day) exposure models designed to mimic recreational vaping (Castañeda & Pinkerton, 2016; Dutta & Sengupta, 2016; Lim et al., 2021; Raucci et al., 2008). Histopathological analyses in these models have revealed oil‐laden macrophages, indicative of VEA accumulation and macrophage activation, and interstitial inflammation consistent with inflammatory lung injury. Complementary in vitro studies show that human alveolar type II (ATII) cells sustain direct VEA‐induced injury, accompanied by increased release of monocyte‐ and neutrophil‐recruiting chemokines. The elevated BALF VEA concentrations observed here likely reflect the severity of pulmonary injury and associated impaired alveolar fluid clearance, while alterations in pulmonary metabolism may also further contribute to heightened, sex‐specific susceptibility, warranting additional investigation.

One of the most compelling findings from our study is the distinct plasma sRAGE profiles observed between male and female mice. RAGE is a transmembrane, multiligand receptor constitutively expressed on type I alveolar epithelial cells and inducibly on alveolar endothelial cells (Lim et al., 2021). Under oxidative stress, RAGE activation triggers intracellular signal transduction via NF‐κB, driving a cascade of pro‐inflammatory cytokine release and tissue injury. Its soluble isoform (sRAGE), generated through alternative splicing or proteolytic cleavage, acts as both a biomarker of RAGE activation and a decoy receptor that dampens ligand‐induced inflammatory signaling (Braley et al., 2016; Lim et al., 2021; Raucci et al., 2008; Sukkar et al., 2012). Although sex‐dependent hormonal regulation of the RAGE pathway has been described in a variety of non‐pulmonary diseases (Bajwa et al., 2023; Fernandez et al., 2010; Mukherjee et al., 2005; Tanaka et al., 2000; Tesarová et al., 2007), we are the first to report sex‐dimorphic sRAGE responses in an EVALI model and, to our knowledge, in any acute lung injury model. It is plausible that estrogen may enhance ADAM10‐mediated RAGE shedding, driving higher circulating sRAGE in females and modulating sex‐specific lung injury (Fernandez et al., 2010). Sex‐specific differences in CCSP expression along the large airway epithelium were also observed, with CCSP also serving a critical immunomodulatory role and essential to epithelial repair (Almuntashiri et al., 2023; Martinu et al., 2023). Together, these findings highlight potential hormonally regulated pathways that may define sex‐specific vulnerability and inform targeted therapies for VEA‐induced lung injury.

Intrinsic sex differences modulate the pathophysiology, incidence, morbidity, and mortality of multiple lung diseases across the lifespan, ranging from neonatal conditions to adult diseases that include asthma, cystic fibrosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, and lung cancer (Silveyra et al., 2021). Male‐biased outcomes have also been observed in viral epidemics, including COVID‐19, SARS‐CoV‐2, and MERS‐CoV (Matute‐Bello et al., 2008). The underlying causes of these differences remain unclear, and it is not fully established to what extent they arise from biological versus environmental factors. In this model, we demonstrate that biological sex differences in EVALI are partly mediated by differential immune dysregulation, likely influenced by hormonally regulated pathways, which may contribute to the sex‐specific disparities observed across the spectrum of lung diseases.

Our study offers several strengths and contributes to the limited body of literature on VEA‐induced lung injury. A major innovation of our model is the balanced inclusion of male and female mice, enabling direct investigation of sex‐based differences in pulmonary response. To our knowledge, this is the first animal model to specifically interrogate biological sex as a determinant of the dysregulated immune response to inhalational VEA exposure. Second, biomarkers of disease remain underexplored in the context of EVALI. Building upon a limited body of mechanistic VEA literature, our study identifies biomarkers of neutrophil‐dependent lung injury, pro‐inflammatory signaling, and epithelial damage, providing new insights into core mechanisms of injury and highlighting emerging therapeutic targets for future investigation. We focused specifically on a young adult mouse model, representing an important demographic with EVALI. This subgroup is known to exhibit a distinct pathophysiologic response compared to older adults, making it particularly relevant for modeling age‐specific disease dynamics. Finally, our study investigated early changes in biomarker profiles and identified evidence of evolving lung injury as early as 3 days following VEA inhalation exposure, equivalent to roughly 3 months of human exposure (Dutta & Sengupta, 2016). These findings underscore the rapid onset of pulmonary toxic effects of recreational VEA use among adolescents and young adults, extending beyond prior literature and signaling a significant public health risk.

Limitations to our study include the fact that while experimental murine models are widely used to investigate human disease, mice differ in anatomy and the innate immune response. Notably, murine toll‐like receptor 4 signaling diverges from humans, and mice lack the CXCL8 (IL‐8) gene, instead producing functional analogs KC (CXCL1) and MIP‐2 (CXCL2) (Matute‐Bello et al., 2008, 2011). Although we used an inbred strain (C57BL/6) mice to limit genetic variability (homozygous at almost all loci), the sex‐stratified analysis did not employ exclusively sex‐matched littermates, which may introduce potential environmental and genetic variability. In addition, some variability was observed in the chamber concentrations of VEA, specifically between the 3‐day and 10‐day female groups, although male versus female exposure levels were not significantly different within each timepoint. Inconsistent aerosol generation by heating coils may explain this variation, especially in the 3‐day female group. While the exposure regimen (3 h/day on weekdays using a third‐generation e‐cigarette) was designed to reflect real‐world standards of operating use, human‐equivalent exposure may still differ. Nonetheless, stable murine vital signs and consistent body weight suggest reliable minute ventilation and delivery, as well as exposure within typical thresholds of exposure. Another limitation is the use of pure VEA, which does not fully replicate the complex chemical matrix of THC/VEA‐containing aerosols; real‐world mixtures may produce synergistic effects on both bioactivity and toxicity not captured here. Furthermore, we did not examine a dose–response range of VEA exposure or the precise mechanisms of VEA‐induced lung injury. While prior in vivo studies have implicated direct cytotoxic effects of VEA on alveolar epithelial cells and hydrolysis of VEA to free tocopherol triggering oxidative stress along the airways (Matsumoto et al., 2022), the full spectrum of injury remains unclear. These may include interference of hydrophobic VEA particles with pulmonary surfactant, the presence of heavy metals in the inhaled aerosol, or thermal degradation of VEA into reactive compounds such as ketene at vaporization temperatures (Massey et al., 1982; Rebuli et al., 2023; Wu & O'Shea, 2020). Lastly, we did not explore sex‐specific molecular pathways beyond the biomarkers assessed, with the role of estrogen mediation on the RAGE axis warranting further investigation.

Following the outbreak of EVALI cases in 2019 and mounting evidence implicating VEA as a primary toxic agent, the FDA advised against the use of VEA in any vaping or e‐cigarette products (U.S. Food and Drug Administration, 2020). Despite this, clinical reports continue to detect VEA in patients with EVALI (Callahan et al., 2024). Although cannabis legalization has increased access to regulated THC products, it may also be inadvertently fueling the illicit market for contaminated e‐liquids and counterfeit cartridges. In a recent review of 15,748 published cases across 161 medical reports of EVALI, 65% of patients were male and 35% were female. Age distribution showed 80% of the EVALI population is under the age of 34 years, with 17% aged between 13 and 17 years of age (Marrocco et al., 2022). Clinical manifestations are nonspecific (Cao et al., 2020), with no validated diagnostic biomarkers or precision‐based therapies available, and the long‐term health consequences of use remain unknown, underscoring a pressing need for mechanistic and translational research (Rebuli et al., 2023).

In conclusion, VEA remains a significant causative agent of EVALI and continues to be a major public health concern. In this adolescent animal model of subacute VEA exposure, we observed histopathologic lung tissue injury and dysregulated inflammation at 10 days of exposure, alongside signs of alveolar barrier dysfunction as early as 3 days, despite the absence of overt vital derangements. Male mice comprised a distinct subgroup exhibiting heightened susceptibility to worsened injury, with sex‐specific differences in immune response, CCSP, and sRAGE expression. To our knowledge, this study is the first to identify sexual dimorphism in lung disease manifestation following short‐term inhaled VEA exposure. These findings may enhance diagnostic precision, inform targeted treatment development, and further advance mechanistic understanding of EVALI pathogenesis. Recognizing sex as a relevant biological variable is essential to understanding the heterogeneity of EVALI‐associated outcomes. A sex‐informed approach to future research may further uncover critical features of immune‐pathogenesis associated with EVALI and further refine public health strategies aimed at mitigating use and exposure, particularly among high‐risk populations.