Chronic Electronic Cigarette Exposure Promotes Atherosclerosis and Chondrogenic Modulation of Smooth Muscle Cells

Electronic cigarettes (E-cigs) represent a rapidly growing public health concern, particularly due to their widespread adoption among adolescents. Recent epidemiological and experimental evidence has linked E-cig use to accelerated atherosclerosis, though the underlying mechanisms remain incompletely understood. Here, we investigated how E-cig exposure promotes atherosclerotic lesion formation and vascular remodeling through phenotypic modulation of vascular smooth muscle cells (SMCs). We utilized SMC lineage-tracing mice crossed onto an ApoE-null hyperlipidemic background and exposed them to pod-based E-cigs (Juul) three times weekly over a 12-week period (n=16 per group), comparing results to control mice exposed to Ambient air. Single-cell RNA sequencing (scRNA-seq) and assay for transposase-accessible chromatin sequencing (scATAC-seq) were conducted on cells isolated from the aortic sinus, complemented by histological assessment of osteogenic markers. Our analyses revealed a significant shift in SMC phenotype toward a chondrogenic lineage (CMC) in e-cigarette-exposed mice, evidenced by markedly increased expression of chondrogenic markers Col2a1 and Tnfrsf11b. Correspondingly, histological evaluations demonstrated increased osteogenic activity in E-cig-exposed animals. Chromatin accessibility profiling further identified a unique cellular cluster enriched for glutamatergic signaling pathways, particularly showing enhanced accessibility at Grin2a, a gene encoding the NMDA receptor subunit GluN2A. Notably, GRIN2A expression was upregulated in HCASMCs upon e-cigarette exposure, and the observed E-cig-induced phenotypic modulation was found to be GRIN2A-dependent, suggesting a robust gene-environment interaction. Collectively, these findings elucidate critical mechanistic pathways through which e-cigarette exposure promotes atherosclerosis, underscoring GRIN2A as a potential therapeutic target to address cardiovascular risks associated with electronic nicotine delivery systems.