Abstract Body: Background: Electronic cigarettes (e-cigs), popular among youth, are linked to increased cardiovascular disease risk, but the molecular mechanisms remain unclear. Coronary smooth muscle cells (SMCs) play a key role in atherosclerosis, with ER stress and the unfolded protein response (UPR) involved in SMC phenotypes. The effect of e-cig exposure on ER stress and UPR in SMCs is unknown.
Methods: Human coronary artery smooth muscle cells (HCASMCs) were exposed to e-cig extract for 72 hours, and ER stress/UPR pathways were analyzed via qPCR. Protein aggregation was assessed with Thioflavin T and Proteostat fluorescence. SDS-insoluble proteins were analyzed with DIA label-free proteomics. For in vivo experiments, SMC-lineage tracing tdTomato reporter mice (Myh11-Cre) on an ApoE-null hyperlipidemic background were fed a high-fat diet for 12 weeks and exposed to e-cig (Juul) vapor daily for 2 weeks. Control mice were exposed to air. Single-cell RNA-seq and ATAC-seq were performed using 10X Genomics.
Results: E-cig exposure significantly induced the UPR in HCASMCs, as indicated by the upregulation of Perk pathway targets, including ATF4 (1.6-fold, p<0.01) and CHOP (1.5-fold, p<0.01). Furthermore, HCASMCs exposed to e-cig showed a substantial increase in protein aggregation (1.7-fold, respectively, p<0.01). Proteomic analysis of the SDS-insoluble fraction identified 523 proteins that aggregated significantly upon e-cig exposure. Functional enrichment analysis of aggregated proteins identified pathways related to MAPK signaling, Parkinson's disease, prion disease, and atherosclerosis. Consistent with these findings, scRNA-seq of aortic cells from e-cig exposed mice (n=9425) vs. controls (n=5282) identified an e-cig-responsive SMC lineage cluster enriched for UPR and heat shock response markers such as Hspb1, Hsp90aa1, Cryab, Hspa1a, and Atf4. scATAC-seq further revealed enhanced chromatin accessibility at heat shock factor (Hsf1/2) motifs in this SMC cluster following e-cig exposure, indicating transcriptional regulation of stress-related pathways.
Conclusion: This study shows that acute e-cigarette exposure induces protein folding stress and aggregation in vascular smooth muscle cells, leading to the activation of UPR and heat shock proteins. These results provide mechanistic insights into the role of e-cigarettes in cardiovascular pathology and highlight potential molecular targets for therapeutic intervention.