Abstract 4147142: Inhaled Benzene Exacerbates Atherogenesis and Compromises Plaque Composition and Stability

Background: Benzene is a ubiquitous environmental pollutant abundant in automobile exhaust, cigarette smoke, forest fires, and present in several household products. It is ranked # 6 on Agency for Toxic Substances and Disease Registry (ATSDR) priority list, however, little is known about its effect on cardiovascular disease, especially atherosclerosis. Hypothesis: Inhaled benzene induces atherogenesis by increasing vascular inflammation in LDL receptor Knockout (LDLR-KO) mice. Methods: Male LDLR-KO or apoE-KO mice were exposed to HEPA-filtered air or benzene (1.0 or 10.0 ppm, 6h/day, 5 days/week) for 24 weeks and maintained on Western diet for the last 12 weeks of exposure. Atherosclerotic lesion composition and nature were determined by liquid chromatography-mass spectrometry (LC-MS), single nuclei RNA sequencing (snRNAseq), and Cytometry by time of flight (CyTOF). Results: Inhaled benzene exposure significantly increased lesions in the aortic valves (>25%, P<0.05). Single cell RNA sequencing of the aortae of benzene-exposed mice revealed 11 major cell types (e.g., vascular smooth muscle cells (VSMCs), endothelial cells, macrophages, T cells, etc.) categorized into 23 sub-populations. snRNAseq and CyTOF analysis showed that benzene exposure robustly increased (P<0.05) CD4 + and CD8 + T-cells, and B-cells in the lesions. Clustering of T cells showed significantly higher expression (P<0.05) of CD4, CD8, and Th17 marker RAR Related Orphan Receptor C (RORc). snRNAseq data also suggested increased cell proliferation and >1.5-fold increase in VSMC number following benzene exposure. LC-MS analysis revealed that benzene exposure differentially regulated the abundance of 930 proteins (at least 2 two unique peptides) in innominate plaque. Importantly, several of the proteins involved in an extracellular matrix organization and elastic fibre formation (e.g. Elastin, Fibronectin , and Latent transforming growth factor beta binding protein 1 ) were downregulated in benzene-exposed mice. These data agree with the snRNAseq data. In vitro , benzene metabolite hydroquinone (HQ, 1 μM) induced ROR-gamma, the transcription factor that regulates Th17, in spleen-derived CD4 + T cells (P<0.05). HQ (0.1 μM) also increased protein kinase C activity and proliferation of human aortic smooth muscle cells (P<0.001). Conclusion: Benzene exposure exacerbates atherosclerosis and compromises plaque cellularity and stability.