Abstract Tu0054: Dioxin increases pro-inflammatory signal of modulated smooth muscle cells leading to upregulation of the complement pathway and increased cross-talk with macrophages

Background: Dioxin (TCDD, 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin) is an active constituent of tobacco smoke and air pollution that links to an increased risk of coronary artery disease (CAD). However, the corresponded molecular mechanisms remain unclear. Smooth muscle cells (SMC) play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity. In this study, we aimed to explore the role of smooth muscle cells through which dioxin contributes to disease progression in atherosclerosis. Methods: We utilized dioxin response element LacZ reporter transgenic mouse model (DRE-LacZ Tg/ApoE KO), performed a single-cell RNA-seq and histology assays of the atherosclerotic aortic sinus on high fat diet (HFD) for 10 weeks and subsequently exposed to vehicle (Control, n=3) or short term dioxin (TCDD, n=3) for 2 weeks with HFD. RNAscope of C3 RNA was performed in the ApoE KO mouse section. In vitro assays using human coronary SMC were performed as validation. Pro-inflammatory gene expression was tested using qPCR. THP1 migration assay and quantitative ROS assay were performed to assess the cell-cell interaction and oxidative stress. Results: We found following exposure to dioxin, the Mod-SMC exhibited increased levels pro-inflammatory gene expression, including Cxcl12, Lgals3, and Gas6. HCASMC treated with TCDD in vitro also showed increase in marker of inflammation, including IL1A. Furthermore, quantitative ROS assay found increase in cellular oxidative stress following dioxin exposure in HCASMC. A CellChat analysis results showed complement signaling pathway has a clear outgoing signaling from modulated SMC interacting with the macrophage populations. Closer look at the genes responsible for this interaction found complement pathway genes, including C1ra, C3, Cfh, Serping1 increase in the modulated SMC following dioxin exposure, while the corresponding complement receptors C3ar1, Itgam, and Itgb2 expression increase in the macrophage populations. The expression of C3 gene was found to be largely restricted to the modulated SMC, and complement receptors expression restricted to macrophages based on the scRNA-seq. RNAscope of C3 RNA in the mouse atherosclerotic lesion showed localization of RNA expression to the media as well as the lesion cap. Conclusion: Dioxin adversely remodels atherosclerotic plaque, and the C3 expression modulated SMCs may contribute to this process through the increased macrophage recruitment and inflammation.