Single-cell sequencing reveals smooth-muscle to fibroblast transition in air pollution mediated atherosclerosis
Authors: Glenn Jonnelle , Vergara-Martel Armando , Cara Elaine Ann , Chalappurath Keerthi , Rengasamy Palanivel , Rajagopalan Sanjay , Dazard Jean-Eudes 
Published: May 2023

Background: Particulate matter air pollution (PM2.5) is the 4th leading cause of mortality globally with > 55% of these deaths directly attributable to atherosclerotic cardiovascular disease(ASCVD). While this implication is now widely accepted, the precise cellular mechanisms especially at the level of the vasculature remain to be determined. Vascular smooth muscle cells (VSMC) are key in all stages of atherosclerosis and have been shown to dedifferentiate to a synthetic fibroblast-like state in atherosclerosis which could potentiate plaque development. We hypothesized that PM2.5 exposure induces VSMC differentiation in aortas of atherosclerosis-prone mice contributing to vascular dysfunction. Methods: Male ApoE-/- mice (n=7/group) were exposed to filtered air (FA, as controls) or PM2.5 using a versatile aerosol concentration enrichment system (VACES) for a total of 36 weeks (5h/day, 6d/wk). After euthanization, we measured atherosclerotic plaque area in aortic roots. Vascular function was assessed using myography, deriving concentration-response curves to phenylephrine (PE 10-10 to 10-4). Remaining aortic tissue was pooled, and cells were processed following the 10X genomics protocol for single-cell RNA sequencing (scRNA-seq). Cell characterization analysis, unsupervised k-nearest neighbor learning algorithm and Uniform Manifold and Approximation Projection (UMAP) were performed. Results: Aortic root atherosclerosis was increased with PM2.5 exposure (mm2: FA: 0.23±0.036; PM: 0.45±0.024; p=0.0006). PM-exposed mice displayed vascular hypercontractility when compared to FA mice (AUC: FA: 145.5 ± 15.42; PM: 277.8 ± 27.32; p=0.0012). For scRNA-seq, we analyzed 25,692 high-quality cells with a mean sequencing depth of 11,900. Although both samples started with the same number of total cells, there was a lower percentage of VSMC in PM2.5 exposed mice (FA: 39%; PM: 46%). With PM2.5 exposure, VSMC had significantly increased gene expression for Cnn1, Acta2, TagIn, and Bgn indicating more fibroblast-like VSMCs. Furthermore, significantly augmented gene ontology terms in VSMC upon PM2.5 exposure include collagen fibril organization, positive regulation of cell motility, blood vessel morphogenesis, and phosphatidylinositol 3-kinase signaling. Collectively, these terms promote VSMC motility, reorganization, and transformation. These findings support our hypothesis that air pollution induces the transition of VSMCs to fibroblasts which could explain the vascular dysfunction we observed. Conclusion: scRNA sequencing combined with statistical and functional analyses revealed transition of vascular smooth muscle cells to fibroblasts that may provide a mechanistic explanation for epidemiologic studies suggesting a link between air pollution exposure and ASCVD. NIH/NIEHS-R35ES031702 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.