Endothelial mesenchymal transition with air pollution mediated atherosclerosis: insights from single cell RNA sequencing and trajectory inference analysis

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Background: Atherosclerotic cardiovascular disease (ASCVD) is the major leading cause of global morbidity and mortality. There is an increasing realization that non-traditional risk factors in the environment may powerfully modulate ASCVD. Air pollution is the world’s leading environmental risk factor implicated extensively in ASCVD with prior studies from our lab and others demonstrating that the fine particulate matter aspect ≤2.5μm (PM 2.5 ) may worsen ASCVD. Recent insights into ASCVD have suggested a fundamental role for endothelial-to-mesenchymal cell transition (EMT) in atherosclerosis. We hypothesize that EMT may play a role in the deleterious effects of PM 2.5 -mediated atherosclerotic progression. Methods: Male ApoE -/- mice (n=7/group) were exposed to PM 2.5 or filtered air (FA, as controls) using a versatile aerosol concentration enrichment system (VACES) for 36 weeks (5h/day, 6d/wk). Concentrations of PM 2.5 averaged 7-10x ambient levels. Animals were euthanized at the end of exposure, followed by assessment of vascular function using myography with derivation of concentration-response curves to acetylcholine (ACh 10 -10 to 10 -4 ). scRNA-sequencing was done using 10x platform. Cell characterization analysis, unsupervised k-nearest neighbor learning algorithm, and Uniform Manifold and Approximation Projection (UMAP) were performed. We used a supervised graph method and UMAP to infer and visualize trajectory of novel cell lineage with lineage root node at endothelial cells. Results. Endothelium-dependent relaxation was impaired in aortas of PM 2.5 -exposed mice (dAUC: FA: 241.2 ± 19.04; PM 2.5 : 125.4 ± 39.21; p=0.0047). Our scRNA-sequencing data revealed 12 unique cell cluster groups in FA and 2 more cluster groups in PM 2.5 . This included an unannotated cell type and a dendritic cell population. Cell lineage inference of FA-exposed mice showed evolution into 2 lineages (vascular smooth muscle cells and monocytes); PM 2.5 -exposed mice diverged into 4 lineages, notably exhibiting differentiation and activation of more fibroblasts and lymphocytes, and an unknown population that had fibroblast-like properties. Importantly, the overlap of biomarkers between the unannotated population and fibroblast cells are mesenchymal cell biomarkers (Col1a1, Col1a2, Mgp, Col1a3, Dcn). This trajectory inference reflected a quasi-continuous transformation along cell lineages, with intermediate cell phenotypes between the annotated populations. Significantly augmented GO terms in EC upon PM 2.5 exposure include upregulation of endothelial cell migration, blood vessel endothelial cell migration, and deregulation of cell population proliferation. Conclusion: Chronic exposure to PM 2.5 results in EMT that may help explain abnormalities in endothelial function and acceleration of ASCVD noted with exposure. Further analysis of cell specific pathways may provide renewed understanding of the mechanisms underlying environmental exposures and progression of atherosclerosis. 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.