Abstract 12775: The Major Contribution and Characteristics of Smooth Muscle Cell Foam Cells in Human Atherosclerotic Lesions Based on Flow Cytometry and Single Cell RNA Sequencing

Abstract

Background and Aims: Smooth muscle cells (SMCs) comprise the majority of cells within human atherosclerotic intima. Our previous study of human coronary atherosclerosis found that ≥50% of foam cells are of SMC origin and not primarily of macrophage origin as previously thought. In a recent investigation we reported that ~70% of all foam cells in ApoE-/- mice fed a Western Diet for 6 weeks are SMC-derived. In the current investigation we utilized flow cytometry and single-cell RNA sequencing to study the contribution and characteristics of SMC-derived foam cells in human atherosclerotic plaques. Hypothesis: SMCs comprise the majority of foam cells in human atherosclerosis and SMC foam cells have a unique gene expression pattern compared to macrophage foam cells. Methods: Formalin-fixed coronary and aortic samples were digested, stained with the lipid dye BODIPY and CD45 antibody and separated into CD45+ (leukocyte) and CD45- (non-leukocyte) foam cell and non-foam cell populations using flow cytometry. Pieces of fresh human coronary artery with adventitia removed were subjected to gentle digestion and isolated cells used for single-cell RNA Sequencing (n=3) using 10X Genomics Chromium Single Cell 3’ Reagent Kits v3 Technology. Results: Flow cytometric analysis indicates that approximately 65% of total foam cells of human coronary and aortic atherosclerotic lesions are of non-leukocyte origin. Unsupervised Seurat-based clustering (R version 4.2.0) singled out multiple cell clusters including SMCs, leukocytes, and endothelial cells in all 3 samples. Combining data resulted in identification of 15 distinct clusters including 8 SMC clusters ranging from well-differentiated to varying degrees of dedifferentiation. Combining multiple markers identified by in vitro and in vivo studies of SMC foam cells we were able to identify 2 possible SMC foam cell clusters with gene expression patterns distinct from macrophage foam cells. Conclusions: Our findings further identify SMCs as the major contributors of foam cells in atherosclerosis, and provide novel tools to investigate the nature of SMC foam cells in human atherosclerosis and their responses to anti-atherosclerotic therapies.