Abstract 524: Single-cell Rna Sequencing Of Early Atherosclerosis In Mice Reveals The Early Emergence Of A Dedifferentiated Smooth Muscle Cell Population

Single Cell RNA Sequencing (scRNA-seq) has proven to be a powerful technique to uncover heterogeneity from otherwise homogeneous tissues. Multiple studies have utilized scRNA-seq to examine the composition of atherosclerotic plaques in both humans and mice. However, most of these studies occurred in advanced atherosclerosis. We sought to expand this knowledge base by performing single-cell RNA sequencing at early atherosclerosis in mice. We crossed the Ldlr-/- hyperlipidemic mouse model to a smooth muscle cell (SMC) lineage tracing mouse (ROSA26:LSL-ZsGreen; MyH11-CreERT2). When treated with tamoxifen, SMCs expressing Myh11 are labeled green. At seven weeks of age, we treated these mice with five IP injections of 40mg/kg of tamoxifen. After one week of rest, we initiated the mice on a western diet. We dissected out the whole aorta at the time of harvest and digested the aorta for one hour with a cocktail of DNase, Hyaluronidase, and Liberase. Subsequently, we FACS sorted out both ZsGreen negative and ZsGreen positive cells and performed single-cell RNA seq using the 10x Genomics pipeline. We performed scRNA-seq with pools of three mice at four time points: after 1, 2, 3, and 4 weeks of western diet feeding. Clustering with CarDEC revealed three SMC populations. At four weeks of western diet, we observed an emergence of a SMC cluster characterized by increased expression of Vcam1, Dcn, Mgp, and Egr1. As expected, Gene Ontology analysis of the top 20 differentially expressed genes revealed this population to contain reduced markers of SMC differentiation. In addition, this population had reduced expression of Pura, a protein known to influence serum response factor and regulate smooth muscle actin expression. In summary, our studies indicate changes within SMCs, and the emergence of a dedifferentiated smooth muscle cell population after four weeks of western diet feeding. This study highlights that remodeling of the vasculature occurs even with short durations of hyperlipidemia. Further ongoing studies include expanding time points to longer durations of western diet and elucidating master regulators of this population with Virtual Inference of Protein activity by Enriched Regulon (VIPER).