Single-Cell RNA-Seq Reveals Adventitial Fibroblast Alterations during Mouse Atherosclerosis

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of mortality in the western world, despite great successes with lipid lowering therapies. This alarming trend highlights the need for novel lipid-independent therapeutic strategies for the treatment of ASCVD. Unbiased genome-wide association studies (GWAS) have identified numerous genes associated with ASCVD that have led to efficacious therapies, indicating that the pathways and genes identified are essential for providing insight into the cellular mechanism behind ASCVD. ASCVD GWAS suggest that vascular cells mediate ASCVD progression, and in vivo studies suggest that some of these genes may function in the adventitia of the vasculature. To further explore a role for adventitial cell types in atherosclerosis, we performed a time course western diet (WD) feeding experiment in Ldlr ^−/− mice, completing single cell RNA-sequencing (scRNA-seq) of the aortic adventitia to determine cellular and transcriptomic changes in this region during disease progression. We found a surprising diversity of adventitial fibroblasts that demonstrate population size changes and gene expression alterations over the course of atherogenesis. These populations included previously highlighted fibroblast populations expressing Anxa3, Cd55, Lox , and Cpe . We next cross-referenced our murine scRNA-seq with published ASCVD GWAS dataset and identified SERPINH1 as a differentially expressed GWAS gene in adventitial fibroblasts during atherosclerosis which is also linked to ASCVD. Further in vitro studies showed that knockdown of SERPINH1 in human adventitial fibroblasts reduced migration and altered expression of fibroblast subcluster marker genes. Our data suggests that reduced SERPINH1 expression in adventitial fibroblasts during atherosclerosis progression is altering the function and identity of adventitial fibroblasts, contributing to disease progression. Collectively, these findings highlight the changing nature of adventitial fibroblasts during atherogenesis, and, specifically, the potential role of SERPINH1 in mediating these processes.