Abstract 15041: Single-Cell RNA Sequencing Reveals Four Distinctive Endothelial Subpopulations Through Mouse Endothelial Aging

Abstract

Introduction: Endothelial cell (EC) dysfunction is a hallmark of vascular aging, contributing to age-related vascular diseases. Elucidating EC aging at the single cell level has significant scientific and therapeutic potential. Research Question: What is the landscape of EC aging at the single cell level? Methods: Mouse aortas were harvested from 1-, 10-, and 20 month-old mice (n=2 males and 2 females at each time point). Pooled single-cell suspensions were prepared, and PECAM1+ cells were enriched by magnetic-activated cell sorting. Results: Single cell RNA sequencing revealed 879 ECs, categorized into six subpopulations (EC 1-6). Of the three canonical EC markers ( Pecam1 , Cdh5, and Kdr ) EC1 and 2 showed higher expression of Pecam1 , whereas EC3 and 4 showed higher expression of Cdh5 and Kdr . ECs from young (1 month-old), middle-aged (10 month-old), and elderly mice (20 month-old) were enriched in EC1, EC3, and EC4, respectively. Gene regulatory network analysis identified specific regulons for each EC subcluster. EC1 displayed progenitor-like features ( Isl1 as one of the top regulons, Procr and Cd34 as highly expressed genes) with high angiogenic potential ( Twist1 as one of the top regulons, Timp2 , Mmp14 , and Mmp2 as highly expressed genes) and the longest G1 phase suggesting its quiescence. EC3 cells exhibited high proliferative status demonstrated by G2M or S phase, accompanied by upregulation of stress-responsive TF Atf3 and NF-κB family members. EC4 demonstrated pronounced Pparg regulon activity. Genes directly regulated by Pparg ( Scarb1 and Cd36 ), and genes involved in fatty acid metabolism and atherogenesis ( Tcf15 , Fabp4 , Gpihbp1 , Lpl , and Meox2) were highly expressed in EC4 and EC3. Conclusions: Our analysis identified four distinct EC subpopulations through the aging process of mouse aortic ECs: EC1, Pecam1 h progenitor-like EC; EC2, Pecam1 h common EC; EC3, Cdh5 h Kdr h inflammatory atherogenic EC; and EC4, Cdh5 h Kdr h non-inflammatory atherogenic EC.