Abstract 11256: Endothelial-to-Mesenchymal Transition (endMT) is a Driver of Endothelial Cell Heterogeneity in Human in vitro and ex vivo Atherosclerotic Plaques

Abstract

Atherosclerosis begins with endothelial cell (EC) dysfunction. However, the relationship between single-cell (sc) EC profiles in vitro and in human atherosclerotic plaques is unexplored. Herein, we exploited sc EC transcriptomes from human plaques and related them to in vitro models of inflammation and angiogenesis, which we expected to be relevant. To curate a gene expression profile of ECs found in human atherosclerotic plaques, we performed a meta-analysis of three publicly available human scRNA-seq datasets comprised of 17 coronary and carotid specimens. In parallel, we generated a Multiome in vitro dataset composed of 6 primary human aortic ECs (HAECs), under three experimental conditions: (1) untreated control, and (2) IL-1B-treated (10ng/mL for 1 hour) HAECs on typical gelatin-coated plastic dishes; and (3) untreated HAECs grown on Matrigel to mimic angiogenesis. Five clusters represented the HAECs from all three in vitro conditions. Pathway enrichment analysis revealed that Matrigel-grown ECs (cluster 1) were largely distinguished by Signaling by Rho GTPases, Miro GTPases and RHOBTB3 (R-HSA-9716542; Log10(P) -45.96), while untreated and IL-1B-treated ECs (clusters 0 and 2) were distinguished by regulation of cell adhesion (GO:0030155; Log10(P) -39.92) and Cell Cycle (R-HSA-1640170; Log10(P) -32.2). Of note, clusters 3 and 4 were comprised of HAECs from all 3 conditions and appeared to represent partial and complete endMT, respectively. Cluster 3 and 4 were characterized by greater expression of mesenchymal markers (COL1A1, COL1A2, COL3A1, TAGLN, VIM, and CDH2), while cluster 4 was additionally characterized by a reduction in EC markers (ERG, CDH5, PECAM1). When in vitro HAEC and ex vivo plaque data were integrated, cells from in vitro cluster 4 clustered with vascular smooth muscle cells from human atherosclerotic plaques. Taken together, these results reveal that HAECs are heterogeneous both in vitro and ex vivo atherosclerotic conditions. Since endMT occurs spontaneously at some minor frequency in vitro (~5%) with expression signatures observed in vivo, these data establish a clinically relevant in vitro model to understand the molecular processes driving endMT.