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Objective: Endothelial cells (ECs) regulate atherogenesis with Endothelial-to-Mesenchymal (EndMT) transition correlating with disease. Single cell (sc) effects of EndMT perturbations in vitro, with quantitative comparison to signatures of human lesions in vivo is lacking. Approach and Results: Multiomic profiling that concurrently measures sc RNA and ATAC was performed on 8 distinct primary cultures of human aortic ECs exposed to 7-day of EndMT-promoting perturbations: IL1B, TGFB2, and ERG knockdown (siERG). Meta-analysis of sc transcriptomes across 17 human arterial specimens was performed and two quantitative measures assessed the similarities of ex vivo versus in vitro molecular profiles. Primary HAEC cultures were reproducibly populated by 4 major clusters, termed EC1-4: EC1-angiogenic; EC2-proliferative; EC3-activated/mesenchymal-like; and EC4-mesenchymal. Independent exposure to siERG, IL1B and TGFB2 elicited mostly distinct transcriptional and chromatin accessible responses. EC1 and EC2, the most canonically 'healthy' EC populations were affected predominantly by siERG; the activated cluster EC3 was most responsive to IL1B; and the mesenchymal population EC4 was most affected by TGFB2. Quantitative comparisons between in vitro and ex vivo transcriptomes confirmed EC1 and EC2 as most canonically EC-like, and EC4 as most mesenchymal with minimal effects elicited by siERG, IL1B and TGFB2. Lastly, accessible chromatin regions unique to EC2 and EC4 were most enriched for CAD-associated SNPs from GWAS suggesting these cell phenotypes harbor CAD-modulating mechanisms. Conclusion: Primary EC cultures contain markedly heterogenous cell subtypes defined by their molecular profiles. Surprisingly, pro-EndMT exposures for 7 days were inadequate to shift cells from one subpopulation to another suggesting relatively stable molecular phenotypes in culture. Interpretations could be that EndMT acts on a modest number of transcripts or that the in vitro systems used herein fail to recapitulate the complex EndMT-promoting microenvironment of human atherosclerotic lesions. Recognizing and leveraging heterogeneity in vitro should improve fidelity of these systems for modeling in vivo biology.

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.