Single cell ‘omic profiles of human aortic endothelial cellsin vitroand human atherosclerotic lesionsex vivoreveals heterogeneity of endothelial subtype and response to activating perturbations

Abstract

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.