Identification of endothelial-to-mesenchymal transition gene signatures in single-cell transcriptomics of human atherosclerotic tissue

Abstract Rationale Endothelial cells can differentiate into mesenchymal-like cells via endothelial to mesenchymal transition (EndoMT). In murine models, cell transitions of EndoMT have been assessed with lineage tracing techniques. Knowledge on molecular mechanisms of EndoMT in human vascular lesions is scarce as studies in human atherosclerosis are limited by observational study designs such as histo-pathological studies. Objective We aim to identify a human EndoMT gene expression signature by combining experimentally induced in vitro EndoMT with lineage-traced pathways from atherosclerotic mice and extrapolate this to human plaque scRNA-seq data. Methods and results First, we stimulated human coronary artery endothelial cells (HCAEC) with TNFα and TFGβ to trigger EndoMT. We executed transcriptomic analyses and defined multiple temporal patterns of gene expression changes during EndoMT. We used Cdh5-Cre ERT2 Rosa-eYFP apoE -/- lineage traced mouse scRNA-seq data to demonstrate that the temporal in vitro gene expression changes are reflected in EndoMT trajectories in mice plaque tissue. Finally, we constructed three candidate EndoMT lineages across multiple subpopulations of ECs and SMCs in human carotid scRNA-seq data (n=46). We examined gene expression over the course of these lineages and identified 73 markers for the presence of EndoMT such as NRG1 and DEPP1 . Conclusion This study reveals the gene expression profile of EndoMT trajectories in human atherosclerotic plaques by combining RNA-seq data from in vitro models with single-cell transcriptomic datasets. Our gene expression atlas of EndoMT in atherosclerosis could serve as a reference for future studies, providing novel inroads to study atherosclerotic mechanisms for the development of novel therapies.