Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction

Endothelial cells (ECs), forming the inner lining of blood vessels, exhibit remarkable plasticity and heterogeneity. During development, they differentiate into arterial, venous, or lymphatic ECs, acquiring organ-specific properties. Endothelial-to-mesenchymal transition (EndMT) contributes to heart and valve development. Throughout life, ECs adapt to various environments, with phenotypic changes contributing to endothelial dysfunction and atherosclerosis. Dynamic tip-stalk cell phenotypes are crucial for blood vessel growth and involve metabolic reprogramming. Single-cell RNA sequencing studies reveal EC heterogeneity in tumors, highlighting metabolic plasticity. Under pathological conditions (e.g., high TGF-β2 levels), ECs can undergo EndMT in adulthood, implicated in atherosclerosis and myocardial fibrosis. However, lineage tracing suggests limited contribution of ECs to cardiac fibroblasts. Partial mesenchymal transition or mesenchymal activation may facilitate vessel growth by promoting EC migration and invasion. This study aims to define the molecular signatures and plasticity of ECs in response to cardiac ischemia in vivo using single-cell RNA sequencing.

The literature extensively documents the plasticity and heterogeneity of endothelial cells (ECs) in both health and disease. Studies have highlighted the role of ECs in developmental processes such as the formation of the vascular plexus and their differentiation into specialized arterial, venous, or lymphatic ECs. The process of EndMT, where ECs transition to mesenchymal cells, has been identified as essential for proper heart and valve development. Furthermore, research has shown the phenotypic changes that ECs undergo in response to various stimuli, including high LDL cholesterol, pro-inflammatory states, and turbulent flow patterns, contributing to endothelial dysfunction and the development of atherosclerotic lesions. The dynamic switching between tip and stalk cell phenotypes during blood vessel growth and its relation to metabolic reprogramming has also been thoroughly investigated. Recent single-cell RNA sequencing studies have further advanced our understanding of EC heterogeneity and metabolic plasticity in various contexts such as tumors and the heart after injury. The controversy surrounding the contribution of ECs to cardiac fibrosis through EndMT has also prompted investigations using fibroblast lineage tracing.

This study employed single-cell RNA sequencing (scRNA-seq) of the non-cardiomyocyte fraction of murine hearts at various time points (day 0, 1, 3, 5, 7, 14, 28) post-myocardial infarction (MI). A publicly available dataset was used for initial analysis (Figs 1–3). For lineage tracing, Cdh5-CreERT2;mT/mG mice were used, with tamoxifen injection inducing GFP expression in ECs. Hearts were harvested at different time points post-MI and processed for scRNA-seq. The scRNA-seq data was analyzed using the Seurat and Monocle software packages in R. Trajectory analysis was used to identify EC states after MI. Gene ontology enrichment analysis, cell cycle scoring, and ligand-receptor interaction analysis were performed. In vitro experiments using human umbilical vein endothelial cells (HUVECs) were conducted to induce and reverse EndMT using TGF-β2 stimulation and withdrawal. qPCR, immunohistochemistry, FACS analysis, and (13)C glucose metabolic flux analysis were also employed. DNA methylation analysis was performed using the Infinium Human-Methylation EPIC BeadChip. Statistical analysis included t-tests, ANOVA, Kruskal-Wallis tests, Chi-squared tests, and Fisher's exact tests.

Single-cell RNA sequencing revealed 19 cell clusters, including 4 EC clusters. Post-MI, EC numbers declined initially, with an influx of immune cells. Early changes in ECs included responses to hypoxia, inflammation, and apoptosis. Between days 1 and 7, genes related to EndMT, extracellular matrix organization, and cell proliferation were enriched. Trajectory analysis identified 5 EC states, with state 4 characterized by mesenchymal gene expression (EndMA). The majority of ECs transiently occupied state 4 (days 1–7), returning to baseline by day 14. Mesenchymal marker expression was confirmed by bulk RNA sequencing of isolated Cdh5+ cells and immunostaining. EndMA cells showed increased glycolysis and reduced fatty acid signaling, representing a metabolic switch. Lineage tracing with Cdh5-CreERT2;mT/mG mice confirmed the transient nature of EndMA. Inhibition of TGF-β signaling reduced EC clonal expansion, suggesting EndMA contributes to neovascularization. In vitro experiments showed that the mesenchymal transition of ECs was reversible after removing the stimuli.

This study demonstrates that ECs undergo a transient, reversible mesenchymal activation (EndMA) after MI, rather than a complete EndMT. This EndMA is associated with metabolic reprogramming and contributes to EC proliferation and expansion, potentially facilitating neovascularization. The transient nature of EndMA, supported by lineage tracing, challenges the concept of EndMT as a permanent cellular fate change in this context. While EndMA may promote beneficial vascular regeneration, it might also indirectly contribute to fibrosis by secreting extracellular matrix proteins and inflammatory mediators. Future research should investigate the role of EndMA in other injury or disease conditions and how factors influencing EC epigenetic stability might affect the reversibility of the process.

This study provides a comprehensive analysis of EC plasticity after MI, showing a transient mesenchymal activation (EndMA) instead of a permanent EndMT. EndMA is a reversible process linked to metabolic changes and contributes to neovascularization. Future studies should explore the broader implications of EndMA in various diseases and investigate the mechanisms regulating its reversibility, potentially leading to novel therapeutic strategies.

The study primarily used a mouse model of MI, limiting the direct generalizability to human hearts. While the in vitro experiments provide mechanistic insights, they may not fully capture the complexity of the in vivo microenvironment. Further research is needed to determine the precise contribution of EndMA to cardiac fibrosis and other pathological processes.