Clin Res Cardiol (2022). https://doi.org/10.1007/s00392-022-02002-5

TWIST family member Tcf15 protects endothelial identity

L. Tombor1, D. Rafii1, L.-M. Kettenhausen1, N. Baumgarten1, W. J. Shim2, J. Xu2, S.-F. Glaser1, M. Merten1, K. Stilz1, V. Larcher1, D. John1, S. I. Bibli3, N. Guimarães-Camboa1, M. Schulz1, N. Palpant2, S. Dimmeler1

1Zentrum für Molekulare Medizin, Institut für Kardiovaskuläre Regeneration, Goethe Universität Frankfurt am Main, Frankfurt am Main; 2Genetics and Genomics Division, Intitute for Molecular Bioscience, Brisbane, AU; 3Institut für Vascular Signalling, Universitätsklinikum Frankfurt, Frankfurt am Main;

Myocardial infarction (MI) and cardiac ischemia activate molecular repair signaling mechanisms in the interstitial cells of the heart. Hereby endothelial cells (ECs) sense low oxygen levels and form new vascular networks in part by clonal expansion to allow oxygenation and healing of the ischemic tissue. Some ECs react to MI-induced microenvironmental changes by temporarily switching from fatty acid to glucose consumption and express mesenchymal, proliferation and migration-related genes. This transient endothelial-to-mesenchymal activation (EndMA) occurs within the early phase after MI and is reversible. Plastic conversions into other lineages are not uncommon for ECs (e.g. in development or tumor formation), but require a special transcription factor activity pattern. We aimed to identify key regulatory genes of cardiac EC identity during post-MI remodeling by using single-cell RNA sequencing data coupled with a computational method, TRIAGE, which identifies cell type-specific regulatory factors governing cell differentiation. We found an endothelial specific TWIST family member (Tcf15), which is downregulated between day 1 and day 7 after MI. Human ECs in vitro respond similarly by a significant TCF15 reduction when cultured in 3% O2 hypoxic conditions (57.8% ± 8.9 % of control) or TGF-b2 supplemented conditions (53.3% ± 7.9% of control). To assess whether the temporal Tcf15 loss is associated with metabolic reprogramming and mesenchymal activation after MI, we analyzed the transcriptional expression patterns in ECs. Cells with low levels of Tcf15 showed higher expression of mesenchymal genes (Col1a2, Vim, Fn1) and lower expression of endothelial marker genes (Tie2, Cdh5, Pecam1) and genes associated with fatty acid uptake (Cd36, Fabp4, Meox2), supporting that reduced TCF15 may be involved in early plasticity changes post-MI. To gain insights into the direct effects of TCF15 on human EC identity, we treated cells with TGF-b2 for 48h and subsequently silenced TCF15 via siRNA mediated knockdown (72.3% ± 3% reduction to control). After 7 days, TCF15 knockdown cells had 1.5-fold higher levels of mesenchymal marker CNN1 compared to control. In contrast, when TCF15 levels were increased by lentiviral overexpression, ECs were protected against TGF-b2 induced mesenchymal gene program activation (3.01-fold less SM22 and 2.8-fold less CNN1). Moreover, TCF15 silencing impaired VEGF induced sprouting potential but prevented cells from apoptosis (3.7-fold less 7AAD + cells and 3.4-fold less AnnexinV + cells vs. control). Metabolite assessment of these cells indicated an increase in free carnitines and reduction in fatty acid availability. Additionally, we identified EGR1 and KLF15 as putative target genes of TCF15 which both have pleiotropic functions in angiogenesis, survival and endothelial identity, suggesting that depletion of TCF15 prepares ECs for plasticity changes. Taken together, our findings demonstrate that TWIST family member TCF15 maintains EC identity. We speculate that temporal loss of TCF15 might facilitate EndMA and enable cell adaptation to injury by preventing apoptosis, angiogenesis and fatty acid uptake pathways.

https://dgk.org/kongress_programme/jt2022/aP1555.html