1Universitätsklinikum Frankfurt Institut für Kardiovaskuläre Physiologie Frankfurt am Main, Deutschland; 2Max-Planck-Institut für Herz- und Lungenforschung Bad Nauheim, Deutschland
Corepressors negatively regulate gene expression by chromatin compaction. Alteration of the gene expression profile could be used as strategy to switch the phenotype of endothelial cells, key players during the process of new blood vessel formation, from a quiescent to a pro-angiogenic state. Here we hypothesized that by specifically targeting corepressor proteins, the endothelial angiogenic function could be improved. To study this, we characterized nuclear repressor complexes in human umbilical vein endothelial cells (HUVECs) as well as in organ culture of mouse aortic tissue.
Single cell data sets revealed that the corepressors NCoR1, SMRT and REST are particularly high expressed in vasculature, especially endothelial cells. Importantly, targeted knockdown of these corepressors in HUVECs demonstrated that exclusively NCoR1 depletion increased the angiogenic capacity as determined by spheroid- and mouse aortic outgrowth assays. The underlying mechanism was explored by RNA- and ATAC-Seq. NCoR1 depletion significantly upregulated the expression of angiogenesis-associated genes, especially tip cell genes, including ESM1, DLL4 and NOTCH4. Confrontation assays comparing cells with and without NCoR1-deficiency revealed that loss of NCoR1 promoted a tip-cell position during spheroid sprouting. Interestingly, a proximity ligation assay identified NCoR1 as a direct binding partner of the transcription factor RBPJk, regulating NOTCH signaling during tip and stalk cell selection. Loss of this interaction, after NCoR1 depletion, increased NOTCH activity as determined by luciferase activity measurements. Finally, targeted NOTCH4 depletion in NCoR1 deficient cells abolished the previously described effects on angiogenic capacity.
NCoR1 is an interesting target allowing the positive modulation of pathophysiological angiogenesis via its direct interaction with the NOTCH specific transcription factor RBPJk. Targeted depletion of NCoR1 promotes a tip cell position and thus increased angiogenic capacity in endothelial cells.