Incongruence between transcriptional and vascular pathophysiological cell states II
The Notch pathway is a major regulator of endothelial transcriptional specification. Targeting the Notch receptors or the ligand Dll4 dysregulates angiogenesis. Here, by analyzing single and compound genetic mutants for all Notch signaling members, we find significant differences in the way ligands and receptors regulate liver vascular homeostasis. Loss of Notch receptors caused endothelial hypermitogenic cell-cycle arrest and senescence. Conversely, Dll4 loss triggered a strong Myc-driven transcriptional switch, inducing endothelial proliferation and the tip-cell state. Myc loss suppressed the induction of angiogenesis in the absence of Dll4, without preventing the vascular enlargement and organ pathology. Similarly, inhibition of other pro-angiogenic pathways, including MAPK/ERK and mTor, had no effect on the vascular expansion induced by Dll4 loss; however, anti-VEGFA treatment prevented it without fully suppressing the transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states, vascular phenotypes, and related pathophysiology. Our findings also suggest that vascular structure abnormalization, rather than neoplasms, causes the reported anti-Dll4 antibody toxicity. Overall design: Different experiments were performed. In the  Experiment 1, endothelial cells from mouse heart, lung, liver and brain with Dll4 loss and control conditions  were isolated by Fluorescence-activated cell sorting (FACS) according to high CD31 expression and were used for bulkRNAseq. In the Experiment 2, mouse liver endothelial cells from livers treated with Dll4 blocking antibodies and mouse IgG (control samples) were isolated by Fluorescence-activated cell sorting (FACS) according to high CD31 expression and were used for bulkRNAseq. In the Experiment 3 Cdh5-CreERT2 + iSuRe-Cre+ mouse liver endothelial cells with and without Rbpj loss were isolated  by FACS according to CD31+ MbTomato expression and were used for bulkRNAseq.