Incongruence between transcriptional and vascular pathophysiological cell states

The Notch pathway is a major regulator of endothelial transcriptional specification. Targeting the Notch receptors or Delta-like ligand 4 (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 the vascular structure abnormalization, rather than neoplasms, causes the reported anti-Dll4 antibody toxicity.

The Notch pathway is a major regulator of endothelial transcriptional specification. Targeting the Notch receptors or Delta-like ligand 4 (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 r el at ed p at ho ph ys io logy. Our findings also suggest that the vascular structure a b n or m a li z a tion, rather than neoplasms, causes the reported anti-Dll4 antibody toxicity.
Notch is a cell-to-cell ligand-receptor signaling pathway that has a major influence on cell transcription and biology 1 , playing important roles in several diseases 2 . General Notch signaling or γ-secretase inhibitors have been used in clinics with undesired side effects, including disruption of the normal intestinal stem-cell differentiation 2,3 . Specific blocking antibodies are now available that target the various ligands and receptors of the Notch pathway [4][5][6][7][8] . Given the specificity of Dll4 expression in endothelial cells (ECs), targeting this ligand was initially thought to be an effective and safe strategy for specifically modulating Notch signaling and angiogenesis in disease, such as during tumor Article https://doi.org/10.1038/s44161-023-  Targeting Dll4 induces heterozonal responses in liver vessels The previous RNA-seq and histological data revealed the adult liver endothelium as the most reactive vascular bed to the targeting of Dll4-Notch signaling. Rats and chimpanzees treated with anti-Dll4 antibodies also developed significant liver vascular neoplasms and disease 5,8 ; therefore, we focused our analysis on this organ. To gain deeper insight, we performed a high-resolution spatiotemporal phenotypic and transcriptomic analysis after targeting Dll4 for 2 days to 3 weeks. In contrast to targeting Dll4 during angiogenesis, targeting Dll4 in liver sinusoidal ECs (LSECs) for 48 h, which abolishes the generation of cleaved N1ICD, did not induce major transcriptomic changes (only 11 differentially expressed genes) or vascular phenotypic changes (Extended Data Fig. 2a-e). Gene set enrichment analysis (GSEA) revealed upregulation of only a few E2F and Myc target genes at this time point (Extended Data Fig. 2f-h). The increase in vascular density after targeting Dll4 was relatively slow and progressive, only becoming noticeable 1 week after genetic deletion (Fig. 2a-c). Endothelial proliferation peaked at day 4 and was sustained after, leading to a progressive increase in vascular density and the total number of ECs ( Fig. 2b-d). Proliferation of neighboring hepatocytes was also increased, peaking after the peak in endothelial proliferation (Fig. 2e), suggesting that Dll4 KO ECs secrete angiocrine factors inducing hepatocyte proliferation, as shown previously during liver regeneration 10 .
The effect of Dll4 targeting was, however, notably heterogeneous and zonal. Only vessels around the central veins and with a known venous identity 11 had a higher number of ECs (Fig. 2f,g), larger nuclei ( Fig. 2h), and expression of cell-cycle (Fig. 2i,j) and apoptosis (Fig. 2k) markers. Therefore, the previously reported anti-Dll4-driven liver histopathology and increase in cell proliferation 8 is now found to be mainly associated to the central-vein sinusoids, which become enlarged and full of blood cells (Extended Data Fig. 2i-m). Paradoxically, the portal-vein sinusoids, which have arterial identity and the highest Dll4 expression and Notch activity (Fig. 2l-n and Extended Data Fig.  2n,o), showed a minor increase in EC proliferation (Fig. 2i) despite a significant loss in the expression of arterial genes ( Fig. 2o and Extended Data Fig. 2p). Besides the cell-cycle marker Ki67, we also analyzed more specific S-phase (EdU) and cell-cycle arrest/senescence (p21) markers. This analysis revealed expression of p21 in 30% of Dll4 iDEC ECs in the venous vessels around the central veins (Fig. 2p). Among Ki67 + ECs, 40% were positive for EdU and 25% were positive for p21 (Fig. 2q). This shows that there is a mix of productive cell division (EdU + ) and arrest (p21 + ) after Dll4 loss in liver ECs. Pulse-chase single-cell ifgMosaic tracking revealed that relatively few of the Ki67 + ECs had the ability to divide and clonally expand after Dll4 targeting, with some cells dividing 6 to 50 times more than their neighbors (Fig. 2r). All of these progenitor cells were located in the sinusoids around central veins (Fig. 2r,iii).

Loss of Notch1 or Rbpj in LSECs induces hypermitogenic arrest
Notch ligands and receptors can be targeted with a range of pharmacological compounds and antibodies [4][5][6][7] , and so far only Dll4-targeting antibodies have been reported to cause major vascular disease 5,8 . In contrast, genetic deletion of Notch1 or Rbpj in mice has been suggested growth 6,7 . However, anti-Dll4 treatment was later shown to induce a loss of endothelial quiescence and vascular neoplasms, which were proposed to be the main cause of pathology in several organs 5,8,9 . This toxicity diminished the clinical appeal of Dll4/Notch blockers in cancer or cardiovascular disease settings.
Here, we characterized the effect of single or compound targeting of all Notch signaling members on adult mice vascular homeostasis. High-resolution single-cell RNA sequencing (scRNA-seq) and threedimensional (3D) confocal microscopy of adult liver vessels revealed very significant differences in the way each Notch member regulates vascular signaling, structure and single-cell states. γ-Secretase inhibitors or removal of Notch receptors did not cause substantial vascular or organ disease. Abnormal proliferating and sprouting single-cell states were generated only after Dll4 targeting. Surprisingly, suppression of these angiogenic cell states by additional genetic or pharmacological targeting was insufficient to prevent vascular and organ disease. Conceptually, our data show that the major transcriptional changes and angiogenic cell states elicited by targeting Dll4 correlate with, but do not cause, the observed vascular pathophysiology. Instead, we propose that it is the unrelated vascular structure abnormalization and malfunction that leads to organ pathology and the reported toxicity of anti-Dll4 treatment 5,8 .

Notch pathway expression and signaling in adult organ ECs
To elucidate the role of Notch signaling in global vascular homeostasis, we first assessed its activity in different organ vascular beds by immunodetection of the activated form of the Notch1 intracellular domain (N1ICD Val1744 ). This epitope was detected in ∼50% of all organ ECs (Fig. 1a,b). Bulk RNA-seq analysis revealed that Dll4 and Notch1 are the most expressed ligand-receptor pair in quiescent vessels of most organs (Fig. 1c,d and Extended Data Fig. 1a), and that Mfng is the most str ongly expressed Notch glycosyltransferase. These enzymes are known to significantly enhance Delta ligand signaling and decrease Jagged ligand signaling 1 . Adult mice with induced deletion of Dll4 in ECs (Dll4 iDEC -Dll4 flox/flox  led to a significant reduction in N1ICD Val1744 and Hey1 signals in most organs' quiescent ECs ( Fig. 1e-i). This indicates that Dll4 is the main functional ligand responsible for triggering Notch activity in most quiescent vessels. We observed compensatory upregulation of Dll1 only in lungs (Fig. 1i). Dll4 deletion elicited remarkably different gene expression signatures among different organ vascular beds, with the adult liver endothelium presenting the most pronounced changes in gene expression (Fig. 1j,k and Extended Data Fig. 1). Despite significant transcriptional changes in most organs' ECs, only the endothelium of the heart, muscle and liver showed an increase in the frequency of cycling or activated Ki67 + cells upon Dll4 deletion (Fig. 1l-n), and these were the only organs with clear alterations in the 3D vascular architecture after the loss of Dll4-Notch signaling (Fig. 1o). The brain underwent significant changes in gene expression (Fig. 1j,k and Extended Data Fig. 1), but these were not accompanied by endothelial proliferation or vascular morphological changes. . c, Schematic representation to illustrate the bulk RNA-seq experiment performed with adult ECs isolated by FACS. d, Heatmap with RNA-seq reads per kilobase per million mapped reads (RPKM). e, Experimental layout for the inducible deletion of Dll4 in Cdh5 + ECs (Dll4 iDEC ) with Cdh5(PAC)-CreERT2. f, Expression of Dll4 protein in CD31 + EMCN + vessels. g,h, Dll4 deletion significantly reduces Notch signaling activity (cleaved Val1744 N1ICD) in all quiescent vascular beds. In brain micrographs, white arrowheads indicate ECs and yellow arrowheads indicate non-ECs. Note that whereas N1ICD is maintained in non-ECs, most N1ICD signal disappears from the ECs in Dll4 iDEC brains. i, Schematic representation to illustrate the bulk RNA-seq experiment performed with adult ECs. Below, a heatmap showing the relative expression of all Notch pathway components and canonical target genes in control and Dll4 iDEC mutant ECs. j, Unsupervised hierarchical clustering showing stronger gene expression changes in Dll4 iDEC liver ECs compared with the other organs. Z-score lcpm, Z-score of the logarithmic counts per million. k, Unsupervised hierarchical clustering showing strong upregulation of Myc target genes in Dll4 iDEC liver ECs compared with the other organs. l-n, Dll4 deletion results in increased EC proliferation (Ki67 + ERG + cells) in some organs but not others. o, 3D reconstruction images from thick vibratome sections show vessel (CD31 + EMCN + ) enlargement in Dll4 iDEC heart and liver but not in brain. Data are presented as mean values ± s.d. For statistics, see Source Data File 1. Scale bars, 100 μm. Article https://doi.org/10.1038/s44161-023-  to cause vascular phenotypes very similar to the genetic deletion of Dll4, during angiogenesis and in adult vessels 9, [12][13][14][15] . Therefore, we investigated if deleting Notch1 or Rbpj, the master regulator of all Notch receptor signaling, induced vascular pathology similar to that induced by the loss of Dll4 (Fig. 3a). Surprisingly, Notch1 and Rbpj deletion for 2 weeks or 4 weeks did not significantly increase EC proliferation and related vascular pathophysiology (Fig. 3b-e and Extended Data Fig. 3a-g), despite these mutant cells having even higher activity of phosphorylated extracellular signal-related kinase (p-ERK) than ECs lacking Dll4 (Fig. 3f- 0  3  6  9  12  15  18  21  24  27  30  33  36  39  42  45  51  1  43  85  127  169  211  253  295  337  379  48   0   5   10   15  40  50  60  70  antibody 4 also lacked the major hallmarks of pathology observed in anti-Dll4-treated livers (Extended Data Fig. 3h). Next, we compared the transcriptome of Dll4 iDEC and Rbpj iDEC vessels. ECs from both mutant lines showed a similar upregulation of genes related to cell-cycle activation and metabolism (Fig. 3i) and had enlarged nuclei (Fig. 3j).
However, compared with Dll4 iDEC livers, Rbpj iDEC livers had significantly less vascular expansion and organ abnormalities ( Fig. 3k and Extended Data Fig. 3c-g) and stronger upregulation of p21 (Fig. 3l), a cell-cycle inhibitor frequently upregulated in senescent or hypermitogenically arrested cells 16 . We also identified a significant increase in the number   of binucleated p21 + ECs, suggestive of replicative stress and G2 arrest of the mutant cells ( Fig. 3m and Extended Data Fig. 3i). RNA-seq analysis revealed signatures of genetic pathways linked to G2/M checkpoints, chromosome segregation, and general replicative stress and senescence in Rbpj iDEC ECs ( Fig. 3n and Extended Data Fig. 3j). To determine the functional effect of p21 upregulation, we analyzed compound Rbpj iDEC p21 KO mice (Fig. 3o). p21 loss did not affect the minor vascular sinusoid dilation seen in Rbpj iDEC livers, but did increase the frequency of cycling (Ki67 + ) and apoptotic (cleaved caspase-3 + ) cells ( Fig. 3p-r), in line with the role of p21 as a cell-cycle and apoptosis inhibitor 17 , particularly in hypermitogenically activated Rbpj KO cells. This dual and paradoxical effect of p21 loss on both cell proliferation and apoptosis may explain the relatively mild increase in EC numbers in Rbpj iDEC p21 KO livers compared with the fully arrested Rbpj iDEC liver vessels. These results suggest that loss of Dll4 induces a reduction in Notch signaling that results in a mixed population of proliferative and arrested ECs, whereas the complete loss of Notch signaling induces mostly hypermitogenic arrest, without productive cell division.

Targeting Dll4 and Notch induces incongruent cell states
Next, we performed scRNA-seq to identify possible differences in vascular single-cell states induced by targeting Dll4, Notch1 or Rbpj. This analysis was performed on cells expressing the Cdh5-CreERT2 and iSuRe-Cre alleles 18 to guarantee endothelium-specific recombination, labeling and full genetic deletion of all of the floxed genes used in this study (Fig. 4a,b and Extended Data Fig. 4a). To reduce batch effects, Tomato + CD31 + ECs were isolated on the same day from multiple control and mutant animals, tagged with different oligonucleotide-conjugated antibodies, and loaded in the same chip. The few mutant cells with mRNA expression of Dll4 and Notch1 were likely contaminants. For Rbpj, only exons 6-7 are deleted, leading to a less stable, but still detectable, 3′ mRNA. Altogether, the scRNA-seq data analysis showed the existence of ten clearly defined cell clusters ( Most of the upregulated genes in the tip-cell cluster were associated with Myc metabolism, increased ribosome biosynthesis, glycolysis, mTORC1 signaling, and fatty acid and oxidative phosphorylation (Extended Data Fig. 4e). Paradoxically, Notch1 iDEC and Rbpj iDEC liver ECs, in which the decrease in Notch signaling was more pronounced (Hes1 expression in Fig. 4b), showed a more moderate metabolic activation, and most of these mutants ECs clustered in either the venous C1v cluster or the activated C3 cluster and did not reach the extreme C4 tip-cell state (Fig. 4c,d).
Histology confirmed that indeed only the Dll4 iDEC mutants had a significant population of Esm1 + tip cells (Extended Data Fig. 4f) and that these were mostly present in the venous sinusoidal capillaries interconnecting the liver central veins (Fig. 4j,k), where EC proliferation and density are the highest (Fig. 2f-i). The upregulation of the global cell-cycle marker Stmn1 in Dll4 iDEC livers ( Fig. 4l) correlated with the sixfold higher frequency of Ki67-protein + cells in these mutants compared with the Notch1 and Rbpj mutants (Fig. 3d). Most Esm1 + tip cells were not Ki67 + , in accordance with their higher sprouting activity and arrested nature, but had proliferating Ki67 + cells as close neighbors (Fig. 4j,m). Notch1 iDEC and Rbpj iDEC ECs showed significant upregulation of the replication-stress/senescence markers p21 (cdkn1a), p53 (trp53) and p16 (cdkn2a) (Fig. 4n). These cells undergo hypermitogenic S/ G2/M arrest (Fig. 3m,n) without becoming Kcne3 + /Esm1 + sprouting tip cells (Fig. 4c,d), which is in contrast to the current understanding of sprouting angiogenesis 16,22 .    alleles to ensure genetic deletion of the floxed alleles. b-d, Increased EC density (ERG + per field) and proliferation (Ki67 + ERG + /ERG + ) were observed only in Dll4 iDEC liver ECs. e, Gross liver pathology is observed exclusively in Dll4 iDEC livers. f-h, p-ERK immunostaining and whole-liver western blot showing that the frequency of p-ERK-expressing ECs and intensity levels increase in the mutants, particularly the Notch1 and Rbpj mutants. i, Heatmap with the normalized enrichment score (NES) from significant hallmark analysis (FDR q value < 0.05) by GSEA from bulk RNA-seq data. FC, fold change. j, Mutant liver ECs have a larger nuclei size than control liver ECs. k, Vascular (CD31 + ) dilation or expansion is more pronounced in Dll4 iDEC mutants. l, p21 expression in ECs (p21 + ERG + ) is more increased in Rbpj iDEC mutants. m, Binucleated cells (white arrowheads) identified in Dll4 iDEC and Rbpj iDEC mutants. High magnification of insets a and b are shown at the bottom. n, GSEA analysis shows a positive and significant enrichment in Chromosome Segregation-related and Cellular Senescence-related genes in Rbpj iDEC mutant liver ECs as shown by the NES. o, Experimental layout for the inducible deletion of Rbpj in a p21 KO background. p, 3D projection of thick vibratome sections showing the endothelial surface marker CD31 and EMCN, and proliferation (Ki67) analysis in ECs (ERG + ). q, Analysis of the apoptosis marker cleaved caspase-3. r, The absence of p21 in a Rbpj iDEC background results in a modest increase in EC density (ERG + ), but both EC proliferation (Ki67 + ERG + ) and apoptosis (cleaved caspase-3, CC3) are significantly increased. Data are presented as mean values ± s.d. For statistics, see Source Data File 1. Scale bars, 100 μm, except e, 1 mm. Article https://doi.org/10.1038/s44161-023-  Notch1 iDEC livers upregulated the expression of Notch4 (Extended Data Fig. 5a), a receptor known to partially compensate for Notch1 deletion 23 . Deletion of Notch1/2/4 in ECs, similarly to Rbpj loss, results in even lower Hes1 expression and higher p21 expression (arrest); however, this does not result in the induction of tip cells (Esm1 + /Kcne3 + ) or proliferating Stmn1 + cells (

Wnt2
Pecam1 We also tested a general γ-secretase inhibitor, DBZ, which is known to block Notch signaling and elicit strong effects on tumor and retina angiogenesis 6 , similarly to anti-Dll4 treatment (Extended Data Fig. 5g). However, this compound had a very weak effect on quiescent vessels, similar to the changes seen in Dll4 heterozygous livers ( Fig. 4r-t). We also observed by scRNA-seq that ECs with full loss of Dll4 signaling for only 4 days had already lost the arterial capillary program (C1a cluster) and become activated (C3 cluster), but had not yet had time to fully differentiate to tip cells (C4 cluster in Extended Data Fig. 5h-j). This suggests that in order to fully activate quiescent ECs and induce significant numbers of tip cells and vascular abnormalization, pronounced and continuous loss of Dll4 signaling must be sustained for about 1 week, which can be achieved with genetic deletion or blocking antibodies 5 but not with small-molecule inhibitors targeting Notch.
The difference between the liver vascular phenotypes of Dll4 and Notch receptor mutants could be also due to a role of the ligand, and not the receptors, on signaling to adjacent liver cells. scRNA-seq analysis of all other liver cell types revealed that hepatocytes did not express significant amounts of Notch receptors (Extended Data Fig. 6a-e). Hepatic stellate cells, Kupffer cells (stellate macrophages) and some other blood cell types expressed Notch receptors, but their target genes were not significantly downregulated by endothelial Dll4 deletion, suggesting that this ligand mainly signals within ECs (Extended Data Fig. 6d-f). Single-cell data analysis revealed a significant increase in leukocytes in Dll4 iDEC livers, particularly monocytes, neutrophils and macrophages (Extended Data Fig. 6c), presumably due to the vascular pathology and the subsequent abnormal blood flow that leads to the accumulation of these cells and an organ pathology signature (Extended Data Figs. 2j,m and 6c,g-j). Remarkably, EC-specific expression of N1ICD rescues the major hallmarks of the Dll4 iDEC vascular pathology at the organ and single-cell levels (Extended Data Fig. 7). These data suggest that it is not the loss of Dll4 signaling to non-ECs that causes the difference between Dll4 iDEC and Notch1/2/4 iDEC or Rbpj iDEC mutants. It also confirms that it is the partial downregulation of the Dll4-Notch transcriptional program in ECs, which is not matched by the complete loss of Notch receptors or Rbpj, that causes the liver vasculature abnormalization and subsequent pathology.

Deletion of all other Notch ligands does not elicit pathology
Besides Dll4, other Notch ligands are also expressed in liver ECs (Fig. 5a). The Notch signaling target Hes1 is more expressed in Dll4 iDEC than in Notch1 iDEC , Rbpj iDEC or Notch1/2/4 iDEC mutants (Fig. 4b,q), suggesting that the other weakly expressed Notch ligands ( Jagged1, Jagged2 and Dll1) may partially compensate the loss of Dll4 and induce residual Notch signaling essential for the induction of the tip-cell state. Notably, Jagged1 mRNA was barely detectable in bulk or scRNA-seq data of quiescent liver ECs (Figs. 1d and 5a), but its protein was clearly expressed in liver vessels (Fig. 5b). Deletion of all three ligands (Jag1, Jag2 and Dll1) did not alter vascular morphology, induce pathology, or increase the frequency of Ki67 + cells, confirming that Dll4 is the main Notch ligand in quiescent vessels ( Fig. 5c-g). Liver blood profiling revealed an increase in the percentage of neutrophils, but this was also seen in circulating blood, suggesting a systemic rather than organ-specific role of these ligands (Fig. 5h,i). In agreement with this, scRNA-seq data analysis confirmed that most mutant ECs remained quiescent and did not become activated or form tip cells (Fig. 5j-l). Moreover, deletion of Jag1, Jag2 and Dll1 in ECs did not compromise the portal sinusoid arterial identity (Fig. 5k,m), instead revealing a slight increase in the Notch signaling target Hes1 and the arterial gene CD34, together with a very pronounced decrease in the expression of the venous-enriched Wnt2 gene (Fig. 5n). This counterintuitive increase in Notch signaling was also observed previously after the loss of Jagged1 during angiogenesis 24 .

Myc loss prevents Dll4 iDEC transcriptional states but not pathology
Next, we aimed to determine the molecular mechanisms responsible for the unique EC activation, tip-cell signature, and vascular pathology induced by targeting Dll4. As mentioned above, Myc and its target Odc1 were among the most strongly upregulated genes in Dll4 mutant ECs, compared with Notch1 and Rbpj mutants. Myc is known to activate important ribosome biogenesis and protein translation pathways, favoring cell growth 25 . Dll4 iDEC livers showed upregulation of a large range of canonical E2F, Myc, mTORC1 and ribosomal (Rpl) genes, particularly in the activated, proliferating and endothelial tip-cell clusters ( Fig. 6a and Extended Data Fig. 4). This hypermetabolic transcriptional status was confirmed by mass spectrometry (MS) analysis of protein lysates obtained from freshly isolated liver ECs (Fig. 6b-f), providing a high-depth proteomic analysis of the endothelial tip-cell state induced by targeting Dll4. We also independently confirmed Myc mRNA and protein upregulation in Dll4 KO vessels (Fig. 6g,h).
Next, we investigated the implication of Myc in the Dll4 iDEC transcriptional program and subsequent vascular-related pathology. Myc loss (in Dll4/Myc iDEC animals) almost entirely blocked the EC activation induced by Dll4 loss, and very few ECs were in the activated (C3) and tip-cell (C4) clusters ( Fig. 6i-l and Extended Data Fig. 8a). Consistent with the scRNA-seq data, frequencies of proliferating (Ki67 + ) and tip (Esm1 + ) cells in Dll4/Myc iDEC mutants were similar to those in wild-type animals ( Fig. 6m and Extended Data Fig. 8b, c). Myc activity is thus essential for the strong metabolic and biosynthetic phenotype of Dll4 KO liver ECs and the appearance of the abnormal cell states. Surprisingly, despite this strong transcriptional and cell-state reversion to a quiescent state, Dll4/ Myc iDEC mutant vessels were still highly abnormal and dilated ( Fig.  6n and Extended Data Fig. 8d). The vascular abnormalities in Dll4/ Myc iDEC mutant livers were not in accordance with their more quiescent scRNA-seq profile (Fig. 6j-l), nor with the significantly lower frequencies of Ki67 + and Esm1 + cells (Fig. 6m). Interestingly, Dll4/ Myc iDEC livers retained hallmarks of tissue hypoxia and inflammation ( Fig. 6l and Extended Data Fig. 8e) and had strong activation of surrounding hepatocytes already 5 days after deletion (Extended Data Fig. 8f), despite having a quiescent endothelium. Altogether, these data indicate that the vascular structure abnormalization observed in Dll4 mutant livers is not driven by the detectable changes in endothelial transcriptional programs or the proliferative and tip EC states.

Anti-VEGFA treatment prevents the Dll4 iDEC pathology with less effect on transcription
Among the few GSEA hallmark pathways whose upregulation in Dll4 mutants was not altered in Dll4/Myc iDEC vessels was the hypoxia pathway and inflammatory response ( Fig. 6l and Extended Data Fig. 8e). Hypoxia is known to induce expression of vascular endothelial growth factor A (VEGFA), which can induce vascular expansion without the need for proliferation 26 . The expression of VEGFA was significantly upregulated in the Dll4 KO venous tip-cell cluster (Extended Data Fig. 9a). Therefore, we explored if anti-VEGFA treatment could prevent the appearance of the activated vascular cell states, vascular enlargement and liver pathology induced by Dll4 deletion. Unlike Myc loss, anti-VEGFA treatment reduced both the vascular expansion and the liver pathology induced by Dll4 deletion (Fig. 7a- These results show that anti-VEGFA treatment prevents not only the appearance of the abnormal single-cell states induced by Dll4 targeting, as Myc loss also does, but also the vascular expansion and blood flow abnormalities associated with organ pathology. However, blocking VEGF had a much lesser effect than Myc loss on the Dll4 KO transcriptional signature (Fig. 7k). Anti    livers attenuated, but did not completely downregulate, many of the genes associated with metabolic and biosynthetic activities (Fig. 7l and Extended Data Fig. 9h, i). This suggests that even though Dll4 iDEC+ anti-VEGFA-treated ECs are transcriptionally and metabolically more active than Dll4/Myc iDEC ECs, only the latter form abnormal and enlarged vessels that result in organ pathology.     Inhibition of major signaling pathways did not prevent Dll4 iDEC pathology VEGFA induces many important endothelial functions that are often difficult to distinguish, such as proliferation, sprouting, cell size, survival and permeability [27][28][29] . VEGF is thought to execute its effects on sprouting and angiogenesis mainly through ERK signaling 30,31 . However, administration of a highly effective ERK/ MEK signaling inhibitor (SL327) had a much more modest effect than anti-VEGFA treatment, and only partially reduced the number of activated and tip ECs (Fig. 7m- 35,36 signaling. However, the inhibition of these pathways also did not prevent the vascular pathophysiology induced by targeting Dll4 (Fig. 8 and Extended Data Fig. 9k,l). Rapamycin effectively prevented the increase in the number of ECs, but not vascular dilation and pathology. Thus, the vascular pathophysiology effects of anti-VEGFA treatment, and anti-Dll4, are broader and independent of the activity of these signaling pathways.
Overall, these results show that the genetic and pharmacological modulation of single-cell states related to endothelial dedifferentiation, activation, proliferation and sprouting often do not correlate with adult vascular phenotypes, function and ultimately organ pathology.

Discussion
Notch is one of the most important pathways for vascular development because it enables the necessary differentiation of ECs during angiogenesis 28,37,38 . Here, we expand on previous observations that Notch also plays an important role in the homeostasis of several organ vascular beds 8,9,12 . Dll4 is active in all organ vascular beds, and its loss affects the transcriptome of most quiescent ECs; however, Dll4 targeting effectively activates vascular growth in only the heart, muscle and liver. Even though the existence of four Notch receptors and five ligands allows for the possibility of multiple quantitative and qualitative signaling combinations and redundancy, our results confirm that Dll4 and Notch1 are clearly the most important Notch ligand-receptor pair for maintaining the global homeostasis of ECs.
Previous work suggested that Dll4 and Notch1/Rbpj have similar functions in vascular development and homeostasis [6][7][8]15,23,24,39 , with only Jagged ligands shown to have opposite functions in Notch signaling and angiogenesis 24 . In this study, we show that Dll4 can have distinct functions from its receptors in vascular biology. It was possible to identify this difference only because of the use of scRNA-seq and high-resolution confocal analysis of liver vessel morphology; bulk RNA-seq analysis did not reveal significant differences between the transcriptomes of Dll4 and Rbpj mutants. The loss of Dll4, unlike the loss of Notch receptors or Rbpj, elicits a unique cascade of changes that culminates in the loss of sinusoidal marker genes and upregulation of Myc, similar to the loss of Gata4 (ref. 19). Dll4 iDEC liver vessels lose all quiescent arterial and venous cell states. The arterial cells become highly activated, and the venous cells show either tip-cell or proliferating cell signatures. Paradoxically, although Dll4 loss induces a weaker loss of Notch signaling than is induced by the loss of Notch receptors or Rbpj, it elicits a much stronger metabolic activation and expansion of the liver endothelium. This may be in part related to the bell-shaped response of ECs to mitogenic stimuli, as we previously showed during retina angiogenesis 16 . Our data indicate that full loss of Notch, or Rbpj, induces stronger ERK signaling and hypermitogenic arrest associated with hallmarks of cellular senescence, whereas Dll4 iDEC vessels retain a residual level of Notch signaling that instead effectively induces strong Myc-driven ribosome biogenesis and a metabolic switch toward active protein synthesis and cell growth that drives both EC proliferation and the generation of tip cells. The pro-proliferative effect of targeting Dll4 in quiescent vessels is in contrast to the hypermitogenic cell-cycle arrest that occurs after targeting Dll4 during embryonic and retina angiogenesis 16,40 , presumably a reflection of the significantly lower levels of growth factors, including VEGF, in adult organs.
Previously, a noncanonical and N1ICD transcription-independent role for Dll4/Notch in inducing Rac1 and maintaining vascular barrier function was proposed 41 . Dll4 deletion could also affect signaling to  other cell types, unlike deletion of the Notch receptors in ECs. However, our data show that the liver vascular abnormalization after targeting Dll4 can be rescued by the expression of N1ICD in ECs. This suggests that the vascular pathology is caused by the absence of Dll4 canonical signaling and transcription within the endothelium, and not due to noncanonical effects on vascular barrier function, or the loss of Notch signaling in other adjacent cell types. The observed lack of pathology in anti-Notch1-treated livers also corroborates this.
High-resolution confocal microscopy revealed the heterozonal effect of Dll4 targeting. The induction of EC proliferation and tip cells was restricted to the most hypoxic liver venous sinusoids, precisely the ones with lower expression of Dll4       expansion and organ pathology to become noticeable. During angiogenesis, this transcriptional and vascular morphology switch is already evident after 24 h of anti-Dll4 treatment 16 . This slow transcriptional reprogramming of quiescent ECs by Dll4 targeting may be related to the much lower levels of growth factors and nutrient availability in adult organs. The slow nature of this reprogramming may also explain the lack of effect of the small-molecule inhibitor DBZ on quiescent ECs. Unlike anti-Dll4 treatment or genetic deletion, which result in continuous loss of signaling, the less stable small-molecule inhibitor DBZ elicited no significant change in the quiescent vascular cell transcriptional states and phenotypes, whereas it is very effective during retina angiogenesis 15,16 . Anti-Notch1 4 also did not cause liver vascular pathology, despite its strong effect on angiogenesis. These findings have implications for selecting the most effective and safest way to target Notch in clinics, including blocking antibodies that target Dll4 versus antibodies that target Notch receptors, or the use of smallmolecule inhibitors. Our data indicate that Notch receptor-targeting antibodies or small-molecule γ-secretase inhibitors do not induce significant liver vascular pathology and should be as effective as anti-Dll4 treatment at dysregulating tumor-related or ischemia-related angiogenesis, which can be beneficial in some therapeutic settings. It has also been shown that is possible to modulate the stability and pharmacokinetics of anti-Dll4 treatment to decrease its toxicity while maintaining its therapeutic and angiogenesis efficacy 5 . Our analysis also confirms the importance of Myc for the biology of ECs in the absence of Dll4. We previously reported that Myc loss rescues the ability of Rbpj KO or Dll4 KO ECs to form arteries 40 . Here, we show that Myc loss abrogates the generation of activated, proliferative and sprouting tip cells after Dll4 targeting, but surprisingly, this return to genetic and phenotypic quiescence is insufficient to prevent Dll4targeting-induced vascular expansion, dysfunction and consequent organ pathology. In contrast, anti-VEGFA treatment did not completely abrogate the Dll4-targeting genetic program, but was able to prevent the associated vascular and organ pathology. However, this effect of anti-VEGFA treatment was not reproduced by inhibition of MAPK/ERK, Rac1, Pi3K/mTOR or NO signaling. This suggests a broader role for anti-VEGFA treatment in preventing pathological vascular enlargement and remodeling when combined with the anti-Dll4 antibody, that could be also related to its effect on liver EC survival. Our data suggest that the action of VEGF on vascular expansion and survival is independent of its direct effect on these signaling pathways 30, [32][33][34][35][36] , and independent of cell proliferation and sprouting, as also previously proposed 26,43 . The sum of these findings also suggests that the recently developed bispecific antibody targeting both Dll4 and VEGF simultaneously (navicixizumab, OncXerna) may be less toxic than the use of anti-Dll4 treatment alone 44 .
Altogether, the data obtained with several compound mutant and pharmacological approaches show that most of the transcriptional changes and angiogenic cell states elicited by targeting Dll4 correlate with, but do not cause, vascular pathophysiology (Extended Data Fig. 10). Therefore, vascular neoplasms are not the cause of the previously reported anti-Dll4 antibody toxicity 8 . Instead, we propose that the unrelated venous sinusoid enlargement and architecture abnormalization lead to vascular malfunction, blood accumulation, inflammation and hypoxia, altogether resulting in organ pathology.
These data also raise questions about the general use of single-cell transcriptional or genetic states to describe and predict functional or dysfunctional vascular phenotypes and ultimately organ pathophysiology. A single-cell transcriptional state is only a small part of a cell's phenotype and function.
All mouse husbandry and experimentation was conducted using protocols approved by local animal ethics committees and authorities showing that the expansion and abnormalization of the liver sinusoids (CD31 + EMCN + ), particularly around CVs, observed after Dll4 deletion, are not prevented by the administration of the indicated compounds. On the right, images show EC (ERG + nuclei) proliferation (Ki67 + ). c, Charts showing quantification of EC density/numbers and proliferation. Note that mTOR inhibitor-treated liver ECs do not proliferate significantly (same ERG + content), despite a fraction being Ki67 + . d, Deletion of Rac1 with Cdh5-CreERT2 in adult liver endothelium (Cdh5 + ) does not prevent the vascular pathology induced by blocking Dll4 with REGN1035. e, The use of the indicated inhibitors in postnatal mouse retina angiogenesis assays for 48 h confirms that they do not prevent the increase in vascular expansion/density (isolectin B4 labeling) induced by anti-Dll4 antibody treatment (7.5 mg/kg). Note that 2 days of angiogenesis growth correspond to the vasculature formed above the red dashed line. Data 18-03029/2020 or the Institutional Animal Care and Use Committee Protocol IS00013945). The mouse colonies were maintained in racks with individual ventilation cages according to current national legislation. Mice had dust-free and pathogen-free bedding, and sufficient nesting and environmental enrichment material for the development of species-specific behavior. All mice had ad libitum access to food and water in environmental conditions of 45-65% relative humidity, temperatures of [21][22][23][24] °C, and a 12 h/12 h light/dark cycle. In addition, to preserve animal welfare, mouse health was monitored with an animal health surveillance program, which follows the Federation of European Laboratory Animal Science Associations (FELASA) recommendations for specific pathogen-free facilities.
We used mice with C57BL/6 or C57BL/6×129SV genetic backgrounds. To generate mice for analysis, we intercrossed mice with an age range of  weeks. Mice used for experiments were 2-5 months old. We do not expect our data to be influenced by mouse sex.

Immunofluorescence on cryosections
Tissues were fixed for 2 h in 4% PFA in PBS at 4 °C. After three washes in PBS for 10 min each, organs were stored overnight in 30% sucrose (Sigma) in PBS. Organs were then embedded in OCT (Sakura) and frozen at −80 °C. Cryosections (35 μm) were cut on a cryostat (Leica), washed three times for 10 min each in PBS, and blocked and permeabilized in PBS containing 10% donkey serum (Millipore), 10% fetal bovine serum (FBS) and 1% Triton X-100. Primary antibodies were diluted in blocking/ permeabilization buffer and incubated overnight at 4 °C. This step was followed by three 10-min washes in PBS and incubation for 2 h with conjugated secondary antibodies (1:200, Jackson Laboratory) and 4,6-diamidino-2-phenylindole (DAPI) in PBS at room temperature. After three washes in PBS, sections were mounted with Fluoromount-G (SouthernBiotech). All antibodies used are listed in Supplementary  Table 2. To detect Ki67 or c-Myc in the same section as ERG, we used rabbit anti-Ki67 or anti-c-Myc together with a Fab fragment Cy3 secondary antibody, which is compatible with the later use of rabbit anti-ERG conjugated to Alexa Fluor 647.

Vibratome section immunofluorescence
Tissues were fixed for 2 h in 4% PFA in PBS and washed as above. Organs were then embedded in 6% agarose low-melting gel (Invitrogen), and organ sections (100 μm) were cut on a vibratome. Sections were permeabilized for 1 h in PBS containing 1% Triton X-100 and 0.5% Tween 20. Sections were then blocked for 1 h in a PBS solution containing 1% Triton X-100, 10% donkey serum and 10% FBS. Primary antibodies were diluted in blocking buffer and incubated with sections overnight at 4 °C. This step was followed by six washes with 1% Triton X-100 in PBS for 15 min and incubation for 2 h with conjugated secondary antibodies (1:200, Jackson Laboratory) and DAPI in PBS at room temperature. After three 15-min washes in PBS, sections were mounted with Fluoromount-G (South-ernBiotech). All antibodies used are listed in Supplementary Table 2.

Whole-mount immunofluorescence of retinas
For postnatal mouse retina immunostaining, eyes were collected and fixed in 4% PFA in PBS for 20 min at room temperature. After microdissection, retinas were fixed in 4% PFA for an additional 45 min, followed by two PBS washes of 10 min each. Retinas were blocked and permeabilized with PBTS buffer (0.3% Triton X-100, 3% FBS and 3% donkey serum) for 1 h. Samples were then incubated overnight at 4 °C in biotinylated isolectin B4 (diluted 1:50; Vector Laboratories, B-1205) and primary antibodies (Supplementary Table 2) diluted in PBTS buffer. After five washes of 20 min each in PBTS buffer diluted 1:2, samples were incubated for 2 h at room temperature with Alexa-conjugated secondary antibodies (Thermo Fisher). After three washes of 30 min each in PBTS buffer (diluted 1:2), and two washes of 10 min each in PBS, retinas were mounted with Fluoromount-G (SouthernBiotech).

Immunofluorescence on paraffin sections
The N1ICD epitope and the Jag1 ligand were detected with the tyramide signal amplification (TSA) kit (NEL774) procedure in paraffin sections after antigen retrieval. In brief, sections were dewaxed and rehydrated, followed by antigen retrieval in sub-boiling sodium citrate buffer (10 mM, pH 6.0) for 30 min. The slides were cooled down to room temperature for 30 min, followed by incubation for 30 min in 3% H 2 O 2 in methanol to quench endogenous peroxidase activity. Next, slides were rinsed in double-distilled H 2 O and washed three times for 5 min each in PBS, followed by blocking for 1 h in PBS containing 3% BSA, 200 mM MgCl 2 , 0.3% Tween 20 and 5% donkey serum. Sections were then incubated with primary antibody in the same solution overnight at 4 °C. After washes, slides were incubated for 2 h with anti-rabbit-HRP secondary antibody at room temperature, and, after washing, the signal was amplified using the TSA fluorescein kit (NEL774). Sections were mounted with Fluoromount-G (SouthernBiotech). All antibodies used are listed in Supplementary Table 2.

In vivo EdU labeling and EC proliferation detection
To detect EC proliferation in adult livers, 20 μg per g body weight EdU (Invitrogen, A10044) was injected intraperitoneally into adult mice 5 h before dissection. Livers were isolated for cryosection analysis. EdU signals were detected with the Click-iT EdU Alexa Fluor 647 or 488 Imaging Kit (Invitrogen, C10340 or C10337). In brief, after all other primary and secondary antibody incubations, samples were washed according to the immunofluorescence staining procedure and then incubated with Click-iT EdU reaction cocktail for 40 min, followed by DAPI counterstaining.

Image acquisition and analysis
Immunostained organ sections were imaged at high resolution with a Leica SP5, SP8 or SP8 Navigator confocal microscope fitted with a ×10, ×20 or ×40 objective for confocal scanning. Individual fields or tiles of large areas were acquired from cryosections, vibratome or paraffin sections. Large Z-volumes of the vibratome samples were imaged for 3D representation. All images shown are representative of the results obtained for each group and experiment. Animals were dissected and processed under exactly the same conditions. Comparisons of phenotypes or signal intensity were made with pictures obtained using the same laser excitation and confocal scanner detection settings. Fiji/ ImageJ was used to threshold, select and quantify objects in confocal micrographs. Both manual and automatic ImageJ public plug-ins and custom Fiji macros were used for quantification.

Latex perfusion and CUBIC clearing
Mice were euthanized in a CO 2 chamber. The abdominal cavity was opened, and the liver portal vein was exposed. With the help of a dissection microscope, latex (Injection Medium, Latex, Red, Laboratory Grade, Carolina, 868703) was injected in the portal vein with a 40G needle as previously described 59 . Perfusion was stopped as soon as latex was visually detectable in the liver surface vessels. Liver dissection was performed only 15 min after the perfusion to ensure latex solidification. The liver was then washed in PBS and put in PFA 4% in Nature Cardiovascular Research Article https://doi.org/10.1038/s44161-023-  PBS at 4 °C overnight. After, three PBS washes for 15 min each were done at room temperature. To clear the organ, livers were incubated at 37 °C in CUBIC1 (ref. 60) solution (25 wt% urea, 25 wt% N′-Tetrakis(2hydroxypropyl)ethylenediamine, 15 wt% Triton X-100) for a total 4 days, with the solution being exchanged every day. After clearing, liver images were captured with an Olympus camera connected to a Leica dissection scope with retroillumination. A magnification of ×0.8 was used.

Western blot analysis
For the analysis of protein expression, livers were transferred to a reagent tube and frozen in liquid nitrogen. On the day of immunoblotting, the tissue was lysed with lysis buffer (Tris-HCl pH 8, 20 mM, EDTA 1 mM, DTT 1 mM, Triton X-100 1% and NaCl 150 mM, containing protease inhibitors (Sigma, P-8340), phosphatase inhibitors (Calbiochem, 524629) and orthovanadate-Na 1 mM) and homogenized with a cylindrical glass pestle. Tissue and cell debris were removed by centrifugation, and the supernatant was diluted in loading buffer and analyzed by SDS-PAGE and immunoblotting. Membranes were blocked with BSA and incubated with the primary antibodies listed in Supplementary Table 2.

EC isolation for transcriptomic and proteomic analysis
The following methods were used to isolate ECs for bulk RNA-seq, and proteomics analysis. At day 14 after the first tamoxifen injection, heart, lungs, liver and brain were dissected, minced and digested with 2.5 mg ml −1 collagenase type I (Thermo Fisher), 2.5 mg ml −1 dispase II (Thermo Fisher) and 50 ng ml −1 DNase I (Roche) at 37 °C for 30 min. Cells were passed through a 70-μm filter. Erythroid cells were removed by incubation with blood lysis buffer (0.15 M NH 4 Cl, 0.01 M KHCO 3 and 0.01 M EDTA in distilled water) for 10 min on ice. Cell suspensions were blocked in blocking buffer (DPBS containing no Ca 2+ or Mg 2+ and supplemented with 3% dialyzed FBS; Thermo Fisher). For EC analysis, cells were incubated at 4 °C for 30 min with APC-conjugated rat anti-mouse CD31 (1:200; BD Biosciences, 551262). DAPI (5 mg ml −1 ) was added to the cells immediately before fluorescence-activated cell sorting (FACS), which was performed with FACSAria (BD Biosciences) or Synergy 4L cell sorters. For bulk RNA-seq experiments, approximately 10,000-20,000 cells for each group of DAPI − APC-CD31 + ECs (for Dll4 loss of function and control) and DAPI − APC-CD31 + /MbTomato + ECs (for Rbpj loss of function and control) were sorted directly to RLT buffer (RNeasy Micro Kit, Qiagen). RNA was extracted with the RNeasy Micro Kit and stored at −80 °C. For proteomic analysis, approximately 3 × 10 6 DAPI − APC-CD31 + ECs per group were sorted directly to blocking buffer. Cells were spun down for 10 min at 350 × g, and the pellet was stored at −80 °C.
To isolate ECs for scRNA-seq experiments, 1.5 mg of tamoxifen was injected on 4 consecutive days. At day 14 after the first tamoxifen injection, livers were dissected, minced and digested for 30 min with prewarmed (37 °C) dissociation buffer (2.5 mg ml −1 collagenase I (Thermo Fisher, 17100017), 2.5 mg ml −1 dispase II (Thermo Fisher, 17105041), 1 μl ml −1 DNase in PBS containing Ca 2+ and Mg 2+ (Gibco)). The digestion tube was agitated every 3-5 min in a water bath. At the end of the 30-min incubation, sample tubes were filled up to 15 ml with sorting buffer (PBS containing no Ca 2+ or Mg 2+ and supplemented with 10% FBS (Sigma, F7524)) and centrifuged (450 × g, 5 min, 4 °C). The supernatant was aspirated, and cell pellets were resuspended in 2 ml of 1x Red Blood Cell (RBC) Lysis Buffer (BioLegend, 420301) and incubated for 5 min on ice. We added 6 ml of sorting buffer to each sample, and samples were then passed through a 70-μm filter. Live cells were counted in a Neubauer chamber using trypan blue exclusion. Cells from each condition (4 × 10 6 per condition) were collected in separate tubes, and cells were incubated for 30 min with horizontal rotation in 300 μl of antibody incubation buffer (PBS + 1% BSA) containing 1 μl of CD31-APC, 1 μl of CD45-APC-Cy7, and 1 μl of hashtag oligo (HTO) conjugated antibodies (BioLegend). HTOs were used to label and distinguish the different samples when loaded on the same 10x Genomics port, thus also guaranteeing the absence of batch effects. After antibody incubation, samples were transferred to 15-ml Falcon tubes, 10 ml of sorting buffer were added, and samples were centrifuged (450 × g, 5 min, 4 °C). The supernatant was aspirated, pellets were resuspended in 1.5 ml of sorting buffer and transferred to Eppendorf tubes, and the resulting suspensions were centrifuged (450 × g, 5 min, 4 °C). The resulting pellets were resuspended in 300 μl of sorting buffer containing DAPI. Cells were sorted with a FACSAria Cell Sorter (BD Biosciences), and CD31 + CD45 − MbTomato + cells were sorted. BD FACSDiva v8.0.1 and FlowJo v10 were used for FACS data collection and analysis.

Next-generation sequencing sample and library preparation
Next-generation sequencing experiments were performed in the Genomics Unit at Centro Nacional de Investigaciones Cardiovasculares (CNIC).
For bulk RNA-seq, control and Dll4 iDEC EC samples, 1 ng of total RNA was used to amplify the cDNA using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech-Takara) following manufacturer's instructions. Then, 1 ng of amplified cDNA was used to generate barcoded libraries using the Nextera XT DNA Library Preparation Kit (Illumina). For control and Rbpj iDEC EC samples, between 400 pg and 3,000 pg of total RNA were used to generate barcoded RNA-seq libraries using the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer's instructions. For control and anti-Dll4 antibody-treated ECs, libraries were generated using the Ovation Single Cell RNA-Seq System (NuGEN) following manufacturer's instructions. All libraries were sequenced on a HiSeq 2500 (Illumina).
For scRNA-seq experiments, single cells were encapsulated into emulsion droplets using the Chromium Controller (10x Genomics). scRNA-seq libraries were prepared according to manufacturer's instructions. The aim for target cell recovery for each port was in general 10,000 cells, with a target cell recovery of 2,000-2,500 cells per experimental condition labeled with a given hashtag antibody. Generated libraries were sequenced on a HiSeq 4000 or NextSeq 2000 (Illumina).

Transcriptomic data analysis
Transcriptomic data were analyzed by the Bioinformatics Unit at CNIC.
For bulk RNA-seq, the number of reads per sample was between 12 million and 42 million. Reads were processed with a pipeline that assessed read quality using FastQC (Babraham Institute, http:// www.bioinformatics.babraham.ac.uk/projects/fastqc) and trimmed sequencing reads using cutadapt 61 , eliminating Illumina and SMARTer adaptor remains and discarding reads with <30 base pairs (bp). More than 93% of reads were kept for all samples. The resulting reads were mapped against the mouse transcriptomes GRCm38.76 and GRCm38.91, and gene expression levels were estimated with RSEM 62 . The percentage of aligned reads was above 83% for most samples. Expression count matrices were then processed with an analysis pipeline that used Bioconductor package limma 63 for normalization (using the trimmed mean of M values (TMM) method) and differential expression testing, taking into account only those genes expressed with at least 1 count per million (CPM) in at least two samples (the number of samples for the condition with the least replicates), and using a random variable to define blocks of samples obtained from the same animal. Changes in gene expression were considered significant if associated with a Benjamini and Hochberg-adjusted P value < 0.05. A complementary GSEA 64 was performed for each contrast, using the whole collection of genes detected as expressed (12,872 genes) to identify gene sets that had a tendency to be more expressed in either of the conditions being compared. We retrieved gene sets representing pathways or functional categories from the Hallmark, Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and BioCarta databases, and Gene Ontology (GO) collections from the Biological Article https://doi.org/10.1038/s44161-023-  Process, Molecular Function and Cellular Component ontologies from MSigDB 65 . Enriched gene sets with a false discovery rate (FDR) < 0.05% were considered of interest. Data were analyzed with Python v2.7, using the Seaborn (https://seaborn.pydata.org) and Pandas (https://pandas. pydata.org) libraries.
The following pipeline was followed for scRNA-seq data processing and in silico EC selection. For alignment and quantification of gene expression, the reference transcriptome was built using mouse genome GRCm38 and Ensembl gene build v98 (https://sep2019.archive. ensembl.org). The phiYFP-sv40pA, MbTomato-2A-Cre-  or CreERT2 transgene sequences expressed in the samples were added to the reference. Gene metadata were obtained from the corresponding Ensembl BioMart archive. Reads from hashtags and transcripts were processed, aligned and quantified using the Cell Ranger v4.0.0 pipeline. Single-cell analysis was based on Scater 66 and Seurat 67 packages. Low-quality cells were filtered out using the following criteria: total counts, >1,500 and <40,000; genes detected, >600; mitochondrial transcripts content, <25%; total counts/median, >0.1; hashtag counts, >100; hemoglobin transcripts, <0.1%; and percentage of counts in the top 50 genes, <65%. Cells were demultiplexed using the sample hashtag antibody signals (BioLegend). Counts were log-normalized and scaled, followed by principal component analysis (PCA) and clustering using the shared nearest-neighbors algorithm and Louvain clustering (settings as defaults except for the 1,000 most variable genes, 10 principal components, and a resolution of 0.5). Clusters and cells were classified based on the SingleR method 68 using Blueprint ENCODE and the Human Primary Cell Atlas cell-type profile collection. This identification was used to select ECs for the analysis and remove minor contaminants (T cells, B cells and monocytes). Hashtag-based doublets were removed, and only ECs were reclustered using the same procedure (with 2,000 variable genes, 7 PCs, a resolution of 0.3, and a random seed for uniform manifold approximation and projection (UMAP) = 123456) to get a final clustering that was later manually refined based on marker expression. Following cluster identification with the starting dataset, the remaining liver EC datasets were mapped using the FindTransfer-Anchors function from the Seurat R package using 30 PCA dimensions with the default settings.
The following pipeline was followed for liver non-EC scRNA-seq. Cells were demultiplexed by applying the cellranger multi pipeline. The following quality-control steps were performed to minimize lowquality cells and improve posterior normalization and analysis: (1) a minimum of normalized counts per cell of 2,000 and a maximum of 30,000; (2) a minimum gene detection filter of 500 genes and a maximum of 6,000; (3) a maximum mitochondria content of 5%; (4) a maximum ribosomal content of 35%; (5) a maximum hemoglobin content of 1%; and (6) only single cells were selected, and doublets were filtered out in the cellranger multi demultiplexing step. Counts were log-normalized and scaled, followed by PCA and clustering using the shared nearest-neighbors algorithm and Louvain clustering (settings as defaults except for the 2,000 most variable genes, 30 principal components and a resolution of 0.8). Clusters and cells were classified based on the SingleR method using Blueprint ENCODE, Human Primary Cell Atlas, and mouse RNA-seq datasets available in the celldex package, as well as a recent liver single-cell dataset 69 , in order to classify each cluster to a different cell type. Final clustering was later manually refined based on marker expression.

Liver EC proteomics
Protein extraction from cell samples was carried out in the presence of SDS as described 70 . Protein concentration was determined by the RC DC Protein Assay (Bio-Rad Laboratories). Samples (100 μg) were subjected to overnight tryptic digestion using filter-aided sample preparation (FASP) technology (Expedeon) 71 . The resulting peptides were desalted on Oasis HLB C18 extraction cartridges (Waters Corporation) and dried down. The cleaned-up peptide samples were subjected to stable isotope labeling using isobaric tags for relative and absolute quantitation (iTRAQ 8-plex, AB Sciex) following the manufacturer's instructions. The differentially tagged samples were then pooled and desalted on Oasis HLB C18 cartridges. A 100-μg aliquot of dried, labeled peptides was taken up in 0.1% trifluoroacetic acid and separated into five fractions by high pH reversed-phase peptide fractionation 72 . The bound peptides were eluted gradually with 12.5%, 15%, 17.5%, 20% and 50% acetonitrile, and the fractions obtained were vacuum-dried and stored at −20 °C for later use. The labeled peptide samples were taken up in 0.1% formic acid and analyzed on an EASY-nLC 1000 liquid chromatograph (Thermo Fisher Scientific) coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). The peptide samples were loaded onto a C18 reversed-phase nano-precolumn (Acclaim PepMap 100; 75-μm internal diameter, 3-μm particle size and 2-cm length; Thermo Fisher Scientific) and separated on an analytical C18 nano-column (EASY-Spray column PepMap RSLC C18; 75-μm internal diameter, 3-μm particle size and 50-cm length; Thermo Fisher Scientific) using a linear gradient: 8-27% B for 240 min, 31-100% B for 2 min, 100% B for 7 min, 100-2% B for 2 min, and 2% B for 30 min (where A is 0.1% formic acid in high-performance liquid chromatography (HPLC)-grade water, and B is 90% acetonitrile, 0.1% formic acid in HPLC-grade water). Full MS spectra were acquired over the 400-1,500 mass-to-charge (m/z) range with 120,000 resolution, 2 × 10 5 automatic gain control, and 50-ms maximum injection time. Data-dependent tandem MS (MS/ MS) acquisition was performed at 5 × 10 4 automatic gain control and 120-ms injection time, with a 2-Da isolation window and 30-s dynamic exclusion. Higher-energy collisional dissociation of peptides was induced with 31% normalized collision energy and analyzed at 35,000 resolution in the Orbitrap. Protein identification was carried out using the SEQUEST HT algorithm integrated in Proteome Discoverer v2.1 (Thermo Fisher Scientific). MS/MS scans were matched against a mouse protein database (UniProtKB release 2017-07) supplemented with pig trypsin and human keratin sequences. Parameters for database searching were as follows: trypsin digestion with a maximum of two missed cleavage sites allowed, precursor mass tolerance of 800 ppm, and a fragment mass tolerance of 0.02 Da. Amino-terminal and Lys iTRAQ 8-plex modifications were set as fixed modifications, whereas Met oxidation, Cys carbamidomethylation, and Cys methylthiolation were set as variable modifications. The identification results were analyzed with the probability ratio method 73 . An FDR for peptide identification was calculated based on searching against the corresponding inverted database using the refined method 74,75 after precursor mass tolerance postfiltering at 20 ppm. Quantitative information was extracted from the intensity of iTRAQ reporter ions in the low-mass region of the MS/ MS spectra 76 . The comparative analysis of protein abundance changes relied on the weighted scan peptide-protein (WSPP) statistical model 77 by means of the SanXoT software package as described 78 . As input, WSPP uses a list of quantifications in the form of log 2 ratios (each cell sample versus the mean of the three wild-type cell samples) with their statistical weights. From these, WSPP generates the standardized forms of the original variables by computing the quantitative values expressed in units of standard deviation around the means (Zq). For the study of coordinated protein alterations, we used the Systems Biology Triangle (SBT) algorithm, which estimates functional category averages (Zc) from protein values by performing the protein-to-category integration, as described 76 . The protein category database was built up using annotations from the GO database.

Statistical analysis and reproducibility
All bar graphs show mean ± s.d. Experiments were repeated with independent animals. Comparisons between two groups of samples with a Gaussian distribution were by unpaired two-tailed Student's t-test. Comparisons among more than two groups were made by one-way or two-way analysis of variance (ANOVA) followed by multiple comparison tests as indicated in the Source Data. All calculations were done in Article https://doi.org/10.1038/s44161-023-  Microsoft Excel, and final data points were analyzed and represented with GraphPad Prism. No randomization or blinding was used, and animals or tissues were selected for analysis based on their genotype, the detected Cre-dependent recombination frequency, and the quality of multiplex immunostaining. Sample sizes were chosen according to the observed statistical variation and published protocols.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
RNA-seq data can be viewed in the Gene Expression Omnibus (GEO) database under accession number GSE231613 (SuperSeries of GSE229793 and GSE231612). Instructions and code to reproduce all scRNA-seq results can be found at https://github.com/RuiBenedito/ Benedito_Lab. Proteomics data can be found in the Proteomics Identifications (PRIDE) database under accession number PXD041349. Unprocessed original photographs of the data are available upon request. All other data supporting the findings in this study are included in the main article and associated files.
Article https://doi.org/10.1038/s44161-023-  Extended Data Fig. 2 | No major genetic and vascular changes after blocking Dll4 signaling in quiescent vessels for 2 days. a, Anti-Dll4 treatment for 48 h starting at postnatal day 5, results in a strong increase in retina vascular density and angiogenesis. b, Experimental layout for the antibody-based blockade of Dll4 ligand function in adult mice. c-d, Confocal micrographs showing that anti-Dll4 blockade for 2 days in adult mice significantly reduces Notch1 activity (cleaved Notch1Val1744), but not EC density (DAPI+ Endomucin+) and EC proliferation (Ki67+DAPI+ Endomucin+ cells) as depicted in chart D from Fig. 2. e, List of the few differential expressed genes (DEG) 2 days after Dll4 blockade in liver endothelium. f, Heatmap with the normalized enrichment score (NES) from significantly deregulated GSEA hallmark pathways (FDR qval< 0.05). g, Heatmap representing logFC of every expressed gene in the indicated conditions versus control livers. h, Comparison of gene expression fold changes between anti-Dll4 for 2 days (short-term) and Dll4 deletion for 2 weeks (longterm). The top20 DEG belonging to the indicated GSEA pathways are shown. i, 3D projection images from confocal scanning of thick vibratome sections show that the vascular malformations observed in Dll4 iDEC livers are located in sinusoids connecting central veins (CV), but not in sinusoids located close to portal veins (PVs). j, Low magnification stereomicroscope images of livers from control and Dll4 iDEC mice showing liver pathology and blood accumulation in the mutants. k, Hematoxylin and Eosin staining images of liver sections from control and Dll4 iDEC mice show sinusoidal dilation in areas surrounding and connecting central veins (CVs). l, Confocal micrographs showing higher EC density (CD31 or EMCN+) and abnormal or enlarged sinusoids around central veins (delimited by yellow dashed lines). m, Immunostaining for CD45 shows strong accumulation of blood cells in the enlarged sinusoids connecting central veins. n, Violin plots showing expression of all canonical Notch pathway genes and downstream targets (Hes1, Hey1, Hey2)     Targeting of the ligand Dll4 triggers incomplete loss of Notch signaling which results in the loss of arterial markers, reduced PV caliber and a strong Myc-driven metabolic activation associated with a well-defined cluster of proliferating and tip cells located in the sinusoids around the central veins. Dll4 mutant ECs have very high ribosome biogenesis, protein synthesis and oxidative phosphorylation favouring cell growth and metabolism. This genetic activation correlates with a significant increase in the number of proliferating and sprouting venous ECs, vascular enlargement and subsequent organ pathology associated to the abnormal blood flow in CV sinusoids. The loss of Notch receptors or Rbpj leads to complete loss of Notch signaling and also the loss of the arterial transcriptional program and reduced PV caliber, but in this case most liver sinusoidal ECs undergo an hypermitogenic cell-cycle arrest and display senescence features. In contrast to Dll4 mutant ECs, Notch or Rbpj mutant ECs do not effectively proliferate or sprout and there is no significant vascular and organ pathology in mutant livers. Loss of all other Notch ligands leads to a mild increase in Notch signaling, without any associated vascular pathophysiology. Loss of Myc prevents most of the Dll4 mutant transcriptional program activation and cellular states. However, even in the absence of proliferating, sprouting and activated cells, Dll4/Myc iDEC mutant livers still have abnormal and expanded CV sinusoids and significant organ pathology. Targeting VEGF only partially reduces the Dll4 mutant genetic programs, but it is enough to prevent most of the activated and tip cell states, being ECs in a quiescent state. Anti-VEGFA also induces the very significant loss of ECs, which overall prevents the vascular enlargement and associated organ pathology. The effect of anti-VEGFA is broader and is not matched by the use of inhibitors targeting the ERK, Rac1 and Pi3k/mTor signaling.
Corresponding author(s): Rui Benedito Last updated by author(s): May 6, 2023 Reporting Summary Nature Portfolio wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Portfolio policies, see our Editorial Policies and the Editorial Policy Checklist.

Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

Software and code
Policy information about availability of computer code Data collection Organ sections were imaged at high resolution with a Leica SP5, SP8, or SP8 Navigator confocal microscopes fitted with 10x, 20x, or 40x objectives for confocal scanning. Leica stereomicroscopes with Olympus Camera were used for whole liver images. Bulk RNAseq and scRNAseq data was collected with Illumina HiSeq2500, HiSeq4000 or NextSeq2000 sequencers software. BD FACS Diva V8.0.1 and Flow JO v10 was utilized for FACS data collection and analysis. Proteomic data was obtained using the SEQUEST HT algorithm integrated in Proteome Discoverer 2.1 (Thermo Fisher Scientific).

March 2021
approximately 3x106 DAPI-negative APC-CD31+ ECs per group were sorted directly to blocking buffer. Cells were spun down for 10 min at 350g and pellet stored at -80°C. To isolate ECs for scRNA-seq experiments, 1.5 mg tamoxifen was injected on 4 consecutive days. At day 14 after the first tamoxifen injection, livers were dissected, minced, and digested for 30 min with pre-warmed (37C) dissociation buffer (2.5mg/ml collagenase I -Thermo Fisher 17100017), 2.5mg/ml dispase II (Thermo Fisher 17105041), 1ul/ml DNAse in PBS containing Ca2+ and Mg2+ (Gibco)). The digestion tube was agitated every 3-5 minutes in a water bath. At the end of the 30 minutes incubation, sample tubes were filled up to 15 ml with sorting buffer (PBS containing no Ca2+ or Mg2+ and supplemented with 10% FBS (Sigma, F7524)) and centrifuged (450g, 5 min, 4°C). The supernatant was aspirated, and cell pellets were resuspended in 2ml 1x RBC lysis buffer (BioLegend, 420301) and incubated for 5 min on ice. To each sample were added 6 ml of sorting buffer, and samples were then passed through a 70um filter. Live cells were counted in a Neubauer Chamber using trypan blue exclusion. Cells from each condition (4x106/condition) were collected in separate tubes, and cells were incubated for 30 min with horizontal rotation in 300μl antibody incubation buffer (PBS + 1% BSA) containing 1 μl CD31-APC, 1 μl CD45-APC-Cy7, and 1μl of hash tag oligo (HTO) conjugated antibodies (Biolegend). HTOs were used to label and distinguish the different samples when loaded on the same 10x genomics port, and in this way also guarantee the absence of batch effects. After the antibodies incubation, samples were transferred to 15 ml Falcon tubes, 10 ml sorting buffer was added, and samples were centrifuged (450g, 5min, 4°C). The supernatant was aspirated, pellets were resuspended in 1.5 ml sorting buffer and transferred to Eppendorf tubes, and the resulting suspensions were centrifuged (450g, 5min, 4°C). The resulting pellets were resuspended in 300 μl sorting buffer containing DAPI. Cells were sorted by FACS with an Aria Cell Sorter (BD Biosciences) and CD31+, CD45-MbTomato+ cells were sorted. BD FACS Diva V8.0.1 and Flow JO v10 was utilized for FACS data collection and analysis.

Gating strategy
The gating strategy is indicated in Extended Data Fig. 1 and 4.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.