γ-Linolenic acid in maternal milk drives cardiac metabolic maturation.

Birth presents a metabolic challenge to cardiomyocytes as they reshape fuel preference from glucose to fatty acids for postnatal energy production1,2. This adaptation is triggered in part by post-partum environmental changes3, but the molecules orchestrating cardiomyocyte maturation remain unknown. Here we show that this transition is coordinated by maternally supplied γ-linolenic acid (GLA), an 18:3 omega-6 fatty acid enriched in the maternal milk. GLA binds and activates retinoid X receptors4 (RXRs), ligand-regulated transcription factors that are expressed in cardiomyocytes from embryonic stages. Multifaceted genome-wide analysis revealed that the lack of RXR in embryonic cardiomyocytes caused an aberrant chromatin landscape that prevented the induction of an RXR-dependent gene expression signature controlling mitochondrial fatty acid homeostasis. The ensuing defective metabolic transition featured blunted mitochondrial lipid-derived energy production and enhanced glucose consumption, leading to perinatal cardiac dysfunction and death. Finally, GLA supplementation induced RXR-dependent expression of the mitochondrial fatty acid homeostasis signature in cardiomyocytes, both in vitro and in vivo. Thus, our study identifies the GLA-RXR axis as a key transcriptional regulatory mechanism underlying the maternal control of perinatal cardiac metabolism.

Metabolic changes contribute to maladaptive right ventricular hypertrophy in pulmonary hypertension beyond pressure overload: an integrative imaging and omics investigation.

Right ventricular (RV) failure remains the strongest determinant of survival in pulmonary hypertension (PH). We aimed to identify relevant mechanisms, beyond pressure overload, associated with maladaptive RV hypertrophy in PH. To separate the effect of pressure overload from other potential mechanisms, we developed in pigs two experimental models of PH (M1, by pulmonary vein banding and M2, by aorto-pulmonary shunting) and compared them with a model of pure pressure overload (M3, pulmonary artery banding) and a sham-operated group. Animals were assessed at 1 and 8 months by right heart catheterization, cardiac magnetic resonance and blood sampling, and myocardial tissue was analyzed. Plasma unbiased proteomic and metabolomic data were compared among groups and integrated by an interaction network analysis. A total of 33 pigs completed follow-up (M1, n = 8; M2, n = 6; M3, n = 10; and M0, n = 9). M1 and M2 animals developed PH and reduced RV systolic function, whereas animals in M3 showed increased RV systolic pressure but maintained normal function. Significant plasma arginine and histidine deficiency and complement system activation were observed in both PH models (M1&M2), with additional alterations to taurine and purine pathways in M2. Changes in lipid metabolism were very remarkable, particularly the elevation of free fatty acids in M2. In the integrative analysis, arginine-histidine-purines deficiency, complement activation, and fatty acid accumulation were significantly associated with maladaptive RV hypertrophy. Our study integrating imaging and omics in large-animal experimental models demonstrates that, beyond pressure overload, metabolic alterations play a relevant role in RV dysfunction in PH.

ISG20L2: an RNA nuclease regulating T cell activation.

ISG20L2, a 3' to 5' exoribonuclease previously associated with ribosome biogenesis, is identified here in activated T cells as an enzyme with a preferential affinity for uridylated miRNA substrates. This enzyme is upregulated in T lymphocytes upon TCR and IFN type I stimulation and appears to be involved in regulating T cell function. ISG20L2 silencing leads to an increased basal expression of CD69 and induces greater IL2 secretion. However, ISG20L2 absence impairs CD25 upregulation, CD3 synaptic accumulation and MTOC translocation towards the antigen-presenting cell during immune synapsis. Remarkably, ISG20L2 controls the expression of immunoregulatory molecules, such as AHR, NKG2D, CTLA-4, CD137, TIM-3, PD-L1 or PD-1, which show increased levels in ISG20L2 knockout T cells. The dysregulation observed in these key molecules for T cell responses support a role for this exonuclease as a novel RNA-based regulator of T cell function.

Cardiovascular Progerin Suppression and Lamin A Restoration Rescue Hutchinson-Gilford Progeria Syndrome.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is a rare disorder characterized by premature aging and death mainly because of myocardial infarction, stroke, or heart failure. The disease is provoked by progerin, a variant of lamin A expressed in most differentiated cells. Patients look healthy at birth, and symptoms typically emerge in the first or second year of life. Assessing the reversibility of progerin-induced damage and the relative contribution of specific cell types is critical to determining the potential benefits of late treatment and to developing new therapies. METHODS: We used CRISPR-Cas9 technology to generate LmnaHGPSrev/HGPSrev (HGPSrev) mice engineered to ubiquitously express progerin while lacking lamin A and allowing progerin suppression and lamin A restoration in a time- and cell type-specific manner on Cre recombinase activation. We characterized the phenotype of HGPSrev mice and crossed them with Cre transgenic lines to assess the effects of suppressing progerin and restoring lamin A ubiquitously at different disease stages as well as specifically in vascular smooth muscle cells and cardiomyocytes. RESULTS: Like patients with HGPS, HGPSrev mice appear healthy at birth and progressively develop HGPS symptoms, including failure to thrive, lipodystrophy, vascular smooth muscle cell loss, vascular fibrosis, electrocardiographic anomalies, and precocious death (median lifespan of 15 months versus 26 months in wild-type controls, P<0.0001). Ubiquitous progerin suppression and lamin A restoration significantly extended lifespan when induced in 6-month-old mildly symptomatic mice and even in severely ill animals aged 13 months, although the benefit was much more pronounced on early intervention (84.5% lifespan extension in mildly symptomatic mice, P<0.0001, and 6.7% in severely ill mice, P<0.01). It is remarkable that major vascular alterations were prevented and lifespan normalized in HGPSrev mice when progerin suppression and lamin A restoration were restricted to vascular smooth muscle cells and cardiomyocytes. CONCLUSIONS: HGPSrev mice constitute a new experimental model for advancing knowledge of HGPS. Our findings suggest that it is never too late to treat HGPS, although benefit is much more pronounced when progerin is targeted in mice with mild symptoms. Despite the broad expression pattern of progerin and its deleterious effects in many organs, restricting its suppression to vascular smooth muscle cells and cardiomyocytes is sufficient to prevent vascular disease and normalize lifespan.

Quantification of Farnesylated Progerin in Hutchinson-Gilford Progeria Patient Cells by Mass Spectrometry.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare fatal disorder characterized by premature aging and death at a median age of 14.5 years. The most common cause of HGPS (affecting circa 90% of patients) is a de novo heterozygous synonymous single-base substitution (c.1824C>T; p.G608G) in the LMNA gene that results in the accumulation of progerin, an aberrant form of lamin A that, unlike mature lamin A, remains permanently farnesylated. The ratio of progerin to mature lamin A correlates with disease severity in HGPS patients, and can be used to assess the effectiveness of therapies aimed at lessening aberrant splicing or progerin farnesylation. We recently showed that the endogenous content of lamin A and progerin can be measured by mass spectrometry (MS), providing an alternative to immunological methods, which lack the necessary specificity and quantitative accuracy. Here, we present the first non-immunological method that reliably quantifies the levels of wild-type lamin A and farnesylated progerin in cells from HGPS patients. This method, which is based on a targeted MS approach and the use of isotope-labeled internal standards, could be applied in ongoing clinical trials evaluating the efficacy of drugs that inhibit progerin farnesylation.

Loss of SRSF3 in Cardiomyocytes Leads to Decapping of Contraction-Related mRNAs and Severe Systolic Dysfunction.

RATIONALE: RBPs (RNA binding proteins) play critical roles in the cell by regulating mRNA transport, splicing, editing, and stability. The RBP SRSF3 (serine/arginine-rich splicing factor 3) is essential for blastocyst formation and for proper liver development and function. However, its role in the heart has not been explored. OBJECTIVE: To investigate the role of SRSF3 in cardiac function. METHODS AND RESULTS: Cardiac SRSF3 expression was high at mid gestation and decreased during late embryonic development. Mice lacking SRSF3 in the embryonic heart showed impaired cardiomyocyte proliferation and died in utero. In the adult heart, SRSF3 expression was reduced after myocardial infarction, suggesting a possible role in cardiac homeostasis. To determine the role of this RBP in the adult heart, we used an inducible, cardiomyocyte-specific SRSF3 knockout mouse model. After SRSF3 depletion in cardiomyocytes, mice developed severe systolic dysfunction that resulted in death within 8 days. RNA-Seq analysis revealed downregulation of mRNAs encoding sarcomeric and calcium handling proteins. Cardiomyocyte-specific SRSF3 knockout mice also showed evidence of alternative splicing of mTOR (mammalian target of rapamycin) mRNA, generating a shorter protein isoform lacking catalytic activity. This was associated with decreased phosphorylation of 4E-BP1 (eIF4E-binding protein 1), a protein that binds to eIF4E (eukaryotic translation initiation factor 4E) and prevents mRNA decapping. Consequently, we found increased decapping of mRNAs encoding proteins involved in cardiac contraction. Decapping was partially reversed by mTOR activation. CONCLUSIONS: We show that cardiomyocyte-specific loss of SRSF3 expression results in decapping of critical mRNAs involved in cardiac contraction. The molecular mechanism underlying this effect likely involves the generation of a short mTOR isoform by alternative splicing, resulting in reduced 4E-BP1 phosphorylation. The identification of mRNA decapping as a mechanism of systolic heart failure may open the way to the development of urgently needed therapeutic tools.

E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine.

Cadherin-based intercellular adhesions are essential players in epithelial homeostasis, but their dynamic regulation during tissue morphogenesis and remodeling remain largely undefined. Here, we characterize an unexpected role for the membrane-anchored metalloproteinase MT2-MMP in regulating epithelial cell quiescence. Following co-immunoprecipitation and mass spectrometry, the MT2-MMP cytosolic tail was found to interact with the zonula occludens protein-1 (ZO-1) at the apical junctions of polarized epithelial cells. Functionally, MT2-MMP localizes in the apical domain of epithelial cells where it cleaves E-cadherin and promotes epithelial cell accumulation, a phenotype observed in 2D polarized cells as well as 3D cysts. MT2-MMP-mediated cleavage subsequently disrupts apical E-cadherin-mediated cell quiescence resulting in relaxed apical cortical tension favoring cell extrusion and re-sorting of Src kinase activity to junctional complexes, thereby promoting proliferation. Physiologically, MT2-MMP loss of function alters E-cadherin distribution, leading to impaired 3D organoid formation by mouse colonic epithelial cells ex vivo and reduction of cell proliferation within intestinal crypts in vivo Taken together, these studies identify an MT2-MMP-E-cadherin axis that functions as a novel regulator of epithelial cell homeostasis in vivo.

ISG15 governs mitochondrial function in macrophages following vaccinia virus infection.

The interferon (IFN)-stimulated gene 15 (ISG15) encodes one of the most abundant proteins induced by interferon, and its expression is associated with antiviral immunity. To identify protein components implicated in IFN and ISG15 signaling, we compared the proteomes of ISG15-/- and ISG15+/+ bone marrow derived macrophages (BMDM) after vaccinia virus (VACV) infection. The results of this analysis revealed that mitochondrial dysfunction and oxidative phosphorylation (OXPHOS) were pathways altered in ISG15-/- BMDM treated with IFN. Mitochondrial respiration, Adenosine triphosphate (ATP) and reactive oxygen species (ROS) production was higher in ISG15+/+ BMDM than in ISG15-/- BMDM following IFN treatment, indicating the involvement of ISG15-dependent mechanisms. An additional consequence of ISG15 depletion was a significant change in macrophage polarization. Although infected ISG15-/- macrophages showed a robust proinflammatory cytokine expression pattern typical of an M1 phenotype, a clear blockade of nitric oxide (NO) production and arginase-1 activation was detected. Accordingly, following IFN treatment, NO release was higher in ISG15+/+ macrophages than in ISG15-/- macrophages concomitant with a decrease in viral titer. Thus, ISG15-/- macrophages were permissive for VACV replication following IFN treatment. In conclusion, our results demonstrate that ISG15 governs the dynamic functionality of mitochondria, specifically, OXPHOS and mitophagy, broadening its physiological role as an antiviral agent.

HDAC6 controls innate immune and autophagy responses to TLR-mediated signalling by the intracellular bacteria Listeria monocytogenes.

Recent evidence on HDAC6 function underlines its role as a key protein in the innate immune response to viral infection. However, whether HDAC6 regulates innate immunity during bacterial infection remains unexplored. To assess the role of HDAC6 in the regulation of defence mechanisms against intracellular bacteria, we used the Listeria monocytogenes (Lm) infection model. Our data show that Hdac6-/- bone marrow-derived dendritic cells (BMDCs) have a higher bacterial load than Hdac6+/+ cells, correlating with weaker induction of IFN-related genes, pro-inflammatory cytokines and nitrite production after bacterial infection. Hdac6-/- BMDCs have a weakened phosphorylation of MAPK signalling in response to Lm infection, suggesting altered Toll-like receptor signalling (TLR). Compared with Hdac6+/+ counterparts, Hdac6-/- GM-CSF-derived and FLT3L-derived dendritic cells show weaker pro-inflammatory cytokine secretion in response to various TLR agonists. Moreover, HDAC6 associates with the TLR-adaptor molecule Myeloid differentiation primary response gene 88 (MyD88), and the absence of HDAC6 seems to diminish the NF-κB induction after TLR stimuli. Hdac6-/- mice display low serum levels of inflammatory cytokine IL-6 and correspondingly an increased survival to a systemic infection with Lm. The impaired bacterial clearance in the absence of HDAC6 appears to be caused by a defect in autophagy. Hence, Hdac6-/- BMDCs accumulate higher levels of the autophagy marker p62 and show defective phagosome-lysosome fusion. These data underline the important function of HDAC6 in dendritic cells not only in bacterial autophagy, but also in the proper activation of TLR signalling. These results thus demonstrate an important regulatory role for HDAC6 in the innate immune response to intracellular bacterial infection.

A Novel Systems-Biology Algorithm for the Analysis of Coordinated Protein Responses Using Quantitative Proteomics.

The coordinated behavior of proteins is central to systems biology. However, the underlying mechanisms are poorly known and methods to analyze coordination by conventional quantitative proteomics are still lacking. We present the Systems Biology Triangle (SBT), a new algorithm that allows the study of protein coordination by pairwise quantitative proteomics. The Systems Biology Triangle detected statistically significant coordination in diverse biological models of very different nature and subjected to different kinds of perturbations. The Systems Biology Triangle also revealed with unprecedented molecular detail an array of coordinated, early protein responses in vascular smooth muscle cells treated at different times with angiotensin-II. These responses included activation of protein synthesis, folding, turnover, and muscle contraction - consistent with a differentiated phenotype-as well as the induction of migration and the repression of cell proliferation and secretion. Remarkably, the majority of the altered functional categories were protein complexes, interaction networks, or metabolic pathways. These changes could not be detected by other algorithms widely used by the proteomics community, and the vast majority of proteins involved have not been described before to be regulated by AngII. The unique capabilities of The Systems Biology Triangle to detect functional protein alterations produced by the coordinated action of proteins in pairwise quantitative proteomics experiments make this algorithm an attractive choice for the biological interpretation of results on a routine basis.

Deficiency of MMP17/MT4-MMP proteolytic activity predisposes to aortic aneurysm in mice.

RATIONALE: Aortic dissection or rupture resulting from aneurysm causes 1% to 2% of deaths in developed countries. These disorders are associated with mutations in genes that affect vascular smooth muscle cell differentiation and contractility or extracellular matrix composition and assembly. However, as many as 75% of patients with a family history of aortic aneurysms do not have an identified genetic syndrome. OBJECTIVE: To determine the role of the protease MMP17/MT4-MMP in the arterial wall and its possible relevance in human aortic pathology. METHODS AND RESULTS: Screening of patients with inherited thoracic aortic aneurysms and dissections identified a missense mutation (R373H) in the MMP17 gene that prevented the expression of the protease in human transfected cells. Using a loss-of-function genetic mouse model, we demonstrated that the lack of Mmp17 resulted in the presence of dysfunctional vascular smooth muscle cells and altered extracellular matrix in the vessel wall; and it led to increased susceptibility to angiotensin-II-induced thoracic aortic aneurysm. We also showed that Mmp17-mediated osteopontin cleavage regulated vascular smooth muscle cell maturation via c-Jun N-terminal kinase signaling during aorta wall development. Some features of the arterial phenotype were prevented by re-expression of catalytically active Mmp17 or the N-terminal osteopontin fragment in Mmp17-null neonates. CONCLUSIONS: Mmp17 proteolytic activity regulates vascular smooth muscle cell phenotype in the arterial vessel wall, and its absence predisposes to thoracic aortic aneurysm in mice. The rescue of part of the vessel-wall phenotype by a lentiviral strategy opens avenues for therapeutic intervention in these life-threatening disorders.

A Single In-Vial Dual Extraction Strategy for the Simultaneous Lipidomics and Proteomics Analysis of HDL and LDL Fractions.

A single in-vial dual extraction (IVDE) procedure for the subsequent analysis of lipids and proteins in the high-density lipoprotein (HDL) and low-density lipoprotein (LDL) fractions derived from the same biological sample is presented. On the basis of methyl-tert-butyl ether (MTBE) extraction, IVDE leads to the formation of three phases: a protein pellet at the bottom, an aqueous phase with polar compounds, and an ether phase with lipophilic compounds. After sample extraction, performed within a high-performance liquid chromatography vial insert, the ether phase was directly injected for lipid fingerprinting, while the protein pellet, after evaporation of the remaining sample, was used for proteomics analysis. Human HDL and LDL isolates were used to test the suitability of the IVDE methodology for lipid and protein analysis from a single sample in terms of data quality and matching composition to that of HDL and LDL. Subsequently, HDL and LDL fractions isolated from ApoE-KO and wild-type mice were used to validate the capacity of IVDE for revealing changes in lipid and protein abundance. Results indicate that IVDE can be successfully used for the subsequent analysis of lipids and proteins with the advantages of time saving, simplicity, and reduced sample amount.

Paraoxonase-1 overexpression prevents experimental abdominal aortic aneurysm progression.

Abdominal aortic aneurysm (AAA) is a permanent dilation of the aorta due to excessive proteolytic, oxidative and inflammatory injury of the aortic wall. We aimed to identify novel mediators involved in AAA pathophysiology, which could lead to novel therapeutic approaches. For that purpose, plasma from four AAA patients and four controls were analysed by a label-free proteomic approach. Among identified proteins, paraoxonase-1 (PON1) was decreased in plasma of AAA patients compared with controls, which was further validated in a bigger cohort of samples by ELISA. The phenylesterase enzymatic activity of PON1 was also decreased in serum of AAA patients compared with controls. To address the potential role of PON1 as a mediator of AAA, experimental AAA was induced by aortic elastase perfusion in wild-type (WT) mice and human transgenic PON1 (HuTgPON1) mice. Similar to humans, PON1 activity was also decreased in serum of elastase-induced AAA mice compared with healthy mice. Interestingly, overexpression of PON1 was accompanied by smaller aortic dilation and higher elastin and vascular smooth muscle cell (VSMC) content in the AAA of HuTgPON1 compared with WT mice. Moreover, HuTgPON1 mice display decreased oxidative stress and apoptosis, as well as macrophage infiltration and monocyte chemoattractant protein-1 (MCP1) expression, in elastase-induced AAA. In conclusion, decreased circulating PON1 activity is associated with human and experimental AAA. PON1 overexpression in mice protects against AAA progression by reducing oxidative stress, apoptosis and inflammation, suggesting that strategies aimed at increasing PON1 activity could prevent AAA.

Selected reaction monitoring assays in mesenchymal stem cells from osteoarthritis patients.

Osteoarthritis (OA) is considered the most prevalent form of arthritis. The aim of this study was to verify potential protein OA biomarkers by applying Selected Reaction Monitoring (SRM) assays to protein extracts obtained from Bone Marrow-Mesenchymal Stem Cells (BM-MSCs) isolated from OA patients. BM aspirates were obtained from the femoral channel of OA patients at the time of surgery and from the femoral channel of hip fracture subjects without OA during hip joint replacement surgery for the treatment of subcapital fracture. SRM results verified the differential expression of several protein biomarkers in BM-MSCs from OA patients.

iSanXoT: A standalone application for the integrative analysis of mass spectrometry-based quantitative proteomics data.

Many bioinformatics tools are available for the quantitative analysis of proteomics experiments. Most of these tools use a dedicated statistical model to derive absolute quantitative protein values from mass spectrometry (MS) data. Here, we present iSanXoT, a standalone application that processes relative abundances between MS signals and then integrates them sequentially to upper levels using the previously published Generic Integration Algorithm (GIA). iSanXoT offers unique capabilities that complement conventional quantitative software applications, including statistical weighting and independent modeling of error distributions in each integration, aggregation of technical or biological replicates, quantification of posttranslational modifications, and analysis of coordinated protein behavior. iSanXoT is a standalone, user-friendly application that accepts output from popular proteomics pipelines and enables unrestricted creation of quantification workflows and fully customizable reports that can be reused across projects or shared among users. Numerous publications attest the successful application of diverse integrative workflows constructed using the GIA for the analysis of high-throughput quantitative proteomics experiments. iSanXoT has been tested with the main operating systems. Download links for the corresponding distributions are available at https://github.com/CNIC-Proteomics/iSanXoT/releases.

Oxidative Post-translational Protein Modifications upon Ischemia/Reperfusion Injury.

While reperfusion, or restoration of coronary blood flow in acute myocardial infarction, is a requisite for myocardial salvage, it can paradoxically induce a specific damage known as ischemia/reperfusion (I/R) injury. Our understanding of the precise pathophysiological molecular alterations leading to I/R remains limited. In this study, we conducted a comprehensive and unbiased time-course analysis of post-translational modifications (PTMs) in the post-reperfused myocardium of two different animal models (pig and mouse) and evaluated the effect of two different cardioprotective therapies (ischemic preconditioning and neutrophil depletion). In pigs, a first wave of irreversible oxidative damage was observed at the earliest reperfusion time (20 min), impacting proteins essential for cardiac contraction. A second wave, characterized by irreversible oxidation on different residues and reversible Cys oxidation, occurred at late stages (6-12 h), affecting mitochondrial, sarcomere, and inflammation-related proteins. Ischemic preconditioning mitigated the I/R damage caused by the late oxidative wave. In the mouse model, the two-phase pattern of oxidative damage was replicated, and neutrophil depletion mitigated the late wave of I/R-related damage by preventing both Cys reversible oxidation and irreversible oxidation. Altogether, these data identify protein PTMs occurring late after reperfusion as an actionable therapeutic target to reduce the impact of I/R injury.

In-gel protein digestion using acidic methanol produces a highly selective methylation of glutamic acid residues.

Mass-tolerant open search methods allow the high-throughput analysis of modified peptides by mass spectrometry. These techniques have paved the way to unbiased analysis of post-translational modifications in biological contexts, as well as of chemical modifications produced during the manipulation of protein samples. In this work, we have analyzed in-depth a wide variety of samples of different biological origin, including cells, extracellular vesicles, secretomes, centrosomes and tissue preparations, using Comet-ReCom, a recently improved version of the open search engine Comet-PTM. Our results demonstrate that glutamic acid residues undergo intensive methyl esterification when protein digestion is performed using in-gel techniques, but not using gel-free approaches. This effect was highly specific to Glu and was not found for other methylable residues such as Asp.

MYH10 activation rescues contractile defects in arrhythmogenic cardiomyopathy (ACM).

The most prevalent genetic form of inherited arrhythmogenic cardiomyopathy (ACM) is caused by mutations in desmosomal plakophilin-2 (PKP2). By studying pathogenic deletion mutations in the desmosomal protein PKP2, here we identify a general mechanism by which PKP2 delocalization restricts actomyosin network organization and cardiac sarcomeric contraction in this untreatable disease. Computational modeling of PKP2 variants reveals that the carboxy-terminal (CT) domain is required for N-terminal domain stabilization, which determines PKP2 cortical localization and function. In mutant PKP2 cells the expression of the interacting protein MYH10 rescues actomyosin disorganization. Conversely, dominant-negative MYH10 mutant expression mimics the pathogenic CT-deletion PKP2 mutant causing actin network abnormalities and right ventricle systolic dysfunction. A chemical activator of non-muscle myosins, 4-hydroxyacetophenone (4-HAP), also restores normal contractility. Our findings demonstrate that activation of MYH10 corrects the deleterious effect of PKP2 mutant over systolic cardiac contraction, with potential implications for ACM therapy.

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.