Mild Hypoxia Accelerates Cerebral Cavernous Malformation Disease Through CX3CR1-CX3CL1 Signaling.

BACKGROUND: Heterogeneity in the severity of cerebral cavernous malformations (CCMs) disease, including brain bleedings and thrombosis that cause neurological disabilities in patients, suggests that environmental, genetic, or biological factors act as disease modifiers. Still, the underlying mechanisms are not entirely understood. Here, we report that mild hypoxia accelerates CCM disease by promoting angiogenesis, neuroinflammation, and vascular thrombosis in the brains of CCM mouse models. METHODS: We used genetic studies, RNA sequencing, spatial transcriptome, micro-computed tomography, fluorescence-activated cell sorting, multiplex immunofluorescence, coculture studies, and imaging techniques to reveal that sustained mild hypoxia via the CX3CR1-CX3CL1 (CX3C motif chemokine receptor 1/chemokine [CX3C motif] ligand 1) signaling pathway influences cell-specific neuroinflammatory interactions, contributing to heterogeneity in CCM severity. RESULTS: Histological and expression profiles of CCM neurovascular lesions (Slco1c1-iCreERT2;Pdcd10fl/fl; Pdcd10BECKO) in male and female mice found that sustained mild hypoxia (12% O2, 7 days) accelerates CCM disease. Our findings indicate that a small reduction in oxygen levels can significantly increase angiogenesis, neuroinflammation, and thrombosis in CCM disease by enhancing the interactions between endothelium, astrocytes, and immune cells. Our study indicates that the interactions between CX3CR1 and CX3CL1 are crucial in the maturation of CCM lesions and propensity to CCM immunothrombosis. In particular, this pathway regulates the recruitment and activation of microglia and other immune cells in CCM lesions, which leads to lesion growth and thrombosis. We found that human CX3CR1 variants are linked to lower lesion burden in familial CCMs, proving it is a genetic modifier in human disease and a potential marker for aggressiveness. Moreover, monoclonal blocking antibody against CX3CL1 or reducing 1 copy of the Cx3cr1 gene significantly reduces hypoxia-induced CCM immunothrombosis. CONCLUSIONS: Our study reveals that interactions between CX3CR1 and CX3CL1 can modify CCM neuropathology when lesions are accelerated by environmental hypoxia. Moreover, a hypoxic environment or hypoxia signaling caused by CCM disease influences the balance between neuroinflammation and neuroprotection mediated by CX3CR1-CX3CL1 signaling. These results establish CX3CR1 as a genetic marker for patient stratification and a potential predictor of CCM aggressiveness.

Exploring the Impact of Saccharin on Neovascular Age-Related Macular Degeneration: A Comprehensive Study in Patients and Mice.

Purpose: We aimed to determine the impact of artificial sweeteners (AS), especially saccharin, on the progression and treatment efficacy of patients with neovascular age-related macular degeneration (nAMD) under anti-vascular endothelial growth factor (anti-VEGF-A) treatment. Methods: In a cross-sectional study involving 46 patients with nAMD undergoing intravitreal anti-VEGF therapy, 6 AS metabolites were detected in peripheral blood using liquid chromatography - tandem mass spectrometry (LC-MS/MS). Disease features were statistically tested against these metabolite levels. Additionally, a murine choroidal neovascularization (CNV) model, induced by laser, was used to evaluate the effects of orally administered saccharin, assessing both imaging outcomes and gene expression patterns. Polymerase chain reaction (PCR) methods were used to evaluate functional expression of sweet taste receptors in a retinal pigment epithelium (RPE) cell line. Results: Saccharin levels in blood were significantly higher in patients with well-controlled CNV activity (P = 0.004) and those without subretinal hyper-reflective material (P = 0.015). In the murine model, saccharin-treated mice exhibited fewer leaking laser scars, lesser occurrence of bleeding, smaller fibrotic areas (P < 0.05), and a 40% decrease in mononuclear phagocyte accumulation (P = 0.06). Gene analysis indicated downregulation of inflammatory and VEGFR-1 response genes in the treated animals. Human RPE cells expressed taste receptor type 1 member 3 (TAS1R3) mRNA and reacted to saccharin stimulation with changes in mRNA expression. Conclusions: Saccharin appears to play a protective role in patients with nAMD undergoing intravitreal anti-VEGF treatment, aiding in better pathological lesion control and scar reduction. The murine study supports this observation, proposing saccharin's potential in mitigating pathological VEGFR-1-induced immune responses potentially via the RPE sensing saccharin in the blood stream.

Macrophage-induced integrin signaling promotes Schlemm's canal formation to prevent intraocular hypertension and glaucomatous optic neuropathy.

Schlemm's canal (SC) functions to maintain proper intraocular pressure (IOP) by draining aqueous humor and has emerged as a promising therapeutic target for glaucoma, the second-leading cause of irreversible blindness worldwide. However, our current understanding of the mechanisms governing SC development and functionality remains limited. Here, we show that vitronectin (VTN) produced by limbal macrophages promotes SC formation and prevents intraocular hypertension by activating integrin αvß3 signaling. Genetic inactivation of this signaling system inhibited the phosphorylation of AKT and FOXO1 and reduced ß-catenin activity and FOXC2 expression, thereby causing impaired Prox1 expression and deteriorated SC morphogenesis. This ultimately led to increased IOP and glaucomatous optic neuropathy. Intriguingly, we found that aged SC displayed downregulated integrin ß3 in association with dampened Prox1 expression. Conversely, FOXO1 inhibition rejuvenated the aged SC by inducing Prox1 expression and SC regrowth, highlighting a possible strategy by targeting VTN/integrin αvß3 signaling to improve SC functionality.

Role of endothelial PDGFB in arterio-venous malformations pathogenesis.

Arterial-venous malformations (AVMs) are direct connections between arteries and veins without an intervening capillary bed. Either familial inherited or sporadically occurring, localized pericytes (PCs) drop is among the AVMs' hallmarks. Whether impaired PC coverage triggers AVMs or it is a secondary event is unclear. Here we evaluated the role of the master regulator of PC recruitment, Platelet derived growth factor B (PDGFB) in AVM pathogenesis. Using tamoxifen-inducible deletion of Pdgfb in endothelial cells (ECs), we show that disruption of EC Pdgfb-mediated PC recruitment and maintenance leads to capillary enlargement and organotypic AVM-like structures. These vascular lesions contain non-proliferative hyperplastic, hypertrophic and miss-oriented capillary ECs with an altered capillary EC fate identity. Mechanistically, we propose that PDGFB maintains capillary EC size and caliber to limit hemodynamic changes, thus restricting expression of Krüppel like factor 4 and activation of Bone morphogenic protein, Transforming growth factor ß and NOTCH signaling in ECs. Furthermore, our study emphasizes that inducing or activating PDGFB signaling may be a viable therapeutic approach for treating vascular malformations.

Systemic Blood Proteome Patterns Reflect Disease Phenotypes in Neovascular Age-Related Macular Degeneration.

There is early evidence of extraocular systemic signals effecting function and morphology in neovascular age-related macular degeneration (nAMD). The prospective, cross-sectional BIOMAC study is an explorative investigation of peripheral blood proteome profiles and matched clinical features to uncover systemic determinacy in nAMD under anti-vascular endothelial growth factor intravitreal therapy (anti-VEGF IVT). It includes 46 nAMD patients stratified by the level of disease control under ongoing anti-VEGF treatment. Proteomic profiles in peripheral blood samples of every patient were detected with LC-MS/MS mass spectrometry. The patients underwent extensive clinical examination with a focus on macular function and morphology. In silico analysis includes unbiased dimensionality reduction and clustering, a subsequent annotation of clinical features, and non-linear models for recognition of underlying patterns. The model assessment was performed using leave-one-out cross validation. The findings provide an exploratory demonstration of the link between systemic proteomic signals and macular disease pattern using and validating non-linear classification models. Three main results were obtained: (1) Proteome-based clustering identifies two distinct patient subclusters with the smaller one (n = 10) exhibiting a strong signature for oxidative stress response. Matching the relevant meta-features on the individual patient's level identifies pulmonary dysfunction as an underlying health condition in these patients. (2) We identify biomarkers for nAMD disease features with Aldolase C as a putative factor associated with superior disease control under ongoing anti-VEGF treatment. (3) Apart from this, isolated protein markers are only weakly correlated with nAMD disease expression. In contrast, applying a non-linear classification model identifies complex molecular patterns hidden in a high number of proteomic dimensions determining macular disease expression. In conclusion, so far unconsidered systemic signals in the peripheral blood proteome contribute to the clinically observed phenotype of nAMD, which should be examined in future translational research on AMD.

Slit2-Robo Signaling Promotes Glomerular Vascularization and Nephron Development.

BACKGROUND: Kidney function requires continuous blood filtration by glomerular capillaries. Disruption of glomerular vascular development or maintenance contributes to the pathogenesis of kidney diseases, but the signaling events regulating renal endothelium development remain incompletely understood. Here, we discovered a novel role of Slit2-Robo signaling in glomerular vascularization. Slit2 is a secreted polypeptide that binds to transmembrane Robo receptors and regulates axon guidance as well as ureteric bud branching and angiogenesis. METHODS: We performed Slit2-alkaline phosphatase binding to kidney cryosections from mice with or without tamoxifen-inducible Slit2 or Robo1 and -2 deletions, and we characterized the phenotypes using immunohistochemistry, electron microscopy, and functional intravenous dye perfusion analysis. RESULTS: Only the glomerular endothelium, but no other renal endothelial compartment, responded to Slit2 in the developing kidney vasculature. Induced Slit2 gene deletion or Slit2 ligand trap at birth affected nephrogenesis and inhibited vascularization of developing glomeruli by reducing endothelial proliferation and migration, leading to defective cortical glomerular perfusion and abnormal podocyte differentiation. Global and endothelial-specific Robo deletion showed that both endothelial and epithelial Robo receptors contributed to glomerular vascularization. CONCLUSIONS: Our study provides new insights into the signaling pathways involved in glomerular vascular development and identifies Slit2 as a potential tool to enhance glomerular angiogenesis.

Specialized endothelial tip cells guide neuroretina vascularization and blood-retina-barrier formation.

Endothelial tip cells guiding tissue vascularization are primary targets for angiogenic therapies. Whether tip cells require differential signals to develop their complex branching patterns remained unknown. Here, we show that diving tip cells invading the mouse neuroretina (D-tip cells) are distinct from tip cells guiding the superficial retinal vascular plexus (S-tip cells). D-tip cells have a unique transcriptional signature, including high TGF-ß signaling, and they begin to acquire blood-retina barrier properties. Endothelial deletion of TGF-ß receptor I (Alk5) inhibits D-tip cell identity acquisition and deep vascular plexus formation. Loss of endothelial ALK5, but not of the canonical SMAD effectors, leads to aberrant contractile pericyte differentiation and hemorrhagic vascular malformations. Oxygen-induced retinopathy vasculature exhibits S-like tip cells, and Alk5 deletion impedes retina revascularization. Our data reveal stage-specific tip cell heterogeneity as a requirement for retinal vascular development and suggest that non-canonical-TGF-ß signaling could improve retinal revascularization and neural function in ischemic retinopathy.

Pathological angiogenesis in retinopathy engages cellular senescence and is amenable to therapeutic elimination via BCL-xL inhibition.

Attenuating pathological angiogenesis in diseases characterized by neovascularization such as diabetic retinopathy has transformed standards of care. Yet little is known about the molecular signatures discriminating physiological blood vessels from their diseased counterparts, leading to off-target effects of therapy. We demonstrate that in contrast to healthy blood vessels, pathological vessels engage pathways of cellular senescence. Senescent (p16INK4A-expressing) cells accumulate in retinas of patients with diabetic retinopathy and during peak destructive neovascularization in a mouse model of retinopathy. Using either genetic approaches that clear p16INK4A-expressing cells or small molecule inhibitors of the anti-apoptotic protein BCL-xL, we show that senolysis suppresses pathological angiogenesis. Single-cell analysis revealed that subsets of endothelial cells with senescence signatures and expressing Col1a1 are no longer detected in BCL-xL-inhibitor-treated retinas, yielding a retina conducive to physiological vascular repair. These findings provide mechanistic evidence supporting the development of BCL-xL inhibitors as potential treatments for neovascular retinal disease.

PRL-2 phosphatase is required for vascular morphogenesis and angiogenic signaling.

Protein tyrosine phosphatases are essential modulators of angiogenesis and have been identified as novel therapeutic targets in cancer and anti-angiogenesis. The roles of atypical Phosphatase of Regenerative Liver (PRL) phosphatases in this context remain poorly understood. Here, we investigate the biological function of PRL phosphatases in developmental angiogenesis in the postnatal mouse retina and in cell culture. We show that endothelial cells in the retina express PRL-2 encoded by the Ptp4a2 gene, and that inducible endothelial and global Ptp4a2 mutant mice exhibit defective retinal vascular outgrowth, arteriovenous differentiation, and sprouting angiogenesis. Mechanistically, PTP4A2 deletion limits angiogenesis by inhibiting endothelial cell migration and the VEGF-A, DLL-4/NOTCH-1 signaling pathway. This study reveals the importance of PRL-2 as a modulator of vascular development.

Endophilin-A2 dependent VEGFR2 endocytosis promotes sprouting angiogenesis.

Endothelial cell migration, proliferation and survival are triggered by VEGF-A activation of VEGFR2. However, how these cell behaviors are regulated individually is still unknown. Here we identify Endophilin-A2 (ENDOA2), a BAR-domain protein that orchestrates CLATHRIN-independent internalization, as a critical mediator of endothelial cell migration and sprouting angiogenesis. We show that EndoA2 knockout mice exhibit postnatal angiogenesis defects and impaired front-rear polarization of sprouting tip cells. ENDOA2 deficiency reduces VEGFR2 internalization and inhibits downstream activation of the signaling effector PAK but not ERK, thereby affecting front-rear polarity and migration but not proliferation or survival. Mechanistically, VEGFR2 is directed towards ENDOA2-mediated endocytosis by the SLIT2-ROBO pathway via SLIT-ROBO-GAP1 bridging of ENDOA2 and ROBO1. Blocking ENDOA2-mediated endothelial cell migration attenuates pathological angiogenesis in oxygen-induced retinopathy models. This work identifies a specific endocytic pathway controlling a subset of VEGFR2 mediated responses that could be targeted to prevent excessive sprouting angiogenesis in pathological conditions.

NCK-dependent pericyte migration promotes pathological neovascularization in ischemic retinopathy.

Pericytes are mural cells that surround capillaries and control angiogenesis and capillary barrier function. During sprouting angiogenesis, endothelial cell-derived platelet-derived growth factor-B (PDGF-B) regulates pericyte proliferation and migration via the platelet-derived growth factor receptor-ß (PDGFRß). PDGF-B overexpression has been associated with proliferative retinopathy, but the underlying mechanisms remain poorly understood. Here we show that abnormal, α-SMA-expressing pericytes cover angiogenic sprouts and pathological neovascular tufts (NVTs) in a mouse model of oxygen-induced retinopathy. Genetic lineage tracing demonstrates that pericytes acquire α-SMA expression during NVT formation. Pericyte depletion through inducible endothelial-specific knockout of Pdgf-b decreases NVT formation and impairs revascularization. Inactivation of the NCK1 and NCK2 adaptor proteins inhibits pericyte migration by preventing PDGF-B-induced phosphorylation of PDGFRß at Y1009 and PAK activation. Loss of Nck1 and Nck2 in mural cells prevents NVT formation and vascular leakage and promotes revascularization, suggesting PDGFRß-Y1009/NCK signaling as a potential target for the treatment of retinopathies.

Lacteal junction zippering protects against diet-induced obesity.

Excess dietary lipid uptake causes obesity, a major global health problem. Enterocyte-absorbed lipids are packaged into chylomicrons, which enter the bloodstream through intestinal lymphatic vessels called lacteals. Here, we show that preventing lacteal chylomicron uptake by inducible endothelial genetic deletion of Neuropilin1 (Nrp1) and Vascular endothelial growth factor receptor 1 (Vegfr1; also known as Flt1) renders mice resistant to diet-induced obesity. Absence of NRP1 and FLT1 receptors increased VEGF-A bioavailability and signaling through VEGFR2, inducing lacteal junction zippering and chylomicron malabsorption. Restoring permeable lacteal junctions by VEGFR2 and vascular endothelial (VE)-cadherin signaling inhibition rescued chylomicron transport in the mutant mice. Zippering of lacteal junctions by disassembly of cytoskeletal VE-cadherin anchors prevented chylomicron uptake in wild-type mice. These data suggest that lacteal junctions may be targets for preventing dietary fat uptake.

SMAD4 Prevents Flow Induced Arteriovenous Malformations by Inhibiting Casein Kinase 2.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is an inherited vascular disorder that causes arteriovenous malformations (AVMs). Mutations in the genes encoding Endoglin ( ENG) and activin-receptor-like kinase 1 ( AVCRL1 encoding ALK1) cause HHT type 1 and 2, respectively. Mutations in the SMAD4 gene are present in families with juvenile polyposis-HHT syndrome that involves AVMs. SMAD4 is a downstream effector of transforming growth factor-ß (TGFß)/bone morphogenetic protein (BMP) family ligands that signal via activin-like kinase receptors (ALKs). Ligand-neutralizing antibodies or inducible, endothelial-specific Alk1 deletion induce AVMs in mouse models as a result of increased PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B) signaling. Here we addressed if SMAD4 was required for BMP9-ALK1 effects on PI3K/AKT pathway activation. METHODS: The authors generated tamoxifen-inducible, postnatal, endothelial-specific Smad4 mutant mice ( Smad4iΔEC). RESULTS: We found that loss of endothelial Smad4 resulted in AVM formation and lethality. AVMs formed in regions with high blood flow in developing retinas and other tissues. Mechanistically, BMP9 signaling antagonized flow-induced AKT activation in an ALK1- and SMAD4-dependent manner. Smad4iΔEC endothelial cells in AVMs displayed increased PI3K/AKT signaling, and pharmacological PI3K inhibitors or endothelial Akt1 deletion both rescued AVM formation in Smad4iΔEC mice. BMP9-induced SMAD4 inhibited casein kinase 2 ( CK2) transcription, in turn limiting PTEN phosphorylation and AKT activation. Consequently, CK2 inhibition prevented AVM formation in Smad4iΔEC mice. CONCLUSIONS: Our study reveals SMAD4 as an essential effector of BMP9-10/ALK1 signaling that affects AVM pathogenesis via regulation of CK2 expression and PI3K/AKT1 activation.

FGF-dependent metabolic control of vascular development.

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are important to these processes. Although much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism, little is understood about the role of fibroblast growth factors (FGFs) in this context. Here we identify FGF receptor (FGFR) signalling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signalling inputs results in decreased glycolysis, leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/Fgfr3 double mutant mice, while HK2 overexpression partly rescues the defects caused by suppression of FGF signalling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development.

The Robo4 cytoplasmic domain is dispensable for vascular permeability and neovascularization.

Vascular permeability and neovascularization are implicated in many diseases including retinopathies and diabetic wound healing. Robo4 is an endothelial-specific transmembrane receptor that stabilizes the vasculature, as shown in Robo4-/- mice that develop hyperpermeability, but how Robo4 signals remained unclear. Here we show that Robo4 deletion enhances permeability and revascularization in oxygen-induced retinopathy (OIR) and accelerates cutaneous wound healing. To determine Robo4 signalling pathways, we generated transgenic mice expressing a truncated Robo4 lacking the cytoplasmic domain (Robo4ΔCD). Robo4ΔCD expression is sufficient to prevent permeability, and inhibits OIR revascularization and wound healing in Robo4-/- mice. Mechanistically, Robo4 does not affect Slit2 signalling, but Robo4 and Robo4ΔCD counteract Vegfr2-Y949 (Y951 in human VEGFR2) phosphorylation by signalling through the endothelial UNC5B receptor. We conclude that Robo4 inhibits angiogenesis and vessel permeability independently of its cytoplasmic domain, while activating VEGFR2-Y951 via ROBO4 inhibition might accelerate tissue revascularization in retinopathy of prematurity and in diabetic patients.

Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer.

The CXCL4 paralog CXCL4L1 is a less studied chemokine that has been suggested to exert an antiangiogenic function. However, CXCL4L1 is also expressed in patient tumors, tumor cell lines, and murine xenografts, prompting a more detailed analysis of its role in cancer pathogenesis. We used genetic and antibody-based approaches to attenuate CXCL4L1 in models of pancreatic ductal adenocarcinoma (PDAC). Mechanisms of expression were assessed in cell coculture experiments, murine, and avian xenotransplants, including through an evaluation of CpG methylation and mutation of critical CpG residues. CXCL4L1 gene expression was increased greatly in primary and metastatic PDAC. We found that myofibroblasts triggered cues in the tumor microenvironment, which led to induction of CXCL4L1 in tumor cells. CXCL4L1 expression was also controlled by epigenetic modifications at critical CpG islands, which were mapped. CXCL4L1 inhibited angiogenesis but also affected tumor development more directly, depending on the tumor cell type. In vivo administration of an mAb against CXCL4L1 demonstrated a blockade in the growth of tumors positive for CXCR3, a critical receptor for CXCL4 ligands. Our findings define a protumorigenic role in PDAC development for endogenous CXCL4L1, which is independent of its antiangiogenic function. Cancer Res; 76(22); 6507-19. ©2016 AACR.

PERK mediates the IRES-dependent translational activation of mRNAs encoding angiogenic growth factors after ischemic stress.

Angiogenesis is induced by various conditions, including hypoxia. Although cap-dependent translation is globally inhibited during ischemia, the mRNAs encoding two important proangiogenic growth factors, vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2), are translated at early time points in ischemic muscle. The translation of these mRNAs can occur through internal ribosome entry sites (IRESs), rather than through cap-dependent translation. Hypoxic conditions also induce the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, leading us to assess the interplay between hypoxia, ER stress, and IRES-mediated translation of FGF-2 and VEGF We found that unlike cap-dependent translation, translation through FGF-2 and VEGF IRESs was efficient in cells and transgenic mice subjected to ER stress-inducing stimuli. We identified PERK, a kinase that is activated by ER stress, as the driver of VEGF and FGF-2 IRES-mediated translation in cells and in mice expressing IRES-driven reporter genes and exposed to hypoxic stress. These results demonstrate the role of IRES-dependent translation in the induction of the proangiogenic factors VEGF and FGF-2 in response to acute hypoxic stress. Furthermore, the PERK pathway could be a viable pharmacological target to improve physiological responses to ischemic situations.

Targeting NCK-Mediated Endothelial Cell Front-Rear Polarity Inhibits Neovascularization.

BACKGROUND: Sprouting angiogenesis is a key process driving blood vessel growth in ischemic tissues and an important drug target in a number of diseases, including wet macular degeneration and wound healing. Endothelial cells forming the sprout must develop front-rear polarity to allow sprout extension. The adaptor proteins Nck1 and 2 are known regulators of cytoskeletal dynamics and polarity, but their function in angiogenesis is poorly understood. Here, we show that the Nck adaptors are required for endothelial cell front-rear polarity and migration downstream of the angiogenic growth factors VEGF-A and Slit2. METHODS AND RESULTS: Mice carrying inducible, endothelial-specific Nck1/2 deletions fail to develop front-rear polarized vessel sprouts and exhibit severe angiogenesis defects in the postnatal retina and during embryonic development. Inactivation of NCK1 and 2 inhibits polarity by preventing Cdc42 and Pak2 activation by VEGF-A and Slit2. Mechanistically, NCK binding to ROBO1 is required for both Slit2- and VEGF-induced front-rear polarity. Selective inhibition of polarized endothelial cell migration by targeting Nck1/2 prevents hypersprouting induced by Notch or Bmp signaling inhibition, and pathological ocular neovascularization and wound healing, as well. CONCLUSIONS: These data reveal a novel signal integration mechanism involving NCK1/2, ROBO1/2, and VEGFR2 that controls endothelial cell front-rear polarity during sprouting angiogenesis.

Glioblastoma invasion and cooption depend on IRE1α endoribonuclease activity.

IRE1α is an endoplasmic reticulum (ER)-resident transmembrane signaling protein and a cellular stress sensor. The protein harbors a cytosolic dual kinase/endoribonuclease activity required for adaptive responses to micro-environmental changes. In an orthotopic xenograft model of human glioma, invalidation of IRE1α RNase or/and kinase activities generated tumors with remarkably distinct phenotypes. Contrasting with the extensive angiogenesis observed in tumors derived from control cells, the double kinase/RNase invalidation reprogrammed mesenchymal differentiation of cancer cells and produced avascular and infiltrative glioblastomas with blood vessel co-option. In comparison, selective invalidation of IRE1α RNase did not compromise tumor angiogenesis but still elicited invasive features and vessel co-option. In vitro, IRE1α RNase deficient cells were also endowed with a higher ability to migrate. Constitutive activation of both enzymes led to wild-type-like lesions. The presence of IRE1α, but not its RNase activity, is therefore required for glioblastoma neovascularization, whereas invasion results only from RNase inhibition. In this model, two key mechanisms of tumor progression and cancer cell survival are functionally linked to IRE1α.

Alk1 and Alk5 inhibition by Nrp1 controls vascular sprouting downstream of Notch.

Sprouting angiogenesis drives blood vessel growth in healthy and diseased tissues. Vegf and Dll4/Notch signalling cooperate in a negative feedback loop that specifies endothelial tip and stalk cells to ensure adequate vessel branching and function. Current concepts posit that endothelial cells default to the tip-cell phenotype when Notch is inactive. Here we identify instead that the stalk-cell phenotype needs to be actively repressed to allow tip-cell formation. We show this is a key endothelial function of neuropilin-1 (Nrp1), which suppresses the stalk-cell phenotype by limiting Smad2/3 activation through Alk1 and Alk5. Notch downregulates Nrp1, thus relieving the inhibition of Alk1 and Alk5, thereby driving stalk-cell behaviour. Conceptually, our work shows that the heterogeneity between neighbouring endothelial cells established by the lateral feedback loop of Dll4/Notch utilizes Nrp1 levels as the pivot, which in turn establishes differential responsiveness to TGF-ß/BMP signalling.