Blood vessel networks expand in a 2-step process that begins with vessel sprouting and is followed by vessel anastomosis. Vessel sprouting is induced by chemotactic gradients of the vascular endothelial growth factor (VEGF), which stimulates tip cell protrusion. Yet it is not known which factors promote the fusion of neighboring tip cells to add new circuits to the existing vessel network. By combining the analysis of mouse mutants defective in macrophage development or VEGF signaling with live imaging in zebrafish, we now show that macrophages promote tip cell fusion downstream of VEGF-mediated tip cell induction. Macrophages therefore play a hitherto unidentified and unexpected role as vascular fusion cells. Moreover, we show that there are striking molecular similarities between the pro-angiogenic tissue macrophages essential for vascular development and those that promote the angiogenic switch in cancer, including the expression of the cell-surface proteins TIE2 and NRP1. Our findings suggest that tissue macrophages are a target for antiangiogenic therapies, but that they could equally well be exploited to stimulate tissue vascularization in ischemic disease. (Blood. 2010;116(5): 829-840) IntroductionBlood vessels are essential for tissue homeostasis in all vertebrates, and new vessel growth, termed neo-angiogenesis, is therefore a critical process in wound repair to counter tissue ischemia. Undesirably, neo-angiogenesis also promotes the expansion of tumors. Moreover, nonproductive neo-angiogenesis, which fails to restore oxygenation of ischemic tissues, promotes disease progression in, for example, diabetic retinopathy. Much current research is therefore focused on the identification of molecular and cellular targets for either pro-or antiangiogenic therapies. We previously elucidated the mechanism by which alternative splice forms of the vascular endothelial growth factor (VEGF) cooperate to promote blood vessel growth. 1,2 This work led to the current model of angiogenesis, in which blood vessel endothelium specializes into tip and stalk cells to promote vascular network expansion by sprouting growth. While the stalk cells form a lumen to transport blood, the tip cells extend filopodia to detect chemotactic growth factor gradients, which are formed by a combination of VEGF isoforms with a differential affinity for the extracellular matrix. Cooperating with VEGF, notch-delta signaling controls the balance of tip versus stalk cell specialization. 3 Even though much progress has been made in elucidating the mechanism of vascular sprout induction and guidance, a fundamental yet unanswered problem is which mechanism promotes the fusion of nascent vessel sprouts to add new circuits to the existing plexus.Macrophages promote pathologic angiogenesis in several diseases. Thus, circulating bone marrow-derived cells differentiate into proangiogenic cells with macrophage characteristics at adult sites of VEGF expression 4 and are recruited to growing tumors to promote tumor vascularization and therefore progression....

SummaryDuring development, the axons of retinal ganglion cell (RGC) neurons must decide whether to cross or avoid the midline at the optic chiasm to project to targets on both sides of the brain. By combining genetic analyses with in vitro assays, we show that neuropilin 1 (NRP1) promotes contralateral RGC projection in mammals. Unexpectedly, the NRP1 ligand involved is not an axon guidance cue of the class 3 semaphorin family, but VEGF164, the neuropilin-binding isoform of the classical vascular growth factor VEGF-A. VEGF164 is expressed at the chiasm midline and is required for normal contralateral growth in vivo. In outgrowth and growth cone turning assays, VEGF164 acts directly on NRP1-expressing contralateral RGCs to provide growth-promoting and chemoattractive signals. These findings have identified a permissive midline signal for axons at the chiasm midline and provide in vivo evidence that VEGF-A is an essential axon guidance cue.

The earliest blood vessels in the mammalian embryo are formed when endothelial cells (ECs) differentiate from angioblasts and coalesce into tubular networks. Thereafter, the endothelium is thought to expand solely by proliferation of pre-existing ECs. Here we show that the earliest precursors of erythrocytes, megakaryocytes and macrophages, the yolk sac-derived erythro-myeloid progenitors (EMPs), provide a complementary source of ECs that are recruited into pre-existing vasculature. Whereas a first wave of yolk sac-resident EMPs contributes ECs to the yolk sac endothelium, a second wave of EMPs colonises the embryo and contributes ECs to intraembryonic endothelium in multiple organs, where they persist into adulthood. By demonstrating that EMPs constitute a hitherto unrecognised source of ECs, we reveal that embryonic blood vascular endothelium expands in a dual mechanism that involves both the proliferation of pre-existing ECs and the incorporation of ECs derived from hematopoietic precursors.

Key Points NRP1 promotes brain angiogenesis cell autonomously in endothelium, independently of heterotypic interactions with nonendothelial cells. NRP1 plays a key role in endothelial tip rather than stalk cells during vessel sprouting in the brain.

Neuropilin 1 regulates angiogenesis in a VEGF-independent manner via association with ABL1, suggesting that Imatinib represents a novel opportunity for anti-angiogenic therapy.

Neuropilin 1 (NRP1) is a receptor for class 3 semaphorins and vascular endothelial growth factor (VEGF) A and is essential for cardiovascular development. Biochemical evidence supports a model for NRP1 function in which VEGF binding induces complex formation between NRP1 and VEGFR2 to enhance endothelial VEGF signalling. However, the relevance of VEGF binding to NRP1 for angiogenesis in vivo has not yet been examined. We therefore generated knock-in mice expressing Nrp1 with a mutation of tyrosine (Y) 297 in the VEGF binding pocket of the NRP1 b1 domain, as this residue was previously shown to be important for high affinity VEGF binding and NRP1-VEGFR2 complex formation. Unexpectedly, this targeting strategy also severely reduced NRP1 expression and therefore generated a NRP1 hypomorph. Despite the loss of VEGF binding and attenuated NRP1 expression, homozygous Nrp1 Y297A/Y297A mice were born at normal Mendelian ratios, arguing against NRP1 functioning exclusively as a VEGF 164 receptor in embryonic angiogenesis. By overcoming the mid-gestation lethality of full Nrp1-null mice, homozygous Nrp1 Y297A/Y297A mice revealed essential roles for NRP1 in postnatal angiogenesis and arteriogenesis in the heart and retina, pathological neovascularisation of the retina and angiogenesis-dependent tumour growth. KEY WORDS: NRP1, VEGF, Angiogenesis, Arteriogenesis, Retina, Hindbrain INTRODUCTIONNRP1 is a transmembrane receptor for the VEGF 165 isoform (VEGF 164 in mice) and the neuronal guidance cue SEMA3A, with essential roles in both vascular and neuronal development (reviewed by Pellet-Many et al., 2008;Raimondi and Ruhrberg, 2013). Accordingly, Nrp1-null mice die before birth with severe cardiovascular and neuronal defects (Kitsukawa et al., 1997;Kawasaki et al., 1999 Centre for Cardiovascular Biology and Medicine, BHF Laboratories, Division of Medicine, University College London, 5 University Street, London WC1E 6JJ, UK. *These authors contributed equally to this work ‡ These authors contributed equally to this work § Authors for correspondence (I.Zachary@ucl.ac.uk; c.ruhrberg@ucl.ac.uk) This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.Received 25 August 2013; Accepted 3 November 2013 whereas mice carrying a mutated extracellular domain that abolishes SEMA3A, not VEGF 164 , binding show defective nerve, but not blood vessel, patterning (Gu et al., 2003;Vieira et al., 2007). These and other genetic, biochemical and cell biological data support a model in which VEGF 165 binding induces complex formation between NRP1 and VEGFR2 (KDR -Mouse Genome Informatics) to enhance VEGFR2 signalling during EC migration in vitro (e.g. Soker et al., 2002;Wang et al., 2003;Evans et al., 2011) and arteriogenesis in vivo (Lanahan et al., 2013).The extracellular NRP1 a1/a2 and b1/b2 domains are crucial f...

Neuropilin 1 (NRP1) is a transmembrane glycoprotein that is essential for blood vessel development in vertebrates. Best known for its ability to bind members of the vascular endothelial growth factor (VEGF) and class 3 semaphorin families through its extracellular domain, it also has a highly conserved cytoplasmic domain, which terminates in a SEA motif that binds the PDZ protein synectin/GIPC1/NIP. Previous studies in zebrafish embryos and tissue culture models raised the possibility that the SEA motif of NRP1 is essential for angiogenesis. Here, we describe the generation of mice that express a form of NRP1 that lacks the cytoplasmic domain and, therefore, the SEA motif (Nrp1cytoΔ/Δ mice). Our analysis of pre- and perinatal vascular development revealed that vasculogenesis and angiogenesis proceed normally in these mutants, demonstrating that the membrane-anchored extracellular domain is sufficient for vessel growth. By contrast, the NRP1 cytoplasmic domain is required for normal arteriovenous patterning, because arteries and veins crossed each other at an abnormally high frequency in the Nrp1cytoΔ/Δ retina, as previously reported for mice with haploinsufficient expression of VEGF in neural progenitors. At crossing sites, the artery was positioned anteriorly to the vein, and both vessels were embedded in a shared collagen sleeve. In human eyes, similar arteriovenous crossings are risk factors for branch retinal vein occlusion (BRVO), an eye disease in which compression of the vein by the artery disrupts retinal blood flow, causing local tissue hypoxia and impairing vision. Nrp1cytoΔ/Δ mice may therefore provide a suitable genetic model to study the aetiology of BRVO.

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