Blood vessel formation during vertebrate development relies on a process called angiogenesis and is essential for organ growth and tissue viability. In addition, angiogenesis leads to pathological blood vessel growth in diseases with tissue ischaemia, such as neovascular eye disease and cancer. Neuropilin 1 (NRP1) is a transmembrane protein that serves as a receptor for the VEGF165 isoform of the vascular endothelial growth factor (VEGF) to enhance cell migration during angiogenesis via VEGF receptor 2 (VEGFR2), and it is also essential for VEGF-induced vascular permeability and arteriogenesis. In addition, NRP1 activation affects angiogenesis independently of VEGF signalling by activating the intracellular kinase ABL1. NRP1 also acts as a receptor for the class 3 semaphorin (SEMA3A) to regulate vessel maturation during tumour angiogenesis and vascular permeability in eye disease. In the present paper, we review current knowledge of NRP1 regulation during angiogenesis and vascular pathology.
- endothelial cell
- vascular endothelial growth factor
- vascular permeability
The cardiovascular system is the first organ system to develop during embryogenesis in vertebrates. Vasculogenesis enables the creation of new blood vessels from angioblasts, whereas angiogenesis through vessel sprouting subsequently expands these blood vessels into a vast network capable of sustaining tissue metabolism. Angiogenic vessel sprouts are composed of endothelial tip cells that lead the growing sprouts and endothelial stalk cells that form the lumen and proliferate (e.g. ). Under physiological conditions, angiogenesis occurs during embryonic and perinatal development. In contrast, the endothelium is usually quiescent in adults and becomes proliferative again only in specific circumstances, for example in the cycling uterus and during pregnancy, or during wound healing and other pathological conditions (reviewed in [2,3]). For example, in diseases with tissue ischaemia, such as diabetic retinopathy, wet age-related macular degeneration or cancer, hypoxic cells trigger the formation of new blood vessels to increase their supply of nutrients and oxygen, which typically involves up-regulation of the vascular endothelial growth factor (VEGF) . However, an excess of VEGF also increases vascular permeability to pathological levels, and VEGF-A was indeed first identified as a vascular permeability factor. Although endothelial permeability can be beneficial for the extravasation of antibodies and immune cell trafficking into damaged tissues, excessive serum leak causes oedema and that may disrupt tissue function.
Domain structure of NRP1 and NRP2
The neuropilins NRP1 and NRP2 are single pass transmembrane glycoproteins with a large extracellular domain of 840 amino acid residues, a short transmembrane domain and a cytoplasmic tail of 40 residues (reviewed in ). NRP1 is thought to lack catalytic activity, but its intracellular domain interacts with an adaptor protein named synectin or GIPC1 (reviewed in ) to promote endocytic trafficking [5–7]. Initially, NRP1 expression was discovered in neurons as an adhesion receptor , but its specific angiogenic adhesion ligands have not yet been identified. Subsequently, NRP1 was shown to be a receptor for the class 3 semaphorin (SEMA3A), a secreted glycoprotein that regulates axon guidance, and then as a receptor for an isoform of VEGF termed VEGF165 (reviewed in ). NRP2 has 44% homology with NRP1, a similar domain organization and binding sites for SEMA3F and VEGF145 (reviewed in ). The role of NRP1 and its ligands in angiogenesis is discussed in detail below.
Essential role for NRP1 in developmental angiogenesis
During embryonic development and in the early postnatal period, NRP1 is prominent on developing blood vessels, for example in the hindbrain and retina [8,9]. A role for NRP1 in vasculature was initially identified through its transgenic overexpression in mice, which increased the number of capillaries, but also caused haemorrhages . Subsequently, loss of NRP1 was shown to impair vessel spouting into the brain and spinal cord of mice [11–13]. Similarly, knockdown of either of two zebrafish NRP1 homologues (Nrp1a or Nrp1b) was reported to impair vessel sprouting and arteriovenous patterning [14–16].
During angiogenesis, NRP1 is not only expressed in endothelial cells, but also in other cell types; for example, neural progenitors and tissue macrophages express NRP1 alongside sprouting vessels in the mouse embryo hindbrain [11,17]. The brain vessel defects of NRP1 knockouts might therefore be due to an additive effect of NRP1 loss from both endothelial and non-endothelial cells. However, experiments using Cre-LoxP technology in mice to create cell-type-specific Nrp1-null mutants revealed that NRP1 is exclusively required in endothelial rather than non-endothelial cells for brain angiogenesis [8,11]. Interestingly, incomplete endothelial Cre-LoxP recombination in these experiments resulted in mosaic hindbrain vessels containing both NRP1-positive and NRP1-negative endothelial cells, with NRP1-retaining cells being enriched in the tip, but not stalk cell position . These observations predict a role for NRP1 during sprouting angiogenesis in endothelial cell migration, as this is a tip cell function, but not proliferation, which is a stalk cell function. In agreement with this idea, NRP1 is not essential for endothelial cell proliferation in the developing yolk sac, even though it is required for yolk sac angiogenesis, at least in C57/Bl6 mice . Moreover, NRP1 has been shown to promote both the VEGF- and extracellular matrix (ECM)-stimulated migration of endothelial cells (e.g. [19,20]; see below). In addition to its role in developmental angiogenesis, NRP1 has been implicated in tumour angiogenesis, with NRP1 expression reported for tumour vasculature and cancer cells (reviewed in ).
NRP1 ligands in angiogenesis: SEMA3A
Even though SEMA3A can bind NRP1 on embryonic blood vessels, mice lacking SEMA3A or semaphorin signalling through both neuropilins have apparently normal developmental angiogenesis (e.g. [22,23]). Moreover, mice lacking both SEMA3A and VEGF165 have similar defects in vascular patterning to mice lacking VEGF165 only . These findings suggest that SEMA3A and semaphorin signalling through neuropilins are not essential for angiogenesis in mouse embryos [22,24]. In contrast, exogenous SEMA3A disrupts VEGFA-induced angiogenesis in the chick chorioallantoic membrane assay by inhibiting the activation of focal adhesion kinase (FAK) and the cellular homologue of v-Src (c-Src) . Knockdown of Sema3a1 also impairs vascular development in zebrafish embryos [26,27]. The reasons that SEMA3A signalling through NRP1 regulates vascular development in fish and chick, but not mouse, are presently unknown.
Despite being dispensable for developmental angiogenesis, SEMA3A regulates pathological angiogenesis in mice, for example in mice with oxygen-induced retinopathy (OIR). In this model, neonatal mice are exposed to a high oxygen atmosphere for 5 days and then returned to normoxia; hyperoxia causes vessel regression in the retina, and the resulting vasculature is unable to sustain retinal metabolism on return to normoxia, causing inflammation and the up-regulation of excessive and abnormal vessel growth. SEMA3A is secreted from neurons in the avascular retina in response to the cytokine interleukin 1β (IL-1β) and creates a repulsive barrier that forces sprouting vessels to grow ectopically towards the vitreous; in agreement, endothelial cells treated with conditioned medium from hypoxic retinal ganglion cells (RGC), which secrete SEMA3A, show cytoskeletal rearrangements and loss of stress fibres . SEMA3A inhibition enables normal neovascularization within the hypoxic retina in this model, promoting regeneration of neural tissue and improving retinal function .
SEMA3A also regulates tumour angiogenesis in cancer models. Initially, SEMA3A expression induces endothelial cell apoptosis, which correlates with inhibition of tumour angiogenesis and cancer growth, but SEMA3A subsequently supports vascular normalization by promoting pericyte coverage of tumour vessels and reducing vessel leakiness [29,30]. SEMA3A can also indirectly induce the maturation of tumour vessels by recruiting NRP1-expressing monocytes, which then secrete growth factors such as transforming growth factor β (TGFβ) and platelet-derived growth factor β (PDGFβ) to attract pericytes involved in vessel maturation [31,32].
NRP1 ligands in angiogenesis: VEGF165
In vitro studies showed that VEGF165 binding to NRP1 enhances VEGF receptor 2 (VEGFR2) signalling to increase endothelial cell proliferation and migration. To test the relevance of VEGF binding to NRP1 for angiogenesis, knockin mice expressing NRP1 carrying a Tyr297 mutation were generated . This mutation had previously been shown to disrupt VEGF binding to NRP1 . Unexpectedly, the Nrp1Y297A/Y297A mice lacking VEGF binding to NRP1 had much milder angiogenesis defects than full NRP1 knockouts or endothelium-specific NRP1 knockouts . In fact, the knockin Nrp1Y297A mutation additionally caused a severe reduction in NRP1 expression, and it is therefore likely that the mild embryonic angiogenesis defects of Nrp1Y297A/Y297A mouse embryos are at least partly caused by low NRP1 expression levels rather than defective VEGF binding to NRP1 .
The absence of severe angiogenesis defects in Nrp1Y297A/Y297A embryos agreed with prior findings in Vegfa120/120 mouse embryos, which lack VEGF164 because they express only the VEGF120 isoform. Thus the Vegfa120/120 mutation causes a milder reduction in vessel branching together with increased vascular diameter in embryonic day (E) 12.5 mouse brain, rather than the near complete loss of vessel branching seen in Nrp1-null mutants [12,35]. In fact, the vascular defects in Vegfa120/120 mouse embryo hindbrains are more likely caused by changes in the extracellular localization of different VEGF isoforms rather than isoform-specific signalling through NRP1. Thus VEGF120 lacks the domains that confer extracellular matrix binding in VEGF165 and the larger VEGF189 isoform, but are important to establish VEGF gradients for chemotaxis and vessel sprouting [1,35].
Even though Nrp1Y297A/Y297A mice have only mild defects in embryonic angiogenesis, they have more severe vessel defects in the perinatal retina and heart , suggesting a role in postnatal vascular development. Moreover, VEGF165 binding to NRP1 may be important for pathological neovascularization, as the OIR response of mouse pups is attenuated in Nrp1Y297A/Y297A mice and their tumour growth is delayed . In summary, these findings suggest that VEGF165 binding to NRP1 is largely dispensable for embryonic angiogenesis, but may be important for postnatal developmental and pathological angiogenesis. The analysis of vascular defects in Nrp1Y297A/Y297A mice and the comparison of Nrp1−/− and Vegfa120/120 mice suggested that NRP1 additionally functions in angiogenesis in a VEGF-independent pathway. In agreement, we recently reported that NRP1 promotes angiogenesis driven by ECM signalling  (see below).
NRP1 signalling in endothelial cells: association with co-receptors
Because NRP1 is a non-catalytic transmembrane protein, it interacts with co-receptors to transduce downstream signals. Thus, in neurons, NRP1 associates with plexins to transduce semaphorin signals (reviewed in ), whereas in endothelial cells, NRP1 interacts with VEGFR1 or VEGFR2 . In circumstances where endothelial cells respond to class 3 semaphorins, it is therefore expected that a plexin co-receptor should be involved in signal transduction. This has, for example, been proposed for SEMA3A/NRP1-mediated vascular permeability signalling . Interestingly, NRP1 can also form a tripartite complex with plexin D1 (PLXND1) and VEGFR2 to transduce semaphorin signals in neurons , but such a complex has not yet been demonstrated in endothelial cells. In VEGF165-stimulated endothelial cells, instead, NRP1 is thought to preferentially complex with VEGFR2 via a VEGF165 bridge.
Conceptually, VEGF165-bound NRP1 may interact with VEGFR2 in cis, when the same endothelial cell co-expresses both receptors, or in trans, when one endothelial cell expresses VEGFR2 and another endothelial or non-endothelial cell expresses NRP1. Accordingly, it has been suggested that endothelial VEGFR2 interacts with NRP1 on tumour cells in trans . A recent study with porcine aortic endothelial (PAE) cells showed that NRP1–VEGFR2 trans interaction decreases the activation of endothelial VEGFR2 and prevents VEGFR2 endocytosis and suppresses tumour angiogenesis; in contrast, cis interaction induces rapid NRP1–VEGFR2 complex formation and initiation of signal transduction through phospholipase Cγ (PLCγ) and extracellular-signal-regulated kinase (ERK) . Moreover, this pathway was important for tumour initiation by regulating the early steps in tumour vascularization, but was not important at the later stages of tumour vascularization . In contrast, our recent work in the mouse embryo hindbrain angiogenesis excluded the possibility of a trans interaction between non-endothelial NRP1 and endothelial VEGFR2 in developmental angiogenesis; thus hindbrain vessels express VEGFR2, but loss of NRP1 function from neural progenitors or macrophages did not impair angiogenesis in this organ (see above; ).
Although VEGF165 is the main VEGF isoform that binds NRP1, VEGF121 can also bind NRP1, at least in vitro and with much lower affinity [40,41]. Unlike VEGF165, VEGF121 binding to NRP1 cannot induce the formation of an extracellular bridge between NRP1 and VEGFR2 to enhance VEGFR2 signal transduction (e.g. [36,38,42,43]). Moreover, the physiological or pathological significance of low-affinity VEGF121 binding to NRP1 is unknown. Importantly, expression of VEGF120 at the expense of VEGF164 in mice causes similar defects as loss of NRP1 in the nervous system (e.g. ), arguing against an essential physiological role for VEGF120 binding to NRP1 in mice.
NRP1 also interacts with integrins in vitro, including the β1 and β3 integrin subunits [45,46]. The interaction of NRP1 with αvβ3 integrin negatively regulates VEGF-mediated angiogenesis by limiting the NRP1 and VEGFR2 interaction, as demonstrated in endothelial cell wound closure assay in vitro, aortic ring microvessel sprouting ex vivo and growth factor-induced angiogenesis in β3-null mice . Indeed, the combined effect of β3 inhibition and NRP1 blockade reduces VEGF-mediated angiogenesis more than inhibiting each molecule individually . Nevertheless, integrin signalling is generally thought to stimulate cell migration and is therefore considered pro-angiogenic. In agreement, we have shown that the integrin ligand fibronectin promotes actin remodelling and endothelial cell migration in an NRP1-dependent mechanism . It is not yet known whether NRP1 interacts with integrins directly to promote these processes, or if they occur indirectly via interaction with other proteins. Yet it is clear that fibronectin-stimulated NRP1 signalling promotes angiogenesis independently of VEGF165 and VEGFR2 .
NRP1 associates with the cytoplasmic adaptor synectin to regulate VEGFR2 trafficking
In vitro studies suggested that the NRP1 cytoplasmic tail and its interactor synectin are required for complex formation between NRP1 and VEGFR2 . To understand the mechanistic role of the NRP1 interaction with VEGFR2, porcine aortic endothelial cells lacking endogenous VEGFR2 expression were co-transfected with expression vectors for VEGFR2 and several different, fluorophore-linked RAB proteins; the analysis of co-localization of the over-expressed proteins showed that synectin binding to the NRP1 cytoplasmic tail promotes VEGF165-stimulated VEGFR2 trafficking into different subsets of vesicles distinguished by specific RAB proteins . In agreement, cultured arterial endothelial cells from mice lacking the NRP1 cytoplasmic tail showed an enrichment of VEGFR2 in RAB5+ endosomes and decreased entry of VEGFR2 into EAA1+ endosomes after VEGF165 stimulation, and this defect was associated with reduced ERK signalling . This NRP1-cytoplasmic tail-dependent pathway was essential for the formation of a normal number of arterioles in several different organs examined . In contrast, the cytoplasmic NRP1 tail is dispensable for both developmental and pathological angiogenesis in mice [7,9].
Although the NRP1 cytoplasmic tail is dispensable for angiogenesis in mice , different results have been obtained in zebrafish studies. Thus NRP1 lacking the SEA (Ser-Glu-Ala) motif of the cytoplasmic domain  cannot rescue the defective dorsal migration of intersomitic vessels after Nrp1 knockdown, and knockdown of synectin causes similar defects . It has not been resolved why intersomitic sprouting in fish might be compromised by loss of the NRP1 cytoplasmic tail, but appears normal in mice. One possibility may be that these vessels in the fish have a stronger arterial character and/or depend on specific VEGFR2 trafficking pathways that are regulated by NRP1.
VEGFR2-independent NRP1 signalling in endothelial cells
Tissue culture studies have suggested that NRP1 promotes cell motility independently of VEGFR2. For example, fusion of the extracellular domain of the epidermal growth factor (EGF) receptor to the transmembrane and cytoplasmic domains of NRP1 creates a chimaeric receptor that promotes the migration of EGF-stimulated endothelial cells . Moreover, NRP1 also modulates ECM interactions independently of VEGFR2 [46,50], for example in angiogenesis  (Figure 1). Specifically, our in vitro studies showed that NRP1 promotes ECM-induced endothelial cell migration and actin remodelling in a VEGFR2-independent mechanism that involves ABL1-dependent phosphorylation of the focal adhesion component paxillin. Moreover, blockade of ABL1/2 signalling impaired both physiological and pathological angiogenesis in the perinatal retina in vivo. A dual role for NRP1 in the ECM-stimulated signalling via ABL1 and VEGF-induced VEGFR2 signalling pathway angiogenesis may explain why the Nrp1Y297A/Y297A mice have milder vascular defects than full NRP1 knockouts .
NRP1 operates in multiple signalling pathways that regulate blood vessel growth, both during normal development and in pathological conditions. Thus stimulating NRP1 signalling may be beneficial for therapeutic angiogenesis to improve the delivery of oxygen and nutrients to ischaemic tissues, whereas NRP1 blockade may provide an anti-angiogenic strategy, for example to curb tumour angiogenesis or ocular neovascularization.
C.R. is supported by a Wellcome Trust Investigator Award [grant number 095623/Z/11/Z]; and A.L. by a Ph.D. studentship from the British Heart Foundation [grant number FS/13/60/30457].
We apologize to all colleagues whose relevant work is not cited owing to space constraints.
Angiogenesis and Vascular Remodelling: New Perspectives: A Biochemical Society Focused Meeting held at University of Chester, U.K., 14–16 July 2014. Organized and Edited by Roy Bicknell (Birmingham University Medical School, U.K.), Michael Cross (University of Liverpool, U.K.), Stuart Egginton (University of Leeds, U.K.), Victoria Heath (University of Birmingham, U.K.) and Ian Zachary (University College London, U.K.).
Abbreviations: ECM, extracellular matrix; EGF, epidermal growth factor; ERK, extracellular-signal-regulated kinase; NRP1, neuropilin 1; OIR, oxygen-induced retinopathy; SEMA3A, semaphorin 3A; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor
- © The Authors Journal compilation © 2014 Biochemical Society