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Originally published In Press as doi:10.1074/jbc.M703554200 on June 16, 2007

J. Biol. Chem., Vol. 282, Issue 33, 24049-24056, August 17, 2007
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Neuropilin-1 Binds to VEGF121 and Regulates Endothelial Cell Migration and Sprouting*

Qi Pan{ddagger}, Yvan Chathery{ddagger}1, Yan Wu§, Nisha Rathore, Raymond K. Tong||, Franklin Peale, Anil Bagri{ddagger}, Marc Tessier-Lavigne**, Alexander W. Koch||2, and Ryan J. Watts{ddagger}3

From the Departments of {ddagger}Tumor Biology and Angiogenesis, §Antibody Engineering, Pathology, ||Protein Chemistry, and **Research Drug Discovery, Genentech, Inc., South San Francisco, California 94080

Received for publication, April 30, 2007 , and in revised form, June 11, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Neuropilin-1 (NRP1) was first described as a receptor for the axon guidance molecule, Semaphorin3A, regulating the development of the nervous system. It was later shown that NRP1 is an isoform-specific receptor for vascular endothelial growth factor (VEGF), specifically VEGF165. Much interest has been placed on the role of the various VEGF isoforms in vascular biology. Here we report that blocking NRP1 function, using a recently described antibody that inhibits VEGF165 binding to NRP1, surprisingly reduces VEGF121-induced migration and sprout formation of endothelial cells. Intrigued by this observation, direct binding studies of NRP1 to various VEGF isoforms were performed. We show that VEGF121 binds directly to NRP1; however, unlike VEGF165, VEGF121 is not sufficient to bridge the NRP1·VEGFR2 complex. Additionally, we show that VEGFR2 enhances VEGF165, but not VEGF121 binding to NRP1. We propose a new model for NRP1 interactions with various VEGF isoforms.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiogenesis, the formation of new blood vessels from existing vessels, plays a central role in a variety of physiological and pathological processes, including embryonic development and tumor growth (1, 2). Vascular endothelial growth factor (VEGF)4 is a key regulator of normal and pathologic angiogenesis. It plays an essential role in the specification, morphogenesis, differentiation, and homeostasis of vessels by regulating the proliferation, migration, and survival of endothelial cells (3). Inactivation of one allele of the Vegf gene leads to embryonic lethality at midgestation from severe cardiovascular defects (4, 5), indicating a tight dose-dependent regulation of embryonic vessel development by VEGF. Inhibiting angiogenesis by blocking VEGF function is also a promising strategy for the treatment of solid tumor cancer and several other diseases of excess vascularization, including age-related macular degeneration (6).

VEGF is encoded by a single gene found on chromosome 6 in humans, which contains eight exons (7, 8). The VEGF messenger RNA undergoes alternative splicing to give rise to at least six isoforms, containing 121, 145, 165, 183, 189, and 206 amino acids, respectively (7-11). All VEGF isoforms are secreted as covalently linked homodimers. Isoforms differ in the presence and absence of sequences in exons 6 and 7, which have been shown to encode heparin-binding domains of the protein and are thought to be responsible for the sequestration of VEGF to ECM (8, 12). Thus, different VEGF isoforms distribute differentially in the environment of a VEGF-secreting cell, depending on the presence or absence of the heparin-binding domain. Among the most prevalent VEGF isoforms, VEGF121 is soluble, whereas VEGF189 is bound to the cell surface or tethered to ECM, and VEGF165, which lacks one of the two heparin-binding domains in VEGF189, has intermediate distribution (13). It is proposed that differential VEGF isoform localization in the extracellular space controls vascular branching pattern, with the heparin-binding isoforms providing spatially restricted stimulatory cues to initiate branch formation (14).

VEGF can bind to two receptor tyrosine kinases to induce signal transduction: VEGFR1 and VEGFR2. They are both characterized by seven extracellular Ig-like domains followed by a membrane-spanning region and a conserved intracellular tyrosine kinase domain interrupted by a kinase insert domain and are activated by ligand-triggered dimerization (15-17). Although VEGFR1 has the greater affinity for VEGF, VEGFR2 is tyrosine-phosphorylated much more efficiently upon ligand binding and is thought to be the major receptor in endothelial cells for VEGF-induced responses (18, 19).

A third receptor, neuropilin-1 (NRP1), was identified as an isoform-specific receptor for VEGF recognizing the exon-7-encoded domain of VEGF, and therefore binds VEGF165, but not VEGF121 (20, 21). NRP1 was originally characterized as a semaphorin receptor regulating axon guidance (22, 23). It was later demonstrated in genetic studies that NRP1 is also important for vascular morphogenesis. These data showed that Nrp1-/- mice die at embryonic day 10.5-12.5 from cardiovascular anomalies (24). Since the cytoplasmic domain of NRP1 is short, it is generally thought that NRP1 has to couple with other signaling proteins to function. However, how NRP1 acts to regulate endothelial cell function is unclear. It is proposed that NRP1 acts as a co-receptor for VEGF165, enhancing VEGF binding to VEGFR2, and thus increases VEGFR2 signaling (21). The extracellular domain of VEGFR2 does not interact directly with NRP1 (25), and instead VEGFR2·NRP1 complexes are formed due to bridging, when VEGF165 binds to both receptors simultaneously (26). In addition, there is evidence that NRP1 may signal independently of VEGFR2 to regulate endothelial cell function. In one study, a chimeric NRP1 receptor, with an EGF-binding extracellular domain, caused migration of endothelial cells in response to EGF (27). In another study, NRP1 was shown to regulate endothelial cell adhesion to ECM proteins independently of VEGFR2 (28).

Here, we present evidence that VEGF121 interacts directly with NRP1 and may directly induce NRP1-mediated signaling events. First, a monoclonal antibody against NRP1, which blocks its interaction with VEGF165 (29), inhibits VEGF121-induced endothelial cell (EC) migration and sprouting. Second, NRP1 interacts directly with VEGF121 in vitro, but a cleaved form of VEGF, VEGF109, does not bind to NRP1. Third, in contrast to VEGF165, VEGF121 does not induce the formation of a VEGFR2·NRP1 complex, suggesting that VEGF121 may signal through NRP1 independently of VEGFR2. Finally, we present data showing that NRP1 qualitatively enhances VEGF165 binding to VEGFR2, an effect not seen with VEGF121. Based on these findings, we present a model for NRP1 interactions with various VEGF isoforms that serves as a foundation to further understand the role of NRP1 in VEGF-mediated processes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—VEGF165 and sVEGFR2 was from R & D Systems (293-VE-010/CF and 357-KD-050/CF, respectively). Except for the materials used for Figs. 2D and 4C, VEGF121 was purchased from PeproTech (100-20A). Additional VEGF121 samples were from Cellsciences (CRV001B and CRV010B) and Calbiochem (676473). VEGF112 was from R & D Systems (298-VS-025/CF). VEGF109 was produced at Genentech, Inc. (25). Human umbilical vein endothelial cells (HUVECs) were purchased from Cambrex and cultured in EGM-2 medium (Cambrex).

Protein Purification—Human soluble neuropilin-1 constructs (sNRP1s, which contain amino acids Met-1 to Ser-641, corresponding to neuropilin ECD without the membrane proximal MAM domain) were cloned into the expression vector pRK5 either fused to a C-terminal histidine tag or to the Fc portion of human IgG1 to facilitate affinity purification. Proteins were produced by transient transfection of Chinese hamster ovary (CHO) cells using DMRIE-C (Invitrogen). sNRP1-Fc was purified to >95% purity by affinity chromatography using protein A-Sepharose (GE Healthcare). sNRP1-His was purified to >95% purity by affinity chromatography using Ni2+-nitrilo-triacetic acid Superflow (Qiagen) followed by a cation exchange chromatography step (SP-Sepharose, GE Healthcare). Protein identities were confirmed by N-terminal sequencing using the Edman degradation method. Concentrations were determined by the BCA assay and by A280 absorption measurements. Purity and homogeneity were assessed by SDS-PAGE, size exclusion chromatography, and laser light scattering.

Surface Plasmon Resonance—Binding experiments were performed by surface plasmon resonance measurements on a Biacore 3000 instrument (Biacore Inc.) at 25 °C. sNRP1-Fc, sNRP1-His, VEGF165, VEGF121 (from Peprotech), and VEGFR2 ("ligands") were immobilized at high surface densities (~10,000 response units) on an activated CM5 chip using standard amine-coupling procedures as described by the manufacturer. Ligands were injected at a concentration of 10 µg/ml in 20 mM sodium acetate, pH 4.5, and at a flow rate of 5 µl/min until desired surface densities, measured in response units, were reached. Unreacted groups were blocked by injecting 1 M ethanolamine. To perform binding assays, "analytes" or mixtures of analytes at different concentrations were injected in 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% (v/v) surfactant P20, at a flow rate of 5 µl/min. Blank surfaces were used for background corrections. Injections of 10 mM glycine, pH 3.0, at 100 µl/min for 1 min were used to regenerate surfaces between two binding experiments. We used steady state analysis to estimate the affinity of VEGF165 and VEGF121 to NRP1. VEGF samples at concentrations between 50 and 2000 nM were injected and allowed to reach equilibrium state before dissociation. Dissociation constants (Kd) in nM were calculated by plotting the equilibrium response versus concentration.

VEGFR2 Phosphorylation Assay—HUVECs were grown in 6-well tissue culture plates to confluence and then starved overnight in basal medium EBM-2 (Cambrex) with 0.2% fetal bovine serum and 0.1% bovine serum albumin. Cells were stimulated with the indicated VEGF isoform for 10 min at 37 °C and then washed once in ice-cold phosphate-buffered saline. Total VEGFR2 and phospho-VEGFR2 levels were determined with the human VEGFR2 DuoSet IC enzyme-linked immunosorbent assay kit (R & D Systems). Enzyme-linked immunosorbent assay plates were coated with mouse anti-human VEGFR2 antibody and detected with goat anti-human VEGFR2 or mouse anti-phosphotyrosine for total VEGFR2 or phosphorylated VEGFR2, respectively.

Cell Migration Assay—Migration assays were performed using a modified Boyden chamber with the 8-µm pore size Falcon 24-multiwell insert system (BD Biosciences). The plates were precoated with 8 µg/ml laminin overnight at 37 °C. Confluent HUVECs were starved overnight, harvested, and resuspended in assay medium (EBM-2, 0.1% bovine serum albumin). 100 µl of cells with or without anti-NRP1B antibody were added into the upper chamber, whereas migration stimuli were added to the lower chamber in 500 µl of assay medium. Cells were allowed to migrate for 16 h at 37 °C. To stop the assay, cells on the upper face of the membrane were removed with a sponge swab, and cells on the lower face were fixed with methanol and stained with Cytox green (Molecular Probes, Inc., Eugene, OR). Images were taken with an inverted fluorescent microscope, and cell number was analyzed with ImageJ.

Bead Outgrowth Assay—Dextran-coated Cytodex 3 microcarrier beads (Amersham Biosciences) were incubated with HUVECs (400 cells/bead) in EGM-2 overnight at 37 °C. HUVEC-coated beads were then washed three times with 5 ml of clotting medium (EGM-2 minus VEGF), and resuspended in clotting medium with 2.5 µg/ml fibrinogen (Sigma) at a density of 200 beads/ml. To induce clotting, 0.5 ml of fibrinogen/bead solution was added into one well of a 24-well tissue culture plate containing 0.625 units of thrombin (Sigma) and incubated for 5 min at room temperature and then for 20 min at 37 °C. The clot was equilibrated in 1 ml of clotting medium for 30 min at 37 °C. The medium was then replaced with 1 ml of clotting medium containing skin fibroblast cells (Detroit 551; ~30,000 cells/ml). Different VEGF variants and antibodies were added to each well as indicated, and the assay was monitored for 8 days with a change in medium every other day. Each condition was repeated in two wells. Images of the beads were captured by an inverted microscope, and concentric circles spaced at 100, 200, and 300 µm were drawn around the bead in each image. The number of vessels crossing each line was counted.


Figure 1
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  		FIGURE 1.
VEGFR2 phosphorylation and HUVEC migration induced by different VEGF variants. A, HUVECs were incubated with gradually increasing concentrations of the indicated VEGF variant for 10 min at 37 °C. VEGFR2 phosphorylation level was determined by enzyme-linked immunosorbent assays using antibodies that recognized either total or tyrosin-phosphorylated VEGFR2. n = 3 for each condition. B, HUVECs were allowed to migrate in the presence of the indicated VEGF variant overnight at 37 °C. n = 6 for each condition.

 
Immunoprecipitation and Immunoblotting—Starved HUVECs were incubated with anti-VEGF or anti-NRP1B antibodies (50 µg/ml) in EBM-2 containing 0.1% bovine serum albumin for 30 min at 37 °C. Cells were then cooled on ice for 15 min, and the indicated VEGF variant was added, followed by a 30-min incubation at 4 °C. Cells were stimulated for 7 min at 37 °C and then washed with ice-cold PBS and lysed in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, phosphatase inhibitor mixture I and II (Sigma), and complete protease inhibitor tablet (Roche Applied Science)). Cell lysates were spun for 1 min at 14,000 rpm at 4 °C. The supernatants were collected and incubated with agarose-conjugated anti-VEGFR2 antibodies (sc-504 AC and sc-315 AC; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 16 h at 4 °C. Agarose beads were washed three times in cold wash buffer (20 mM Tris, pH 7.5, 100 mM NaCl) and then resuspended in 2x SDS sample buffer and incubated for 5 min at 95 °C under nonreducing conditions. Samples were resolved in Tris-glycine gels and immunoblotted with anti-NRP1 (sc-7239; Santa Cruz Biotechnology) or anti-VEGFR2 (Genentech, Inc.).


Figure 2
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  		FIGURE 2.
Blocking NRP1 function inhibits VEGF165- and VEGF121-induced HUVEC migration. The effects of 25 µg/ml anti-NRP1B on HUVEC migration induced by VEGF165, VEGF121, and VEGF109 are shown in A-C, respectively. *, p < 0.004 when comparing the same concentrations of VEGF isoform with or without anti-NRP1B. D, 25 µg/ml anti-NRP1B reduced HUVEC migration in response to 1 nM VEGF121 from different suppliers: VEGF121 #1, Peprotech (100-20A; the same VEGF121 as used in B); VEGF121 #2, Cellsciences (CRV001B); VEGF121 #3, Cellsciences (CRV010B); VEGF121 #4, Calbiochem (676473).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
VEGF121 Is Comparable with VEGF165 in Inducing VEGFR2 Phosphorylation and Endothelial Cell Migration—We initially compared the potency of VEGF121 with VEGF165 in inducing VEGFR2 phosphorylation and EC migration. Activation of VEGFR2 signaling was examined in HUVECs. ECs were incubated with different VEGF variants at concentrations that varied from 0.05 to 250 nM, and the ratio of phospho-VEGFR2 to total VEGFR2 under each condition was determined by Duo Set enzyme-linked immunosorbent assays. As shown in Fig. 1A, at the same molar concentration, VEGF121 and VEGF165 led to comparable levels of VEGFR2 phosphorylation. They both led to 50% of maximal receptor phosphorylation at around 1 nM and reached peak activation at about 25 nM. We also tested a truncated form of VEGF, VEGF109 (25). In the VEGFR2 phosphorylation assay, VEGF109 displayed reduced potency compared with VEGF121 and VEGF165.

We then studied VEGF variants in their ability to induce EC migration. At 1 nM, VEGF121 and VEGF165 led to similar levels of HUVEC migration (Fig. 1B), whereas VEGF109 induced significantly less migration.


Figure 3
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  		FIGURE 3.
HUVEC sprouting induced by different VEGF variants. A, HUVEC-coated microcarrier beads were embedded in fibrin gel and incubated in EGM-2 containing the indicated VEGF variant. Sprouts were fixed with 4% paraformaldehyde after 8 days, and images were taken for analysis. To study the effect of blocking NRP1 function on sprout formation, anti-NRP1B or control antibody anti-VEGF was added at 50 µg/ml to the culture. B, to quantifysproutformation, circles centered on the beads were drawn, and the number of capillary-like tubes crossing each circle was counted. Quantification of the results from 10 beads under each condition is shown.

 
Anti-NRP1B Inhibits VEGF121-induced EC Migration and Sprout Formation—We next tested whether blocking NRP1 function would alter the bioactivity of the various VEGF isoforms. As reported previously (29), a monoclonal antibody against NRP1 (anti-NRP1B), which blocks VEGF165 binding to NRP1, significantly reduced EC migration in response to 1 and 5 nM VEGF165 (Fig. 2A). Interestingly, anti-NRP1B also significantly reduced HUVEC migration in response to VEGF121 at all concentrations of VEGF121 tested (Fig. 2B). In contrast, VEGF109-induced EC migration was not inhibited by anti-NRP1B (Fig. 2C). Significant reduction of VEGF121-induced migration was also observed with VEGF samples from a number of different sources (Fig. 2D). The specificity of anti-NRP1B blocking both VEGF165- and VEGF121-induced migration, but not VEGF109, is further supported by our previous studies showing that anti-NRP1B does not block basic fibroblast growth factor- or human growth factor-induced endothelial cell migration (29).

To dissect the activity of these VEGF variants in capillary formation, we carried out a bead outgrowth assay in fibrin gels (30). Since VEGF165 and VEGF121 were similar in their ability to activate VEGFR2, whereas VEGF109 was less potent (Fig. 1), the three VEGF variants were used at 0.1, 0.1, and 0.5 nM, respectively, to achieve similar levels of sprouting. We used a 10-fold lower overall VEGF concentration in the sprouting assay, as compared with the VEGFR2 phosphorylation and migration assays, based on an empirically identified optimal concentration, as determined by dose response studies in this assay (data not shown). Cultures were monitored over the course of 8 days, and images were taken at the end of the assay. As a positive control, anti-VEGF completely inhibited capillary-like tube formation induced by all three VEGF isoforms (Fig. 3A, right). When anti-NRP1B was added at a saturating concentration (50 µg/ml) to the bead outgrowth assay, it greatly reduced sprout formation in the presence of either VEGF165 or VEGF121. However, different from the inhibition executed by anti-VEGF, a few short sprouts were still observed around the carrier bead, and the vessel diameter was greatly increased with anti-NRP1B treatment (arrows; Fig. 3A, middle). We concluded that this might be due to the fact that anti-NRP1B greatly reduces VEGF-induced EC migration but has little effect on cell proliferation as we have previously shown (29). On the other hand, anti-NRP1B only slightly decreased sprout formation in the presence of VEGF109 (Fig. 3A; quantification in Fig. 3B). We speculate that this slight reduction might be attributed to the small amounts of VEGF165 or VEGF121 produced by fibroblasts, which were grown on top of the fibrin gel as support cells for the bead outgrowth assay. Also worth noting is the qualitative phenotype we observed with VEGF109-treated beads, which results in very wide vessels regardless of the presence of anti-NRP1B (arrows; Fig. 3A, lower panels).

Since it is generally believed that VEGF121, and by inference VEGF109, do not interact with NRP1, we considered two explanations as to why blocking NRP1 function would disrupt VEGF121-induced EC behavior. First, blocking NRP1 function may indirectly alter VEGF121 interactions with VEGFR2 and thus reduce VEGF121 function. Second, NRP1 may interact directly with VEGF121. We therefore tested the interactions of the various VEGF isoforms with NRP1 in vitro.

VEGF121 Interacts with NRP1 in Vitro—We employed surface plasmon resonance as a direct binding assay to study the interaction between the two VEGF isoforms and NRP1. VEGF165 showed strong, specific binding to sNRP1-Fc immobilized on a sensor chip at high density (Fig. 4A), confirming earlier results with a similar neuropilin construct (25). Once again, as a negative control we used a truncated VEGF, VEGF109, which did not bind NRP1. Interestingly, VEGF121 showed a significant, concentration-dependent binding to sNRP1-Fc (Fig. 4B). Both VEGF isoforms also exhibited binding to other NRP constructs, such as sNRP1-His (data not shown). This is remarkable, since, compared with VEGF165, VEGF121 lacks the heparin-binding domain encoded by exon 7 and has been previously reported not to bind NRP1 (31, 32). Steady state binding analysis revealed that VEGF165 and VEGF121 have comparable binding affinities to NRP1, with KD values of 120 and 220 nM for VEGF165 and VEGF121, respectively. We were also able to confirm the interaction between VEGF121 and NRP1 for VEGF samples from a number of different sources (Fig. 4C). All but one VEGF121 sample showed specific binding to NRP1 with very similar association and dissociation profiles and only slight differences in the maximum relative response. The one VEGF121 sample that did not show any binding to sNRP1-Fc (Fig. 4C) or sNRP1-His (data not shown) is in fact a truncated version of VEGF121. This particular VEGF121 sample is lacking nine C-terminal amino acids, as stated in the manufacturer's specification sheet (R & D Systems), which we confirmed by our own mass spectrometry analysis (data not shown), and should therefore be referred to as VEGF112. Mass spectrometry analysis was also used to confirm the proper size of the VEGF121 sample that did indeed show significant binding (Fig. 4B), which was used for all of our cell-based experiments. All VEGF isoforms and two truncated VEGFs bind to VEGFR2 immobilized on a sensor chip (Fig. 4D). We conclude that although both VEGF121 and truncated VEGF variants display similar binding to VEGFR2, only full-length VEGF121 is able to interact with NRP1. Last, we demonstrated the specificity of these interactions by showing that anti-NRP1B blocks NRP1 binding to immobilized VEGF165 and VEGF121 (Fig. 4, E and F, respectively).


Figure 4
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  		FIGURE 4.
Binding of different VEGF isoforms to neuropilin-1 and VEGFR2 by surface plasmon resonance. Ligands were immobilized onto Biacore CM5 sensor chips, and binding sensorgrams were recorded for analytes or a mixture of analytes as indicated. The arrows depict the beginning of the association and dissociation phase (beginning and end of injections). A, binding of VEGF165 (at three different concentrations) and of VEGF109 (at 100 nM) to immobilized sNRP1-Fc. B, binding of VEGF121 to immobilized sNRP1-Fc. C, binding of VEGF121 samples from different suppliers (all at ~100 nM) to sNRP1-Fc. Number codes are the same as in Fig. 2D. VEGF112 was from R & D Systems (298-VS-025/CF). D, binding of different VEGF isoforms and two truncated VEGF variants (all at 100 nM) to immobilized VEGFR2. E, binding of sNRP1-Fc (at 100 nM) and a 1:2 mixture with anti-NRP1B to immobilized VEGF165. F, binding of sNRP1-Fc (at 100 nM) and a 1:2 mixture with anti-NRP1B to immobilized VEGF121.

 
VEGF121 Is Not Sufficient to Bridge the Formation of the VEGFR2·NRP1 Complex—Having confirmed that NRP1 can interact directly with VEGF121, we next explored what effects this would have on VEGF121/VEGFR2 interactions. It is well known that VEGF165 mediates the formation of complexes containing VEGFR2 and NRP1, which enhances the affinity of VEGF165 for VEGFR2 and therefore VEGFR2 signaling (26). We wondered whether VEGF121 also functions on ECs through bridging the formation of VEGFR2·NRP1 complex. Consistent with previous observations (26), anti-VEGFR2 antibodies immunoprecipitated NRP1 in the presence of VEGF165 but not VEGF121 (Fig. 5, lanes 2 and 5, NRP1 blot), indicating that VEGF121 does not promote VEGFR2·NRP1 complex formation. As expected, VEGF109 is also not sufficient to promote VEGFR2·NRP1 complex formation (lane 8, NRP1 blot).

Observing that VEGF121 does not promote VEGFR2·NRP1 complex formation highlights the possibility that VEGF121 may signal through NRP1 independently of VEGFR2 to regulate endothelial cell function.


Figure 5
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  		FIGURE 5.
VEGF165, but not VEGF121, mediates the formation of complexes containing NRP1 and VEGFR2 on endothelial cells. Serum-starved HUVECs were treated with the anti-VEGF (lanes 3, 6, and 9) or anti-NRP1B (lanes 4, 7, and 10) at 25 µg/ml for 30 min and then incubated with the indicated VEGF variant for 30 min on ice and then for 8 min at 37 °C. Cell lysates were subjected to immunoprecipitation using agarose-conjugated anti-VEGFR2 and immunoblotted with antibodies specific for VEGFR2 or NRP1. Immunoblotting against total cell lysate indicated equal sample loading.

 


Figure 6
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  		FIGURE 6.
Binding of VEGF isoform·VEGFR2 complexes to neuropilin by surface plasmon resonance. sNRP1-Fc was immobilized onto a Biacore CM5 sensor chip, and binding data were collected for VEGF·VEGFR2 complexes. The arrows indicate the beginning of the association and dissociation phase (beginning and end of injections). A, sensorgrams for VEGF165 in a 1:1 complex with VEGFR2 and for VEGF165 and VEGFR2 alone (all at 100 nM). B, sensorgrams for VEGF121 in a 1:1 complex with VEGFR2 and for VEGF121 and VEGFR2 alone (all at 150 nM).

 
VEGFR2 Enhances the Interaction between NRP1 and VEGF165 but Not between NRP1 and VEGF121—Again we used surface plasmon resonance measurements as direct binding assays to determine if we would be able to discern a difference between the roles of VEGF165 and of VEGF121 in VEGFR2·NRP1·VEGF complex formation. Therefore 1:1 (mol/mol) mixtures of either VEGF165 or VEGF121 with VEGFR2 were allowed to interact with immobilized NRP1. Association and dissociation profiles were compared with the ones where VEGFR2 and VEGFs were allowed to interact with NRP1 separately. The sensorgram for VEGFR2/VEGF165 binding to NRP1 is qualitatively different from the sensorgram for VEGF165 binding alone in both association and dissociation phases (Fig. 6A). In particular, the dissociation rate is markedly decreased. The differences in the association and dissociation processes can be explained by VEGF165 binding to VEGFR2 and NRP1 simultaneously, therefore bridging between the two receptors. A mixture of VEGF121 with VEGFR2, on the other hand, does not display significantly different binding to NRP1 compared with VEGF121 alone (Fig. 6B). Thus, our in vitro binding data support the notion that VEGF121, while being able to bind to both VEGFR2 and NRP1, is unable to bridge the two receptors. Interestingly, we were also able to detect binding between VEGFR2 and NRP1 (Fig. 6, A and B), but the much lower maximum response units for this interaction, despite the same concentration of analyte, suggest much weaker interactions compared with NRP1/VEGF or VEGFR2/VEGF interactions. We conclude that VEGFR2 enhances the interaction between VEGF165 and NRP1, but not between VEGF121 and NRP1, which appears to be explained by only VEGF165 being able to bridge the two receptors.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It is generally believed that only the heparin-binding VEGF isoforms (mainly VEGF165) can bind to and function through NRP1 on endothelial cells (Fig. 7A). In the current study, we present new evidence that a heparin-noninteracting VEGF isoform, VEGF121, also possess the ability to bind to NRP1. Furthermore, we show that this interaction is biologically relevant, since a function-blocking antibody targeting NRP1 inhibits VEGF121-induced endothelial cell migration and sprouting.

It is worth noting that recent studies using a small four-amino acid peptide known as Tufstin, which shares homology to the tail region of both VEGF165 and VEGF121, but not VEGF109, directly interacts with NRP1 (33). These findings suggest that the C-terminal region of VEGF may be sufficient to interact with NRP1. However, the concentrations of peptide used in this study were very high; therefore, it remains to be determined how much this particular region of VEGF121 contributes to NRP1 binding. Nevertheless, we speculate that the primary reason others have failed to observe VEGF121 binding directly to NRP1 is that they are using inadvertently cleaved VEGF121. This possibility is further supported by the fact that commercially available VEGF121 may in fact be a cleaved form, and thus mass spectrometry should be used to verify the particular isoform (see R & D insert on VEGF121, which is actually VEGF112 and does not bind to NRP1; Fig. 4C).

Unlike VEGF165, VEGF121 does not induce the formation of a VEGFR2·VEGF·NRP1 complex. As illustrated in Fig. 7B, VEGF121 binds to NRP1 directly and may regulate endothelial cell function through a VEGFR2-independent or parallel pathway. It is also possible that VEGF121 initially binds to NRP1 and that as a result of the high copy number of NRP1 on the cell surface compared with VEGFR2 (21), NRP1 subsequently presents VEGF121 to VEGFR2, thus enhancing VEGFR2 signaling. These two possibilities are not mutually exclusive, and we address both mechanisms below.


Figure 7
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  		FIGURE 7.
Model for signaling through their receptors. VEGF165, VEGF109 (A), and VEGF121 (B). See text for discussion.

 
Evidence supporting a role for NRP1 independent of or in parallel with VEGFR2 signaling include a previous study, where it was shown that EGF can induce migration of endothelial cells expressing a fusion protein consisting of the extracellular domain of EGFR fused to the transmembrane and intracellular domain of NRP1 (27). This experiment suggested that the intracellular domain of NRP1 signals independently of VEGFR2 activation. In addition, we have previously shown that NRP1 plays a unique role in endothelial cell motility, best illustrated by the fact that anti-NRP1 antibodies block VEGF165-induced endothelial cell migration, but have little effect on VEGF165-induced proliferation or permeability (29). Furthermore, here we observe that VEGF121 binds to NRP1 but does not form a NRP1·VEGF·VEGFR2 complex, in contrast to what is observed with VEGF165 (Fig. 5). This observation combined with fact that anti-NRP1B blocks VEGF121 binding to NRP1 (Fig. 4F) and also greatly reduces VEGF121-mediated endothelial cell migration (Fig. 2, B and D) and sprouting (Fig. 3), supports the idea that VEGF may act directly through NRP1 to regulate endothelial cell motility.

Insight can also be derived from the comparison of the various VEGF isoforms in each of the bioactivity assays. Notably, VEGF109, which does not bind to NRP1 (Fig. 4A), has a lower maximal activation of endothelial cell migration (Fig. 1), even at the same levels of VEGFR2 phosphorylation (compare 1 nM for VEGF165 and VEGF121 with 5 nM for VEGF109). Also worth noting is the comparison of the VEGF isoforms in the bead outgrowth assay (Fig. 3). All three VEGF isoforms induce bead outgrowth, but in the case of VEGF109, the sprouts appear to be much wider. Interestingly, these wide sprouts are in some ways similar to the endothelial cell masses observed adjacent to VEGF165 and VEGF121 beads treated with anti-NRP1B (Fig. 3A, arrows). Having previously shown that NRP1 is not essential for VEGF-induced endothelial cell proliferation (29) but is necessary for migration, we hypothesize that blocking NRP1 function would diminish sprouting, whereas subsequently endothelial cells would continue to divide. In support of this prediction, when endothelial sprouts are treated with anti-NRP1B after being established, the vessels begin to widen, whereas further sprouting is inhibited.5

A second, although less attractive hypothesis may explain the effects we observe with VEGF121. This hypothesis is based on the premise that NRP1 acts to enhance VEGF binding to VEGFR2, subsequently modulating VEGFR2 signaling. This hypothesis is supported by data presented in the hallmark paper by Soker et al. (21). In that study, the authors showed enhanced VEGF binding, VEGFR2 activation, and endothelial cell migration in porcine aortic endothelial cells expressing both NRP1 and VEGFR2, as compared with porcine aortic endothelial cells only expressing VEGFR2. Additionally, we have previously described a modest yet significant decrease in VEGFR2 phosphorylation when blocking VEGF165 binding to NRP1 (29). In this study, we demonstrate a qualitative enhancement of VEGF165 binding to NRP1 in the presence of VEGFR2 (Fig. 6A) but not VEGF121 (Fig. 6B), consistent with the idea that VEGF121 is not sufficient to bridge the NRP1·VEGF·VEGFR2 complex (Fig. 5). Last, direct support for the possibility that NRP1 may present VEGF121 to VEGFR2 is provided by the observation that maximal VEGFR2 phosphorylation is higher with VEGF121 when compared with VEGF109 (Fig. 1A), whereas both isoforms show similar binding to VEGFR2 (Fig. 4D). Because VEGF121 binds to NRP1 and VEGF109 does not (Fig. 4), this result suggests that NRP1 enhances VEGF121 activation of VEGFR2. These observations are also consistent with a recent study showing that NRP1 can enhance VEGF121-induced VEGFR2 activation without the formation of a NRP1·VEGF· VEGFR2 complex (32). However, in contrast to this study, we speculate that these findings may be primarily explained by VEGF121 interacting directly with NRP1.

In conclusion, we show for the first time that NRP1 can interact directly with VEGF121 in a biologically relevant fashion, but a cleaved form of VEGF, either VEGF112 or VEGF109, does not bind to NRP1. These data suggest that unless VEGF is proteolytically cleaved, blocking NRP1 function should greatly diminish VEGF·NRP1-mediated events, particularly its effect on endothelial cell motility. We present a model for the various VEGF isoforms binding to NRP1 (Fig. 7), and conclude that additional experiments will be required to fully understand the role of NRP1 in both VEGF and vascular biology.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Biomedical Sciences Graduate Program, University of California, 533 Parnassus Ave., San Francisco, CA 94143. Back

2 To whom correspondence may be addressed: Dept. of Protein Chemistry, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080. Tel.: 650-467-7432; Fax: 650-225-5945; E-mail: akoch{at}gene.com. 3 To whom correspondence may be addressed: Dept. of Tumor Biology and Angiogenesis, Genentech, Inc., 1 DNA Way, South San Francisco, California 94080. Tel.: 650-467-8197; Fax: 650-467-3562; E-mail: rwatts{at}gene.com.

4 The abbreviations used are: VEGF, vascular endothelial growth factor; NRP1, neuropilin-1; EC, endothelial cell; HUVEC, human umbilical vein endothelial cell. Back

5 Q. Pan and R. J. Watts, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Gloria Meng for helpful discussions and Germaine Fuh for discussions and for providing anti-VEGF and VEGF109.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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