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Published as doi: 10.1096/fj.06-6277com.
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(The FASEB Journal. 2007;21:915-926.)
© 2007 FASEB

Neuropilin-1 and neuropilin-2 enhance VEGF121 stimulated signal transduction by the VEGFR-2 receptor

Niva Shraga-Heled*, Ofra Kessler*, Claudia Prahst{dagger}, Jens Kroll{dagger}, Hellmut Augustin{dagger} and Gera Neufeld*,1

* Cancer and Vascular Biology Research Center, Rappaport Research Institute in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel; and

{dagger} Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, Freiburg, Germany

1Correspondence: Cancer and Vascular Biology Research Center, Rappaport Research Institute in the Medical Sciences, Bruce Rappoport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa 31096, Israel. E-mail: gera{at}tx.technion.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The neuropilin-1 (np1) receptor binds the 165 amino-acid form of vascular endothelial growth factor165 (VEGF165) and functions as an enhancer that potentiates VEGF165 signaling via the VEGFR-2 tyrosine-kinase receptor. To study the mechanism by which neuropilins potentiate VEGF activity we produced a VEGF165 mutant (VEGF165KF) that binds to neuropilins but displays a much lower affinity toward VEGFR-1 and VEGFR-2. VEGF165KF failed to induce VEGFR-2 phosphorylation in cells lacking neuropilins. However, in the presence of np1, VEGF165KF bound weakly to VEGFR-2, induced VEGFR-2 phosphorylation, and activated ERK1/2. Interestingly, VEGF165KF did not promote formation of VEGFR-2/np1 complexes nor did high concentrations of VEGF165KF inhibit VEGF165 induced formation of such complexes, suggesting that VEGF165 does not stabilize VEGFR-2/np1 complexes by forming bridges spanning VEGFR-2 and np1. VEGF121 is a VEGF form that does not bind to neuropilins. Surprisingly, both np1 and neuropilin-2 (np2) enhanced VEGF121-induced phosphorylation of VEGFR-2 and VEGF121-induced proliferation of endothelial cells. The enhancement of VEGF121 activity by np1 was accompanied by a 10-fold increase in binding affinity to VEGFR-2 and was not associated with the formation of new VEGFR-2/np1 complexes. These observations suggest that neuropilins enhance the activity of VEGF forms that do not bind to neuropilins, indicate that np2 is a functional VEGF receptor, and imply that spontaneously formed VEGFR-2/np1 complexes suffice for efficient neuropilin mediated enhancement of VEGF activity.—Shraga-Heled, N., Kessler, O., Prahst, C., Kroll, J., Augustin, H., Neufeld, G. Neuropilin-1 and neuropilin-2 enhance VEGF121 stimulated signal transduction by the VEGFR-2 receptor.


Key Words: angiogenesis • semaphorin • endothelial cells • vascular endothelial growth factor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
VEGF IS AN IMPORTANT INDUCER of vasculogenesis and angiogenesis, and it plays a central role in the etiology of angiogenic diseases (1 , 2) . Several major VEGF forms differing in the representation of the heparin binding peptides encoded by exons 6 and 7 are produced as a result of alternative splicing. VEGF121 lacks both exons and does not bind to heparin, while VEGF189 contains both, and VEGF145 and VEGF165 display intermediate affinities for heparin and contain exon-6 and exon-7, respectively (1 , 3 4 5) . All VEGF isoforms bind to the tyrosine-kinase receptors VEGFR-1 (flt-1) (6) and VEGFR-2 (KDR/flk-1) (7 , 8) . The binding of VEGF to VEGFR-2 initiates intracellular signal transduction and is correlated with the induction of endothelial cell proliferation, migration, and in vivo angiogenesis (9 10 11) . By contrast, the binding of VEGF to VEGFR-1 does not seem to result in the induction of cell proliferation, and it is believed that its role is mainly inhibitory (12 , 13) .

Endothelial cells also express VEGF receptors that bind VEGF165 but not VEGF121 (14) . These splice form-specific VEGF receptors are the products of the np1 and np2 genes (15 , 16) . The neuropilins also function in addition as receptors for members of the class-3 semaphorin subfamily of axon guidance factors (17) . Gene targeting experiments have revealed that mice lacking functional np1 receptors die during embryogenesis as a result of faulty heart and blood vessel development, indicating a central regulatory role for this receptor in developmental angiogenesis (18) . In contrast, mice lacking functional np2 receptors develop normally, although they are smaller and suffer from minor defects in their lymphatic system (19) . Interestingly, these mice do not respond to VEGF in retinal angiogenesis experiments (20) . Np1 and np2 double-null mice are more severely affected than mice lacking just np1. These mice die early during embryogenesis, blood vessels do not develop, and the phenotype resembles the severe phenotype of mice lacking functional VEGFR-2 receptors (10 , 36) . These experiments therefore provide circumstantial evidence that np1 and np2 are critical signal transducing receptors that fulfill important functions during developmental angiogenesis.

The molecular mechanisms by which neuropilins affect VEGF induced signal transduction have not been studied in depth. It had been reported that VEGFR-2-mediated cell migration induced by VEGF165, but not by VEGF121, is strongly enhanced in the presence of np1 receptors (15 , 21) . It was suggested that this effect is mediated by the formation of VEGFR-2/np1 complexes and that in these complexes VEGF165 binds simultaneously to VEGFR-2 and to np1, forming a bridge that stabilizes the complexes (22) . However, in another report, it was claimed that the formation of VEGFR-2/np1 complexes is not dependent on the presence of VEGF165 (23) . It is assumed, although not formally established, that the formation of complexes between np1 and VEGFR-2 is responsible for the activity-enhancing effects of np1. With regard to the possible role of np2 in VEGF signaling, it is not known if np2 can potentiate VEGF signaling as does np1 and whether np2 forms complexes with VEGFR-2.

We have created a VEGF165 mutant that binds with much reduced affinity if at all to VEGFR-1 and VEGFR-2 but binds well to neuropilins. Using this mutant, we show that VEGF165 does not need to bind to neuropilins in order to enable potentiation of VEGFR-2-mediated signal transduction by neuropilins and that the enhancing effects of neuropilins do not require formation of VEGF165 bridges linking neuropilins and VEGFR-2. We strengthen these conclusions by showing that both neuropilins are able to enhance VEGF121 signaling mediated by VEGFR-2, even though VEGF121 does not bind to neuropilins, and present evidence indicating that np2 also functions as an enhancer of VEGFR-2-mediated signal transduction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Antibodies directed against np1, np2, ERK-2, phosphorylated ERK-1/2, VEGFR-2, and phosphorylated Y-951 of VEGFR-2 were purchased from Santa-Cruz Biotechnology (Santa Cruz, CA). Antibodies directed against phosphorylated Y-1175 of VEGFR-2 were obtained from Cell Signaling Technologies (Beverly, MA, USA). Small interfering RNA (siRNA) directed against np1, np2, or control siRNA were obtained from Qiagen (Valencia, CA, USA). VEGF121 was produced using the baculovirus system and purified, as described previously (24) . Media and sera for cell culture were from Invitrogen-Life Technologies (Carlsbad, CA, USA), except for the M199 medium that was from Biological Industries (Kibbutz Beth-Haemek, Israel). Oligofectamine was obtained from Invitrogen.

Cell lines
Human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial (PAE) cells were cultured as described previously (16 , 25) . HUVEC were not used beyond passage 8. The generation of PAE cells expressing recombinant np1 (PAE/np1), np2 (PAE/np2), VEGFR-1 (PAE/VEGFR-1), and VEGFR-2 (PAE/VEGFR-2) was described previously (15 , 16 , 26) . To generate PAE cells expressing VEGFR-2 and either np1 or np2, we cotransfected PAE/VEGFR-2 cells with a np1 expression vector (pcDNA3/np1) or with a np2 expression vector (PECE/np2) (16) . These plasmids were cotransfected into the cells along with the pBabePuro plasmid, which carries resistance to puromycin. Cells expressing combinations of two receptors were selected with G418 (0.5 mg/ml) and puromycin (2 µg/ml).

Cell proliferation experiments and use of siRNA
The np1 specific siRNA r(AAGGAAACCUUGGUGGGAU)d(TT) and the np2 specific r(CCAGAAGAUUGUCCUC AAC)d(TT) siRNA or control nonsilencing siRNA r(UUCUCCGAACGUGUCACGU)dTdT were transfected into HUVEC at a final concentration of 120 nM using oligofectamine. The cells were trypsinized one day after transfection, and reseeded. For phosphorylation experiments, the cells were seeded in 6-well plates at a concentration of 5 x 105 cells/well in M199 medium containing 10% FCS. The experiment was performed 24 h after seeding. For proliferation experiments, 2 x 104 cells were seeded in 24-well dishes in M199 containing 10% FCS. Proliferation experiments were performed as described previously (27) .

Generation of a cDNA encoding a VEGF165 mutant that displays reduced affinities to VEGFR-1 and VEGFR-2 but retains its neuropilin binding ability (VEGF165KF)
In order to reduce the VEGFR-1 binding ability, we introduced the following three-point mutations into VEGF165: D63S, G65M, and L66R. To reduce the VEGFR-2-binding ability, we introduced the following four-point mutations: I43A, I46A, Q79A, and I83A. These point mutations were based on previously published data (28 , 29) and were introduced through consecutive polymerase chain reaction (PCR) reactions.

Construction and expression of lentiviruses that direct expression of VEGF165 and VEGF165KF
Full-length VEGF165 and VEGF165KF encoding cDNAs were subcloned between the BamHI and XhoI sites of the pTK208 plasmid containing the cytomegalovirus (CMV) promoter (generously provided by Dr. Tal Kafri, Gene Therapy Center, University of North Carolina, Chapel Hill, NC, USA). The virus was produced by calcium-mediated cotransfection of the lentiviral vector (20 µg), packaging vector pCMVdR8.91 (15 µg), and a plasmid encoding the vesicular stomatitis virus coat envelope pMD2-VSVG (10 µg) into HEK-293 cells. VEGF165 and VEGF165KF were purified from serum free-conditioned medium using heparin-sepharose affinity chromatography, as described previously (30) .

Phosphorylation assays
HUVEC or PAE cells were seeded at a concentration of 5 x 105 cells in 6-well gelatinized dishes in growth medium containing 10% FCS. The cells were allowed to attach and incubated for 16 h in a humidified incubator at 37°C. The PAE cells were serum starved for 16 h before the experiment. The experiment was initiated by the addition of various VEGF forms at the designated concentrations to the serum-free medium. The experiment was terminated after 10 min at room temperature by a wash with ice-cold PBS. Cells were lysed with 0.03 ml of lysis buffer containing HEPES (50 mM, pH 7.4), 4 mM EDTA, 1% Triton-X-100, 0.5 mg/ml Na3VO4, 4.5 mg/ml Na4P2O7 and fresh protease inhibitors (PMSF 0.2 mg/ml, leupeptin 2 µg/ml, and aprotinin 2 µg/ml). The cells were scraped off, and nonsoluble debris was removed by centrifugation at 15,000 g at 4°C for 20 min. Aliquots of supernatant containing 40 µg protein (determined using the Bradford protein quantification kit (Bio-Rad), according to vendor instructions) were loaded on an SDS/PAGE gel, blotted onto a nitrocellulose filter, and probed with antibodies directed against phosphorylated VEGFR-2 or phosphorylated ERK-1/2. Bound antibody (Ab) was detected as described previously (27) . The blot was then stripped by incubation with reblot plus mild solution (Chemicon, Temecula, CA, USA). The blot was then reprobed with an Ab directed against VEGFR-2 (total VEGFR-2) or against ERK-2 (total ERK).

Binding and cross-linking experiments
Binding and cross-linking experiments using various 125I-labeled VEGF forms were performed as described previously (16) . The determination of dissociation constants and receptor densities in saturation-binding experiments was determined using the GraphPad Prism-4 software (San Diego, CA, USA).

Coimmunoprecipitation experiments
Various receptor-expressing PAE cells were seeded at 1.2 x 106 cells per 6 cm gelatin-coated dishes. Before the start of the experiment, the cells were starved for 16 h. Various factors were added, and the cells were incubated for 30 min. at 4°C with gentle agitation. The cells were then shifted to 37°C for 15 min. In experiments involving competition, the competitors were added to the dishes 10 min before the addition of the other factors. The experiment was terminated by a wash with ice-cold PBS, followed by the addition of lysis buffer (20 mM Tris/HCl pH-7, 0.3 M NaCl, 1% Triton-X-100, 1 mM Na3VO4, 5 mM NaF, 10 mM Na4P2O7, 4 mM EDTA, and fresh protease inhibitors (PMSF 0.2 mg/ml, leupeptin 2 µg/ml, and aprotinin 2 µg/ml). For immunoprecipitation, extracts containing 0.35-mg protein were incubated with appropriate antibodies (0.2 µg/ml) overnight at 4°C. The extracts were incubated with protein-A or protein-G coated sepharose beads 2 h at 4°C. Beads were washed twice for 5 min at 4°C with lysis buffer, followed by two washes with PBS. They were then boiled for 5 min in SDS containing loading buffer, and the proteins were separated on a 6% SDS/PAGE gel.

Western blot analysis
Western blot analysis was performed using horseradish peroxidase conjugated secondary antibodies, as described previously (27) . Bound Ab was visualized using the enhanced chemiluminescence (ECL) kit of Amersham (Piscataway, NJ, USA), according to the instructions of the vendor. Quantification of band intensity was performed using a phosphor-imager (Fuji Film, LAS-3000), and the ratio between phosphorylated protein and the total amount of a target protein was determined using the Multi-Gauge program.

In vitro angiogenesis assay
This assay was performed essentially as described previously (31 , 32) . In brief, HUVEC cells were harvested, and a defined number of cells was suspended in ECGM medium containing 0.25% (w/v) methylcellulose (Sigma, Taufkirchen, Germany). Spheroids were generated by pipetting 450 endothelial cells in a hanging drop of 25 µl on plastic dishes to allow overnight aggregation. Spheroids were suspended at room temperature in 0.5 ml endothelial cell basal media (ECBM; Promocell, Heidelberg, Germany) containing 20% FCS and 1% (w/v) methylcellulose before polymerization of the rat tail-derived collagen gel. The ice-cold collagen stock solution (8 vol.) was mixed with 10 x M199 (Sigma, Taufkirchen, Germany; 1 vol.) and 0.1 N NaOH (approximately 1 vol.) to adjust the pH to 7.4. The neutralized collagen solution (0.5 ml) was rapidly mixed with 0.5 ml spheroid suspension and allowed to polymerize in prewarmed 24-well plates. After polymerization, 100 µl basal medium with 10 x concentrated test substances was added on top of each gel. After 24 h, 3D in vitro angiogenesis was digitally quantified by measuring the length of the sprouts that had grown out of each spheroid (ocular grid at 40 x magnification; CSL: cumulative sprout length) using the digital imaging software analySIS (Soft imaging system, Muenster, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of a VEGF165 mutant (VEGF165KF) that binds to neuropilins but not to VEGFR-1 or VEGFR-2
To investigate the mechanism by which neuropilins enhance VEGF-induced signaling via the VEGFR-2 receptor, we produced a VEGF165 mutant displaying a strongly reduced affinity to VEGFR-2 and VEGFR-1 (VEGF165KF) by introducing point mutations into the respective VEGFR-1 and VEGFR-2 binding sites of VEGF165 based on previously published data (28 , 29) . To compare the binding characteristic of VEGF165 and VEGF165KF, we used PAE cells that do not express endogenous VEGF receptors (15 , 16 , 33) in which we have stably expressed each of the known VEGF receptors (16) to conduct binding/competition experiments. These experiments revealed that the dissociation constant of VEGF165KF to VEGFR-2 is ~4,000-fold higher than that of VEGF165 (Fig. 1 A) and that it does not bind at all to VEGFR-1 (Fig. 1B ). In the case of VEGFR-2, it was difficult to obtain exact values because at the highest VEGF165KF concentrations used, there is already significant displacement of 125I-labeled VEGF165 from cell surface heparan-sulfates, making it difficult to distinguish between binding to VEGFR-2 and heparan-sulfates. Therefore, the true VEGFR-2 dissociation constant of VEGF165KF may be higher than our estimate. By contrast, VEGF165KF competed efficiently with 125I-labeled VEGF165 for binding to PAE cells expressing either np1 or np2, with an affinity that was only marginally lower than the affinity of VEGF165 (Fig. 1C, D ).


Figure 1
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  		Figure 1. Characterization of the receptor binding properties of vascular endothelial growth factor165KF (VEGF165KF). 125I-labeled VEGF165 (5 ng/ml) was bound for 90 min. at 4°C to porcine aortic endothelial (PAE)/VEGF receptor-2 (VEGFR-2) (A), PAE/VEGFR-1 (B), PAE/np1 (C), or PAE/np2 (D) cells in the presence of increasing concentrations of VEGF165 (open circles) or VEGF165KF (solid circles) as described (16) . Bound 125I-labeled VEGF165 was quantified using a gamma counter.

VEGF165KF induces tyrosine phosphorylation of VEGFR-2 in the presence of np1
On the basis of the binding properties of VEGF165KF, we hypothesized that VEGF165KF will lack the ability to induce VEGFR-2-mediated signal transduction. We also thought that an excess of VEGF165KF should inhibit the formation of VEGF165-induced VEGFR-2/np1 complexes (22) and as a result inhibit the np1-mediated enhancement of VEGF165-induced signal transduction by VEGFR-2. VEGF165KF did not induce VEGFR-2 phosphorylation in PAE cells expressing VEGFR-2 but no neuropilins or in PAE cells expressing VEGFR-2 and np2 even at concentrations as high as 1 µg/ml (Fig. 2 C, B). However, we found that 0.2 µg/ml of VEGF165KF already promoted phosphorylation of VEGFR-2 in PAE/VEGFR-2/np1 cells (Fig. 2A ), and even 10 ng/ml of VEGF165KF already induced a clear low level phosphorylation of VEGFR-2 in these cells (data not shown). Both Y-951 (Fig. 2A ) and Y-1175 (data not shown) were phosphorylated in response to VEGF165KF. The VEGF165KF-induced phosphorylation of VEGFR-2 in PAE/VEGFR-2/np1 cells also resulted in induction of downstream signaling as manifested by the induction of ERK1/2 phosphorylation (Fig. 2D , lane 6). In accordance with these observations, we found that at a concentration of 50 ng/ml, VEGF165KF was also able to induce significant in vitro angiogenesis in a sprout formation assay (31 , 32) done using HUVEC cells that express VEGFR-2 and both neuropilins (Fig. 3 ). The effects of VEGF165KF and VEGF165 in this assay depended on neuropilins, since siRNA directed against np1, as well as siRNA directed against np2 inhibited the sprouting that was induced by VEGF165 by ~50–60% and inhibited completely the sprouting response to VEGF165KF (data not shown), indicating that both np1 and np2 play an important regulatory role in the regulation of in vitro angiogenesis.


Figure 2
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  		Figure 2. VEGF165KF induces np1 dependent phosphorylation of VEGFR-2 and extracellular regulated kinase 1/2 (ERK1/2). PAE/VEGFR-2/np1 cells (A), PAE/VEGFR-2/np2 cells (B) or PAE/VEGFR-2 cells (C) were incubated in the absence of added growth factors (lane 1) in the presence of 0.2 µg/ml (lanes 3 and 4) or 1 µg/ml (lanes 5 and 6) of VEGF165KF (KF) for 10 min. at room temperature. After this preincubation, VEGF165 (165) (20 ng/ml) was added to the designated cell cultures (lanes 2, 3, and 5), and all of the dishes were incubated 10 more min. at room temperature. Phosphorylation of Y-951 of VEGFR-2 was determined by Western blot analysis using antibodies specific for the phosphorylated tyrosine 951 residue of VEGFR-2, as described in Materials and Methods. The blots were stripped and reprobed with an Ab directed against VEGFR-2 (Total VEGFR-2). ERK1/2 phosphorylation (D) in the three cell types was determined after stimulation with 50 ng/ml of VEGF165 or 50 ng/ml VEGF165KF, as described previously (27) . The experiment was performed 3 times with similar results.


Figure 3
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  		Figure 3. VEGF165KF induces sprouting angiogenesis of human umbilical vein endothelial cells (HUVEC). A) HUVEC spheroids were embedded in collagen gels and stimulated for 24 h with 25 ng/ml VEGF165, 50 ng/ml VEGF165KF, or 25 ng/ml VEGF165 and 1 µg/ml VEGF165KF, as described in Material and Methods. Purified supernatants from mock transfected cells were used as a control. After 24 h, in vitro angiogenesis was digitally quantified by measuring the cumulative length of the sprouts that had grown out of the spheroid. B) The mean and SD of the cumulative sprout length from 10 randomly selected spheroids per gel are shown. The experiment was performed two times with similar results.

The PAE/VEGFR-2, PAE/VEGFR-2/np2 and the PAE/VEGFR-2/np1 cell lines express very similar levels of VEGFR-2 ( Fig. 5B , C). As expected, np1 potentiated substantially the VEGF165 induced phosphorylation of VEGFR-2 (compare Fig. 2A lane 2 and Fig. 2C lane 2). However, contrary to our expectations, VEGF165KF did not inhibit the VEGF165 induced phosphorylation of VEGFR-2 in PAE/VEGFR-2/np1 cells and did not limit it to the levels observed in PAE/VEGFR-2 cells (Fig. 2A , lane 5), indicating that the enhancement of VEGFR-2 phosphorylation by np1 does not depend on simultaneous binding of VEGF165 to VEGFR-2 and np1 to form a bridge linking the two receptors. In agreement, an excess of VEGF165KF did not inhibit VEGF165 induced in vitro angiogenesis either (Fig. 3) . Interestingly, VEGF165KF did not induce VEGFR-2 or ERK1/2 phosphorylation in the presence of np2 (Figs. 2B, D ) although np2 seemed to potentiate VEGFR-2 phosphorylation in response to VEGF165 (compare Fig. 2B , lane 2 and Fig. 2C , lane 2).


Figure 4
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  		Figure 4. The influence of neuropilins on the interaction of VEGF165KF with VEGFR-2. A) PAE/VEGFR-2/np1 cells were grown to confluence in 6-cm dishes as described. 125I-labeled VEGF165 (10 ng/ml) was bound to the cells at 4°C for 90 min. in the absence of any additions (lanes 1 and 2) or in the presence of increasing concentrations of VEGF165 (lanes 3–7) or VEGF165KF (lanes 8–10). The cells were subsequently washed and bound. 125I-labeled VEGF165 was cross-linked to the cells using BS3 as described. Cross-linked complexes were separated on a 6% SDS/PAGE and autoradiographed to visualize labeled bands, as described under Materials and Methods. The experiment was performed two times with similar results. B) PAE/VEGFR-2/np1 (lanes 1 and 2), PAE/VEGFR-2/np2 (lane 3), or PAE/VEGFR-2 (lanes 4–6) cells were incubated with 125I-labeled VEGF165 (0.2 ng/ml, lanes 1 and 4) or 125I-labeled VEGF165KF at 10 ng/ml (lanes 2 and 5) or at 100 ng/ml (lanes 3 and 6) for 90 min. at 4°C in the presence of 10 µg/ml heparin. Covalent cross-linking of 125I-labeled VEGFs to the cells and visualization of cross-linked complexes were performed as described under A. C) 125I-VEGF165 (lanes 2–4) or 125I-VEGF165KF (lanes 1, 5, and 6) were bound at the indicated concentrations to PAE cells expressing VEGFR-2 (VR2) alone or cells expressing in addition np1 or np2 as indicated. Cross-linked complexes were detected using autoradiography following SDS/PAGE as described.


Figure 5
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  		Figure 5. Both neuropilins enhance VEGFR-2-mediated signal transduction induced by VEGF121 and VEGF165. A) Increasing concentrations of 125I-labeled VEGF121 were bound to PAE/VEGFR-2 or to PAE/VEGFR-2/np1 cells that were grown to confluence in 24-well dishes for 90 min at 4°C. Nonspecific binding was determined in the presence of 1 µg/ml unlabeled VEGF121. At the end of the binding reaction, the cells were washed twice with cold PBS and lysed, and the amount of bound 125I-labeled VEGF121 measured using a gamma counter. Scatchard analysis of the binding was performed using the GraphPad Prism-4 software. B, C) PAE/VEGFR-2, PAE/VEGFR-2/np1, and PAE/VEGFR-2/np2 cells were incubated for 10 min in serum-free medium at room temperature in the absence of added growth factors (lanes 1, 4, and 7) or in the presence of 10 ng/ml of either VEGF165 (lanes 3, 6, and 9) or VEGF121 (lanes 2, 5, and 8). Phosphorylation of VEGFR-2 on Y-951 (B) or on Y-1175 (C) was then determined by Western blot analysis using antibodies specific for these two phosphorylated tyrosine residues, as described in Materials and Methods. The blot was stripped and reprobed with an Ab directed against VEGFR-2 (Total VEGFR-2). The ratio between the amount of phosphorylated tyrosine-951 or tyrosine-1175 and total VEGFR-2 was determined as described in Materials and Methods. This ratio is termed normalized band density in the histograms, which were derived from the gels. The experiment was performed three times with similar results.

The effect of neuropilins on the binding of 125I-labeled VEGF165KF to VEGFR-2
The observations described above suggest that np1 may perhaps increase substantially the affinity of VEGFR-2 to VEGF165KF, enabling VEGF165KF binding to VEGFR-2 despite the reduced affinity that VEGF165KF displays toward VEGFR-2 in the absence of neuropilins. We, therefore, conducted binding/cross-linking experiments to PAE/VEGFR-2/np1 cells in order to determine whether neuropilins enable the binding of VEGF165KF to VEGFR-2 (Fig. 4 ). In binding/cross-linking experiments conducted in PAE/VEGFR-2/np1 cells, we found that 0.25 µg/ml VEGF165 inhibited completely the binding of 125I-labeled VEGF165 to the VEGFR-2 receptor and to np1 (Fig. 4A , lane 5). In comparison, even 4 µg/ml of VEGF165KF did not inhibit completely the binding of 125I-labeled VEGF165 to VEGFR-2, although partial competition was observed (Fig. 4A , lane 10). In contrast, the binding of 125I-labeled VEGF165 to np1 was inhibited by VEGF165 and VEGF165KF at similar concentrations (Fig. 4A ).

Because the competition experiment described above is not sufficiently sensitive to detect low levels of VEGF165KF binding to VEGFR-2, we also used 125I-VEGF165KF in binding/cross-linking experiments (Fig. 4B ). Heparin potentiates the binding of 125I-labeled VEGF165 to neuropilins and to VEGFR-2 by acting as an extracellular chaperone for VEGF165 (14) . It was recently claimed that np1 functions as a heparin mimetic (34) . We reasoned that if that hypothesis is correct, then it can be predicted that in the presence of a saturating concentration of heparin, the neuropilins will not be able to potentiate the binding of VEGF165 to VEGFR-2. To test this assumption, we also included a saturating concentration of heparin (10 µg/ml) in the following binding/cross-linking experiments shown in Figs. 4B, C . Despite the presence of heparin, in the absence of neuropilins, there was no detectable binding of a low concentration of 125I-labeled VEGF165 (0.2 ng/ml) to VEGFR-2 in cells expressing only VEGFR-2 (Fig. 4B , lane 4) nor did heparin enable the binding of higher concentrations of 125I-labeled VEGF165KF (10 and 100 ng/ml) to VEGFR-2 in these cells (Fig. 4B , lanes 5 and 6). However, in the presence of np1, the low 125I-labeled VEGF165 concentration that we used was already high enough to enable significant binding of 125I-VEGF165 to VEGFR-2 (Fig. 4B , lane 1). It follows that np1 has an enhancing effect even in the presence of heparin, suggesting that np1 does not function as a simple heparin mimetic. Interestingly, in the presence of np1, we detected binding of 125I-labeled VEGF165KF to VEGFR-2, even at the relatively low 125I-VEGF165KF concentration of 10 ng/ml (Fig. 4B , lane 2), thus providing a possible explanation to the VEGFR-2 phosphorylation-enhancing effects of VEGF165KF in these cells. Np2 also enabled the binding of 125I-labeled VEGF165KF to VEGFR-2, although we could detect the binding only in the presence of 100 ng/ml 125I-labeled VEGF165KF (Fig. 4B , lane 3). These results indicate that we should have been able to detect VEGFR-2 phosphorylation in response to 1 µg/ml VEGF165KF in cells coexpressing VEGFR-2 and np2, but we still do not know why we could not detect VEGFR-2 phosphorylation under these conditions (Fig. 2B , lane 6).

Surprisingly, we noticed that while the low 125I-labeled VEGF165 concentration used is sufficient to promote significant binding to VEGFR-2 in cells coexpressing VEGFR-2 and np1, there is hardly any detectable binding to np1 (Fig. 4B , lane 1) suggesting that np1 may perhaps be able to enhance the binding of VEGF165 to VEGFR-2 even in its unoccupied state. To test this hypothesis, we repeated the experiment using a slightly higher concentration of 125I-labeled VEGF165 (0.3 ng/ml). It can be seen that in the presence of np1, there is significant binding of 125I-VEGF165 to VEGFR-2 but no binding to np1 (Fig. 4C , lane 3), whereas in cells expressing VEGFR-2 alone, there is no binding to VEGFR-2 (Fig. 4C , lane 4). In cells coexpressing np2 and VEGFR-2, there was also binding to VEGFR-2, and here again, there was no detectable binding of 125I-labeled VEGF165 to np2 (Fig. 4C , lane 2). Analysis of 125I-labeled VEGF165KF binding to np1 and np2 in these cells (Fig. 4C , lanes 5 and 6) indicates that the concentration of np2 in PAE/VEGFR-2/np2 cells is similar to that of np1 in PAE/VEGFR-2/np1 cells following deduction of nonspecific binding (the concentration of np2 was 30% higher). The concentration of VEGFR-2 in the different cell lines was compared using anti-VEGFR-2 antibodies and appeared to be more or less similar in all of the cell lines we have used (Fig. 5 B).

These results strongly suggest that a neuropilin that has an unoccupied VEGF binding site may be capable of enhancing the binding of VEGF to VEGFR-2. These observations suggested that np1 may also enhance the binding and biological activity of VEGF forms that do not bind to np1. To test this possibility, we conducted saturation binding experiments in which we bound 125I-labeled VEGF121 to VEGFR-2 in cells expressing only VEGFR-2 and in cells coexpressing VEGFR-2 and np1 (Fig. 5A ). Scatchard analysis of these binding experiments revealed that the concentration of VEGFR-2 in the PAE/VEGFR-2, and the PAE/VEGFR-2/np1 cells was similar (12,000 vs. 14,000 receptors/cell, respectively). However the affinity of VEGFR-2 toward 125I-labeled VEGF121 was ~10-fold higher in the presence of np1 (KD = 0.4 nM) than in the absence of np1 (3.5 nM) (Fig. 5A ). This difference was statistically significant and was observed in two independent experiments. Similar 125I-VEGF121-binding experiments designed to characterize the effects of np2 on the interaction of VEGFR-2 with VEGF121 showed a small increase of two-fold in affinity. However, even though this change was seen in two independent experiments, it was not statistically significant (data not shown).

Np1 and np2 enhance VEGF121 and VEGF165-induced phosphorylation of VEGFR-2 in PAE cells
The binding experiments suggested that neuropilins may be able to enhance the activity of VEGF121, even though VEGF121 does not bind to neuropilins. To determine directly whether neuropilins affect VEGF121 activity, we stimulated PAE cells expressing recombinant VEGFR-2 and neuropilins with either VEGF121 or VEGF165. The phosphorylation of VEGFR-2 on Y-951, as well as on Y-1175 was strongly enhanced by VEGF121 and by VEGF165 in the presence of np1 as compared to the phosphorylation levels obtained under identical conditions in PAE cells, expressing only VEGFR-2 (Fig. 5B, 5C ; compare lanes 2 and 3 with lanes 5 and 6). The enhancement was seen in the presence or absence of heparin (data not shown). A significant though smaller enhancement of VEGF121 and VEGF165-induced phosphorylation was also noted in cells coexpressing VEGFR-2 and np2 (Fig. 5B, C , lanes 8 and 9). This experiment indicates that np2 also functions as an enhancer of VEGF121, as well as VEGF165 signaling mediated by VEGFR-2. Furthermore, this experiment indicates that neuropilins can enhance VEGFR-2-mediated VEGF signaling, even when the neuropilin binding sites are not occupied by VEGF as in the case of VEGF121. To make sure that VEGF121 is unable to bind to np1 in the presence of VEGFR-2, we performed binding/cross-linking experiments, in which we bound 125I-VEGF121 to PAE/VEGFR-2/np1 and PAE/VEGFR-2/np2 cells. However, we could not detect binding of 125I-VEGF121 to np1 or to np2 in these cells, even when a 125I-labeled VEGF121 concentration as high as 100 ng/ml was used although cross-linked 125I-labeled VEGF121/VEGFR-2 complexes were easily detectable (data not shown).

Np1 and np2 regulate VEGF121 and VEGF165 induced signal transduction by the VEGFR-2 receptor of HUVEC cells
To find out whether the conclusions obtained using the model system described above also hold true in primary endothelial cells, we used specific siRNAs to inhibit the expression of neuropilins in HUVEC (Fig. 6 A). We then proceeded to examine the effects of these siRNA species on VEGF-induced proliferation. As expected, the np1-directed siRNA inhibited VEGF121, as well as VEGF165-induced cell proliferation, although the inhibition was not complete (Fig. 6B ). In contrast, siRNA directed against np2 produced only marginal inhibition of VEGF121 and VEGF165 activity (Fig. 6B ). This may be due to the relatively low expression levels of np2 in HUVEC, which are about one-third of the expression levels of np1 (16) . However, a combination of both siRNAs consistently inhibited both VEGF121 and VEGF165 induced cell proliferation somewhat more potently than the np1-directed siRNA on its own, indicating that np2 does contribute to VEGF121, as well as VEGF165 signal transduction in HUVEC (Fig. 6B ).


Figure 6
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  		Figure 6. The effect of small interfering RNAs (siRNAs) directed against np1 and np2 on VEGF-induced proliferation and on VEGF-induced phosphorylation of VEGFR-2 in HUVEC. HUVEC were transfected with a control nonsilencing siRNA (C), with a np1-specific siRNA (si-Np1), with a siRNA-directed against np2 (Si-Np2), or cotransfected with both neuropilin directed siRNAs (Si-Np1+2) as described. The cells were subsequently trypsinized and seeded for different experiments. A) Cells were seeded in 6-well dishes and cultured 16 h at 37°C as described. The cells were then lysed and 40 µg protein from the cell lysates separated on an SDS/PAGE gel, blotted onto nitrocellulose, and probed with antibodies directed against np1 or np2. To verify that the amount of protein in each lane is similar, the membrane was stripped and reprobed with antibodies directed against ß-actin. B) Cells were seeded in 24-well dishes and cultured as described. VEGF121 and VEGF165 were added to a final concentration of 10 ng/ml. On the third day after seeding, the growth factors were added again. Cells were counted using a Coulter counter after 5 days, as described previously (27) . C) One day after transfection with siRNAs, HUVEC were seeded in 6-well dishes and incubated 16 h at 37°C, as described in experimental procedures. At the start of the experiment, some dishes received no additions (lanes 1, 4, 7, and 10), while others were stimulated with VEGF165 (10 ng/ml) (lanes 3, 6, 9, and 12) or with VEGF121 (10 ng/ml) (lanes 2, 5, 8, and 11). The cells were incubated 10 min at room temperature. The experiment was terminated, and the cells were lysed. The lysates were separated on an SDS/PAGE and blotted onto nitrocellulose filters. Western blot analysis was performed using an Ab directed against phosphorylated Y-951 of VEGFR-2 as described. The blots were stripped and reprobed with antibodies directed against VEGFR-2 (Total VEGFR-2). Quantification of band intensity and determination of the ratio of phosphorylated vs. total VEGFR-2 were done as described. The ratio is designated in the histogram as the normalized band density.

Similar results were obtained in experiments in which the effects of the two siRNAs on VEGF121 and VEGF165 induced phosphorylation of VEGFR-2 of HUVEC were assessed. The np1 siRNA inhibited VEGF121 and VEGF165 induced phosphorylation of VEGFR-2 at Y-951 (Fig. 6C ) and Y-1175 (Data not shown). On its own, the np2 siRNA did not inhibit significantly the phosphorylation induced by both VEGF forms. However, a combination of the np1 and np2-directed siRNA species, which inhibits the expression of both neuropilins inhibited VEGF165, as well as VEGF121-induced phosphorylation of VEGFR-2 more potently than the inhibition obtained using the np1-directed siRNA alone (compare Fig. 6C , lanes 3 and 9). Similarly, we have observed that sprouting induced by VEGF165, as well as sprouting induced by VEGF121 in an in vitro angiogenesis assay (see Fig. 3 ), was inhibited by 50–60% by siRNA directed against either np1 or np2 but not by a control siRNA, indicating that neuropilins enhance VEGF121-induced sprouting, as well as VEGF165-induced sprout formation (data not shown).

The effects of various VEGF forms on complex formation between VEGFR-2 and neuropilins
It was suggested that the effects of np1 on VEGF-induced VEGFR-2 signaling depends on the formation of stable VEGF-induced complexes between VEGFR-2 and np1 (22) . We have indeed observed that VEGF165 promotes the formation of stable VEGFR-2/np1 complexes that survive coimmunoprecipitation experiments (Fig. 7 A, lane 2). Inclusion of a high VEGF165KF concentration did not inhibit the formation of the VEGF165-induced complexes supporting the notion that in these complexes VEGF165 need not be bound to np1 (Fig. 7A , lane 3). However, even though np1 enhances VEGF121 induced phosphorylation of VEGFR-2 almost as potently as VEGF165 induced phosphorylation (Fig. 5) , we could not detect significant VEGF121-induced de novo formation of stable VEGFR-2/np1 complexes (Fig. 7A , lane 5). Only in the presence of a nonphysiological concentration of 1 µg/ml VEGF121 could we detect an increase in the concentration of these complexes above basal levels(Fig. 7A , lane 7). Interestingly, a high VEGF165KF concentration (1 µg/ml) that produces significant phosphorylation of VEGFR-2 in the presence of np1 (Fig. 2A ) did not induce formation of de novo VEGFR-2/np1 complexes (Fig. 7A , lane 4), indicating independently that the enhancement of VEGF induced biological activity by neuropilins is not necessarily associated with the creation of de novo VEGF-induced VEGFR-2/neuropilin complexes. These observations suggest that the concentration of spontaneously formed complexes found in nonstimulated cells (Fig. 7A , lane 1) may suffice to support neuropilin-mediated enhancement of VEGF binding to VEGFR-2 and VEGF-induced phosphorylation of VEGFR-2. We also found that VEGF165 (Fig. 7B , lane 2), as well as VEGF121 (Fig. 7B , lanes 4 and 6) were able to promote a consistent increase in the concentration of VEGFR-2/np2 complexes above basal levels. In this respect, np2 differs somewhat from np1.


Figure 7
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  		Figure 7. Formation of VEGFR-2/np1 and VEGFR-2/np2 complexes in response to various forms of VEGF. To detect complexes of neuropilins and VEGFR-2 PAE/VEGFR-2/np1 cells (A) or PAE/VEGFR-2/np2 cells (B) were seeded in 6-cm dishes as described. The cells in both experiments were incubated without added growth factors (lane 1) or stimulated with 50 ng/ml VEGF165 (lanes 2 and 3 in A and lane 2 in B), 50 ng/ml VEGF121 (lanes 5 and 6 in A or lanes 4 and 5 in B) or 1 µg/ml VEGF121 (lanes 7 and 8 in A or lanes 6 and 7 in B) or with 1 µg/ml VEGF165KF (lane 4 in A or lane 3 in B). Some plates were coincubated in addition to VEGF121 or VEGF165 with 1 µg/ml VEGF165KF (lanes 3, 6, and 8 in A or lanes 5 and 7 in B). Coimmunoprecipitation of VEGFR-2/np1 and VEGFR-2/np2 complexes was performed using antibodies directed against np1 (A) or np2 (B) for immunoprecipitation and antibodies directed against VEGFR-2 for detection of coimmunoprecipitated VEGFR-2, as described in Materials and Methods. The blots were then stripped and reprobed with antibodies against np1 (A) or np2 (B) to make sure that the efficiency of immunoprecipitation was similar in all of the experimental systems. The experiment was performed 3 times with similar results.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The current model describing the mechanism by which np2 enhances VEGF165 signaling assumes that VEGF165 binds first to np1. This concentrates VEGF165 on the cell surface and "presents" VEGF165 to adjacent VEGFR-2 receptors, leading to the formation of a VEGFR-2/np1 complex that is stabilized by VEGF165 binding to both receptors, thereby forming a "bridge" (22) . This model assumes that the formation of the bridge is important for the enhancing effect of np1 and predicts that np1 will not be able to enhance VEGF121-induced signaling via VEGFR-2, as VEGF121 does not bind to neuropilins (14) . To investigate further the mechanism by which neuropilins modulate VEGF signaling, we produced VEGF165KF, a VEGF mutant that binds to neuropilins but not to tyrosine-kinase VEGF receptors. We thought that VEGF165KF would inhibit the association of VEGF165 with neuropilins, prevent the presentation of VEGF165 to VEGFR-2 by np1, and possibly by np2, and disrupt the formation of VEGF165 cross-bridges. Indeed, in the absence of neuropilins, the affinity of VEGF165KF to both VEGFR-1 and VEGFR-2 was so low we could not measure it accurately if at all (at least 4 orders of magnitude lower). Surprisingly, in the presence of np1 and to a much lesser extent also in the presence of np2, VEGF165KF regained its ability to bind VEGFR-2, albeit with a much reduced affinity compared to VEGF165. This experiment indicates that np1 enhances VEGF binding to VEGFR-2 so potently that it enables even VEGF165KF to bind to VEGFR-2 at a detectable level. The increase in affinity induced by np1 apparently enables VEGF165KF to initiate signal transduction via VEGFR-2 at relatively high concentrations. Np2 also enabled VEGF165KF binding to VEGFR-2 but only at a much higher VEGF165KF concentration, and in contrast with np1 did not enable VEGF165KF signaling through VEGFR-2. This difference between np1 and np2 may be due, at least in part, to the relatively inefficient enhancement of VEGF165KF binding to VEGFR-2 by np2. Surprisingly, VEGF165KF did not inhibit the formation of VEGF165-induced VEGFR-2/np1 complexes, regardless of the VEGF165KF concentrations used. Because VEGF165KF inhibits the binding of VEGF165 to neuropilins but not to VEGFR-2, it follows that that the formation of VEGF165 bridges is not required for the formation or stabilization of VEGFR-2/np1 complexes. Furthermore, VEGF165KF was not able to inhibit the enhancing effect that np1 exerts on VEGF165 induced signal transduction via VEGFR-2, suggesting that np1 may be able to enhance VEGF165-induced signaling via VEGFR-2, even under conditions in which the binding of VEGF165 to np1 is inhibited.

Interestingly, and in agreement with this last observation, we noticed that np1, and to a lesser extent np2, enhanced the binding of very low concentrations of 125I-labeled VEGF165 to VEGFR-2, even though we could not detect any binding of VEGF165 to neuropilins at these concentrations. Looking back, we found that we had made similar observations in experiments, in which we cross-linked increasing concentrations of 125I-labeled VEGF165 to HUVEC, although we did not grasp their significance at the time (35) . These observations also suggest that VEGF165 may not need to bind to np1 in order for np1 to be able to enhance the binding of VEGF165 to VEGFR-2 and enhance VEGF165 signaling via VEGFR-2. In agreement with this hypothesis, we have observed that np1 is able to enhance VEGFR-2 phosphorylation in response to VEGF121 and that the enhancement is associated with a substantial np1-induced increase in the affinity of VEGFR-2 to VEGF121. These results were strengthened by experiments that showed that inhibition of np1 expression in primary endothelial cells inhibited their VEGF121-induced proliferation, indicating that np1 can enhance signal transduction induced by VEGF forms that do not bind np1. These observations are in apparent contradiction with previous experiments, in which we found that VEGF121-induced cell migration, is not enhanced by np1 (15) . Our experiments, therefore, suggest that the binding of VEGF to np1 is not required for certain np1-mediated activities, such as the enhancement of VEGF-induced cell proliferation, while enhancement of some activities, such as cell migration may require an interaction of VEGF with np1. These differences will need to be studied in more detail in the future.

We also observed that np2 is also able to enhance VEGFR-2-mediated VEGF121 and VEGF165 signal transduction. However, our experiments indicate that np2 is a less potent enhancer as compared to np1. It is not clear how np2 potentiates VEGF-induced signaling mediated by VEGFR-2. Although we noticed in two experiments, a small np2-mediated increase in the affinity of VEGF121 to VEGFR-2, this increase was not sufficiently large to be statistically significant. Thus, it is not clear whether in the case of np2, an increase in VEGFR-2 affinity toward VEGF represents the major mechanism by which np2 affects VEGFR-2-mediated signaling. Nevertheless, our experiments indicate that np2 clearly contributes to VEGF signaling in endothelial cells, since inhibition of the expression of both neuropilins in HUVEC abrogates completely the proliferation/survival responses to VEGF, while inhibition of np1 expression alone does not inhibit completely the response to VEGF121 or VEGF165. This observation is in agreement with prior reports in which it was found that the phenotype of double knockout mice lacking both np1 and np2 is similar to the phenotype of mice that lack functional VEGFR-2 receptors (36) .

The enhancing effects of np1 have been reported previously to depend on the formation of VEGFR-2/np1 complexes. Nevertheless, different reports do not agree on whether these complexes exist before stimulation with VEGF165 or whether these complexes form strictly in response to a VEGF165 challenge (22 , 23) . Our findings indicate that in cells expressing relatively high concentrations of VEGFR-2 and np1, such complexes can form spontaneously, although VEGF165 is able to induce formation of additional complexes above basal levels. In contrast, VEGF121 did not induce formation of VEGFR-2/np1 complexes above basal levels. Because we find that np1 can enhance VEGF121-induced signal transduction via VEGFR-2, we hypothesize that enhancement of VEGF121 activity by np1 will depend on the relative concentrations of VEGFR-2 and np1 in target cells, since these concentrations will determine the concentration of preformed complexes. Our hypothesis, therefore, predicts that in cells expressing low concentrations of VEGFR-2 and/or np1, we may not be able to see an enhancement of VEGF121 activity by np1 because of low concentrations of preformed complexes. In contrast we expect that VEGF165 activity will be enhanced by np1 in such cells because VEGF165 is expected to promote the formation of de novo complexes.

To conclude, our experiments indicate that np1 is able to enhance VEGF-induced signal transduction via VEGFR-2, even when the VEGF binding site of np1 remains unoccupied. In the case of np1, this effect is at least partially due to a np1-induced increase in the affinity of VEGFR-2 to VEGF. Our experiments also provide evidence indicating that np2 also enhances VEGF-induced signaling via VEGFR-2, although the mechanism used by np2 to enhance VEGF signaling is not yet completely clear. Furthermore, our experiments show that np1 and np2 collaborate to enhance VEGF-induced signaling via VEGFR-2 in endothelial cells. These observations indicate that both np1 and np2 should perhaps be considered as potential targets for the development of drugs that target VEGF signal transduction.


   ACKNOWLEDGMENTS
 
We thank Sharon Soueid for invaluable help with binding experiments. This work was supported by grants from the Israel Science Foundation (ISF) (to G.N.), by the German-Israeli Binational Foundation (GIF) (to G.N. and H.A.), by the International Union Against Cancer (AICR) (to G. N.), and by the by the Rappaport Family Institute for Research in the Medical Sciences of the Faculty of Medicine at the Technion, Israel Institute of Technology (to G. N.).

Received for publication April 5, 2006. Accepted for publication October 2, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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