Vascular Endothelial Growth Factor 165 (VEGF165) Activities Are Inhibited by Carboxymethyl Benzylamide Dextran That Competes for Heparin Binding to VEGF165 and VEGF165·KDR Complexes

We have previously shown that carboxymethyl dextran benzylamide (CMDB7), a heparin-like molecule, inhibits the growth of tumors xenografted in nude mice, angiogenesis, and metastasis by altering the binding of angiogenic growth factors, including platelet-derived growth factor, transforming growth factor β, and fibroblast growth factor 2, to their specific receptors. In this study, we explore the effect of CMDB7 on the most specific angiogenic growth factor, vascular endothelial growth factor 165 (VEGF165). We demonstrate here that CMDB7 inhibits the mitogenic effect of VEGF165 on human umbilical vein endothelial cells (HUV-ECs) by preventing the VEGF165-induced VEGF receptor-2 (KDR) autophosphorylation and consequently a specific intracellular signaling. In competition experiments, the binding of 125I-VEGF165 to HUV-ECs is inhibited by CMDB7 with an IC50 of 2 μm. Accordingly, CMDB7 inhibits the cross-linking of125I-VEGF165 to the surface of HUV-ECs, causing the disappearance of both labeled complexes, 170–180 and 240–250 kDa. We show that CMDB7 increases the electrophoretic mobility of VEGF165, thus evidencing formation of a stable complex with this factor. Moreover, CMDB7 reduces the125I-VEGF165 binding to coated heparin-albumin and prevents a heparin-induced increase in iodinated VEGF165 binding to soluble 125I-KDR-Fc chimera. Concerning KDR, CMDB7 has no effect on 125I-KDR-Fc electrophoretic migration and does not affect labeled KDR-Fc binding to coated heparin-albumin. In the presence of VEGF165,125I-KDR-Fc binding to heparin is enhanced, and under these conditions, CMDB7 interferes with KDR binding. These data indicate that CMDB7 effectively inhibits the VEGF165activities by interfering with heparin binding to VEGF165and VEGF165·KDR complexes but not by direct interactions with KDR.

Neovascularization is essential for tumor growth and is regulated by tumor cell-produced factors that have mitogenic and chemotactic effects on vascular endothelial cells (1,2). Several studies have recently indicated that vascular endothelial growth factor (VEGF-A) 1 expression in tumor cells may play a major role in tumor angiogenesis (3)(4)(5)(6). VEGF-A was observed in vivo to act as a potent angiogenic factor and blood vessel permeabilizing agent (7,8).
VEGF-A is an homodimeric glycoprotein, the monomer of which exists in five forms, VEGF 121 , VEGF 145 , VEGF 165 , VEGF 189 , and VEGF 206 , as a result of alternative splicing from a single gene (9). These various isoforms of VEGF-A differ in their affinity for heparin and extracellular matrix components. VEGF-A forms induce endothelial cell proliferation and migration and are essential for embryonic vessel development (10). The best characterized VEGF-A is the heparin-binding 165amino acid-long form, VEGF 165 (11,12). The binding of VEGF 165 to its receptors requires cell surface heparan sulfates and can be modulated by the addition of exogenous heparin (13).
Two tyrosine kinase receptors have been identified as VEGF-A receptors, the VEGFR-1 (fms-like tyrosine kinase, Flt-1) (14) and the VEGFR-2 (kinase domain region (KDR) in human, and the homologous fetal liver kinase-1 (Flk-1) in mouse) (15). These transmembrane proteins with apparent molecular masses of about 200 and 220 kDa, respectively, have been shown to bind VEGF-A with high affinity. Both KDR/ Flk-1 and Flt-1 contain seven immunoglobulin-like domains in the extracellular regions and large insert sequences in their intracellular kinase-domains (16). Recently, an additional binding site, neuropilin-1 (NRP1), with a molecular mass of about 130 kDa, was identified and shown to be expressed on the surface of endothelial and tumor cells. NRP1 can act as a co-receptor, forming a complex with VEGFR-2 and thus enhancing VEGF 165 -induced activities mediated by VEGFR-2 (17).
Several molecules with anti-angiogenic activity have been described in the last decade (18,19). The mechanisms involved in action of most of these molecules are unclear, but several of these angiogenesis inhibitors were found to be either heparin analogs or heparin-binding substances (20,21). These observations and recent results showing that heparin-degrading enzymes can inhibit the tumor angiogenesis (22) suggest that heparan sulfates may play an important regulatory role in the angiogenic processes.
Carboxymethyl dextran benzylamide (CMDB7) is a noncytotoxic substituted dextran. Its chemical derivatization involves statistical distribution of chemical groups linked to the 1-6 glucosyl units forming the macromolecular chains (23). CMDB7 mimics some properties of heparin, such as the inter-actions with angiogenic growth factors, including FGF2 (24,25), transforming growth factor ␤, and platelet-derived growth factor (26), but it has no anticoagulant or anticomplement effects (27,28). We have shown that CMDB7 specifically inhibits the mitogenic effect and receptor binding of FGF2, plateletderived growth factor, and transforming growth factor ␤, and thus prevents the endothelial cell proliferation and migration as observed in vitro (25). In addition, CMDB7 inhibits in vitro the growth of breast tumor cells (24 -26). In vivo studies demonstrated that breast HH9 (25) and MCF-7ras (26) xenograft growth in nude mice is blocked by CMDB7 treatment. In parallel, we observed the decrease of angiogenesis within these tumors. Furthermore, CMDB7 is able to inhibit by 88% the incidence of lung micrometastasis from breast carcinoma MDA-MB435 cell implants in the mammary fad pad of nude mice (29).
In the present study, we evaluated for the first time, in our knowledge, the possible effects of CMDB7 on activities of the most specific angiogenic growth factor, VEGF 165 . We show that CMDB7 inhibits (a) mitogenic activity of VEGF 165 on endothelial cells, (b) VEGF 165 binding to its specific receptors, and (c) VEGF 165 signaling by KDR in human umbilical vein endothelial cells. We further demonstrate the CMDB7 ability to bind to VEGF 165 and to compete for heparin binding to VEGF 165 and VEGF 165 ⅐KDR complexes.

EXPERIMENTAL PROCEDURES
Dextran Derivative Preparation-A water-soluble dextran derivative (CMDB7) was prepared as previously described (23). Its chemical composition, determined by acidimetric titration and elementary analysis of nitrogen, is 0% dextran, 70% carboxymethyl and 30% benzylamide. Average molecular weight was estimated as 80,000 g/mol.
Cell Line and Cell Culture-Human umbilical vein endothelial cells (HUV-ECs) were purchased from the American Type Culture Collection (Rockville, MD). HUV-ECs were routinely grown in M199 (Life Technologies, Inc.) and were cultured at 37°C in a 5% CO 2 -humidified atmosphere. Culture medium is supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50units/ml penicillin, and 50 mg/ml streptomycin (all obtained from Life Technologies, Inc.).
Cell Proliferation Assay-HUV-ECs were seeded at a density of 2 ϫ 10 4 cells/well into 24-well tissue culture plates (Falcon, Strasbourg, France) in M199 -10% FCS. After 24 h, the cells were growth-arrested by serum starvation for another 24 h and then incubated for 72 h with VEGF 165 (R&D Systems Europe, Abingdon, United Kingdom) at various concentrations (60 -500 pM) or at a fixed VEGF 165 concentration of 250 pM in the presence or absence of CMDB7 at various concentrations. Cells were washed with PBS, dissociated with 0.025% trypsin-EDTA (Life Technologies) and counted using a Coulter counter (Coultronics, Margency, France). All experiments were performed in triplicate and data illustrate the mean cell numbers Ϯ S.E. provided from one representative of three independent experiments.
Western Blot Analysis and Anti-phosphotyrosine Assay-5 ϫ 10 5 HUV-ECs were plated into six-well tissue culture plates (Falcon) in M199 -10% FCS. After 24 h, the cells were washed in PBS and then incubated in serum-free medium containing 0.01% bovine serum albumin. After an overnight incubation at 37°C, the cells were washed with PBS and then incubated in serum-free medium containing 0.01% bovine serum albumin and 0.1 mM sodium orthovanadate for 15 min at 37°C. The media were then removed, and the cells were incubated for 5 min at 37°C with the serum-deprived medium containing 1.2 nM VEGF 165 in the presence or absence of 40 M CMDB7. The incubations were terminated by aspiration of the medium, two washings with cold PBS containing 1 mM sodium orthovanadate, followed by the addition of 200 l of cold lysis buffer (20 mM Tris-HCl (pH 7.5), 1% Igepal CA-630, 10% (v/v) glycerol, 100 mM NaCl, 5 mM EDTA, 50 mM sodium fluoride, 1/10 (v/v) protease inhibitor mixture (Sigma), 0.2 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate) at 4°C for 10 min. Insoluble material was removed by centrifugation at 4°C for 15 min at 14,000 ϫ g and the protein concentrations of soluble fraction were determined with the BCA protein assay kit (Sigma). Cellular proteins were resolved by SDS-polyacrylamide gel electrophoresis (6%) and transferred to nitrocellulose membranes (R&D Systems). Immunoblots were probed with the following antibodies: an anti-phosphotyrosine mouse monoclonal antibody PY99 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (1:1000 dilution), and an anti-KDR rabbit polyclonal (Sigma) (1:500 dilution) for 1 h and then washed in Tris-buffered saline containing 0.05% Tween-20. Antigen-antibody complexes were revealed with horseradish peroxidase-coupled secondary antibodies and the enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech).
VEGF 165 Binding to HUV-ECs-For displacement binding assays, HUV-ECs were grown to 80% confluence in 24-well tissue culture plates (Falcon). After an overnight incubation in serum-free medium and two washings with ice-cold binding buffer (PBS/0.2% gelatin), the cells were incubated at 4°C for 2 h in 0.3 ml of binding buffer containing 7 pM 125 I-VEGF 165 (Amersham Pharmacia Biotech) in the presence or absence of CMDB7 at various concentrations. Incubation was terminated by gently removing the medium and washing the cell monolayer three times with ice-cold binding buffer. The bound radioactivity was measured using a ␥-counter (LKB 1261 Multigamma) after cell lysis in 0.3 ml of 0.5 N NaOH for 30 min. Nonspecific binding was determined in the presence of an excess (5 nM) of unlabeled VEGF 165 (R&D Systems). For the Scatchard analysis, the binding was accomplished using increasing concentrations (0 -5000 pM) of unlabeled VEGF 165 (R&D Systems) and 7 pM 125 I-VEGF 165 (Amersham Pharmacia Biotech) at 4°C for 2 h. Parallel experiments were performed in the presence of 10 and 40 M CMDB7. Each curve was analyzed according to the Scatchard procedure or by fitting to a logistic curve. All experiments were carried out in triplicate and were repeated at least three times.
Affinity Cross-linking of VEGF 165 -Subconfluent cell cultures in sixwell plates (Falcon) were washed twice with binding buffer (PBS/0.2% gelatin) and then incubated for 2 h at 4°C with 100 pM 125 I-VEGF 165 (Amersham Pharmacia Biotech) in the presence of a 1000-fold excess of unlabeled VEGF 165 (R&D Systems) or 40 M CMDB7. Cells were then washed with ice-cold binding buffer and cross-linked for 15 min at room temperature with 0.5 mM disuccinimidyl suberate (Pierce Perbio Science France, Bezons, France). The reaction was quenched by addition of an excess of 1 M Tris-HCl (pH 7.5) for 2 min. Cells were lysed with ice-cold lysis buffer (10 mM Tris-HCl, pH 7.0, 1 mM EDTA, 1% Nonidet P-40 (v/v), 1/10 (v/v) protease inhibitor mixture (Sigma)) for 10 min. Cell extracts were then clarified by centrifugation, and supernatants mixed with 2ϫ sample buffer (100 mM Tris-HCl, 4% SDS, 10% glycerol, 0.05% bromphenol blue) were boiled for 3 min before loading on a SDS-6% polyacrylamide gel. After sample running, gels were dried and exposed (7-20 days) to a Kodak X-Omat film at Ϫ80°C for autoradiography.
Agarose Gel Electrophoresis-The CMDB7 effects on the electrophoretic mobility of 125 I-VEGF 165 (Amersham Pharmacia Biotech) and 125 I-KDR-Fc (labeled in laboratory) were analyzed by nondenaturant agarose gel electrophoresis as described by Lee and Lander (30). After a 1-h incubation of 125 I-VEGF 165 (10 5 cpm/3 ng) or 125 I-KDR-Fc (10 5 cpm/3 ng) with CMDB7 at various concentrations at 4°C, the mixtures (10 l) were analyzed in 1% agarose gel at pH 7.0 using running buffer containing 125 mM sodium acetate, 50 mM 3-(N-morpholino)-2-hydroxypropane sulfonic acid, 6% sucrose, and 0.5% (w/v) bromphenol, and typical electrophoresis was performed at 60 -70 V for 2 h. Gels were dried and then exposed to Kodak X-Omat film (Amersham Pharmacia Biotech). If the negatively charged CMDB7 binds to essentially electrobasic VEGF 165 or to KDR, an anodic shift in the migration of the tracer should be observed.
VEGF 165 Binding to Soluble KDR Receptors-The surface of flatbottomed polystyrene wells (Disposable Immulon 1 Remowawell, Dynatech, Cambridge, MA) were coated overnight at 4°C with 200 l of 2 g/ml anti-Fc IgG (Sigma) in PBS buffer. The nonspecific interactions were saturated with PBS containing 0.2% gelatin and 0.01% Tween-20 (PGT buffer) for an additional overnight at 4°C. Blocking buffer was then removed, and the plates were washed three times with 300 l of PGT buffer. Then, 2 g/ml purified recombinant human KDR-Fc chimera (R&D Systems), 40 pM 125 I-VEGF 165 (Amersham Pharmacia Biotech), and unlabeled VEGF 165 (R&D Systems) at increasing concentrations (0 -2500 pM) were added successively to a final volume of 200 l in PGT buffer. The incubations were performed in the presence or absence of CMDB7 or heparin (Sigma). After an overnight incubation at 4°C, wells were washed three times with 300 l of PGT buffer, and the radioactivity of each well measured in a ␥-counter (LKB 1261 Multigamma).
VEGF 165 and KDR Binding to Heparin-Albumin-Heparin-albumin (Sigma) at concentration 17 g/ml in 100 l of PBS was coated on the surface of polystyrene wells (Disposable Immulon 1 Remowawell) as described above for anti-Fc IgG. The binding assays to coated heparinalbumin were performed in total volume of 90 l overnight at 4°C. We studied the binding of 40 pM 125 I-VEGF 165 (Amersham Pharmacia Biotech) in the presence or absence of CMDB7 and the binding of 125 I-KDR (1.5 ϫ 10 5 cpm/5 ng) in the presence or absence of CMDB7 in combination or not with unlabeled VEGF 165 (R&D Systems). After washing, the radioactivity in each well was measured in a ␥-counter (LKB 1261 Multigamma).
Iodination of KDR-Radioiodination was carried out utilizing the chloramine B method. Briefly, 5 g of carrier-free human recombinant KDR-Fc chimera (R&D Systems) was suspended in 45 l of 10ϫ PBS-0.1% Triton X-100. To the reaction tube, 0.5 Ci of Na 125 I (Amersham Pharmacia Biotech) was added, followed by one IODO-BEAD (Pierce Perbio Science). Incubation was performed for 12 min and then stopped by addition of 20 l of 2 mg/ml KI, followed by 430 l of PBS-0.05% bovine serum albumin. The mixture was transferred to preequilibrated PD-10 column (Amersham Pharmacia Biotech) for separation from free iodine. The specific activity of iodinated KDR was 3 ϫ 10 7 cpm/g. The radiolabeled KDR was used to perform a KDR binding to heparinalbumin and affinity electrophoresis in agarose. 165 Mitogenic Activity-The inhibitory effect of CMDB7 was evaluated using HUV-ECs. First, we established the optimal VEGF 165 concentration at which mitogenic effect on those endothelial cells can be observed. HUV-EC number was increased by VEGF 165 at concentrations from 60 to 500 pM, with a maximal 3-fold augmentation observed in the presence of 250 pM VEGF 165 (Fig. 1A). CMDB7 (0.1-20 M) inhibited the 250 pM VEGF 165 -induced proliferation of HUV-ECs in a dose-dependent manner with an IC 50 of 2 M (Fig. 1B). Starting from concentration 5 M, CMDB7 completely prevented the VEGF 165 effect on HUV-ECs (Fig. 1B). In the absence of VEGF 165 , no significant effect of CMDB7 on the cell growth was observed.

CMDB7 Inhibits the VEGF
CMDB7 Inhibits the VEGF 165 -induced Tyrosine Phosphorylation of Receptors on HUV-ECs-Next, we asked whether CMDB7 is able to inhibit the VEGF 165 signal transduction in HUV-ECs. Binding of VEGF-A results in the conformational changes in KDR and Flt-1, followed by their dimerization and autophosphorylation on tyrosine residues (31), inducing tyro-sine phosphorylation of other numerous proteins, as observed in bovine brain capillary endothelial cells (32) and porcine aortic endothelial cells (33). HUV-EC treatment with VEGF 165 for 5 min induced the tyrosine phosphorylation of proteins with apparent molecular masses of 210 and 140 kDa (Fig. 2A, lane  2), as revealed by Western blot analysis with anti-phosphotyrosine antibody. These two bands were not detected in extracts from control cells (Fig. 2A, lane 1) or in cells treated with 40 M CMDB7 alone (Fig. 2A, lane 3). CMDB7 prevented the VEGFinduced tyrosine phosphorylation of both 210-and 140-kDa bands (Fig. 2A, lane 4). To confirm the identity of the phosphorylated proteins, a parallel immunoblot was probed with specific antibodies against KDR and Flt-1. The 210-kDa tyrosinephosphorylated band was recognized by the antibody against KDR (Fig. 2B) but not by antibody against Flt-1 (data not shown). These two antibodies did not stain a 140-kDa band (data not shown), suggesting that it does not corresponds to VEGF receptors. This protein probably participates in downstream signaling events (32,33). The KDR antibody revealed no changes in staining intensity with all treatments (Fig. 2B), demonstrating that the loss in tyrosine phosphorylation was not due to decrease in KDR protein quantity. These results show that the inhibitory effect of CMDB7 on VEGF-induced HUV-EC growth is accompanied by a decrease in tyrosine phosphorylation of KDR by VEGF.
CMDB7 Inhibits the VEGF 165 Binding to Endothelial Cells-Because CMDB7 inhibited the VEGF 165 -induced KDR phosphorylation, we tested the effect of CMDB7 on the specific binding of VEGF 165 to the HUV-ECs. First, the cells were incubated with radiolabeled VEGF 165 at a fixed concentration and CMDB7 at increasing concentrations ranging from 0.1 to 50 M. Fig. 3A shows that CMDB7 decreases the 125 I-VEGF 165 binding to its receptors in a dose-dependent manner with an IC 50 of 2 M. For the Scatchard analysis (Fig. 3B), the cells were incubated with radiolabeled VEGF 165 at a fixed concentration and unlabeled VEGF 165 at increasing concentrations in the presence or absence of CMDB7. Under control conditions (in the absence of CMDB7), two classes of high-affinity binding sites were observed, in agreement with Soker et al. (34) and Li et al. (35). The higher affinity class is characterized by a K d of 326 pM and the lower one by a K d of 27 nM. The addition of CMDB7 does not affect the ligand binding affinity of the two site classes, but it results in a dose-dependent decrease in lower affinity site number. In the presence of 40 M CMDB7, 125 I-VEGF 165 is displaced from all lower affinity receptors. These data show that CMDB7 inhibits the binding of VEGF 165 to HUV-ECs.
CMDB7 Inhibits the Formation of VEGF 165 -Receptor Complexes-To better characterize the VEGF 165 binding sites on HUV-ECs affected by CMDB7, the affinity-labeling experiments were performed. Fig. 4, lane 1, shows two bands with apparent molecular masses of 240 -250 and 170 -180 kDa. The addition of a 200-fold excess of unlabeled VEGF 165 prevented the formation of these bands (Fig. 4, lane 2), evidencing their specificity. CMDB7 (40 M) decreased the intensity of two complexes (Fig. 4, lane 3), demonstrating an inhibition of VEGF 165 binding to its specific receptors with apparent molecular masses of 200 -210 and 130 -140 kDa, calculated by subtraction of the molecular mass of VEGF 165 . The first protein corresponds to KDR monomer. Like Tao et al. (36), we did not detect the complexes with KDR dimers. This observation can be explained by the inefficiency of cross-linker used in our study to covalently bind KDR dimers.
CMDB7 Binds Directly to VEGF 165 and Inhibits Its Binding to Heparin-In order to explore whether CMDB7 is able to interfere directly with VEGF 165 , we used an affinity-electro-  Fig. 5, lane 1, shows that 125 I-VEGF 165 , being weakly cationic under nondenaturing conditions, remains close to loading well. The presence of negatively charged CMDB7 increases the migration of labeled growth factor evidencing a formation of 125 I-VEGF 165 ⅐CMDB7 complex. This shift toward the anode is already visible in the presence of 1 M CMDB7 (Fig. 5, lane 2), and migration dis-tances increase with enhanced CMDB7 concentrations (Fig. 5,  lanes 3-5). These results demonstrate that CMDB7 binds directly to VEGF 165 .
To test whether CMDB7 could alter the VEGF 165 binding to heparin, we have measured the binding of 125 I-VEGF 165 to coated heparin-albumin in the presence of CMDB7 at various concentrations (Fig. 6). Specific 125 I-VEGF 165 binding to hepa-    5. CMDB7 increases the VEGF 165 electrophoretic mobility. 125 I-VEGF 165 (10 5 cpm/3 ng) was mixed with CMDB7 at various concentrations in running buffer and incubated for 2 h at 4°C. Lane 1, no CMDB7; lanes 2-5, 1, 2.5, 5, and 10 M CMDB7, respectively. Electrophoresis in a 1% agarose gel was performed under nondenaturing and nonreducing conditions in neutral running buffer. The gel was dried and then exposed to autoradiography. The arrow indicates the direction of migration in the gel toward the anode. rin-albumin was inhibited by heparin with an IC 50 of 5 g/ml (data not shown) and in the presence of 10 M CMDB7 by 44% (Fig. 6). These results show that CMDB7 competes with heparin for heparin binding to VEGF 165 .
CMDB7 Does Not Bind to KDR and Does Not Compete for Heparin Binding to Receptors-To study the interactions of CMDB7 with VEGF receptors, we radioiodinated the KDR-Fc chimera. The possible direct interactions of CMDB7 and KDR were explored using an affinity-electrophoretic technique (Fig.  7). 125 I-KDR-Fc remains close to the loading well (Fig. 7, lane  1), and its migration does not change in the presence of CMDB7 at concentrations 2.5 to 20 M (lanes 2-5), indicating the absence of CMDB7⅐KDR complexes. To study the effect of CMDB7 on heparin binding to KDR, we have used coated heparinalbumin (Fig. 8). The binding of 125 I-KDR to heparin was not affected by 0.1, 1, or 10 M CMDB7.
CMDB7 Competes to Heparin Binding to VEGF 165 ⅐KDR Complexes-Finally, we studied the CMDB7 effects on formation of VEGF 165 ⅐KDR⅐heparin complexes. Fig. 9A shows that 125 I-KDR-Fc binding to the coated heparin-albumin is enhanced by VEGF 165 in a dose-dependent manner. This indicates that under our experimental conditions, we can reproduce, at least in part, the formation of triple complexes. Although CMDB7 has no effect on KDR-heparin interactions (Fig. 8), it does efficiently inhibit the 125 I-KDR binding to heparin in the presence of 3 nM VEGF 165 (Fig. 9B). This inhibitory effect is dose-dependent and significantly measured at CMDB7 concentration of 0.1 M. Further support for CMDB7 action on VEGF 165 ⅐KDR⅐heparin complexes was supplied by results of another experimental design. This time, the KDR-Fcs were coated and the binding of 125 I-VEGF 165 was measured in the presence or absence of heparin and/or CMDB7 (Fig. 10). In the presence of heparin, when only KDR and VEGF 165 are present, 40 M CMDB7 has no effect on of 125 I-VEGF 165 binding. In the presence of 0.1 g/ml heparin, the VEGF 165 binding is augmented in agreement with the findings of Kaplan et al. (37). Under these conditions, 40 M CMDB7 is able to inhibit the heparin-induced increase in 125 I-VEGF 165 binding. These data suggest that CMDB7 competes for heparin binding to VEGF 165 ⅐KDR complexes. Displacement of heparin by CMDB7 causes the inhibition of both 125 I-VEGF 165 and 125 I-KDR binding. DISCUSSION The role of angiogenesis in tumor progression and invasiveness is well documented now (38 -40), and anti-angiogenesis is becoming a promising new therapeutic approach for the treatment of cancer (41). As the high expression of VEGF and its receptors have been closely correlated to tumor vascularity, progression, and metastasis (42,43), targeting of VEGF/VEGFreceptors is an excellent anti-angiogenic strategy. Different anti-VEGF strategies have been used with success, but direct targeting of the heparin binding sites on VEGF and/or KDR by a heparin analog have never been proposed. In this study, we demonstrated that the dextran derivative CMDB7 is capable of neutralizing the VEGF 165 activities by direct interactions with VEGF 165 and by heparin displacement from VEGF 165 and VEGF 165 ⅐KDR complexes.
We have shown here that CMDB7 efficiently inhibited the VEGF 165 -induced proliferation of endothelial cells in a dose-dependent manner. Furthermore, CMDB7 inhibited the formation of 125 I-VEGF 165 ⅐receptor complexes, namely, 240 -250-and 170 -180-kDa complexes containing KDR and NRP1 receptors, respectively. This is in agreement with the findings of Soker et al. (34), who reported that VEGF binds to endothelial cells via these two receptors. In addition, by studying a receptor autophosphorylation, we demonstrated that CMDB7 suppressed the VEGF 165 signal transduction in endothelial cells. These findings are in agreement with our previous results showing that CMDB7 inhibited in vitro the migration and proliferation of endothelial cells and inhibited in vivo the tumor angiogenesis (24 -26).
Concerning the mechanism of action, we observed that CMDB7 displaced the VEGF 165 from its specific binding sites on HUV-ECs. It is noteworthy that CMDB7 was originally designed as a heparin analog and might therefore behave as an antagonist or partial agonist. Heparin can interfere with VEGF 165 action either by binding to a specific domain on   7. CMDB7 has no effect on KDR electrophoretic mobility. 125 I-KDR-Fc (10 5 cpm/3 ng) was mixed with CMDB7 at various concentrations in running buffer and incubated for 2 h at 4°C. Lane 1, no CMDB7; lanes 2-5, 1, 5, 10, and 20 M CMDB7, respectively. Electrophoresis in a 1% agarose gel was performed under nondenaturing and nonreducing conditions in neutral running buffer. The gel was dried and then exposed to autoradiography. The arrow indicates the direction of migration in the gel toward the anode. VEGF 165 (44) or by interacting with its cellular binding sites, KDR or NRP1 (44). Consequently, CMDB7 might affect both the ligand and its cellular binding sites.
We show here that CMDB7 interacts directly with VEGF165 (Fig. 5). This is in agreement with our previous findings that CMDB7 inhibited the activity of other heparin binding growth factors, such as platelet-derived growth factor, transforming growth factor ␤, FGF2, and FGF4, by binding them and thus altering the conformation of ligand⅐receptor complexes (24 -26). Consequently, we did not observe any effect of CMDB7 on heparin-binding growth factors, including EGF and IGF1 (24 -26).
Because of structural similarity to heparin, CMDB7 could bind to VEGF 165 requiring the heparin binding sites. Our results support this hypothesis as we demonstrate the dosedisplacement of VEGF 165 from heparin-albumin by CMDB7 (Fig. 6). This suggests that CMDB7 and heparin can compete for the same site on VEGF 165 . But the fact that CMDB7 inhibits the VEGF 165 binding to heparin-albumin with IC 50 higher than heparin doses, 10 M versus 0.3-1.7 M, suggests that the affinity of this site is weaker for CMDB7 than for heparin.
In contrast to VEGF 165 , KDR does not directly interact with CMDB7. Indeed, we showed that CMDB7 did not change the electrophoretic migration of KDR-Fc (Fig. 7), suggesting that the CMDB7⅐KDR complexes are not formed. One can think that CMDB7 negative charge is not enough strong to move a molecule as big and heavy as KDR-Fc, which is characterized by a molecular mass of 380 kDa. Our hypothesis that CMDB7 does not act directly on KDR is supported by the fact that CMDB7 does not displace the radiolabeled KDR from heparin-albumin (Fig. 8). The binding of heparin to KDR is well established now (45,46). However, in the light of our observations, it seems that heparin-binding site on KDR differs from that on VEGF 165 because CMDB7 is able to interact with the growth factor but not with the receptor.
CMDB7 binding to VEGF 165 could change the growth factor conformation in a different manner then heparin does, as we previously showed for FGF2 (47). This could perturb the VEGF 165 interactions with KDRs. Indeed, we observed that CMDB7 inhibits the formation of VEGF 165 ⅐KDR complexes on heparin-albumin (Fig. 9B). Interestingly, when the ligand⅐ receptor complexes are formed without heparin, as in the case of VEGF 165 binding to coated KDR-Fcs, CMDB7 has no effect (Fig. 10). This argues for the hypothesis that the mechanism of CMDB7 action involves the displacement of heparin from VEGF 165 and VEGF 165 ⅐KDR complexes.
In conclusion, CMDB7 prevents the binding of VEGF 165 to its cell surface receptors, inducing an inhibition of the receptor phosphorylation and consequently the endothelial cell proliferation arrest. It acts by displacing heparin from VEGF 165 and VEGF 165 ⅐receptor complexes. Being completely nontoxic and very efficient in tumor animal models, this dextran derivative could be used to increase the efficiency of conventional anticancer treatments.