Glypican-1 Is a VEGF165 Binding Proteoglycan That Acts as an Extracellular Chaperone for VEGF165*

Stela GengrinovitchDagger , Bluma BermanDagger , Guido David§, Larry Witte, Gera NeufeldDagger , and Dina RonDagger parallel

From the Dagger  Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel, the § Center for Human Genetics, University of Leuven and Flanders Interuniversity, Institute for Biotechnology, Campus Gasthuisberg O & N6, Herestraat, B-3000 Leuven, Belgium, and the  ImClone Systems Inc., New York, New York 10014

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glypican-1 is a member of a family of glycosylphosphatidylinositol anchored cell surface heparan sulfate proteoglycans implicated in the control of cellular growth and differentiation. The 165-amino acid form of vascular endothelial growth factor (VEGF165) is a mitogen for endothelial cells and a potent angiogenic factor in vivo. Heparin binds to VEGF165 and enhances its binding to VEGF receptors. However, native HSPGs that bind VEGF165 and modulate its receptor binding have not been identified. Among the glypicans, glypican-1 is the only member that is expressed in the vascular system. We have therefore examined whether glypican-1 can interact with VEGF165. Glypican-1 from rat myoblasts binds specifically to VEGF165 but not to VEGF121. The binding has an apparent dissociation constant of 3 × 10-10 M. The binding of glypican-1 to VEGF165 is mediated by the heparan sulfate chains of glypican-1, because heparinase treatment abolishes this interaction. Only an excess of heparin or heparan sulfates but not other types of glycosaminoglycans inhibited this interaction. VEGF165 interacts specifically not only with rat myoblast glypican-1 but also with human endothelial cell-derived glypican-1. The binding of 125I-VEGF165 to heparinase-treated human vascular endothelial cells is reduced following heparinase treatment, and addition of glypican-1 restores the binding. Glypican-1 also potentiates the binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1. Furthermore, we show that glypican-1 acts as an extracellular chaperone that can restore the receptor binding ability of VEGF165, which has been damaged by oxidation. Taken together, these results suggest that glypican-1 may play an important role in the control of angiogenesis by regulating the activity of VEGF165, a regulation that may be critical under conditions such as wound repair, in which oxidizing agents that can impair the activity of VEGF are produced, and in situations were the concentrations of active VEGF are limiting.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glypican-1 is the prototype member of the glycosylphosphatidilinositol anchored cell surface heparan sulfate proteoglycans (HSPGs)1 implicated in cell adhesion and migration, lipoprotein metabolism, anticoagulation, and modulation of growth factor activities (1, 2). The glypican family includes the human and rat glypican (3, 4), rat cerebroglycan (5), rat OCI-5 (6) and its human homologue glypican-3 (7), K-glypican (8), glypican-5 (9), and the Drosophila dally (10). Mutations in dally affect cell division in the Drosophila visual system and lead to morphological defects in the eyes, antenna, genitalia, and wings (10). In addition, mutations in glypican-3 are responsible for the Simpson-Golabi-Behmel syndrome, a disease that is associated with specific congenital malformations and a predisposition to tumors (2, 7). Recent studies in mammalian cells indicated that glypican-1 binds to members of the fibroblast growth factor (FGF) family. Thus, OCI-5 binds to FGF-2, and glypican-1 interacts with at least three FGFs: FGF-1, FGF-2, and FGF-7 (11-13). Furthermore, glypican-1 modulates the biological activities of these growth factors, and this modulation can be stimulatory or inhibitory depending on the FGF type (11, 12). Taken together, these findings indicate that the glypicans play an important regulatory role in the control of cellular growth, differentiation, and morphogenesis.

Vascular endothelial growth factors (VEGFs) are mitogens for endothelial cells and are potent angiogenic factors in vivo. Five VEGF isoforms, designated VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206, are generated via an alternative splicing mechanism from a unique gene (14-16). The active form of the VEGFs is a homodimer, but active heterodimers have also been observed. Two VEGF tyrosine-kinase receptor types have been characterized. These tyrosine-kinase receptors do not differentiate between the various VEGF forms. The KDR/flk-1 receptor mediates the mitogenic activity of VEGF, whereas flt-1 stimulates VEGF-induced cell migration (17). In addition, endothelial cells express VEGF165-specific receptors of unknown function. These receptors do not bind VEGF121 or VEGF145 (16, 18).

The best characterized VEGF forms are VEGF121 and VEGF165 (165- and 121-amino acid-long polypeptide, respectively). VEGF165 contains the peptide encoded by exon-7 of the VEGF gene, whereas VEGF121 lacks this peptide. The presence of exon-7 confers on VEGF165 the ability to bind heparin-like molecules. Removal of heparan sulfates (HS) from the surface of endothelial cells by heparinase digestion reduces the binding of 125I-VEGF165 to its receptors, and addition of heparin restores 125I-VEGF165 binding. We have recently shown that part of the effect of heparin involves restoration of 125I-VEGF165 binding to KDR/flk-1 by a chaperone-like effect that recuperates the biological activity of VEGF165 that was damaged by oxidizing agents (18). Such a role of heparin-like molecules may be important under conditions like wound repair, hypoxia-induced angiogenesis, or inflammation, in which oxidants or free radicals are produced and may damage VEGFs (19-21). Unlike VEGF165, VEGF121 is irreversibly inactivated by oxidizing reagents, and heparin cannot restore its receptor binding ability (18).

Recent evidence strongly suggests that VEGFs play a critical role in the process of tumor angiogenesis. This process is essential for tumor progression and for the subsequent process of tumor metastasis. A number of molecules that display some structural similarity to heparin such as suramin and pentosan sulfate exert an anti-angiogenic effect. These observations, together with the findings that HS-degrading enzymes can inhibit tumor angiogenesis (22), suggest that HSPGs play an important role in the angiogenic process. So far, HSPGs that can bind VEGFs and modulate their biological activities have not been identified. Among glypicans, glypican-1 is the only member expressed in the vascular system (23, 24). We have therefore examined whether glypican-1 can interact with VEGFs and modulate their interaction with VEGF receptors. We show here that purified glypican-1 binds VEGF165 with high affinity and supports its binding to heparinase-treated endothelial cells. Furthermore, glypican-1 can also restore the receptor binding ability of oxidized VEGF165 and can therefore be viewed as a native, cell surface-localized proteoglycan that displays a chaperone-like activity.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Human recombinant VEGF165 and VEGF121 were produced and purified from Sf-9 insect cells as described previously (25-27). Human recombinant FGF-2, FGF-1, PF4, and FGF-7 were produced in bacteria and purified as described previously (28-30). Bovine brain FGF-1 was purchased from R & D. Heparinase type I and III was kindly provided by IBEX Technologies (Montreal, Canada). Intestinal mucosa-derived heparin and chondroitin sulfates A and C and hyaluronic acid were from Sigma. Heparan sulfate from bovine lung and heparin-sepharose were obtained from Amersham Pharmacia Biotech. Carrier-free Na125I and [35S]Na2SO4 were purchased from NEN Life Science Products. Microtiter ELISA plates were from Corning. Tissue culture media, sera, and cell culture supplements were from Beth-Haemek Biological Industries (Israel) or from Life Technologies, Inc. Disuccinimidyl suberate was obtained from Pierce. All other chemicals were purchased from Sigma.

Cells-- Human umbilical vein-derived endothelial cells (HUVEC) were isolated and cultured in M199 medium containing 20% fetal calf serum, as described previously (18). Rat myoblast cells (L6E9) were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum as described previously (31).

Purification of Glypican, Enzymatic Deglycosylation, and Isolation of Glypican-1-associated GAG Side Chains-- Glypican-1 was purified from salt extracts of subconfluent cultures of the rat myoblast cell line L6E9 by anion exchange chromatography with DEAE-Sephacel followed by FGF affinity purification as described previously (11). Enzymatic deglycosylation was carried out in PBS containing 125I-glypican-1 and 0.5 units/ml of heparinase I+III. Incubation was for 2 h at 37 °C. Degradation of the core protein of glypican-1 was carried out overnight with proteinase K (0.5 mg/ml). To ensure that digestion was complete, a parallel incubation was carried out in the presence of radioiodinated glypican-1, and digestion was monitored by SDS-polyacrylamide gel electrophoresis and autoradiography. The GAG side chains were then separated from the protease and degradation products by DEAE-Sephacel chromatography as described (11).

Quantitation of GAG and HSPG-- The Safranin O dye that reacts with carboxyl and sulfate groups and can detect ng amounts of sulfated GAGs (32) was used to estimate GAG content.

The concentrations of glypican-1 are relative to its HS concentration, which accounts to about two-thirds of the total PG concentration (11). Prior to performing the study we examined the biological activity of glypican-1 by testing its ability to enhance binding of FGF-1 and FGF-2 to FGF receptor 1 and to modulate the biological activities of FGF-7 and FGF-1 as described previously (11).

Radioiodination of Glypican-1 and VEGFs-- Radioiodination was carried out utilizing the chloramine T method as described previously (11, 33, 34). 125I-VEGF165 was separated from free iodine using a heparin-Sepharose column, and 125I-VEGF121 was separated from free iodine using size exclusion chromatography on Sephadex-G25 as described (34). The specific activities of the 125I-VEGF165 and the 125I-VEGF121 were about 105 cpm/ng. Radiolabeled glypican-1 was separated from free iodine by chromatography on DEAE-Sephacel (11). Specific activities of iodinated glypican-1 were in range of 6-12 × 106 cpm/µg.

Binding of 125I-Glypican-1 to VEGF-coated Wells-- Binding of radioiodinated glypican-1 to wells coated with VEGF165 and various other proteins was done essentially as described (35). Briefly, 0.2 µg of each protein in 100 µl of coating buffer were adsorbed to 96-well ELISA plates for 2 h at room temperature, and binding was performed with 125I-glypican-1 for 2 h at room temperature. Free 125I-glypican-1 was removed by three washes with wash buffer (35). Estimation of bound 125I-glypican-1 was done following solubilization with 0.2 M NaOH. All the experiments were done in triplicate and were repeated at least three times. Nonspecific binding was estimated as described under "Results," and it was less than 10% of the total binding. Standard deviation between replicates in all the experiments was less than 10%.

Reverse Immunodot Assay on Nitrocellulose Filters-- 35S-HSPGs were partially purified from conditioned medium of metabolically labeled HUVEC cells as described (11). Various proteins were spotted onto nitrocellulose filters at 2 µg/spot. The filters were blocked with coating buffer (35) containing 1% BSA, washed three times with coating buffer, and incubated with binding buffer (35) containing HUVEC-derived 35S-HSPGs (15,000 cpm/cm2) for 2 h at room temperature. Following extensive washes, one filter was air dried and taken for autoradiography to confirm binding of radiolabeled HSPGs, and the remaining filters were incubated with a monoclonal antibody directed against human glypican-1 (monoclonal antibody S1) or with a anti-phosphotyrosine monoclonal antibody (Upstate Biotechnology Inc.) as described (36). Detection of bound antibodies was performed with enhanced chemiluminescence reagents.

Heparinase Digestion and Receptor Binding Assays-- Binding and cross-linking of VEGFs to a soluble extracellular domain of flk-1 fused to secreted human alkaline phosphatase (designated flk/SEAP) was done as described previously (18, 26). Binding and cross-linking to HUVEC cells was essentially performed as described previously, except that instead of using adherent cells, the binding was performed using cells in suspension. The cells were first treated with heparinase (0.5 units/ml) for 1 h at 37 °C and then detached from the tissue culture dishes using PBS solution containing 2 mM EDTA. The cells were collected, washed twice with PBS and once with binding buffer, and then subjected to the binding assay. Following binding the cells were washed extensively with PBS, and cross-linking was performed in solution. Equal amounts of total cell lysates were loaded onto each lane.

Inactivation of VEGF165 by Oxidation-- Oxidation of VEGF165 was performed essentially as described (18). Briefly, 2 µg of growth factor in 100 mM sodium phosphate buffer (pH 7.2) were incubated for 1 min with 0.1% of H2O2 in a final reaction volume of 40 µl. The reaction was terminated by sodium metabisulfite, and the growth factor was separated from the H2O2 and sodium metabisulfite using heparin-Sepharose affinity chromatography (recovery was over 90%).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Glypican-1 Binds to VEGF165-- A solid phase assay was utilized to investigate the ability of glypican-1 to bind VEGF165 and VEGF121. Increasing concentrations of radioiodinated glypican-1 were bound to microtiter plates precoated with 0.2 µg/well of VEGF165 or VEGF121 as well as FGF-7, which was previously reported to bind glypican (11). As shown in Fig. 1, only VEGF165 and FGF-7 bind similarly to 125I-glypican, whereas VEGF121 or BSA did not exhibit any binding capacity. Binding was saturable and Scatchard analysis yielded an apparent dissociation constant of 1.2 × 10-10 M for VEGF165 and 3 × 10-10 M for FGF-7 (Fig. 1B).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1.   Binding of glypican-1 to VEGF. A, binding saturation of 125I-glypican-1 to VEGF. 125I-Labeled glypican-1 (specific activity of 12 × 106 cpm/µg) was bound to VEGF165-coated (), VEGF121-coated (black-down-triangle ), and FGF-7-coated (open circle ) wells as described under "Experimental Procedures." After 2 h of incubation at room temperature, the wells were extensively washed, and quantification of bound 125I-glypican-1 was done as described under "Experimental Procedures." B, Scatchard analysis of glypican-1 binding to VEGF165 () and FGF-7 (open circle ). The Ligand program was used for the analysis (55).

To test the specificity of this interaction we measured the ability of a panel of proteins to compete with 125I-glypican for binding to VEGF165. As shown in Fig. 2, all three FGF members inhibited binding. This finding is with accordance with the previously reported capacity of these growth factors to bind glypican (11). By contrast, VEGF121, insulin, transferrin, and BSA could not compete with 125I-glypican for binding to VEGF165.


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 2.   Inhibition of the binding of 125I-glypican-1 to VEGF165 by heparin-binding growth factors. 125I-Glypican-1 (5 ng/well) was bound to VEGF165-coated wells in the presence of the above indicated growth factors and proteins (1 µg/ml of each). Binding and subsequent quantification of bound 125I-glypican-1 was done as described under "Experimental Procedures." V165, VEGF165; V121, VEGF121; INS, insulin; TR, transferrin.

Binding of Glypican-1 to VEGF165 Is Mediated by Its HS Chain-- To determine whether glypican-1-derived GAGs are involved in VEGF165 binding, we prepared heparinase-digested or protease-digested glypican. Heparinase treatment abolished the ability of glypican-1 to compete with intact 125I-glypican-1 for binding to VEGF165, whereas the free glypican-1-derived GAGs competed with 125I-glypican-1 as efficiently as intact glypican-1 (Fig. 3A). These findings suggest that the interaction of glypican-1 with VEGF is mediated by its GAG side chains. To further verify these findings we compared the ability of intact glypican, glypican-1-derived GAGs, heparin, commercial HS, and non-HS GAGs to inhibit the binding of glypican-1 to VEGF165. Glypican-1-derived HS inhibited the binding in a concentration-dependent manner (Fig. 3B). Half-maximal inhibition was obtained at a concentration of about 180 ng/ml glypican-1-derived HS. This concentration was 8-fold higher as compared with the heparin concentration required for half-maximal inhibition but about 10-fold lower than that of commercial HS. By contrast neither chondroitin sulfate nor hyaluronic acid were able to compete with glypican-1 for binding to VEGF165. These results establish that the HS side chains of glypican-1 mediate binding to VEGF165.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   Binding of glypican-1 to VEGF165 is mediated by its HS side chains. A, VEGF does not interact with glypican-1 core protein. 125I-Glypican-1 (5 ng/well) was bound to VEGF165-coated wells in the absence or presence of 1 µg/ml of unlabeled intact glypican-1 (Gly), glypican-1-free GAGs (Gly-HS) obtained by proteinase K digestion, or glypican-1 core protein (Gly-core) obtained by heparinase digestion. B, inhibition of the binding of 125I-glypican-1 to VEGF165 by glycosaminoglycans. Binding was performed in the presence of the indicated concentrations of heparin (black-square), glypican-1-derived HS (down-triangle), commercial HS (black-down-triangle ), chondroitin sulfate (open circle ), and hyaluronic-acid (). The wells were washed, and the amount of bound radioactivity was quantified.

Glypican-1 Enhances the Binding of VEGF165 to VEGF Receptors-- VEGF165 binds to three VEGF receptors on HUVEC-derived endothelial cells (28). The larger of these receptors is KDR/flk-1, whereas the identity of the other two is not yet known (18). Heparin enhances the binding of VEGF165 to all three receptors (28). Heparin is not a normal constituent of endothelial cells, whereas glypican-1 is known to be expressed in these cells (24).2 We have therefore examined whether glypican-1 can modulate the binding of VEGF165 to cell surface receptors in untreated and in heparinase-digested endothelial cells. Digestion of the endothelial cells by heparinase inhibited almost completely the binding of 125I-VEGF165 to the two smaller receptors, whereas the binding to KDR/flk-1 (18) was inhibited by about 80% (Fig. 4A, lane 3). Exogenously added glypican-1 restored the binding to all three receptors (Fig. 4A, lane 4). Glypican-1 also potentiated the binding of 125I-VEGF165 to the two smaller receptors in untreated cells (Fig. 4A, lane 2).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of glypican-1 on the binding of 125I-VEGF165 to its receptors. A, glypican-1 restores binding of VEGF165 to heparinase-treated HUVEC cells. Cells were treated with heparinase and detached from the plate using PBS solution containing EDTA as described under "Experimental Procedures." Cells were allowed to bind 125I-VEGF165 (10 ng/ml) for 2 h at 4 °C, the cells were then washed, and bound 125I-VEGF165 was cross-linked to cell surface receptors. Cross-linked complexes were visualized following SDS-polyacrylamide gel electrophoresis and autoradiography. Lane 1, untreated cells; lanes 2, untreated cells bound to VEGF in the presence of 3 µg/ml of glypican; lane 3, heparinase-treated cells; lane 4, heparinase-treated cells bound to VEGF in the presence of 3 µg/ml of glypican. B, glypican-1 potentiates the binding of VEGF165 to a soluble form of KDR/flk-1. 125I-VEGF165 (20 ng/ml) was bound to flk-1/SEAP-coated wells in the presence of increasing concentrations of glypican-1 (open circle ) or heparin (), with or without 1 µg/ml unlabeled VEGF165, as described under "Experimental Procedures." Specific binding was determined by subtracting normalized cpm of samples incubated with 1 µg/ml unlabeled VEGF165 from the normalized cpm bound in the absence of unlabeled ligand.

The ability of glypican-1 to potentiate the binding of VEGF165 to KDR/flk-1 was also confirmed utilizing a soluble extracellular domain of this receptor (flk-1/SEAP) in a cell-free assay (26). The potentiation of 125I-VEGF165 binding to the soluble receptor was concentration-dependent and was maximal at about 1 µg/ml of glypican-1 (Fig. 4B). These findings were also confirmed in cross-linking experiments (data not shown).

The ability of myoblast-derived glypican-1 to restore binding of VEGF165 to heparinase-treated HUVEC to a level that is higher than the binding observed in untreated cells strongly suggests that glypican-1 from endothelial cells also fulfills a similar function. We have therefore partially purified glypican-1 from HUVEC cells and have tested its ability to interact with VEGF165. VEGF165 was bound to nitrocellulose membranes and incubated with partially purified 35S-HSPGs. As shown in Fig. 5A, VEGF165 and FGF-2 bound 35S-HSPGs, whereas VEGF121, BSA, transferrin, and insulin did not exhibit any HSPG binding ability. Moreover, a monoclonal antibody directed against human glypican-1 interacted specifically with the VEGF165 and FGF-2-bound HSPGs (Fig. 5B). These observations strongly suggest that HUVEC-associated glypican-1 is a candidate modulator of VEGF165 activity in these cells.


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 5.   Specific binding of HUVEC-derived glypican-1 to VEGF165. VEGF165 (V165), FGF-2, BSA, VEGF121 (V121), transferrin (TR), and insulin (INS) at concentrations of 2 µg/spot were dot-blotted on to a nitrocellulose filter. Binding of soluble 35S-HSPGs from HUVEC cells to the filters and immunodetection of bound glypican were carried out as described under "Experimental Procedures." A, specific binding of soluble 35S-HSPGs to nitrocellulose filters. B and C, immunodetection of bound glypican by monoclonal antibody S1 (B) or with a monoclonal anti-phosphotyrosine antibody (C). The filters were also autoradiographed at the end of the assay to confirm that bound 35S-HSPGs are detectable in both filters. D, a scheme indicating the positions of the various proteins on the filters.

Glypican-1 Restores the Activity of Oxidized VEGF165-- We have previously shown that oxidation of VEGF165 with agents such as H2O2 impairs its ability to bind to the KDR/flk-1 receptor and that heparin restores the receptor binding capacity of VEGF165 (18). Because glypican-1 binds efficiently to VEGF165, we examined whether it displays a similar restorative capacity. As shown in Fig. 6, H2O2-treated VEGF165 lost the ability to efficiently compete with VEGF121 for binding to flk-1/SEAP, and addition of glypican-1 restored the receptor binding capacity of the oxidized VEGF165.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6.   Glypican-1 restores the receptor binding ability of oxidized VEGF165. 125I-VEGF121 (20 ng/ml) was bound to ELISA dishes coated with flk-1/SEAP. The binding was performed in the absence or presence of 0.5 µg/ml of unlabeled VEGF121 (V121), VEGF165 (V165), H2O2-treated VEGF165 (*V165), and H2O2-treated VEGF165 plus 3 µg/ml of glypican (*V165+Gly).

PF4 Inhibits the Binding of Glypican-1 to 125I-VEGF165 and Abrogates the Stimulatory Effect of Glypican-1 on 125I-VEGF165 Receptor Binding-- PF4 is a heparin-binding protein that is synthesized by megakaryocytes, sequestered in platelets, and released from alpha -granules as a complex with chondroitin 4-sulfate proteoglycan (37, 38). PF4 displays an anti-angiogenic activity in vivo that is attributed in part to its heparin binding capacity. Thus, a peptide derived from the heparin-binding carboxyl-terminal domain of PF4 possesses anti-angiogenic properties (29). The receptor binding ability of VEGF165 and its mitogenic activity are inhibited by PF4 (35). Our results indicate that glypican may be one of the endothelial cell-associated heparan sulfate proteoglycans that bind VEGF165 and mediate its biological activity. We reasoned that PF4 may inhibit angiogenesis by preventing the interaction between VEGF and glypican. We have therefore examined whether PF4 can inhibit the binding of VEGF165 to glypican-1 and abrogate the stimulatory effect of glypican-1 on VEGF receptor binding. As shown in Fig. 7A, PF4 efficiently inhibited the binding of glypican-1 to VEGF165-coated wells. Half-maximal inhibition was obtained at a PF4 concentration of 100 ng/ml. Moreover, PF4 nullifies the stimulatory effect of glypican-1 on the binding of 125I-VEGF165 to flk-1/SEAP (Fig. 7B).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7.   PF4 inhibits binding of glypican-1 to VEGF165 and abrogates the stimulatory effect of glypican-1 on binding of VEGF to flk-1/SEAP. A, effect of PF4 on the binding of glypican-1 to VEGF165. 125I-Labeled glypican-1 (specific activity of 6 × 106 cpm/µg) was bound to ELISA dishes coated with VEGF165 in the presence of increasing concentrations of PF4. Binding was quantified as described under "Experimental Procedures." B, PF4 abrogates the stimulatory effect of glypican-1 on VEGF165 binding to flk-1/SEAP receptor. Binding of 125I-VEGF165 (20 ng/ml) to flk-1/SEAP-coated wells was performed in the absence (open bars) or presence (shaded bars) of 2 µg/ml of PF4. When indicated, 1 µg/ml of glypican-1 (Gly) or heparin (Hep) were added.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heparin had been previously shown to bind to VEGF165 and to act as an accessory receptor that enhances the interaction of VEGF165 with its signaling receptors. Furthermore, heparin restores the bioactivity of damaged VEGF165 (18). However, because cells express several types of HSPGs on their surfaces but not heparin, it was important to identify native HSPGs that bind VEGF and modulate its activity. In the present study we have demonstrated for the first time an interaction of VEGF with the lipid anchored cell surface heparan sulfate proteoglycan glypican-1. Glypican-1 was found to interact with VEGF165 and to modulate its receptor binding properties. We show that glypican-1 binds to VEGF165 via its heparan sulfate chains and that the binding is specific and saturable. The affinity of glypican-1 to VEGF165 is high and has an apparent dissociation constant of 1.2 × 10-10 M. Glypican-1 not only binds to VEGF165 but is also capable of enhancing the interaction of VEGF165 with its signaling receptors both in cell-free binding assays and in heparinase-treated endothelial cells.

VEGF165 is a major angiogenic factor that is active in processes such as wound repair, hypoxia-induced angiogenesis, and inflammation (15), processes associated with the generation of oxidizing agents and free radicals. VEGF165 is inactivated by these agents (18), and therefore their presence may inhibit VEGF-induced angiogenesis. Therefore, repair mechanisms allowing restoration of VEGF activity could fulfill an important biological role. Glypican-1 restores the biological activity of oxidized VEGF and is expressed on the cell surface of the endothelial cells that are the natural target for VEGF (24). The presence of glypican on the surface of endothelial cells could therefore be a fail-safe mechanism ensuring that every VEGF165 molecule that reaches the endothelial cells will be eventually able to interact and activate signaling VEGF receptors such as the KDR receptor. This function of glypican-1 may be critical under conditions in which the concentrations of VEGF are limited or under conditions in which the activity of VEGF is compromised.

Studies in recent years have strongly indicated that HSPGs are important modulators of the activity of heparin-binding growth factors, an issue that is particularly well studied for FGFs (39, 40). FGFs bind to cell surface and extracellular matrix-associated HSPGs. The mechanism by which these HSPGs regulate FGF activity is complex and can be manifested at several levels. These molecules protect FGFs from protease digestion or from heat/acid inactivation, and it was shown that they can restore the activity of heat inactivated FGF (41). HSPGs are also thought to provide a reservoir from which FGFs can be rapidly released in response to specific triggering events (42). In addition to these effects, cell-associated HSPGs can increase the affinity of FGFs to their signaling receptors by stabilizing ligand-receptor complexes (43-45). VEGFs interact with several receptor types, some of which may also be able to interact directly with heparan sulfates (46-48). It is therefore possible that in addition to its chaperone-like role, glypican-1 may be able to directly modulate the interaction between VEGF and these receptors using similar mechanisms.

The defined structure of the HSPGs side chains is regulated in a cell type-dependent manner. For example, human lung fibroblast-derived glypican-1 cannot enhance the biological activity of FGF-2 (49). On the other hand, glypican-1 derived from rat myoblasts or expressed ectopically in K562 cells can stimulate the binding of FGF-2 to FGF-receptor 1 and potentiates cellular responses to FGF-2 (11, 12). The ability of glypican-1 from rat myoblasts and from human endothelial cells to interact with VEGF165, together with the finding that rat myoblast glypican-1 can restore VEGF receptor binding in heparinase-treated HUVEC cells, strongly suggests that the HS side chains attached to glypican-1 in both cell types contain similar VEGF165-binding domains. Glypican-1 exists on the surface of cells both as a lipid anchored form and as a peripheral membrane proteoglycan. The peripheral form is generated following cleavage of the lipid anchored form by a specific phospholipase and can be shed from cell surfaces as a soluble proteoglycan (50). The fact that soluble myoblast-derived glypican-1 can bind and modulate VEGF165 activity suggests that HSPGs derived from one cell type can act in a paracrine manner to affect biological responses to heparin-binding growth factors.

Beside glypican-1, endothelial cells express other HSPGs such as fibroglycan, perlecan, and syndecan (24, 51, 52). Studies conducted with growth factors of the FGF family revealed that distinct core proteins can bear HS side chains with similar function (12, 49). A similar situation might exist for growth factors of the VEGF family. The ability of glypican-1 to fully restore VEGF receptor binding to heparinase-treated endothelial cells indicates that even if endothelial cells do express more than one type of stimulatory HSPG species for VEGF, it is likely that they bear HS with a fine structure similar to that of glypican-1 side chains. In addition, in heparinase-digested endothelial cells, glypican-1 enhances VEGF165 binding to its receptors to a level that is higher than that observed in untreated cells incubated with glypican-1 (compare lanes 2 and 4, Fig. 4A). These findings could also imply that endothelial cells express HSPG species that inhibit the binding of VEGF to its receptors.

Heparin-like molecules have long been implicated in the control of angiogenesis. The earliest indication that heparin may be involved in regulation of the angiogenic process was the finding that mast cells accumulate at the site of tumor angiogenesis before capillary ingrowth, that conditioned medium from mast cells induces angiogenesis in vitro, and that this activity can be abolished by protamine and heparan sulfate degrading enzymes (53, 54). It was subsequently found that many heparin-binding growth factors, including members of the FGF and VEGF family, are highly angiogenic. It soon became apparent that heparin and HS play an important role in modulating the biological activity of the growth factors which bind to them. However, only recently were efforts taken to identify native HSPGs that bind and modulate the activity of these growth factors (11-13, 49). Our previous studies have shown that glypican-1 modulates the biological activity of FGF-2, suggesting a role for glypican-1 in the regulation of angiogenesis (11). The present study lends support to this hypothesis and further suggests that glypican-1 may play a more general modulatory role in angiogenesis by regulating the stability and activity of VEGFs.

    ACKNOWLEDGEMENT

We thank Dr. Ronit Reich-Slotky for help in glypican purification.

    FOOTNOTES

* This work was supported by an angiogenesis research center grant from the Israel Science Foundation (to G. N. and D. R.), by a grant from the German Israeli Binational Foundation (to G. N.), and by a grant from the German Foundation for Biotechnological Research (to D. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed. Tel.: 972-4-8294217; Fax: 972-4-8225-153; E-mail: dinar{at}techunix.technion.ac.il.

2 D. Ron, unpublished results.

    ABBREVIATIONS

The abbreviations used are: HSPG, heparan sulfate proteoglycan; BSA, bovine serum albumin; FGF, fibroblast growth factor; FGF-1, acidic fibroblast growth factor; FGF-2, basic fibroblast growth factor; FGF-7, keratinocyte growth factor; GAGs, glycosaminoglycans; HS, heparan sulfate; PBS, Dulbecco's phosphate-buffered saline; VEGF, vascular endothelial growth factor; ELISA, enzyme-linked immunosorbent assay; HUVEC, human umbilical vein endothelial cell; PF4, platelet factor 4.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. David, G. (1993) FASEB J. 7, 1023-1030[Abstract/Free Full Text]
  2. Weksberg, R., Squire, J. A., and Templeton, D. M. (1996) Nat. Genet. 12, 225-227[Medline] [Order article via Infotrieve]
  3. David, G., Lories, V., Decock, B., Marynen, P., Cassiman, J. J., and Van den Berghe, H. (1990) J. Cell Biol. 111, 3165-3176[Abstract]
  4. Karthikeyan, L., Maurel, P., Rauch, U., Margolis, R. K., and Margolis, R. U. (1992) Biochem. Biophys. Res. Commun. 188, 395-401[Medline] [Order article via Infotrieve]
  5. Stipp, C. S., Litwack, E. D., and Lander, A. D. (1994) J. Cell Biol. 124, 149-160[Abstract]
  6. Filmus, J., Shi, W., Wong, Z. M., and Wong, M. J. (1995) Biochem. J. 311, 561-565[Medline] [Order article via Infotrieve]
  7. Pilia, G., Hughesbenzie, R. M., Mackenzie, A., Baybayan, P., Chen, E. Y., Huber, R., Neri, G., Cao, A., Forabosco, A., and Schlessinger, D. (1996) Nat. Genet. 12, 241-247[Medline] [Order article via Infotrieve]
  8. Watanabe, K., Yamada, H., and Yamaguchi, Y. (1995) J. Cell Biol. 130, 1207-1218[Abstract]
  9. Veugelers, M., Vermeesch, J., Reekmans, G., Steinfeld, R., Marynen, P., and David, G. (1997) Genomics 40, 24-30[CrossRef][Medline] [Order article via Infotrieve]
  10. Nakato, H., Futch, T. A., and Selleck, S. B. (1995) Development 121, 3687-3702[Abstract/Free Full Text]
  11. Bonneh-Barkay, D., Shlissel, M., Berman, B., Shaoul, E., Admon, A., Vlodavsky, I., Carey, D. J., Asundi, V. K., Reich-Slotky, R., and Ron, D. (1997) J. Biol. Chem. 272, 12415-12421[Abstract/Free Full Text]
  12. Steinfeld, R., Vandenberghe, H., and David, G. (1996) J. Cell Biol. 133, 405-416[Abstract]
  13. Song, H. H., Shi, W., and Filmus, J. (1997) J. Biol. Chem. 272, 7574-7577[Abstract/Free Full Text]
  14. Leung, D. W., Cachianes, G., Kuang, W. J., Goeddel, D. V., and Ferrara, N. (1989) Science 246, 1306-1309[Medline] [Order article via Infotrieve]
  15. Neufeld, G., Cohen, T., Gitay-Goren, H., Poltorak, Z., Tessler, S., Gengrinovitch, S., and Levi, B. (1996) Cancer Metastasis Rev. 15, 153-158[Medline] [Order article via Infotrieve]
  16. Poltorak, Z., Cohen, T., Sivan, R., Kandelis, Y., Spira, G., Vlodavsky, I., Keshet, E., and Neufeld, G. (1997) J. Biol. Chem. 272, 7151-7158[Abstract/Free Full Text]
  17. Barleon, B., Sozzani, S., Zhou, D., Weich, H. A., Mantovani, A., and Marme, D. (1996) Blood 87, 3336-3343[Abstract/Free Full Text]
  18. Gitay-Goren, H., Cohen, T., Tessler, S., Soker, S., Gengrinovitch, S., Rockwell, P., Klagsbrun, M., Levi, B., and Neufeld, G. (1996) J. Biol. Chem. 271, 5519-5523[Abstract/Free Full Text]
  19. Schraufstatter, I. U., Halsey, W. A., Hyslop, P. A., and Cochrane, C. G. (1988) Methods Enzymol. 163, 328-339[Medline] [Order article via Infotrieve]
  20. Heck, D. E., Laskin, D. L., Gardner, C. R., and Laskin, J. D. (1992) J. Biol. Chem. 267, 21277-21280[Abstract/Free Full Text]
  21. Ruef, J., Hu, Z. Y., Yin, L. Y., Wu, Y., Hanson, S. R., Kelly, A. B., Harker, L. A., Rao, G. N., Runge, M. S., and Patterson, C. (1997) Circ. Res. 81, 24-33[Abstract/Free Full Text]
  22. Sasisekharan, R., Moses, M. A., Nugent, M. A., Cooney, C. L., and Langer, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1524-1528[Abstract]
  23. Rosenberg, R. D., Shworak, N. W., Liu, J., Schwartz, J. J., and Zhang, L. J. (1997) J. Clin. Invest. 99, 2062-2070[Free Full Text]
  24. Mertens, G., Cassiman, J.-J., Van den Berghe, H., Vermylen, J., and David, G. (1992) J. Biol. Chem. 267, 20435-20443[Abstract/Free Full Text]
  25. Cohen, T., Gitay-Goren, H., Neufeld, G., and Levi, B. (1992) Growth Factors 7, 131-138[Medline] [Order article via Infotrieve]
  26. Tessler, S., Rockwell, P., Hicklin, D., Cohen, T., Levi, B., Witte, L., Lemischka, I. R., and Neufeld, G. (1994) J. Biol. Chem. 269, 12456-12461[Abstract/Free Full Text]
  27. Cohen, T., Gitay-Goren, H., Sharon, R., Shibuya, M., Halaban, R., Levi, B., and Neufeld, G. (1995) J. Biol. Chem. 270, 11322-11326[Abstract/Free Full Text]
  28. Gitay-Goren, H., Soker, S., Vlodavsky, I., and Neufeld, G. (1992) J. Biol. Chem. 267, 6093-6098[Abstract/Free Full Text]
  29. Maione, T. E., Gray, G. S., Petro, J., Hunt, A. J., Donner, A. L., Bauer, S. I., Carson, H. F., and Sharpe, R. J. (1990) Science 247, 77-79[Medline] [Order article via Infotrieve]
  30. Ron, D., Bottaro, D. P., Finch, P. W., Morris, D., Rubin, J. S., and Aaronson, S. A. (1993) J. Biol. Chem. 268, 2984-2988[Abstract/Free Full Text]
  31. Ron, D., Reich, R., Chedid, M., Lengel, C., Cohen, O. E., Chan, A. M. L., Neufeld, G., Miki, T., and Tronick, S. R. (1993) J. Biol. Chem. 268, 5388-5394[Abstract/Free Full Text]
  32. Lammi, M., and Tammi, M. (1988) Anal. Biochem. 168, 352-357[Medline] [Order article via Infotrieve]
  33. Neufeld, G., and Gospodarowicz, D. (1985) J. Biol. Chem. 260, 13860-13868[Abstract/Free Full Text]
  34. Vaisman, N., Gospodarowicz, D., and Neufeld, G. (1990) J. Biol. Chem. 265, 19461-19466[Abstract/Free Full Text]
  35. Gengrinovitch, S., Greenberg, S. M., Cohen, T., Gitay-Goren, H., Rockwell, P., Maione, T. E., Levi, B., and Neufeld, G. (1995) J. Biol. Chem. 270, 15059-15065[Abstract/Free Full Text]
  36. de Boeck, H., Lories, V., David, G., Cassiman, J. J., and Van den Berghe, H. (1987) Biochem. J. 247, 765-771[Medline] [Order article via Infotrieve]
  37. Ravid, K., Beeler, D. L., Rabin, M. S., Ruley, H. E., and Rosenberg, R. D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1521-1525[Abstract]
  38. Levine, S. P., Knieriem, L. K., and Rager, M. A. (1990) Blood 75, 902-910[Abstract]
  39. Rapraeger, A. C., Guimond, S., Krufka, A., and Olwin, B. B. (1994) Extracellular Matrix Components 245, 240
  40. Vlodavsky, I., Miao, H. Q., Medalion, B., Danagher, P., and Ron, D. (1996) Cancer Metastasis Rev. 15, 177-186[Medline] [Order article via Infotrieve]
  41. Gospodarowicz, D., and Cheng, J. (1986) J. Cell. Physiol. 128, 475-484[Medline] [Order article via Infotrieve]
  42. Vlodavsky, I., Bar-Shavit, R., Ishai-Michaeli, R., Bashkin, P., and Fuks, Z. (1991) Trends Biochem. Sci. 16, 268-271[CrossRef][Medline] [Order article via Infotrieve]
  43. Nugent, M. A., and Edelman, E. R. (1992) Biochemistry 31, 8876-8883[Medline] [Order article via Infotrieve]
  44. Roghani, M., Mansukhani, A., Dell'Era, P., Bellosta, P., Basilico, C., Rifkin, D. B., and Moscatelli, D. (1994) J. Biol. Chem. 269, 3976-3984[Abstract/Free Full Text]
  45. Moscatelli, D. (1992) J. Biol. Chem. 267, 25803-25809[Abstract/Free Full Text]
  46. Kendall, R. L., and Thomas, K. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10705-10709[Abstract]
  47. Chiang, M. K., and Flanagan, J. G. (1995) Growth Factors 12, 1-10[Medline] [Order article via Infotrieve]
  48. Dougher, A. M., Wasserstrom, H., Torley, L., Shridaran, L., Westdock, P., Hileman, R. E., Fromm, J. R., Anderberg, R., Lyman, S., Linhardt, R. J., Kaplan, J., and Terman, B. I. (1997) Growth Factors 14, 257-268[Medline] [Order article via Infotrieve]
  49. Aviezer, D., Hecht, D., Safran, M., Eisinger, M., David, G., and Yayon, A. (1994) Cell 79, 1005-1013[Medline] [Order article via Infotrieve]
  50. Carey, D. J., and Evans, D. M. (1989) J. Cell Biol. 108, 1891-1897[Abstract]
  51. Kojima, T., Shworak, N. W., and Rosenberg, R. D. (1992) J. Biol. Chem. 267, 4870-4877[Abstract/Free Full Text]
  52. Kinsella, M. G., and Wight, T. N. (1988) Biochemistry 27, 2136-2144[Medline] [Order article via Infotrieve]
  53. Folkman, J., and Shing, Y. (1992) Adv. Exp. Med. Biol. 313, 355-364[Medline] [Order article via Infotrieve]
  54. Azizkhan, R., Azizkhan, J., Zetter, B., and Folkman, J. (1980) J. Exp. Med. 152, 931-944[Abstract]
  55. Munson, P. J., and Rodbard, D. (1980) Anal. Biochem. 107, 220-239[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.