©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Selective Binding of VEGF to One of the Three Vascular Endothelial Growth Factor Receptors of Vascular Endothelial Cells (*)

(Received for publication, June 15, 1995; and in revised form, October 27, 1995)

Hela Gitay-Goren (1)(§) Tzafra Cohen (1)(§) (2) Shoshana Tessler (1) Shay Soker (4) Stela Gengrinovitch (1) Patricia Rockwell (3) Michael Klagsbrun (4) Ben-Zion Levi (2) Gera Neufeld (1)(¶)

From the  (1)Department of Biology, Technion, Israel Institute of Technology, Haifa 32000, Israel, the (2)Department of Food Engineering and Biotechnology, Technion, Israel Institute of Technology, Haifa 32000, Israel, (3)ImClone Systems Inc., New York, New York 10014, and the (4)Department of Biological Chemistry, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

VEGF and VEGF are vascular endothelial growth factor splice variants that promote the proliferation of endothelial cells and angiogenesis. VEGF contains the 44 additional amino acids encoded by exon 7 of the VEGF gene. These amino acids confer upon VEGF a heparin binding capability which VEGF lacks. I-VEGF bound to three vascular endothelial growth factor (VEGF) receptors on endothelial cells, while I-VEGF bound selectively only to the flk-1 VEGF receptor which corresponds to the larger of the three VEGF receptors. The binding of I-VEGF to flk-1 was not affected by the removal of cell surface heparan sulfates or by heparin. Both VEGF and VEGF inhibited the binding of I-VEGF to a soluble extracellular domain of the flk-1 VEGF receptor in the absence of heparin. However, heparin potentiated the inhibitory effect of VEGF by 2-3-fold. These results contrast with previous observations which have indicated that the binding of I-VEGF to the flk-1 receptor is strongly dependent on heparin-like molecules. Further experiments showed that the receptor binding ability of VEGF is susceptible to oxidative damage caused by oxidants such as H(2)O(2) or chloramine-T. VEGF was also damaged by oxidants but to a lesser extent. Heparin or cell surface heparan sulfates restored the flk-1 binding ability of damaged VEGF but not the receptor binding ability of damaged VEGF. These observations suggest that alternative splicing can generate a diversity in growth factor signaling by determining receptor recognition patterns. They also indicate that the heparin binding ability of VEGF may enable the restoration of damaged VEGF function in processes such as inflammation or wound healing.


INTRODUCTION

Four vascular endothelial growth factor (VEGF) (^1)forms are produced by alternative splicing from the VEGF gene(1, 2, 3) . The 121-amino acid form (VEGF) lacks a heparin binding ability, while VEGF, VEGF, and VEGF bind efficiently to heparin. All forms are mitogenic to vascular endothelial cells and induce permeabilization of blood vessels(4, 5) . VEGF induces angiogenesis in vivo(6) and plays a central role in the process of tumor angiogenesis(7, 8, 9) . Binding and cross-linking experiments have shown that VEGF binds to three VEGF receptors on the cell surface of vascular endothelial cells(10, 11, 12) . Two VEGF receptors have recently been identified and cloned(13, 14, 15, 16) . These include the mouse flk-1 receptor and its human homologue KDR, and the human flt-1 receptor. The flk-1 receptor was shown to transduce a VEGF mitogenic signal(9, 17) , while activation of the flt-1 receptor does not seem to result in a similar mitogenic response(16, 18) .

The binding of I-VEGF to various VEGF receptors and the effects of heparin on the binding have been characterized extensively(10, 11, 12, 19) , while the interaction of VEGF with the VEGF receptors of vascular endothelial cells has not yet been studied. We show that VEGF binds selectively to the larger of the three VEGF receptors of human umbilical vein-derived endothelial cells (HUE) and that this receptor probably corresponds to the human homologue of the flk-1 VEGF receptor. We also show that both VEGF and VEGF are susceptible to oxidative damage and that heparin restores the receptor binding ability of damaged VEGF, but not the receptor binding ability of damaged VEGF.


EXPERIMENTAL PROCEDURES

Materials

VEGF and VEGF were produced using the baculovirus system as described for VEGF(20) . VEGF and VEGF were purified as described previously(19, 21) . The flk-1/SEAP soluble receptor was produced as described(19) . Anti-flk-1 and anti-flt-1 antibodies directed against peptides derived from the intracellular domains of the respective receptors were purchased from Santa Cruz Biotechnology Inc. Anti-alkaline phosphatase antibodies were purchased from Dako. Tissue culture media and reagents were from Biological Industries (Beth-Haemek Biological Industries, Kibbutz Beth Haenek, Israel) and tissue culture plasticware from Nunc. Disuccinimidyl suberate was from Pierce Chemical Co., I-sodium from DuPont NEN, and heparin-Sepharose from Pharmacia. Recombinant bacterial heparinase-1 was kindly provided by Dr. Zimermann (Ibex Technologies, Montreal, Canada). High molecular mass protein markers were obtained from Bio-Rad. Porcine mucosa-derived heparin (H-7005) was purchased from Sigma as were all of the other chemicals.

Cell Culture

HUE cells were grown in gelatin-coated dishes in M-199 medium supplemented with 20% fetal calf serum, 4 mM glutamine, antibiotics, and 1 ng/ml bFGF which was added to the cells every other day.

Binding and Cross-linking Experiments

Iodination of human recombinant VEGF or VEGF was performed using either the chloramine-T method or the IODOGEN method with similar results as described previously(10, 21, 22) . However, while I-VEGF was separated from free iodine using a heparin-Sepharose column as described(10) , I-VEGF was separated from free iodine using size exclusion chromatography on Sephadex G-25(21) . The specific activities of the I-VEGF and the I-VEGF were about 10^5 cpm/ng.

The binding and the cross-linking of I-VEGF to endothelial cells was done as described previously(10, 11) , and the binding of I-VEGF was done similarly. Nonspecific binding was determined in the presence of 1-2 µg/ml unlabeled VEGF. The level of nonspecific binding ranged between 10 and 20% of the total binding. The binding and cross-linking of I-VEGF to a flk-1/SEAP fusion protein containing the extracellular domain of the flk-1 receptor was done as described previously for I-VEGF(19, 23) .

Immunoprecipitation Experiments

I-VEGF (5 ng/ml) or I-VEGF (10 ng/ml) were bound and cross-linked to confluent HUE cells grown in 6-cm dishes. The cells were lysed with 1 ml of lysis buffer containing 1% Nonidet P-40, 0.5% deoxycholate, and protease inhibitors. The lysates were centrifuged briefly to remove insoluble debris, and aliquots containing I-VEGF-receptor complexes were taken for immunoprecipitation using various antibodies. Cell lysates were precleared by a 1-h incubation at 4 °C with Sepharose CL-4B. The cell lysates were then incubated for 1 h at 4 °C with 1 µg/ml concentrations of various antibodies. Antibody-I-VEGF-receptor complexes were precipitated for 1 h at 4 °C using protein G-Sepharose. The beads were washed 3 times with lysis buffer, and I-VEGF-receptor complexes were then detached from the beads by boiling in SDS-PAGE sample buffer. I-VEGF-receptor complexes were separated on a 6% gel and visualized as described.

Inactivation of VEGF by Oxidation

VEGF or VEGF (2 µg) in 100 mM sodium phosphate buffer (pH 7.2) were incubated for 1 min with 0.1 mg/ml chloramine-T or 0.1-1% H(2)O(2) in a final reaction volume of 40 µl. At the end of the incubation, sodium metabisulfite (5 µl) was added to a final concentration of 0.5 mg/ml. After 2 min, bovine serum albumin was added to a final concentration of 1 mg/ml.


RESULTS AND DISCUSSION

I-VEGF Binds to One VEGF Receptor on HUE Cells, and the Binding Is Not Affected by Heparin-like Molecules

VEGF and VEGF were produced and purified from the conditioned medium of recombinant baculovirus-infected Sf9 cells and iodinated as described(19, 21) . The purified labeled VEGF forms (Fig. 1) were used further in binding and cross-linking experiments. When a saturating concentration of I-VEGF (20 ng/ml) was bound and cross-linked to HUE cells, only one I-VEGF-receptor complex of about 225 kDa could be detected. The formation of the 225-kDa I-VEGF-receptor complex was completely inhibited when the binding of I-VEGF was performed in the presence of either 2 µg/ml unlabeled VEGF or 2 µg/ml unlabeled VEGF (Fig. 2, lanes 2 and 3), indicating that this receptor binds both VEGF and VEGF. Addition of exogenous heparin (1 µg/ml) to the binding reaction, or removal of cell surface-associated heparan sulfates using heparinase-1, did not affect significantly the formation of the I-VEGF-receptor complex (Fig. 2, lanes 4 and 5, respectively). Additional I-VEGF-receptor complexes were not detected when heparin was added to the binding reaction. In contrast, when 5 ng/ml I-VEGF were bound to the cells in the presence of 1 µg/ml heparin, two smaller I-VEGF-receptor complexes were observed (Fig. 2, compare lane 7 to lane 8). I-VEGF also bound to the two smaller VEGF receptors in the absence of added exogenous heparin provided that higher I-VEGF concentrations were used (11) . In addition, larger I-VEGF containing cross-linked complexes of about 400 kDa were observed (Fig. 2, lane 8). Such high molecular weight complexes were observed by us in the past(23) , and they may represent dimerized receptors. Competition with 2 µg/ml VEGF inhibited the formation of the 225-kDa I-VEGF-receptor complex but did not affect the formation of the two smaller I-VEGF-receptor complexes (Fig. 2, lane 10, and Fig. 3). The formation of the two smaller I-VEGF-receptor complexes could not be inhibited significantly even when VEGF concentrations as high as 20 µg/ml were added to a binding reaction in which I-VEGF was bound to the cells in the presence of heparin (not shown). It therefore seems that VEGF is not able to bind to the two smaller VEGF receptors, or alternatively, that the affinity of VEGF to these receptors is much lower than that of VEGF. The experiments therefore suggest that VEGF and VEGF differ not only with regard to their heparin binding ability, but also in their ability to recognize various species of VEGF receptors.


Figure 1: Iodination of VEGF and VEGF. VEGF variants were iodinated to a specific activity of 10^5 cpm/ng using the chloramine-T method. I-VEGF (121) or I-VEGF (165) were reduced by boiling for 3 min in the presence of 0.1 M dithiothreitol. The iodinated proteins were chromatographed on a 12% SDS-PAGE gel and autoradiographed.




Figure 2: The effect of heparin and the effect of heparinase treatment on the binding and cross-linking of I-VEGF and I-VEGF to HUE cells. HUE cells were grown to confluence on gelatin-precoated 10-cm dishes. Cells were washed once with phosphate-buffered saline at 37 °C and were incubated in binding buffer (20 mM HEPES, pH 7.2, 0.1% gelatin in Dulbecco's modified Eagle's medium) for 1 h at 37 °C with (lanes 5, 6, 11, and 12) or without (lanes 1-4 and 7-10) 0.05 unit/ml heparinase 1. The cells were subsequently washed twice with cold phosphate-buffered saline, and 2.4 ml of cold binding buffer containing 10 ng/ml I-VEGF (lanes 1-6) or 5 ng/ml I-VEGF (lanes 7-12) were added to respective dishes. Other additions were: heparin (1 µg/ml), lanes 4, 6, 8, 10, and 12; unlabeled VEGF (2 µg/ml), lanes 2, 9, and 10; and unlabeled VEGF (2 µg/ml), lane 3. The binding, the subsequent cross-linking of bound growth factor to the cells using 0.25 mM disuccinimidyl suberate, SDS-PAGE of cross-linked samples, and the visualization of cross-linked products were done as described. Equal amounts of protein from cell lysates were chromatographed in each lane.




Figure 3: VEGF inhibits selectively the binding of I-VEGF to the 225-kDa VEGF receptor of HUE cells. I-VEGF (5 ng/ml) was bound to confluent HUE cells grown in 10-cm dishes in the presence of 1 µg/ml heparin and the following concentrations of unlabeled VEGF (µg/ml): lane 1, 0; lane 2, 0.05; lane 3, 0.1; lane 4, 2. After 2 h at 4 °C, the cells were washed, bound I-VEGF was cross-linked to cell surface receptors, and I-VEGF-receptor complexes were visualized as described in the legend to Fig. 2.



The binding of I-VEGF to the larger VEGF receptor of the endothelial cells was not affected by the removal of cell surface heparin-like molecules (Fig. 2, lane 5). This was perhaps to be expected as I-VEGF does not bind to heparin. This observation also indicates that heparin-like molecules do not affect the VEGF binding ability of the larger VEGF receptor of the endothelial cells. However, not all of the VEGF receptors that are capable of I-VEGF binding behave similarly. The I-VEGF binding ability of the VEGF receptors of YU-ZAZ6 melanoma cells is inhibited upon the removal of cell surface heparin-like molecules by heparinase digestion or by the addition of exogenous heparin, suggesting that heparin-like molecules can modulate the VEGF binding ability of the YU-ZAZ6 VEGF receptors (21) .

Antibodies Directed against the flk-1 VEGF Receptor Immunoprecipitate I-VEGF-Receptor Complexes

In order to identify the I-VEGF-receptor complexes seen in the cross-linking experiments, immunoprecipitation experiments were performed. The larger 225-kDa I-VEGF- or I-VEGF-labeled receptor was specifically immunoprecipitated by antibodies that recognize the intracellular domain of the flt-1 receptor (Fig. 4A, lane 4, and Fig. 4B, lane 2, respectively). In contrast, none of the labeled complexes could be precipitated by antibodies that recognize the intracellular domain of the flk-1 receptor or by an irrelevant antibody (Fig. 4). The inability to immunoprecipitate I-VEGF-receptor complexes with anti-flt-1 antibodies was expected since the mRNA encoding the flt-1 VEGF receptor is not very abundant in HUE cells(21) . The two smaller I-VEGF-receptor complexes were not precipitated by any of these antibodies and may represent either novel VEGF receptors or truncated versions of known VEGF receptors which are not recognized by the antibodies used.


Figure 4: Immunoprecipitation of I-VEGF-receptor cross-linked complexes with anti-flk-1 or with anti-flt-1 antibodies. A, I-VEGF (5 ng/ml) was bound and cross-linked to confluent HUE cells grown in 6-cm dishes. The cells were lysed, and aliquots containing I-VEGF-receptor complexes were examined by SDS-PAGE (lane 1) or immunoprecipitated using various antibodies as described under ``Experimental Procedures'' (lanes 2-4). The antibodies used (1 µg/ml each) were: anti-flt-1 (lane 2), anti- HB-EGF (lane 3), and anti-flk-1 (lane 4). Antibody-I-VEGF-receptor complexes were precipitated for 1 h at 4 °C using protein G-Sepharose. Immunoprecipitated I-VEGF-receptor complexes were detached from the beads by boiling in SDS-PAGE sample buffer, separated on a 6% gel, and visualized as described under ``Experimental Procedures.'' B, I-VEGF (10 ng/ml) was bound and cross-linked to confluent HUE cells as described above for I-VEGF. Following cross-linking, the cells were lysed. I-VEGF-receptor complexes in cell lysates were visualized immediately (lane 1) or immunoprecipitated using anti-flk-1 antibodies (lane 2), anti-flt-1 antibodies (lane 3), or anti-HB-EGF antibodies (lane 4) as described under Fig. A. I-VEGF-receptor complexes were separated using SDS-PAGE and visualized as described under Fig. A.



I-VEGF Binds to a Soluble Fusion Protein Containing the Extracellular Domain of flk-1, and the Binding Is Inhibited by VEGF and VEGF Even in the Absence of Heparin

The 225-kDa I-VEGF-receptor complex of HUE cells appears to contain the KDR/flk-1 receptor. To verify that I-VEGF can indeed bind to the flk-1 VEGF receptor, the interaction of VEGF and VEGF with a soluble fusion protein containing the entire extracellular domain of flk-1 fused to soluble alkaline phosphatase (flk-1/SEAP) was examined (Fig. 5, A-C)(19) . This soluble fusion protein was adsorbed to ELISA dishes coated with an antibody to alkaline phosphatase and used in quantitative binding assays. As expected, I-VEGF bound to flk-1/SEAP, and the binding was inhibited by unlabeled VEGF (Fig. 5B). The binding was effective even in the absence of exogenous heparin, and the addition of increasing concentrations of heparin did not affect the binding (not shown). However, to our surprise, we have found that unlabeled VEGF was able to compete with I-VEGF for binding to the flk-1/SEAP fusion protein in the absence of exogenous heparin (Fig. 5A). Addition of heparin modulated the concentration at which a half-maximal displacement of bound I-VEGF was observed, shifting it to VEGF concentrations 2-3-fold lower than those seen in the absence of heparin (not shown). Similar results were obtained when similar binding experiments were performed using heparinase-digested HUE cells (Fig. 5D). These observations are not in agreement with experiments which have indicated that unless very high concentrations of I-VEGF are used, I-VEGF does not bind to flk-1/SEAP in the absence of heparin(19) . These experiments also disagree with observations which have shown that the binding of I-VEGF to cell surface receptors on vascular endothelial cells requires cell surface heparan sulfates (Fig. 2, lanes 11 and 12)(11) .


Figure 5: Heparin restores the receptor binding ability of oxidized VEGF, but not the receptor binding ability of oxidized VEGF. A, I-VEGF (25 ng/ml) was bound to ELISA dishes coated with flk-1/SEAP fusion protein in a final volume of 50 µl as described under ``Experimental Procedures.'' The binding was performed in the presence () or absence (box, ) of heparin (1 µg/ml) and in the presence of increasing concentrations of either untreated VEGF (box) or VEGF which was treated with chloramine-T as described under ``Experimental Procedures'' (, ). The binding was performed for 2 h at room temperature, after which the dishes were washed 3 times with buffer containing 0.1% Tween 20 as described. Bound I-VEGF was solubilized using 0.5 N NaOH, and aliquots were counted in a -counter. 100% of I-VEGF binding corresponds to 5500 cpm. B, I-VEGF (25 ng/ml) was bound to ELISA dishes coated with flk-1/SEAP fusion protein in a final volume of 50 µl as described. The binding was performed in the presence () or absence (box, ) of heparin (1 µg/ml) and in the presence of increasing concentrations of either untreated VEGF (box) or VEGF which was treated with chloramine-T as described (, ). The binding was performed as described under A. 100% of I-VEGF binding corresponds to 10,000 cpm. C, I-VEGF (25 ng/ml) was bound to ELISA dishes coated with flk-1/SEAP fusion protein as described. The binding was performed in the presence (hatched columns) or in the absence (empty columns) of 1 µg/ml heparin. The VEGF and VEGF concentration used for competition was 1 µg/ml. VEGF and VEGF were treated or not with H(2)O(2) (1%) as described under ``Experimental Procedures.'' D, I-VEGF (20 ng/ml) was bound and cross-linked to confluent heparinase 1-digested HUE cells grown in 10-cm dishes in the absence (lanes 1 and 3) or in the presence (lanes 2 and 4) of 1 µg/ml heparin. Unlabeled VEGF was added to a final concentration of 2 µg/ml to some of the binding reactions (lanes 3 and 4). The heparinase digestion and the visualization of cross-linked complexes were done as described in Fig. 2.



These experiments imply that the receptor binding ability of VEGF may be impaired during iodination. Subsequent experiments have indicated that the flk-1/SEAP binding ability of VEGF is sensitive to oxidants. VEGF damaged by oxidizing agents such as chloramine-T or H(2)O(2) was not able to compete with I-VEGF for binding to flk-1/SEAP in the absence of heparin (Fig. 5, A and C). However, the ability to compete with I-VEGF for binding to flk-1/SEAP was partially restored by the addition of 1 µg/ml heparin to the binding reaction (Fig. 5, A and C). Similar results were also obtained in analogous experiments performed with heparinase-treated HUE cells (not shown). Oxidized VEGF also lost some of its ability to compete with I-VEGF for binding to flk-1/SEAP, although VEGF seemed to be somewhat more resistant than VEGF to oxidation (Fig. 5, B and C). However, in contrast to VEGF, the addition of heparin did not restore the flk-1/SEAP binding ability of damaged VEGF (Fig. 5, B and C). The relative insensitivity of VEGF to oxidative damage may explain why I-VEGF, in contrast to I-VEGF, is still able to bind to flk-1/SEAP and to the flk-1 receptor of the vascular endothelial cells in the absence of heparin-like molecules.

The potentiating effect that heparin exerts on the receptor binding ability of untreated VEGF could reflect oxidative damage sustained before or during VEGF purification. Alternatively, it could mean that heparin has a real ability to potentiate the binding of undamaged VEGF to the flk-1 VEGF receptor and perhaps to other types of VEGF receptors as well. Our results imply that the heparin binding ability of VEGF may be required under conditions in which oxidizing agents and free radicals are produced. Such conditions can be encountered in biological processes such as wound healing, hypoxia-induced angiogenesis, or inflammation, processes in which VEGF was shown to play an important role(24, 25, 26, 27, 28) . Under such conditions, cell surface heparin-like molecules could restore the activity of damaged VEGF molecules. This restorative function of heparin-like molecules could be of critical importance under conditions in which the initial concentration of VEGF is low to begin with. Heparin-like molecules are also able to restore the activity of damaged bFGF and aFGF and to protect them from inactivation by heat and oxidation(29, 30) . The protective and restorative effects of heparin could perhaps account for some of the opposing conclusions that were obtained in experiments designed to assess the importance of heparin-like molecules in the interaction of bFGF with FGF receptors(31, 32, 33) . Such a restorative effect would be harder to detect in the case of bFGF since an active bFGF homologue lacking a heparin binding ability (like VEGF) is unavailable.

In conclusion, our experiments indicate that both VEGF and VEGF bind to the 180-kDa VEGF receptor of HUE cells forming 220-230-kDa complexes after covalent cross-linking. However, only VEGF is capable of binding to the two smaller VEGF receptors of the endothelial cells. To the best of our knowledge, this is the first time that splice variants of a growth factor are found to differ in receptor recognition patterns. We have also performed immunoprecipitation experiments which indicate that the 220-kDa I-VEGF-receptor complex contains the KDR/flk-1 VEGF receptor. Competition experiments using a soluble fusion protein containing the extracellular domain of flk-1 have also revealed that heparin is not essential for the binding of VEGF or VEGF to flk-1 receptors. These experiments also suggest that the ability to bind heparin-like molecules may help to preserve the biological function of VEGF under conditions in which oxidants and free radicals are produced.


FOOTNOTES

*
This work was supported by a grant from the German-Israel Binational Foundation (GIF), by a grant from the Israel Cancer Research Fund, and by an angiogenesis research center grant from the Israel Academy of Sciences and Humanities (to G. N.). It was also supported by the Technion-Otto Meyerhof Biotechnology Laboratories (to B.-Z. L. and G. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first two authors contributed equally to this manuscript.

To whom correspondence should be addressed. Tel.: 972-4-829-4216; Fax: 972-4-822-5153; gera{at}techunix.technion.ac.il.

(^1)
The abbreviations used are: VEGF, vascular endothelial growth factor; VEGF, 165-amino acid form of vascular endothelial growth factor; VEGF, 121-amino acid form of vascular endothelial growth factor; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; flk-1, mouse homologue of the human VEGF receptor KOR; HUE, human umbilical vein-derived endothelial cells; PAGE, polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay.


ACKNOWLEDGEMENTS

We thank Drs. Judah Folkman, Dina Ron, and Israel Vlodavsky for critically reading this manuscript.


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