VEGF145, a Secreted Vascular Endothelial Growth Factor Isoform That Binds to Extracellular Matrix*

(Received for publication, August 2, 1996, and in revised form, November 20, 1996)

Zoya Poltorak Dagger , Tzafra Cohen Dagger §, Revital Sivan Dagger , Yelena Kandelis , Gadi Spira , Israel Vlodavsky par , Eli Keshet ** and Gera Neufeld Dagger Dagger Dagger

From the Dagger  Department of Biology and the § Department of Food Engineering and Biotechnology and the  B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 32000, Israel, the par  Department of Oncology, Hadassah University Hospital, Jerusalem 91120, Israel, and the ** Department of Molecular Biology, The Hebrew University-Hadassah Medical School, Jerusalem 91010, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

A vascular endothelial growth factor (VEGF) mRNA species containing exons 1-6 and 8 of the VEGF gene was found to be expressed as a major VEGF mRNA form in several cell lines derived from carcinomas of the female reproductive system. This mRNA is predicted to encode a VEGF form of 145 amino acids (VEGF145). Recombinant VEGF145 induced the proliferation of vascular endothelial cells and promoted angiogenesis in vivo. VEGF145 was compared with previously characterized VEGF species with respect to interaction with heparin-like molecules, cellular distribution, VEGF receptor recognition, and extracellular matrix (ECM) binding ability. VEGF145 shares with VEGF165 the ability to bind to the KDR/flk-1 receptor of endothelial cells. It also binds to heparin with an affinity similar to that of VEGF165. However, VEGF145 does not bind to two additional endothelial cell surface receptors that are recognized by VEGF165 but not by VEGF121. VEGF145 is secreted from producing cells as are VEGF121 and VEGF165. However, VEGF121 and VEGF165 do not bind to the ECM produced by corneal endothelial cells, whereas VEGF145 binds efficiently to this ECM. Basic fibroblast growth factor (bFGF)-depleted ECM containing bound VEGF145 induces proliferation of endothelial cells, indicating that the bound VEGF145 is active. The mechanism by which VEGF145 binds to the ECM differs from that of bFGF. Digestion of the ECM by heparinase inhibited the binding of bFGF to the ECM and released prebound bFGF, whereas the binding of VEGF145 was not affected by heparinase digestion. It therefore seems that VEGF145 possesses a unique combination of biological properties distinct from those of previously characterized VEGF species.


INTRODUCTION

The vascular endothelial growth factor (VEGF)1 isoforms display a limited structural similarity to platelet-derived growth factor and are important regulators of angiogenesis and blood vessel permeability (1-3). The human VEGF isoforms are generated by alternative splicing from a single gene (4-6). The domain encoded by exons 1-5 contains information required for the recognition of the known VEGF receptors KDR/flk-1 and flt-1 (7) and is present in all VEGF isoforms. The amino acids encoded by exon 8 are also present in all the VEGF splice variants. The VEGF isoforms are distinguished by the presence or the absence of the peptides encoded by exons 6 and 7 of the VEGF gene. VEGF121 is 121 amino acids long and lacks both exons. VEGF165 contains the exon 7-encoded peptide, whereas VEGF189 contains both exon 6- and exon 7-encoded peptides (6, 8, 9). VEGF121 and VEGF165 promote angiogenesis, cause permeabilization of blood vessels, and induce proliferation of vascular endothelial cells (10-14). VEGF189 has not yet been purified, but studies with cells expressing VEGF189 indicate that it may induce endothelial cell proliferation (8, 9). Low levels of a mRNA corresponding in size to a mRNA encoding a putative VEGF variant of 145 amino acids (VEGF145) containing exon 6 but lacking exon 7 were detected previously in reverse PCR experiments, but the protein encoded by this mRNA has not yet been characterized (15, 16).

The different VEGF isoforms differ in their heparin binding ability. VEGF121 does not bind to heparin, whereas VEGF165 and VEGF189 do (8, 17-19). The heparin binding affinity of VEGF189 was reported to be higher than that of VEGF165, suggesting that exon 6 contributes to the heparin binding ability of VEGF189 (8). VEGF165 and VEGF121 are secreted efficiently from producing cells and do not bind efficiently to the ECM produced by CEN4 cells (8, 9). In contrast, VEGF189 is retained on the cell surface and in the ECM, from which it can be released by prolonged incubation with heparin. (9). The peptide encoded by exon 7 also seems to affect the receptor recognition patterns of VEGF isoforms. VEGF121 recognizes a single VEGF receptor in endothelial cells that was identified as the KDR/flk-1 VEGF receptor (20, 21). VEGF165 also binds to this receptor but recognizes two additional VEGF receptors of unknown structure that are found in endothelial cells and in several transformed cell types (21, 22).

We have observed that VEGF145 is one of the main VEGF isoforms expressed by several cell lines derived from carcinomas of the female reproductive system. We have characterized VEGF145 and have compared its biological properties with those of other VEGF forms. Recombinant VEGF145 expressing the exon 6-derived peptide binds to heparin but behaves like VEGF121 with regard to its receptor recognition ability. In contrast to VEGF121 and VEGF165, it binds to a basement membrane like ECM produced by corneal endothelial cells in a biologically active form. VEGF145 therefore represents a VEGF form possessing distinct biological characteristics.


EXPERIMENTAL PROCEDURES

Materials

Human recombinant VEGF165 and VEGF121 were purified from Sf-9 insect cells, whereas recombinant human bFGF was produced in bacteria as described previously (19, 23, 24). A rabbit polyclonal antibody directed against VEGF165 (23) and a mouse monoclonal IgM antibody (M-35) directed against full-length VEGF165 were produced in our laboratory using standard techniques. Heparinases type I, II, and III were kindly donated by Dr. J. Zimermann (Ibex Technologies, Montreal, Canada). The bicystronic mammalian expression vector MIRB was kindly provided to us by Dr. Craig MacArthur (Washington University, St. Louis, MO) (25). Anti-VEGF monoclonal antibody clone 26503.11 and peroxidase- and alkaline phosphatase-conjugated anti-rabbit IgG antibodies were from Sigma. Disuccinimidyl suberate was from Pierce. Sodium alginate was from Fluka. Heparin-Sepharose was purchased from Pharmacia Biotech Inc. 125I-Sodium was obtained from New England Nuclear. Anti-mouse IgM antibodies conjugated to alkaline phosphatase were from Southern Biotechnology Associates Inc. (Birmingham, AL). Tissue culture plasticware was from Nunc, and 96-well dishes for enzyme-linked immunosorbent assays were bought from Corning. Grace's medium was obtained from Life Technologies, Inc. All other tissue culture reagents were from Biological Industries Inc. (Kibbutz Beth Haemek, Israel).

Identification of VEGF145 mRNA in Cancer Cells

Total mRNA was prepared from OC-238 human epithelial ovarian carcinoma cells (26). Complementary DNA was synthesized from 300 ng of total RNA using oligo(dT) as a primer and avian myeloblastosis virus reverse transcriptase. PCR amplification was carried out in the presence of a [32P]dCTP tracer (2 µCi in a 100-µl reaction volume), 1 mM of each dNTP, 2.5 mM MgCl2, and 2.5 units of Taq polymerase. 25 amplification cycles were used, each consisting of a 1-min incubation at 94 °C, a 2-min incubation at 65 °C, and a 3-min incubation at 72 °C. The VEGF-specific oligonucleotides used were GGAGAGATGAGCTTCCTACAG and TCACCGCCTTGGCTTGTCACA, corresponding to amino acids 92-98 and to the six carboxyl-terminal amino acids of VEGF, respectively. A pair of primers from the L19 ribosomal protein were included in the reactions as an internal control. Amplified fragments were resolved in a 6% nondenaturing polyacrylamide gel and were visualized by autoradiography. The band corresponding to the VEGF145 mRNA (see Fig. 1B) was excised, reamplified, and sequenced.


Fig. 1. A, the structure of the VEGF splice variants. The peptides encoded by the various exons of the human VEGF gene are shown in boxes but are not drawn to scale. The number of amino acids in each of the exon-encoded peptides is shown at the bottom. The exon structure of VEGF145 is shaded. B, expression of VEGF145 mRNA in OC-238 human epithelial ovarian carcinoma cells. Total RNA from OC-238 cells was translated into cDNA and amplified by PCR using radioactively labeled nucleotides. PCR products were separated on a polyacrylamide gel as described under "Experimental Procedures." Shown is an autoradiogram of the gel. The amplified species of VEGF and L19 cDNA are indicated. C, expression of VEGF145 in A431 and HeLa cells. Total RNA from HeLa and A431 cells was translated into cDNA and amplified by PCR using radioactively labeled nucleotides as described under "Experimental Procedures." Plasmids containing the VEGF121 cDNA, the VEGF165 cDNA, and the VEGF145 recombinant cDNA were included in separate PCR reactions using the primers described under "Experimental Procedures." Shown is an autoradiogram of the gel.
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A similar procedure using different primers was used to detect VEGF145 mRNA in A431 and HeLa cells. The VEGF-specific primers used here were derived from the 5' of the coding region (amino acids 13-20) and from the 3' region encompassed by the last 6 amino acids of VEGF including the translation stop codon of the VEGF sequence.

Construction of VEGF145 Encoding Expression Vectors

In order to produce recombinant VEGF145, we prepared a VEGF145 cDNA construct by deleting the oligonucleotides encoded by exon 7 out of the VEGF189 cDNA. Primers used to amplify exons 1-6 of the VEGF cDNA were the external primer GCTTCCGGCTCGTATGTTGTGTGG, corresponding to a puc118 sequence, and the internal primer ACGCTCCAGGACTTATACCGGGA, corresponding to a sequence at the 3' end of exon 6. Primers used to amplify the 3' end of the VEGF cDNA were complementary to the puc118 sequence GGTAACGCCAGGGTTTTCCCAGTC and to the 3' end of the exon 6 sequence (underlined) and to the start of exon 8 (<UNL>CGGTATAAGTCCTGGAGCGT</UNL>ATGTGACAAGCCGAGGCGGTGA). Following amplification, the PCR products were precipitated, and the products were reamplified using only the puc118-derived external primers. The product was gel purified, subcloned into the PCR-II vector, and sequenced using the sequenase-II kit from U. S. Biochemical Corp. This cDNA was further used for protein expression studies.

Production and Purification of Recombinant VEGF145

The VEGF145 cDNA was subcloned into the BamHI site of the MIRB expression vector (25). Following transfection into BHK-21 cells and selection with 0.6 mg/ml G418, VEGF145-expressing cells were identified using anti-VEGF antibodies. The VEGF145 cDNA was also subcloned into the transfer plasmid pVL-1393 (Invitrogen) downstream from the polyhedrin promoter to yield pVL-1393/v145. This plasmid and baculovirus wild type DNA were co-transfected into Sf9 cells using the calcium-phosphate co-precipitation method, and recombinant baculoviruses were isolated as described (23).

VEGF145 was produced in Sf9 cells as described for VEGF165 (23). The conditioned medium contained approximately 5 mg of VEGF145/liter. The conditioned medium was concentrated by precipitation with 70% ammonium sulfate at 4 °C for 12 h. The precipitate was solubilized in 20 mM Tris, pH 7, and 0.1 M NaCl, dialyzed extensively against this buffer at 4 °C, and applied to a heparin-Sepharose column. The column was washed with the same buffer containing 0.3 M salt, followed by elution with the same buffer containing 0.8 M NaCl. A small residual amount also eluted at 2 M NaCl. The 0.8 M salt eluant was further purified by reverse phase high pressure liquid chromatography on an Applied Biosystems Brownlee C-8 column using a linear gradient of acetonitrile (20-80%) containing 0.1% trifluoroacetic acid. VEGF145 was eluted at 48% acetonitrile. The trifluoroacetic acid in the eluant was neutralized using Tris base, and the acetonitrile was removed using a SpeedVac evaporator at room temperature.

Enzyme-linked Immunosorbent Assays

Increasing concentrations of VEGF in 50 µl of coating buffer (20 mM K2HPO4, 10 mM KH2PO4, 1 mM EDTA, 0.8% NaCl, pH 7.2) were adsorbed to 96-well dishes for 3 h at 25 °C. Free VEGF was aspirated, and the wells were blocked with coating buffer containing 1% bovine serum albumin for 1 h. The wells were extensively washed with wash buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, and 0.1% Tween 20), incubated with the anti-VEGF M-35 monoclonal antibody for 2 h at 25 °C, washed again, and incubated for 1 h with an alkaline phosphatase-conjugated secondary antibody. After final washing, the amount of bound antibody was determined using para-nitrophenylphosphate as substrate.

Cell Culture and Production of ECM-coated Dishes

Human umbilical vein-derived endothelial cells (HUVECs) were prepared from umbilical veins and cultured as described previously in M199 medium supplemented with 20% fetal calf serum, vitamins, 1 ng/ml bFGF, and antibiotics (21, 27). Proliferation assays using HUVECs were done as described previously (11). Bovine corneal endothelial (BCE) cells were isolated from steer eyes and cultured as described previously (28). ECM-coated dishes were prepared from cells grown in the presence or the absence of 30 mM chlorate as described previously (28, 29).

Binding of VEGFs to HUVECs and to ECM-coated Dishes

The binding and the cross-linking of 125I-VEGF165 to confluent layers of HUVECs grown in 5-cm dishes in the presence or the absence of various competitors was done essentially as described. VEGF165 was purified from infected Sf9 cells and iodinated as described (19, 21, 23, 30).

Binding of VEGFs to ECM-coated 96- or 24-well dishes was performed at room temperature. The ECM-coated wells were washed with rinse buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, and 0.1% Tween 20). Nonspecific sites were blocked with binding buffer (20 mM K2HPO4, 10 mM KH2PO4, 1 mM EDTA, 0.8% NaCl, 1 mg/ml bovine serum albumin, pH 7.2) for 1 h at room temperature. The binding buffer was aspirated and iodinated, or unlabeled growth factors were incubated with the ECM-coated wells in binding buffer for 2 h at 24 °C. Free growth factors were removed by aspiration, and the ECM was washed twice with rinse buffer. In binding experiments, 0.2 N NaOH was added to dissociate bound growth factors, and aliquots were counted in a gamma  counter or neutralized using Tris base and analyzed by SDS-PAGE followed by autoradiography. Alternatively, the wells were further incubated with various anti-VEGF monoclonal antibodies in binding buffer for 2 h and washed. Bound antibody was detected with appropriate secondary antibodies coupled to alkaline-phosphatase, using para-nitrophenylphosphate as substrate. Our M-35 anti-VEGF monoclonal antibody and commercial anti-VEGF monoclonal antibodies produced identical results in these assays.

For biological activity experiments, 15,000 HUVECs were seeded on ECM containing various amounts of adsorbed VEGF in a final volume of 1 ml of growth medium. Cells were trypsinized and counted after 3 days in a coulter counter. All experiments were repeated at least twice with similar results.


RESULTS

VEGF145 Is Expressed as a Major VEGF Splice Variant in Several Tumorigenic Cell Lines Originating in the Female Reproductive System

Reverse PCR analysis of mRNA from OC-238 human epithelial ovarian carcinoma cells (Fig. 1B), HeLa cells, and A431 cells (Fig. 1C) detected a VEGF mRNA containing a coding region smaller than that of VEGF165 but larger than that of VEGF121. The size of the coding region corresponded to the expected size of a mRNA encoding a VEGF form containing exons 1-6 and 8 and should lead to the production of a VEGF form containing 145 amino acids (VEGF145) (15). In all these cell lines the VEGF145 cDNA seemed to be expressed at levels comparable with those of VEGF165. The VEGF145 mRNA was not detected in several other transformed cell lines including C6 glioma cells and U937 cells. Sequence analysis of the PCR product from the OC-238 cells showed that this mRNA was indeed generated by alternative splicing and that it contains exons 6 and 8 but not exon 7 of the VEGF gene.

To study the properties of VEGF145, we have expressed the VEGF145 cDNA in Sf9 insect cells using the baculovirus expression system (23). Most of the VEGF145 produced by the infected Sf9 cells was found in the conditioned medium as a homodimer of ~41 kDa, with small amounts of monomeric VEGF145 (Fig. 2B). The VEGF145 dimers dissociated into monomers upon reduction with dithiotreitol (Fig. 2A). VEGF145 was partially purified by heparin-Sepharose affinity chromatography. VEGF145 was eluted from heparin-Sepharose columns using a stepwise salt gradient. Most of the VEGF145 eluted at 0.6-0.7 M NaCl, indicating that the heparin binding affinity of VEGF145 is similar to that of VEGF165 (data not shown) (17, 23). The recombinant VEGF145 was biologically active and induced the proliferation of HUVECs. The ED50 of VEGF145 was 30 ng/ml, whereas VEGF165 was 6-fold more active than VEGF145 in this assay (Fig. 3).


Fig. 2. Production of VEGF145 in Sf9 cells. VEGF145 and VEGF165 were produced in Sf9 insect cells as described under "Experimental Procedures." Conditioned medium containing recombinant VEGF was collected, and 10-µl aliquots were either reduced using 0.1 M dithiothreitol (A) or not reduced (B). Proteins were separated by SDS-PAGE (12% gel) and transferred by electroblotting to nitrocellulose. Filters were blocked for 1 h at room temperature with buffer containing 10 mM Tris-HCl, pH 7, 0.15 M NaCl, and 0.1% Tween 20 (TBST) supplemented with 10% low fat milk. The filters were incubated for 2 h at room temperature with rabbit anti-VEGF polyclonal antibodies in TBST (23), washed three times with TBST, and incubated with anti-rabbit IgG peroxidase-conjugated antibodies for 1 h at room temperature. Bound antibody was visualized using the ECL detection system.
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Fig. 3. VEGF145 stimulates the proliferation of endothelial cells. HUVECs were seeded in 24-well dishes (20,000 cells/well), and increasing concentrations of VEGF121 (diamond ), VEGF145 (black-square), and VEGF165 (square ) were added every other day as described under "Experimental Procedures." Cells were counted in a Coulter counter after 4 days.
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VEGF145 Induces Angiogenesis in Vivo

To determine whether VEGF145 can induce angiogenesis in vivo, the VEGF145 cDNA was subcloned into the BamHI site of the mammalian bicystronic expression vector MIRB (25). The MIRB/VEGF145 plasmid was transfected into BHK-21 cells (31), and stable cell lines producing VEGF145 were isolated. The VEGF145 produced by the mammalian cells was biologically active and was secreted into the growth medium. A stable clone producing 0.1 µg of VEGF145 per 106 cells was isolated. The VEGF145 expressing cells were embedded in alginate beads, and the beads were implanted under the skin of BALB/c mice (32). The pellets containing the alginate beads were removed after 4 days and photographed. Clusters of alginate beads containing VEGF145 expressing cells were dark red with blood, whereas beads containing cells transfected with vector alone had a much lower content of blood (Fig. 4). When examined under higher magnification, pellets containing VEGF145 producing cells appeared much more vascularized than pellets containing control cells.


Fig. 4. VEGF145 stimulates angiogenesis in vivo. The angiogenic activity of VEGF145 was determined using the alginate assay (32). Stable clones of BHK-21 cells transfected with the MIRB expression vector (MIRB) or with the VEGF145 expression vector MIRB/VEGF145 were trypsinized and suspended in Dulbecco's modified Eagle's medium to a concentration of 2.7 × 107 cells/ml. Sodium alginate (1.2%, 0.66 ml) was mixed with 1.33 ml of cell suspension. Beads of 1-µl diameter were formed by contact with a solution of 80 mM CaCl2. The beads were washed three times with saline. Each BALB/c mouse out of a group of four was injected subcutaneously with 400 µl of packed beads containing a given cell type. Clusters of beads were excised after 4 days and photographed. Blood-rich areas appear as dark areas in the photograph.
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VEGF145 Binds to the KDR/flk-1 Receptor but Not to the Two Smaller VEGF Receptors of HUVECs

125I-VEGF165 forms high molecular weight complexes with three types of VEGF receptors following cross-linking to HUVECs (Fig. 5, lane 1), whereas 125I-VEGF121 only binds to the larger of these receptors. The common receptor to which both VEGF121 and VEGF165 bind is the KDR/flk-1 VEGF receptor (Fig. 5, open arrow) (21). In order to compare the receptor recognition pattern of VEGF145 with those of VEGF165, 125I-VEGF165 was bound to HUVECs in the presence of 1 µg/ml of heparin and increasing concentrations of VEGF145. Bound 125I-VEGF165 was subsequently covalently cross-linked to the VEGF receptors. VEGF145 inhibited the binding of 125I-VEGF165 to the KDR/flk-1 receptor of the HUVECs (Fig. 5). This result was verified in a cell-free binding experiment in which VEGF145 competed with 125I-VEGF165 for binding to a soluble fusion protein containing the extracellular domain of the flk-1 receptor (data not shown) (33). In contrast, VEGF145 did not effectively inhibit the binding of 125I-VEGF165 to the two smaller VEGF receptors of the HUVECs (Fig. 5, filled arrow), indicating that the affinity of VEGF145 toward these two receptors is substantially lower than that of VEGF165. This behavior resembles the behavior of VEGF121 (21) and indicates that the presence of exon 6 is not sufficient to enable efficient binding of VEGF145 to these two receptors, despite the heparin binding properties that exon 6 confers on VEGF145.


Fig. 5. Effect of VEGF145 on 125I-VEGF165 binding to endothelial cells. 125I-VEGF165 (10 ng/ml) was bound to confluent HUVECs grown in 5-cm dishes for 2 h at 4 °C in the presence of 1 µg/ml heparin and the following concentrations of VEGF145 (µg/ml): lane 1, 0; lane 2, 0.05, lane 3, 0.1; lane 4, 0.25; lane 5, 0.5; lane 6, 1; lane 7, 2; lane 8, 3. Lane 9 received 2 µg/ml of VEGF121. Bound 125I-VEGF165 was subsequently cross-linked to the cells using disuccinimidyl suberate, and cross-linked complexes were visualized by autoradiography.
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VEGF145 Binds to the ECM Produced by Corneal Endothelial Cells

VEGF189 binds efficiently to the ECM produced by CEN4 cells, whereas VEGF165 binds to it very weakly (9). The fact that VEGF189 binds heparin with high affinity led to the suggestion that the interaction of VEGF189 with the ECM is mediated by heparan sulfate proteoglycans (8, 9). The heparin binding affinities of VEGF145 and VEGF165 are similar and substantially lower than the heparin binding affinity of VEGF189 (8). We therefore expected VEGF145 to bind poorly to ECM. Unexpectedly, experiments in which VEGF145 was bound to an ECM produced by bovine corneal endothelial cells (28, 34) showed that VEGF145 bound efficiently, whereas the binding of VEGF165 was marginal (Fig. 6A). In these experiments the binding was monitored with anti-VEGF antibodies, but similar results were obtained when binding to the ECM was assayed directly using 125I-VEGF145 (30 ng/ml) or 125I-VEGF165 (50 ng/ml) (Fig. 6B, first and third lanes). The binding of 125I-VEGF145 to the ECM was substantially but not completely inhibited by 10 µg/ml heparin (Fig. 6B, second lane). The 125I-VEGF145 used in these experiments contained some impurities (Fig. 6C), but the major iodinated protein that was recovered from the ECM had a mass corresponding to that of 125I-VEGF145 (Fig. 6B, first lane). To make sure that 125I-VEGF145 binds to the ECM and not to exposed plastic surfaces, the ECM was scraped off and washed by centrifugation, and the amount of adsorbed 125I-VEGF145 in the pellet was determined. The ECM contained ~70% of the adsorbed 125I-VEGF145. It therefore appears that the presence of the exon 6-derived peptide in VEGF145 enables efficient binding to the ECM, whereas the exon 7 derived peptide of VEGF165 does not suffice to confer this ability on VEGF165.


Fig. 6. Binding of VEGF145 and VEGF165 to ECM-coated dishes. A, ECM-coated 96-well dishes were incubated with increasing concentrations of VEGF145 (black-square) or VEGF165 (square ). The amount of ECM-bound VEGF was quantified using the M-35 anti-VEGF monoclonal antibody as described under "Experimental Procedures." B, 125I-VEGF145 (first and second lanes, 30 ng/ml) or 125I-VEGF165 (third lane, 50 ng/ml) was bound to ECM-coated wells. Heparin (10 µg/ml) was added with the VEGF145 in the second lane. The binding and the subsequent extraction of bound growth factors were done as described under "Experimental Procedures." Extracted growth factors were subjected to SDS-PAGE (12% gel) followed by autoradiography. C, the 125I-VEGF145 used in the experiment shown in B (0.2 ng) was chromatographed under reducing conditions on a 12% SDS-PAGE gel. Shown is an autoradiogram of the gel.
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VEGF145 Binds to the ECM Using a Mechanism That Is Not Dependent on ECM-associated Heparan Sulfates

The interaction of bFGF with the ECM is mediated by the heparan sulfate moieties of ECM associated proteoglycans (35). It was of interest to determine if VEGF145 uses a similar mechanism. When 125I-VEGF145 was bound to ECM-coated dishes in the presence of 10 µg/ml heparin, the binding was inhibited by ~60% (Fig. 7A). Under the same conditions the binding of 125I-bFGF to the ECM was inhibited by 80% (Fig. 7A). The binding of 125I-VEGF145 to the ECM was also inhibited by 80% in the presence of 0.8 M salt, indicating that the interaction is probably not hydrophobic (data not shown). These results are compatible with the expected behavior of proteins that bind to the ECM via heparin-like molecules. However, 125I-VEGF145 also bound efficiently to an ECM that was digested with heparinase-II (36). In contrast, there was almost no binding of 125I-bFGF to heparinase-II-digested ECM (Fig. 7A) (37, 38).


Fig. 7. The effects of heparinase-II and heparin on the binding of 125I-VEGF145 and 125I-bFGF to ECM-coated wells. A, effect of heparin and heparinase on growth factor binding. ECM-coated wells were incubated with or without 0.1 unit/ml heparinase-II in binding buffer for 2 h at 37 °C. Subsequently, 125I-VEGF145 (40 ng/ml) or 125I-bFGF (114 ng/ml) was added to the wells in the presence or the absence of 10 µg/ml heparin. Following incubation for 3 h at 25 °C, the wells were washed, and ECM-associated iodinated growth factors were dissociated by digestion with trypsin for 15 min at 37 °C. The amount of bound growth factor was determined using a gamma  counter (100% binding was 15,000 and 25,000 cpm/well for 125I-VEGF145 and 125I-bFGF, respectively). B, effect of heparin and heparinase-II on the release of bound growth factors from the ECM. 125I-VEGF145 or 125I-bFGF were bound to ECM-coated wells as described above. The wells were washed and reincubated in binding buffer alone, with 10 µg/ml heparin, or with 0.1 units/ml heparinase-II in a final volume of 50 µl. Following 12 h of incubation at 25 °C, the integrity of the ECM was verified by microscopy, and 45-µl aliquots were taken for counting in a gamma  counter. NaOH was then added to the wells, and the amount of ECM-associated growth factors was determined. The experiment was carried out in parallel to the experiment described in A above. The experiments in A and B were carried out in duplicate, and variation did not exceed 10%. Shown are the mean values. The experiments were repeated four times with similar results.
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In order to further investigate the mode of interaction of VEGF145 with the ECM, we measured the heparin- or heparinase-II-induced release of prebound VEGF145 or bFGF from the ECM. When the ECM-coated wells were incubated for 2 h at 37 °C with buffer, only 20% of the bound 125I-bFGF and 13% of the bound 125I-VEGF145 dissociated from the ECM (Fig. 7B). This background release may be attributed in part to a proteolytic activity residing in the ECM (8, 39). When 10 µg/ml heparin were included in the buffer, only 33% of 125I-VEGF145 was released from the matrix, as compared with the release of 78% of the prebound 125I-bFGF. An even sharper difference was observed when heparinase-II was added to the buffer. The enzyme released 72% of the bound 125I-bFGF, but only 17% of the bound 125I-VEGF145 was released (Fig. 7B). Similar results were obtained when the experiment was performed with unlabeled VEGF145, using a commercial monoclonal anti-VEGF antibody to detect VEGF associated with the ECM (data not shown).

To determine the efficiency of the heparinase-II digestion, the ECM was metabolically labeled with [35S]sulfate and subsequently digested with heparinase-II. The digestion released 80-85% of the labeled sulfate residues (data not shown). To determine whether VEGF145 can bind to ECM depleted of all types of sulfated glycosaminoglycans, BCE cells were grown in the presence of 30 mM chlorate, an inhibitor of glycosaminoglycan sulfation (29). These ECMs were further digested with a mixture of heparinases I, II, and III (36, 40). Neither of these treatments significantly inhibited the binding of VEGF145 to the ECM, despite a >95% decrease in the content of ECM-associated sulfate moieties (data not shown).

Because endothelial cells do not proliferate when they are seeded on ECM produced in the presence of chlorate (29), we examined whether VEGF145 bound to such ECM retains its biological activity. Wells coated with ECM produced in the presence of chlorate were incubated with increasing concentrations of either VEGF145 or VEGF165. The wells were subsequently washed extensively and HUVECs were seeded in the wells. ECM incubated with VEGF145 induced proliferation of vascular endothelial cells, whereas ECM incubated with VEGF165 did not (Fig. 8). We therefore conclude that the ECM-associated VEGF145 is biologically active.


Fig. 8. VEGF145 bound to the ECM produced by BCE cells promotes proliferation of endothelial cells. Wells of 24-well dishes were coated with an ECM produced by BCE cells cultured in the presence of 30 mM chlorate as described (29). The ECM-coated wells were incubated with increasing concentrations of VEGF145 (black-square) or VEGF165 (square ) as indicated and washed extensively as described. HUVECs (15,000 cells/well) were seeded in the ECM-coated wells in growth medium lacking growth factors. Cells were trypsinzed and counted after 3 days. The numbers represent the average number of cells in duplicate wells. The experiment was repeated twice with similar results. Variation within duplicates did not exceed 10%.
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DISCUSSION

Alternative splicing represents an important mechanism for the generation of diversity in growth factors and in their receptors. The alternative splice forms generated from the VEGF gene share angiogenic properties and are active as mitogens for endothelial cells. The VEGF145 mRNA has been previously detected as a rare VEGF mRNA species in placenta (15, 16). We have found expression of the VEGF145 mRNA in several tumorigenic cell types originating from the female reproductive system at levels comparable with the expression levels of VEGF165. Several transformed cell lines from other sources did not express this mRNA, indicating that the expression of VEGF145 may be more restricted compared with other VEGF forms (6). However, it remains to be seen whether production of VEGF145 is restricted to the female reproductive system.

To study the properties of VEGF145, we have produced recombinant VEGF145 in mammalian and in insect cells. VEGF145 was found to induce endothelial cell proliferation and in vivo angiogenesis, in agreement with previous studies that have indicated that these functions are not dependent on the presence of either exon 6 or exon 7 (10) and seem to be associated with the ability to bind to the KDR/flk-1 VEGF receptor (41, 42). However, VEGF145 seemed to be somewhat less active than VEGF165, and it is possible therefore that the presence of exon 6 in VEGF145 can subtly alter the conformation of the protein at the KDR/flk-1 binding site (7). The VEGF145 protein was secreted into the growth medium of producing cells. VEGF121, VEGF145, and VEGF165 are secreted into the medium by producing cells, whereas VEGF189 and VEGF206 are sequestered by cell surface heparan sulfates (8). It therefore seems that the simultaneous presence of both exon 6 and exon 7 is required for efficient binding of VEGF to cell surfaces, whereas the presence of each of these exons on its own does not confer this property on VEGF.

Unlike VEGF121, VEGF145 was able to bind to heparin-Sepharose columns, indicating that the exon 6 encoded peptide acts as an independent heparin binding domain. The affinity of VEGF145 for heparin was similar to that of VEGF165, even though the structures of the heparin binding domains of the two isoforms differ. All the VEGF isoforms tested to date bind to the KDR/flk-1 receptor of endothelial cells (21), and VEGF145 was no exception. However, unlike VEGF165, VEGF121 and VEGF145 do not bind efficiently to two additional VEGF receptors on HUVECs (21). The fact that VEGF121 and VEGF145 are both mitogenic and angiogenic indicates that these two receptors do not play a central role in the angiogenic and mitogenic response of endothelial cells. The biological function and the molecular structure of these two additional receptors is unknown. They appear to be novel VEGF receptors because they are not recognized by VEGF121, whereas both KDR/flk-1 and flt-1 are recognized by VEGF121.2 In addition these two receptors are not immunoprecipitated by antibodies directed against the intracellular domains of the known VEGF receptors (21), and they can be detected in tumor cells that do not express detectable levels of KDR/flk-1 or flt-1 mRNA (22). Our results suggests that specific exon 7 sequences that are not present in the exon 6-derived peptide are required for the interaction of VEGF165 with these two receptors. The heparin binding ability conferred on VEGF165 by the exon 7 peptide may not play a central role in the recognition of these receptors by VEGF165 because VEGF145 and VEGF165 bind with similar affinities to heparin yet differ in their ability to recognize these two receptors.

VEGF189 is not found in the conditioned medium of producing cells and is sequestered on cell surface heparan sulfates and in the ECM. In view of the strong interaction of VEGF189 with heparin, it was suggested that VEGF189 binds tightly to cell surface and ECM localized heparan sulfates, allowing the sequestration of VEGF189 in the ECM and on cell surfaces. This was supported by experiments that showed that heparin can dissociate bound VEGF189 from the ECM, although long incubation times and high heparin concentrations were required (9). In contrast, VEGF165 interacts weakly at best with cell surface heparan sulfates and with the ECM and is released into the medium of producing cells (8, 9). We expected VEGF145 to bind weakly to cell surfaces and to the ECM because VEGF165 and VEGF145 appear to bind to heparin with similar affinities. Indeed, we have found that VEGF145 is secreted as expected into the medium of VEGF145 producing cells. However, VEGF145 bound to the ECM produced by corneal endothelial cells much better than either VEGF165 or VEGF121. The lowest VEGF145 concentration at which binding to the ECM was observed was about an order of magnitude lower than the concentration at which binding of VEGF165 to the ECM was detected. In addition we have observed that VEGF145 that is bound to ECM is able to promote proliferation of endothelial cells. These observations prompted us to compare the ECM binding behavior of VEGF145 with that of bFGF, a growth factor that binds specifically to ECM-associated heparan sulfate moieties (38). Unexpectedly, our observations suggested that VEGF145 and bFGF do not bind to common binding sites on the BCE cell-derived ECM. Digestion of the ECM with heparinases releases bound bFGF but does not release bound VEGF145. It is possible that VEGF145 binds to a heparan sulfate subpopulation that is not recognized by bFGF or by the heparinases used in the present study. However, VEGF145 was also able to bind efficiently to an ECM produced in the presence of chlorate, an inhibitor of glycosaminoglycan sulfation (43) and to ECM digested with a mixture of three different heparinases resulting in a greater than 95% depletion of ECM-associated sulfate groups. These experiments therefore indicate that VEGF145 can bind to ECM components distinct from the heparan sulfate side chains of proteoglycans. A similar observation was reported for transforming growth factor-beta 1, a heparin binding protein (44) that binds to the core protein of the ECM-associated chondroitin sulfate/dermatan sulfate proteoglycan decorin (45) rather than to ECM-associated heparan sulfate moieties.

All the splice variants induce angiogenesis in vivo, so why are five VEGF variants produced? Angiogenesis is often initiated under adverse conditions, such as the conditions encountered during wound healing. Many cell types produce several VEGF forms simultaneously (6, 46), and it is possible that each form offers advantages in different situations. The simultaneous production of several different VEGF forms may therefore ensure a balanced angiogenic response under diverse circumstances. When the properties of the VEGF variants are examined, it is apparent that the differences in their heparin binding abilities may affect their diffusion from a VEGF producing source to target blood vessels. VEGF121 does not bind to either heparan sulfates or to the ECM and should therefore diffuse more readily than the heparin and ECM binding VEGF forms. The ECM may serve as a storage depot for the VEGF forms that bind efficiently to the ECM, and these forms may dissociate slowly from the ECM providing prolonged angiogenic stimulation or be released from the ECM as a result of the activity of proteases (8). The balance may tip toward the production of preferred VEGF isoforms under certain conditions (46). Production of VEGF145 may be such a case because the variety of cell types that produce VEGF145 appears to be limited compared with the range of cell types producing VEGF121 or VEGF165. However, the mechanism that determines what VEGF forms should be produced by a given cell type remains to be elucidated.


FOOTNOTES

*   This work was supported by a Joint Angiogenesis Research Center grant from the Israel Academy of Sciences (to G. N., I. V., and E. K.), a grant from the German-Israeli Binational Foundation, and a grant from the Israel Ministry of Health (to G. N.).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.
Dagger Dagger    To whom correspondence should be addressed.
1   The abbreviations used are: VEGF, vascular endothelial growth factor; BCE, bovine corneal endothelial; bFGF, basic fibroblast growth factor; ECM, extracellular matrix; HUVEC, human umbilical vein-derived endothelial cell; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.
2   G. Neufeld, unpublished results.

Acknowledgments

We thank Yael Shiffenbauer and Dr. Michal Neeman for help in reverse PCR experiments. We thank Dr. Dina Ron and Dr. Dan Cassel for critically reading this manuscript and for helpful discussions.


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