Inhibition of Vascular Endothelial Growth Factor (VEGF)-induced Endothelial Cell Proliferation by a Peptide Corresponding to the Exon 7-Encoded Domain of VEGF165 *

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells (EC) in vitro and a major regulator of angiogenesis in vivo. VEGF121 and VEGF165 are the most abundant of the five known VEGF isoforms. The structural difference between these two is the presence in VEGF165 of 44 amino acids encoded by exon 7 lacking in VEGF121. It was previously shown that VEGF165 and VEGF121 both bind to KDR/Flk-1 and Flt-1 but that VEGF165 binds in addition to a novel receptor (Soker, S., Fidder, H., Neufeld, G., and Klagsbrun, M. (1996)J. Biol. Chem. 271, 5761–5767). The binding of VEGF165 to this VEGF165-specific receptor (VEGF165R) is mediated by the exon 7-encoded domain. To investigate the biological role of this domain further, a glutathioneS-transferase fusion protein corresponding to the VEGF165 exon 7-encoded domain was prepared. The fusion protein inhibited binding of125I-VEGF165 to VEGF165R on human umbilical vein-derived EC (HUVEC) and MDA-MB-231 tumor cells. The fusion protein also inhibited significantly125I-VEGF165 binding to KDR/Flk-1 on HUVEC but not on porcine EC which express KDR/Flk-1 alone. VEGF165had a 2-fold higher mitogenic activity for HUVEC than did VEGF121. The exon 7 fusion protein inhibited VEGF165-induced HUVEC proliferation by 60% to about the level stimulated by VEGF121. Unexpectedly, the fusion protein also inhibited HUVEC proliferation in response to VEGF121. Deletion analysis revealed that a core inhibitory domain exists within the C-terminal 23-amino acid portion of the exon 7-encoded domain and that a cysteine residue at position 22 in exon 7 is critical for inhibition. It was concluded that the exon 7-encoded domain of VEGF165 enhances its mitogenic activity for HUVEC by interacting with VEGF165R and modulating KDR/Flk-1-mediated mitogenicity indirectly and that exon 7-derived peptides may be useful VEGF antagonists in angiogenesis-associated diseases.

Angiogenesis, the process in which new blood vessels sprout from pre-existing vessels, normally occurs during reproduction, embryonic development, and wound repair. On the other hand, pathological processes such as tumor progression may lead to aberrant angiogenesis (reviewed in Refs. [1][2][3][4]. The discovery that tumor growth is angiogenesis-dependent has led to the identification of a number of angiogenesis-promoting factors such as basic (bFGF) 1 and acidic fibroblast growth factor, vascular endothelial growth factor (VEGF), tumor necrosis factor-␣, transforming growth factor-␤, platelet-derived endothelial cell growth factor, and interleukin-8 (reviewed in Refs. 2, 4, and 5). Concomitant with the discovery of positive regulators of angiogenesis, inhibitors of angiogenesis have been identified including thrombospondin-1, interferon-␥, thalidomide, AGM-1470, the 16-kDa fragment of prolactin, cartilage-derived inhibitor, angiostatin, and endostatin (reviewed in Refs. 2, 4, and 5).
There is mounting evidence that VEGF may be a major regulator of angiogenesis (reviewed in Refs. 6 -8). VEGF was initially purified from the conditioned media of folliculostellate cells (9) and from a variety of tumor cell lines (10,11). VEGF was found to be identical to vascular permeability factor, a regulator of blood vessel permeability that was purified from the conditioned medium of U937 cells at the same time (12). VEGF is a specific mitogen for endothelial cells (EC) in vitro and a potent angiogenic factor in vivo. The expression of VEGF is up-regulated in tissues undergoing vascularization during embryogenesis and the female reproductive cycle (13,14). High levels of VEGF are expressed in various types of tumors, but not in normal tissue, in response to tumor-induced hypoxia (15)(16)(17)(18). Treatment of tumors with monoclonal antibodies directed against VEGF resulted in a dramatic reduction in tumor mass due to the suppression of tumor angiogenesis (19).
VEGF exists in five different isoforms that are produced by alternative splicing from a single gene containing eight exons (6, 20 -22). Human VEGF isoforms consist of monomers of 121, 145, 165, 189, and 206 amino acids, each capable of making an active homodimer (22,23). The VEGF 121 and VEGF 165 isoforms are the most abundant. VEGF 121 is the only VEGF isoform that does not bind to heparin and is totally secreted into the culture medium. VEGF 165 is functionally different than VEGF 121 in that it binds to heparin and cell surface heparan sulfate proteoglycans (HSPGs) and is only partially released into the culture medium (24,25). The remaining isoforms are entirely associated with cell surface and extracellular matrix HSPGs (24,25).
VEGF receptor tyrosine kinases, KDR/Flk-1 and/or Flt-1, are expressed by EC and by several types of non-EC such as NIH 3T3, Balb/c 3T3, human melanoma, and HeLa cells (26 -30). It appears that VEGF activities such as mitogenicity, chemotaxis, and induction of morphological changes are mediated by KDR/ Flk-1 but not Flt-1, even though both receptors undergo phosphorylation upon binding of VEGF (31)(32)(33)(34). Recently, we have characterized a new VEGF receptor which is expressed on EC and various tumor-derived cell lines such as breast cancerderived MDA-MB-231 (231) cells (35). Although both VEGF 121 and VEGF 165 bind to KDR/Flk-1 and Flt-1, only VEGF 165 binds to the new receptor. Thus, this is an isoform-specific receptor and has been named as the VEGF 165 receptor (VEGF 165 R). VEGF 165 R has a molecular mass of approximately 130 kDa, and it binds VEGF 165 with a K d of about 2 ϫ 10 Ϫ10 M, compared with approximately 5 ϫ 10 Ϫ12 M for KDR/Flk-1. In structurefunction analysis, it was shown directly that VEGF 165 binds to VEGF 165 R via its exon 7-encoded domain which is absent in VEGF 121 (35).
VEGF 165 is a more potent mitogen for EC than is VEGF 121 (36). One possible explanation is that the interaction of VEGF 165 with VEGF 165 R enhances KDR/Flk-1-mediated VEGF 165 bioactivity. To address this hypothesis, a glutathione S-transferase (GST) fusion protein containing a peptide corresponding to the 44 amino acids encoded by exon 7 (amino acids 116 -159 of VEGF 165 ) was prepared. The GST-exon 7 fusion protein inhibited the binding of 125 I-VEGF 165 to receptors on human umbilical cord vein-derived EC (HUVEC) and on 231 cells. The inhibitory activity was localized to the C-terminal portion of the exon 7-encoded domain. Furthermore, the fusion protein inhibited VEGF-induced proliferation of HUVEC. These results suggest that the exon 7-encoded domain contributes to the enhanced VEGF 165 mitogenic activity for HUVEC and that exon 7-derived peptides are potential antagonists of VEGF mitogenic activity for EC.

EXPERIMENTAL PROCEDURES
Materials-Human recombinant VEGF 165 and VEGF 121 were produced in Sf-21 insect cells infected with recombinant baculovirus encoding human VEGF 165 or VEGF 121 as described previously (35,37). VEGF 165 was purified from the conditioned medium of infected Sf-21 cells by heparin affinity chromatography, and VEGF 121 was purified by anion exchange chromatography. Basic FGF was kindly provided by Dr. Judith Abraham (Scios, Sunnyvale, CA). Cell culture media were purchased from Life Technologies, Inc. 125 I-Sodium was purchased from NEN Life Science Products. Disuccinimidyl suberate and IODO-BEADS were purchased from Pierce. Glutathione-agarose, NAP-5 columns, and pGEX-2TK plasmid were purchased from Pharmacia Biotech Inc. TSK-heparin columns were purchased from TosoHaas (Tokyo, Japan). Molecular weight marker was purchased from Amersham Corp. IL). Porcine intestinal mucosal-derived heparin was purchased from Sigma.
Cell Culture-Human umbilical vein endothelial cells (HUVEC) were obtained from the American Type Culture Collection (ATCC) (Rockville, MD) and grown on gelatin-coated dishes in M-199 medium containing 20% fetal calf serum (FCS) and a mixture of glutamine, penicillin, and streptomycin (GPS). Basic FGF (1 ng/ml) was added to the culture medium every other day. Porcine endothelial cells (PAE), parental and transfected to express KDR/Flk-1 (PAE-KDR), were kindly provided by Dr. Lena Claesson-Welsh and grown in F12 medium containing 10% FCS and GPS as described (32). MDA-MB-231 (231) cells were obtained from ATCC and grown in Dulbecco's modified Eagle's medium containing 10% FCS and GPS.
Endothelial Cell Proliferation Assay-HUVEC were seeded in gelatin-coated 96-well dishes at 4,000 cells/200 l/well in M-199 containing 5% FCS and GPS. After 24 h, VEGF isoforms and VEGF exon 7-GST fusion proteins were added to the wells at the same time. The cells were incubated for 72 h, and [ 3 H]thymidine (1 Ci/ml) was added for 10 -12 h. The medium was aspirated, and the cells were trypsinized and harvested by an automatic cell harvester (TOMTEC) and loaded onto Filtermats (Wallac). The Filtermats were scanned and cpm were deter-mined by a MicroBeta counter (Wallac). The results represent the average of samples assayed in triplicate, and the standard deviations were determined. All experiments were repeated at least three times and similar results were obtained.
Radioiodination of VEGF-The radioiodination of VEGF 165 and VEGF 121 was carried out using IODO-BEADS according to the manufacturer's instructions. Briefly, one IODO-BEAD was rinsed with 100 l of 0.1 M sodium phosphate, pH 7.2, dried, and incubated with 125 Isodium (0.2 mCi/g protein) in 100 l of 0.1 M sodium phosphate, pH 7.2, for 5 min at room temperature. VEGF (1-3 g) was added to the reaction mixture, and after 5 min the reaction was stopped by removing the bead. The solution containing 125 I-VEGF was adjusted to 2 mg/ml gelatin and purified by size exclusion chromatography using a NAP-5 column that was pre-equilibrated with PBS containing 2 mg/ml gelatin. Aliquots of the iodinated proteins were frozen on dry ice and stored at Ϫ80°C. The specific activity ranged from 40,000 to 100,000 cpm/ng protein.
Binding and Cross-linking of 125 I-VEGF-Binding and cross-linking experiments using 125 I-VEGF 165 and 125 I-VEGF 121 were performed as described previously (29,35). VEGF binding was quantified by measuring the cell-associated radioactivity in a ␥-counter (Beckman, Gamma 5500). The counts represent the average of three wells. All experiments were repeated at least three times, and similar results were obtained. 125 I-VEGF cross-linked complexes were resolved by 6% SDS-PAGE, and the gels were exposed to a phosphor screen and scanned after 24 h by a PhosphorImager (Molecular Dynamics). Subsequently, the gels were exposed to x-ray film.
Preparation of GST-VEGF Exon 7 and 8 Fusion Proteins-Different segments of exons 7 and 8 of VEGF were amplified by the polymerase chain reaction from human VEGF cDNA using the following primers: exon 7 ϩ 8 (Ex 7ϩ8), CGGGATCCCCCTGTGGGCCTTGCTC and GG-AATTCTTACCGCCTCGGCTTGTC; exon 7 (Ex 7), CGGGATCCCCCT-GTGGGCCTTGCTC and GGAATTCTTAACATCTGCAAGTACGTT; exon 7 with residues 1-10 deleted (Ex 7d-(1-10)), CGGGATCCCATTT-GTTTGTACAAGAT and GGAATTCTTAACATCTGCAAGTACGTT; exon 7 with residues 1-21 deleted (Ex 7d-(1-21)), CGGGATCCTGTT-CCTGCAAAAACACAG and GGAATTCTTAACATCTGCAAGTACGTT; exon 7 with residues 1-22 deleted (Ex 7d-(1-22)), CGGGATCCTGCA-AAAACACAG and GGAATTCTTAACATCTGCAAGTACGTT, and exon 7 with residues 30 -44 deleted (Ex 7d-(30 -44)), CGGGATCCCCCTGT-GGGCCTTGCTC and GGAATTCTAGTCTGTGTTTTTGCA. The amplified products were digested with BamHI and EcoRI restriction enzymes and cloned into the vector pGEX-2TK (Pharmacia Biotech Inc.) encoding GST (38) to yield the plasmid p2TK-exon 7ϩ8 and its derivatives. Escherichia coli (DH5␣) were transformed with p2TK-exon 7ϩ8 and derivatives to produce GST fusion proteins (see Fig. 5B for sequences). Bacterial lysates were subsequently separated by a glutathione-agarose affinity chromatography (38). Samples eluted from glutathione-agarose were analyzed by 15% SDS-PAGE and silver staining. GST fusion proteins were further purified on a TSK-heparin column as described previously (35). 165 and VEGF 121 differ in their ability to interact with VEGF receptors expressed on HUVEC (35,39). VEGF 121 binds to KDR/Flk-1 to form a 240-kDa labeled complex (Fig. 1, lane 2), whereas VEGF 165 , in addition to forming this size complex, also forms a lower molecular mass complex of 165-175 kDa (Fig. 1, lane 1). This isoform-specific receptor has been named the VEGF 165 receptor (VEGF 165 R). These differential receptor binding properties suggest that VEGF 165 and VEGF 121 might also have differential mitogenic activities. Accordingly, the ability of the two VEGF isoforms to stimulate HUVEC proliferation was tested. VEGF 165 was a more potent mitogen for HUVEC than was VEGF 121 (Fig. 2). VEGF 165 stimulated half-maximal DNA synthesis at 1 ng/ml and maximal stimulation at 4 ng/ml resulting in an 8-fold increase over control. On the other hand, 2 ng/ml VEGF 121 were required for half-maximal stimulation and 20 ng/ml for maximal stimulation resulting in a 4-fold increase in HUVEC proliferation over control. Thus, twice as much VEGF 121 compared with VEGF 165 was needed to attain half-maximal stimulation, and VEGF 121 -induced proliferation was saturated at about one-half the level induced by VEGF 165 . Taken together, these results suggest that there might be a correlation between the enhanced mitogenic activity of VEGF 165 for EC compared with VEGF 121 and the ability of VEGF 165 to bind to an additional receptor (VEGF 165 R) on HUVEC.

Differential Receptor Binding and Mitogenic Activities of VEGF 165 and VEGF 121 for HUVEC-VEGF
A  (35). This finding suggested that an excess of exon 7-encoded peptide might inhibit VEGF 165 binding to VEGF 165 R. GST fusion proteins containing a peptide encoded by VEGF exon 7 or by VEGF exons 7 and 8 were prepared. The 6 amino acids encoded by exon 8 which is C-terminal to exon 7 were included to facilitate the preparation of the fusion protein but did not affect the results in any way (data not shown). The exon 7 fusion protein binds directly to VEGF 165 R on 231 cells (35). It also binds directly to VEGF 165 R on HUVEC but not to KDR/FLK-1 on HUVEC (Fig. 1, lane 3). The ability of the GST-VEGF 165 exons 7-and 8-encoded peptide (GST-Ex 7ϩ8) to compete with 125 I-VEGF 165 binding to HUVEC, which express both KDR/Flk-1 and VEGF 165 R, to PAE-KDR cells which express only KDR/ Flk-1 (32), and to 231 cells which express only VEGF 165 R (35) was tested (Fig. 3). Increasing concentrations of GST-Ex 7ϩ8 markedly inhibited the binding of 125 I-VEGF 165 to HUVEC by about 85-95% (Fig. 3A) and to 231 cells by 97-98% (Fig. 3B). However, the fusion protein did not inhibit the binding of 125 I-VEGF 165 to PAE-KDR cells which do not express any VEGF 165 R (Fig. 3C). GST protein alone even at concentrations of 20 g/ml had no significant effect on the binding of 125 I-VEGF 165 to any of the cell types. Taken together, these binding studies suggested that GST-Ex 7ϩ8 competes for 125 I-VEGF 165 binding by interacting directly with VEGF 165 R but not with KDR.
These binding experiments were extended to analyze the effects of GST-Ex 7ϩ8 on 125 I-VEGF 165 binding to the individual VEGF receptor species by cross-linking (Fig. 4). Crosslinking of 125 I-VEGF 165 to 231 cells resulted in the formation of labeled complexes with VEGF 165 R (Fig. 4, lane 3). The formation of these complexes was markedly inhibited in the presence of 15 g/ml GST-Ex 7ϩ8 (Fig. 4, lane 4). 125 I-VEGF 165 crosslinking to HUVEC resulted in the formation of labeled complexes of higher molecular mass with KDR/Flk-1 and lower molecular mass complexes with VEGF 165 R (35) (Fig. 4, lane 1). GST-Ex 7ϩ8 markedly inhibited the formation of the 165-175-kDa labeled complexes containing VEGF 165 R (Fig. 4, lane 2). Unexpectedly, GST-Ex 7ϩ8 also inhibited markedly the formation of the 240-kDa labeled complex in HUVEC containing KDR/Flk-1 (Fig. 4, lane 2). On the other hand, the fusion protein did not inhibit cross-linking of 125 I-VEGF 165 to KDR/ Flk-1 on the PAE/KDR cells (not shown). Taken together, since (i) VEGF 165 binds to KDR/Flk-1 via the amino acids encoded by exon 4 (40), (ii)) VEGF 165 binds to VEGF 165 R via the amino acids encoded by exon 7, and (iii) GST-Ex 7ϩ8 binds to VEGF 165 R but not to KDR ( Fig. 1 and Fig. 3), these results suggested that by interfering directly with the binding of 125 I-VEGF 165 to VEGF 165 R, GST-Ex 7ϩ8 also inhibits indirectly the binding of 125 I-VEGF 165 to KDR/Flk-1.
Localization of the Core Inhibitory Region within the Exon 7-encoded Domain-The GST-Ex 7 fusion protein encompasses the entire 44 amino acid exon 7-encoded domain. To determine whether a core inhibitory region exists, deletions were made at the N and C termini of exon 7, and the effects on 125 I-VEGF 165 binding to HUVEC were measured (Fig. 5). In these experiments a fusion protein containing the exon 7-encoded domain plus the cysteine residue at position 1 of exon 8 was used as the parental construct. The cysteine residue of exon 8 was included to keep the number of cysteine residues in the VEGF portion of the fusion protein even. The GST-Ex 7 fusion protein inhibited 125 I-VEGF 165 binding to HUVEC by 80% at 2 g/ml fusion protein (Fig. 5). Inhibition of 125 I-VEGF 165 binding to HUVEC and 231 cells was comparable to that of GST-Ex 7ϩ8 (data not shown). Deletion of the first 10 (GST-Ex 7d-(1-10)) or 21 (GST-Ex 7d-(1-21)) N-terminal amino acids did not reduce the inhibitory activity of the fusion proteins. Actually, 1 g/ml of GST-Ex 7d-(1-21) had a greater inhibition activity than the same concentration of GST-Ex 7 suggesting that there may be a region within exon 7 amino acids 1-21 that interferes with the inhibitory activity. On the other hand, deletion of the cysteine residue at position 22 in exon 7 (GST-Ex 7 d-(1-22)) resulted in a complete loss of inhibitory activity. Deletion of the 15 C-terminal amino acids (GST-Ex 7 d- (30 -44)) also resulted in a complete loss of inhibitory activity (Fig. 5). These results indicated that the inhibitory core is found within amino acids 22-44 of exon 7. Moreover, it seems that the cysteine residue at position 22 in exon 7, which is Cys 137 in VEGF, is crucial for maintaining a specific structure required for the inhibition.
GST-Ex 7ϩ8 Inhibits VEGF 165 -induced Proliferation of HUVEC-The inhibition of VEGF 165 binding to KDR/Flk-1 by the GST-Ex 7ϩ8 fusion protein as shown in Fig. 4 suggested that it might also be an inhibitor of VEGF 165 mitogenicity since KDR/Flk-1 mediates VEGF mitogenic activity (32). Addition of 1-5 ng/ml VEGF 165 to HUVEC resulted in a 5.5-fold increase in the proliferation rate, peaking at 2.5 ng/ml (Fig. 6). When 15 g/ml GST-Ex 7ϩ8 was added in addition to VEGF 165 , HUVEC proliferation was reduced by about 60%. GST protein prepared in a similar way did not inhibit HUVEC proliferation even at 25 g/ml indicating that the inhibitory effect was due solely to the presence of the exon 7ϩ8-encoded domain within the fusion protein. It was concluded that exon 7ϩ8 peptide-mediated inhibition of VEGF 165 binding to VEGF receptors on HUVEC correlates with the inhibition of HUVEC proliferation.  1 and 2) and MDA-MB-231 cells (lanes 3 and 4) in 6-cm dishes. The binding was carried out in the presence (lanes 2 and 4) or the absence (lanes 1 and 3) of 15 g/ml GST-Ex 7ϩ8. Heparin (1 g/ml) was added to each dish. At the end of a 2-h incubation, 125 I-VEGF 165 was chemically crosslinked to the cell surface. The cells were lysed, and proteins were resolved by 6% SDS-PAGE. The gel was dried and exposed to x-ray film.

FIG. 5. Localization of a core inhibitory region within exon 7.
GST-Ex 7 fusion proteins containing full-length exon 7-encoded domain or truncations at the N-terminal and C-terminal ends were prepared as described under "Experimental Procedures." A, 125 I-VEGF 165 (5 ng/ml) was bound to subconfluent HUVEC cultures, as described in Fig. 3, in the presence of increasing concentrations of the GST fusion proteins. At the end of a 2-h incubation, the cells were washed and lysed, and the cell-associated radioactivity was determined with a ␥ counter. The counts obtained are expressed as percentage of the counts obtained in the presence of PBS without fusion protein. B, the amino acid sequences of VEGF exon 7 derivatives. These derivatives were prepared to contain the first cysteine residue of exon 8 at their C termini to keep an even number of cysteine residues.
FIG. 6. GST-Ex 7؉8 fusion protein inhibits VEGF 165 -stimulated HUVEC proliferation. HUVEC were cultured in 96-well dishes (5,000 cell/well) as in Fig. 2. Increasing concentrations of VEGF 165 (open circles), together with 15 g/ml GST-Ex 7ϩ8 (closed circles) or 25 g/ml GST (squares), were added to the medium, and the cells were incubated for 4 more days. DNA synthesis was measured in HUVEC as described in Fig. 2. The results represent the average counts of three wells, and the standard deviations were determined. duced mitogenicity (Fig. 7). GST-Ex 7ϩ8, at 15 g/ml, also inhibited VEGF 121 -mediated HUVEC proliferation, by about 2-fold. This was an unexpected result considering that VEGF 121 does not contain exon 7. To understand better the nature of the VEGF 121 inhibition, the effect of GST-Ex 7ϩ8 on the binding of 125 I-VEGF 121 to VEGF receptors was analyzed by cross-linking studies. Cross-linking of 125 I-VEGF 121 to HUVEC resulted in the formation of 240-kDa labeled complexes (Fig. 8, lane 1), which have been shown to contain VEGF 121 and KDR/Flk-1 (35,39). Formation of these complexes was significantly inhibited by GST-Ex 7ϩ8 at 15 g/ml (Fig. 8, lane 2). It was concluded that GST-Ex 7ϩ8 inhibits VEGF 121 -induced mitogenicity possibly by inhibiting its binding to KDR/Flk-1.

DISCUSSION
The most abundant of the VEGF isoforms are VEGF 165 and VEGF 121 . An important question in terms of understanding VEGF biology is whether these isoforms differ in their biochemical and biological properties. To date, it has been demonstrated that VEGF 165 , but not VEGF 121 , binds to cell-surface HSPG (23)(24)(25) and that VEGF 165 is a more potent EC mitogen than is VEGF 121 (36) (Fig. 2). In addition, we recently characterized a novel 130-kDa VEGF receptor found on the surface of HUVEC and tumor cells that is specific in that it binds VEGF 165 but not VEGF 121 (35). VEGF 165 binds to this receptor, termed VEGF 165 R, via the 44 amino acids encoded by exon 7, the exon which is present in VEGF 165 but not VEGF 121 . In contrast KDR/Flk-1 and Flt-1 bind both VEGF 165 and VEGF 121 and do so via the VEGF exons 4 and 3, respectively (40). Our goal in the present study was to determine whether exon 7 modulated VEGF 165 activity, in particular mitogenicity for HUVEC, and by what mechanism. To do so, we developed a strategy of inhibiting the binding of VEGF 165 to VEGF 165 R using a GST fusion protein containing the exon 7-encoded domain and examining any subsequent effects on HUVEC proliferation. Cross-linking experiments demonstrated, as expected, that the exon 7 fusion protein could bind to VEGF 165 R but not to KDR/Flk-1. The exon 7 fusion protein was found to be a potent inhibitor of 125 I-VEGF 165 binding to 231 cells which express VEGF 165 R alone, by 98%, and to HUVEC which express both KDR/Flk-1 and VEGF 165 R, by 85-95%. It did not, however, inhibit at all the binding of 125 I-VEGF 165 to PAE-KDR cells which express KDR/Flk-1 but not VEGF 165 R. GST protein alone did not inhibit binding to any of the cell types demonstrating that the inhibition was due solely to the exon 7 portion of the fusion protein. Cross-linking analysis, which demonstrated the formation of specific 125 I-VEGF 165 ⅐receptor complexes, confirmed that GST-Ex 7ϩ8 markedly inhibited the binding of 125 I-VEGF 165 to VEGF 165 R on HUVEC and 231 cells. Taken together, these results indicate that the exon 7 fusion protein interacts directly with VEGF 165 R and can act as a competitive inhibitor of binding of 125 I-VEGF 165 to this receptor.
The GST-Ex 7ϩ8 fusion protein inhibited VEGF 165 -induced proliferation of HUVEC by about 60%, to a level equivalent to that induced by VEGF 121 suggesting that activation of the KDR/Flk-1 tyrosine kinase receptor was somehow being adversely affected. Indeed, cross-linking analysis showed that the fusion protein not only inhibited cross-linking of 125 I-VEGF 165 to VEGF 165 R but to KDR/Flk-1 as well. This result was unexpected since our cross-linking studies show that the exon 7 fusion protein does not bind directly to KDR/Flk-1 consistent with the previous demonstration that VEGF 165 interacts with KDR/Flk-1 via its exon 4-encoded domain (40). Thus it appears that the binding of 125 I-VEGF 165 to VEGF 165 R via the exon 7-encoded domain modulates indirectly the interaction of the growth factor with KDR/Flk-1. A possible mechanism for this inhibitory effect of GST-Ex 7ϩ8 on HUVEC proliferation is that KDR/Flk-1 and VEGF 165 R are co-localized in close proximity on the cell surface. In this model, a VEGF 165 dimer interacts simultaneously with KDR/Flk-1 via the exon 4 domain and with VEGF 165 R via the exon 7 domain, generating a three-component complex. The GST-Ex 7ϩ8 fusion protein by competing directly with the binding of VEGF 165 to VEGF 165 R impairs indirectly the ability of VEGF 165 to bind to the signaling receptor, KDR/Flk-1. Thus, an efficient binding of VEGF 165 to KDR/Flk-1 might be dependent in part on successful interaction with VEGF 165 R. An alternative possibility is that the exon 7-encoded domain contains a heparin-binding domain (35) and that an excess of GST-Ex 7ϩ8 prevents VEGF 165 from binding to cell-surface HSPGs that are required for efficient binding of VEGF 165 to its receptors (29).
Surprisingly, GST-Ex 7ϩ8 also inhibited the mitogenic activity of VEGF 121 for HUVEC, by about 50%, even though VEGF 121 does not bind to VEGF 165 R (35). A possible explanation is that VEGF 165 R and KDR/Flk-1 are in proximity on the cell surface and that excess GST-Ex 7ϩ8 bound to VEGF 165 R g/ml GST-Ex 7ϩ8. Heparin (1 g/ml) was added to each dish. At the end of a 2-h incubation, 125 I-VEGF 121 was chemically cross-linked to the cell surface. The cells were lysed, and proteins were resolved by 6% SDS-PAGE. The gel was dried and exposed to x-ray film. sterically inhibits access of VEGF 121 to KDR/Flk-1. Cross-linking analysis did indeed show diminished binding of 125 I-VEGF 121 to KDR/Flk-1 in the presence of GST-Ex 7ϩ8 which does not bind directly to KDR/Flk-1, suggesting an indirect effect of the fusion protein on the binding of VEGF 121 to KDR/Flk-1.
GST-Ex 7ϩ8 also inhibits VEGF 165 binding to 231 breast cancer cells, which express VEGF 165 R and not KDR/Flk-1. However, VEGF is not mitogenic for these cells and at present we do not know the consequence of inhibiting VEGF 165 binding to these tumor cells.
The coordinate binding of VEGF 165 to a higher and to a lower affinity receptor (KDR/Flk-1 and VEGF 165 R, respectively) on HUVEC (35) and the inhibitory effects of GST-Ex 7ϩ8 fusion protein on the binding of VEGF 165 to these two receptors suggest that there is a dual receptor system at work in mediating VEGF 165 activity. Several other growth factors have been shown to bind to high and low affinity receptors. Transforming growth factor-␤ generates a complex with three receptors; two of them, receptors I and II, are the signaling receptors, whereas transforming growth factor-␤ receptor III/betaglycan is a lower affinity accessory binding molecule (41). The low affinity receptor for the nerve growth factor family, p75, is part of a complex with the signaling TRK receptors (42). A different type of dual receptor recognition is the binding of bFGF to cell-surface HSPGs and to its signaling receptors (43,44). It has been suggested that the binding of bFGF to its low affinity receptors (HSPGs) may induce conformational changes in bFGF so that the HSPG-bound bFGF could be efficiently presented to its high affinity, signaling receptors (43,44). Thus, the binding of VEGF 165 to both VEGF 165 R and KDR/Flk-1 appears to be part of a general mechanism wherein two different types of receptors are used to modulate growth factor activity.
Receptor binding studies were used to identify an inhibitory core within the 44 amino acids encoded by exon 7. Deletions were made in both the N-terminal and C-terminal regions of exon 7, and the inhibitory activity was localized to the 23amino acid C-terminal portion of exon 7 (amino acids . Of these 23 amino acids, 5 are cysteine residues. The high proportion of cysteine residues suggests that this domain has a defined three-dimensional structure required for efficient binding to VEGF 165 R. The cysteine residue at position 22 of the exon 7 domain is critical for inhibitory activity, probably for maintenance of a necessary three-dimensional structure. A study that examined the role of cysteine residues at different positions in VEGF 165 showed that a substitution of Cys 146 , which lies within the core inhibitory domain of exon 7 (at position 31 in exon 7), by a serine residue resulted in a 60% reduction in VEGF 165 permeability activity and a total loss of EC mitogenicity (45). The Cys 146 mutation had no effect on the dimerization of VEGF (45). Thus, it appears that this cysteine residue is not involved in the formation of interdisulfide bonds between two VEGF monomers but might rather involve intradisulfide bonding within the monomer. These results support our hypothesis that a three-dimensional structure stabilized by cysteine residues exists in the C-terminal half of exon 7 that contributes to VEGF 165 biological activity, such as interaction with VEGF 165 R. Interestingly, a fusion protein corresponding to a deletion of the N-terminal 21 amino acid residues encoded by exon 7 was a more potent inhibitor than the intact exon 7-encoded peptide. It may be that the N-terminal portion interferes in part with the interaction of the C-terminal portion with VEGF 165 R and therefore a deletion of the N-terminal portion results in enhanced binding to VEGF 165 R and yields a better competitor of VEGF 165 .
Since the identification of VEGF as a major angiogenesis factor and contributor to tumor pathology, numerous attempts had been made to design specific VEGF antagonists. These antagonists include anti-VEGF antibodies (19) and soluble KDR/Flk-1 and Flt-1 ectodomains (46 -48). We now add to this group the peptide encoded by exon 7 of VEGF and possibly a smaller core inhibitory peptide. Since the exon 7-encoded peptide inhibits both VEGF 165 -and VEGF 121 -induced mitogenicity for HUVEC, it and its derivatives may be useful as general VEGF inhibitors. The VEGF exon 7-encoded domain is an example of a portion of an EC mitogen being an EC inhibitor. Previously, it has been shown that fragments of SPARC (secreted protein, acidic and rich in cysteine) inhibit EC proliferation while the intact SPARC maintains angiogenic activity (49). Several other EC inhibitors are fragments of larger proteins, which in themselves are devoid of inhibitory activity. These include the 16-kDa fragment of prolactin (50), fragments of laminin (51), plasmin-cleaved fragments of fibronectin (52), angiostatin which is a fragment of plasminogen (53), and endostatin which is a fragment of collagen XVIII (54). Thus, it seems that there are numerous examples of EC inhibitors being generated from larger proteins. Our identification of the NEGF exon 7-encoded domain as an EC antagonist is based on the analysis of VEGF and VEGF receptor structure-function relationships. In the future, further analysis of the exon 7 domain might be useful for the design of small pharmacological peptides that would serve as VEGF antagonists in angiogenesis-related diseases.