A Novel Type of Vascular Endothelial Growth Factor, VEGF-E (NZ-7 VEGF), Preferentially Utilizes KDR/Flk-1 Receptor and Carries a Potent Mitotic Activity without Heparin-binding Domain*

Vascular endothelial growth factor (VEGF) mediates endothelial cell proliferation, angiogenesis, and vascular permeability via the endothelial cell receptors, KDR/Flk-1 and Flt-1. Recently, a gene encoding a polypeptide with about 25% amino acid identity to mammalian VEGF was identified in the genome of Orf virus (OV), a parapoxvirus that affects sheep and goats and occasionally, humans, to generate lesions with angiogenesis. In this study, we examined the biological activities and receptor of OV-derived NZ-7 VEGF (VEGF-E). VEGF-E was found to be a dimer of about 20 kDa with no basic domain nor affinity for heparin column, similar to VEGF121 subtype. VEGF121 has 10–100-fold less endothelial cell mitotic activity than VEGF165 due to lack of a heparin-binding basic region. Interestingly, however, VEGF-E showed almost equal levels of mitotic activity on primary endothelial cells and vascular permeability activity as VEGF165. Furthermore, VEGF-E bound KDR/Flk-1 (VEGFR-2) and induced its autophosphorylation to almost the same extent as VEGF165, but did not bind Flt-1 (VEGFR-1) nor induce autophosphorylation of Flt-1. These results indicate that VEGF-E is a novel type of endothelial growth factor, utilizing only one of the VEGF receptors, and carrying a potent mitogenic activity without affinity to heparin.

Vascular endothelial growth factor (VEGF), 1 also known as vascular permeability factor has a potent mitotic activity specific to vascular endothelial cells and a significant vascular permeability activity (1,2). A wide variety of studies strongly suggest that VEGF plays an important role in most of the pathological angiogenesis associated with diseases such as diabetic retinopathy, rheumatoid arthritis, and solid tumors (3)(4)(5).
Recently, several VEGF-related genes have been isolated and characterized. Placenta growth factor is suggested to be involved in the growth and maintenance of placental tissue, and VEGF-C was shown to carry a lymphangiogenic activity different from VEGF (6,7). VEGF-D is also suggested to have an activity similar to VEGF-C (8). However, other genes such as VEGF-B and NZ-type VEGFs encoded in the open reading frames of Orf virus NZ-2 and NZ-7 strains are not well characterized (9).
Human VEGF has at least four subtypes due to an alternative splicing, VEGF 121 , VEGF 165 , VEGF 189 , and VEGF 206 (10,11). Among these, VEGF 165 has the most potent biological activity and is the most abundant subtype in vivo with a few exceptions such as in placenta. VEGF 121 is also well expressed in many normal and pathological tissues, however, its biological activity has been shown to be 10 -100-fold weaker than that of VEGF 165 . The only difference between these two VEGF subtypes is that VEGF 165 carries an exon 7-derived 44-amino acid stretch near the carboxyl-terminal region, which is enriched with basic amino acid residues and has affinity for heparin and heparan sulfates (12). Since VEGF 165 but not VEGF 121 has affinity for cell surface and since a low concentration of heparin could augment the biological activity of VEGF, this basic stretch is considered to be crucial for generation of strong mitotic signals via VEGF 165 (13)(14)(15).
VEGF binds two endothelial cell-specific receptors, Flt-1 and KDR/Flk-1 tyrosine kinases (16 -19). In addition, the exon 7-derived basic stretch in VEGF 165 has been shown to bind a 130-kDa cell surface molecule (20). Thus, this molecule might also play a role in VEGF signaling, although the mechanism of this action has yet to be elucidated. We are interested in Orf virus NZ strain-encoded VEGFs since the possible gene products have only about 25% amino acid identity with VEGF and have no apparent basic domain, but seem to be involved in the process of pathological angiogenesis in virus-infected lesions.
OV is a linear double-stranded DNA virus and a member of the parapoxvirus genus of the Poxvirus family. It causes contagious pustular dermatitis in sheep and goats and is transmissible to humans by direct contact. Lesions appear after an incubation period of approximately 1 week as hemorrhagic bullae. They may reach several centimeters in diameter, but fade spontaneously after several weeks. Histologically, the lesions are highly vascular and edematous showing an increase in the number of vessels by proliferation of endothelial cells, and contain severe inflammatory infiltrates of mixed character. Viral particles were, however, found only in the cytoplasm of degenerating keratinocytes (21,22). The lesions induced in sheep and humans after infection with OV show extensive dermal vascular responses which are likely to be a direct effect of the expression of the VEGF-like gene (9,23).
In this study we characterized an OV NZ-7-derived VEGFlike sequence, and found that this gene product utilizes only one of the VEGF receptors, KDR/Flk-1, but shows a potent endothelial cell growth stimulatory activity and vascular permeability activity similar to those of VEGF 165 without the heparin-binding region. These results clearly indicate that the NZ-7-derived protein is a new type of VEGF family in the biochemical point of view. Its exonless cDNA structure in the viral genome suggests a phylogenetic origin in vertebrate genome. Since VEGF-A to -D have already been described, we propose the name, "VEGF-E" for this protein.

EXPERIMENTAL PROCEDURES
Cells and Culture Conditions-Sf9 cells were purchased from Invitrogen (California) and cultured in EX-Cell 400 medium (JRH Biosciences, Lenexa, KS). NIH3T3 cell lines overexpressing human Flt-1 (NIH3T3-Flt-1) or human KDR/Flk-1 (NIH3T3-KDR) were used for ligand binding and signal transduction studies. Parental NIH3T3 cells were used for PDGF and FGF stimulation assays. NIH3T3-KDR cells and NIH3T3-Flt-1 cells were established as described previously (24). These cell lines as well as parental NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Nissui, Tokyo) supplemented with 10% calf serum, 2 mM L-glutamine, 40 g/ml kanamycin, and 200 g/ml G418 sulfate (Geneticin; Life Technologies, Inc., Grand Island, NY). NIH3T3 cell lines overexpressing Flt-4, another member of the VEGF receptor gene family, were kindly provided by Dr. K. Alitalo (University of Helsinki, Finland) and cultured in DMEM containing 10% calf serum and 200 g/ml geneticin G418 medium. Sinusoidal endothelial cells were isolated from rat liver as described previously and grown in endothelial growth-UV medium (Kurabo, Osaka) supplemented with VEGF (10 ng/ml) (25). Recombinant human VEGF 165 was prepared as described (24) and recombinant human VEGF 121 was obtained from R&D Systems (Minneapolis, MN). Human PDGF B/B was obtained from Boehringer Mannheim (Germany) and recombinant human basic FGF was obtained from R&D.
VEGF-E cDNA-The nucleotide sequence of the open reading frame identified in the genome of Orf virus NZ-7 strain (9) was synthesized and used for this study. To facilitate oligonucleotide synthesis, three point mutations without change of amino acid sequences were introduced and PstI and KpnI restriction enzyme cleavage sites were generated at codon 65/66 and codon 111, respectively. The three portions were independently synthesized with or without polymerase chain reaction and finally joined to establish the full-length cDNA for NZ-7derived protein (tentatively designated as VEGF-E) on pUC 18 vector.
Polyclonal and Monoclonal Antibodies-Polyclonal antisera against VEGF-E were raised in rabbits using a carboxyl-terminal 20-amino acid sequence as antigen. The antiserum to peptide specifically recognized VEGF-E by Western blot analysis. Polyclonal antibody against the amino-terminal region of the human Flt-1 was kindly provided by Dr. K. Ohsumi (Mitsubishi-Yuka BCL, Tokyo, Japan). Anti-human KDR/ Flk-1 rabbit antiserum (B2) was prepared by using a synthetic oligopeptide for human KDR/Flk-1 residue numbers 1295 to 1317 as previously reported (26). A monoclonal antibody specific to phosphotyrosine (PY20) was obtained from ICN Biochemicals (Costa Mesa, CA). Anti-PLC␥ and anti-phospho mitogen-activated protein kinase antibodies were obtained from Upstate Biotechnology Inc. (Lake Placid, NY) and from Promega (Madison, WI), respectively. Secondary antibodies conjugated to horseradish peroxidase were purchased from Amersham (Arlington Heights, IL).
Preparation of VEGF-E using Baculovirus Expression System-The full-length coding region for the VEGF-E was subcloned into the BamHI-EcoRI site on the multi-cloning region of pVL1393, designated as pVEGF-E. This plasmid was used for cotransfection into Sf9 cells along with linearized Baculovirus DNA "BaculoGold" (Phamingen, San Diego, CA).
Amplification of the recombinant viruses was carried out at 3-day intervals. The titer of the recombinant viruses was checked by agarose gel titration. Sf9 cells grown in serum-free Ex-Cell 400 medium (JRH Biosciences, KS) were used for a large scale preparation of VEGF-E protein, and were infected with viruses at a multiplicity of infection of about 10. One h after infection, the supernatant was removed and fresh serum-free medium was added to the culture plates. Three days after infection, the supernatant was collected and analyzed by SDS-PAGE on a 12% gel followed by Western blotting using anti-VEGF-E antiserum.
Purification of VEGF-E-About 150 ml of the supernatant of VEGF-E recombinant virus-infected Sf9 cells was collected, concen-trated, and dialyzed to 20 mM sodium phosphate buffer, containing 10 mM NaCl and 20% glycerol. The sample was applied to a cation-exchange S-P column (Pharmacia, Uppsala, Sweden). Proteins bound were eluted with 10 mM NaCl-containing 20 mM sodium phosphate buffer (pH 6.0). VEGF-E was eluted as a more than 80% pure protein after most bulk proteins in the sample were eluted in the flow-through fraction. For detection of VEGF-E protein, aliquots of fractions were analyzed by Western blotting using anti-VEGF-E antiserum and by silver staining. The purity of VEGF-E was above 80%.
Endothelial Cell Growth Assay-Human umbilical vein endothelial cells (HUVEC) (Morinaga, Tokyo) were grown in HUVE culture medium (Morinago, Tokyo) and used for endothelial cell growth assay. HUVEC were seeded at 1000 cells/well on 96-well plates in growth factor-free HUVE culture medium. Four h after plating, growth factor (VEGF-E, VEGF 165 , or VEGF 121 ) containing medium was added After 4 days, the cell numbers were determined using a Coulter counter or the MTT (cell proliferation) assay (Sigma). For MTT assay, 20 l of 1 mg/ml 3-(4,5-dimethylthiazolo-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) was added to each well. After the wells were incubated with MTT for 4 h at 37°C, the supernatant was removed completely from each well, then, 150 l of dimethyl sulfoxide was added and OD 540 values of the samples were determined. Rat liver non-parenchymal cells, which contain about 90% sinusoidal endothelial cells, were prepared by perfusion of rat liver with collagenase followed by differential centrifugation (25). Nonparenchymal cell fractions (NP cells) were grown in endothelial cell growth-UV medium (Kurabo, Japan) without growth factor, or with growth factors VEGF 165 or VEGF-E.
Vascular Permeability Assay-Vascular permeability activity of VEGF-E was determined using the Miles assay (27). Anesthetized guinea pigs were shaved on the back and injected intracardinally with 1 ml of 0.5% Evans Blue (Sigma). About 30 min later, 200 l of several samples or control phosphate-buffered saline (PBS) were injected intracutaneously into the back of the guinea pig. After 30 min, leakage of dyes was detected by the presence of blue spots at the injection sites.
Iodination of VEGF-E-Iodination of growth factors was performed essentially as described previously (28). The specific activity of the product 125 I-VEGF 165 was 100,000 cpm/ng and that of 125 I-VEGF-E was 70,000 to 80,000 cpm/ng.
Ligand Binding and Competition Assay-NIH3T3-Flt-1 and NIH3T3 KDR/Flk-1 cells were seeded at 1 ϫ 10 5 cells/well on 24-well collagencoated plates 24 h prior to experimental use. The cells were preincubated with binding buffer (DMEM, 10 mM HEPES (pH 7.2), 0.1% BSA) at 4°C for 30 min. Then the medium was replaced with 0.3 ml of binding medium (DMEM, 10 mM HEPES (pH 7.2), 0.5% BSA) containing radiolabeled ligands, and the binding reactions were allowed to proceed at 4°C for 90 min. All experiments were determined in triplicate wells. After incubation, the cells were washed three times with ice-cold binding buffer then twice with ice-cold PBS containing 0.1% BSA. Subsequently, the cells were solubilized by the addition of 0.5 ml of 0.5 N NaOH per well at 37°C for 10 min, followed by an additional wash with 0.5 ml of PBS per well, and the radioactivity was counted in a ␥-counter. Values were analyzed according to Scatchard's procedure (29,30).
Competition assay was done by incubating the cells with fixed ( 125 I-VEGF 165 1 ng/ml; 125 I-VEGF-E 1 to 2 ng/ml) and increasing concentrations of unlabeled ligands. Nonspecific binding was determined by parallel binding assay with NIH3T3-neo cells or by competition assay in the presence of excess unlabeled ligands with NIH3T3-Flt-1 or NIH3T3-KDR/Flk-1.

Autophosphorylation of Receptors Flt-1 and KDR/Flk-1-For in vivo
phosphorylation, NIH3T3-Flt-1 cells were grown to near confluence, and starved overnight in 0.1% serum-containing medium and stimulated at 37°C with 10 ng/ml VEGF 165 , VEGF-E, or BSA for 5 min. In the case of NIH3T3-KDR/Flk-1, the cells were stimulated after being starved overnight in 0.5% serum medium because of the relatively strong autophosphorylation of KDR/Flk-1. The cells were washed in ice-cold PBS with 0.1 mM Na 3 VO 4 twice and lysed in 1% Triton X-100 lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl 2 , 2% aprotinin, 1 mM PMSF, 50 mM sodium fluoride, 10 mM Na 4 P 2 O 7 , and 2 mM Na 3 VO 4 ). The lysates were clarified by centrifugation (15,000 rpm ϫ 10 min). Protein concentrations were measured using Bio-Rad protein assay (Richmond, CA) and the same amounts of protein of each sample were used for analysis. For immunoblotting, the cell lysates were subjected to 7.5% SDS-PAGE and transferred to a nitrocellulose sheet. The blots were incubated with a blocking solution (5% BSA containing washing buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.3% Tween 20)) and probed with the primary antibody diluted in the blocking solution. The signal was visualized using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL, Amersham) according to the manufacture's instructions.
For immunoprecipitation, cell lysates were incubated with the primary antibody at 4°C overnight. The resulting immune complexes were collected by precipitation with protein A (for anti-KDR/Flk-1 antiserum) or protein G (for anti-PLC-␥ antibodies) Sepharose beads (Pharmacia). The immunoprecipitates were then washed three times with 1% Triton X-100 lysis buffer. For the experiments involving complex formation, 0.1% Triton X-100 lysis buffer was used for washing. Samples were then separated by electrophoresis on 7.5% polyacrylamide gels and analyzed as above with appropriate antibodies, anti-receptors, or anti-phosphotyrosine (PY20) from ICN Biochemicals (Costa Mesa, CA).
Effects of VEGF-E on Receptors PDGFR and FGFR--NIH3T3 cells were seeded at 7 ϫ 10 4 cells per 3.5-cm plate in 1% serum containing DMEM. Four h after plating, 10 ng/ml growth factor (PDGF-B/B, basic FGF or VEGF-E) containing medium was added. After the 4-day culture, the cells were detached with 0.5% trypsin and the cell numbers were counted.
In Vivo Angiogenesis and Histological Analysis of Gels-Recombinant human VEGF 165 or purified VEGF-E was mixed with cold liquid Matrigels and injected subcutaneously into the flanks of 6-week-old ICR and BALB/c nude male mice. Five days after injection, animals were sacrificed and the gels were recovered. The gels were observed macroscopically and under stereoscopic microscope. For histological examination, the gels were fixed in 4% formalin and embedded in paraffin. Sections of gels were either stained with hematoxylin and eosin or reacted with a mouse biotinylated monoclonal antibody (diluted 1:1000) to von Willebrand factor (Dakopatts, Glostrup, Denmark). After applying the biotinylated antibody, the immunoreaction was visualized by the avidin-peroxidase method and by a light counter-staining with hematoxylin and eosin.

Homodimer Structure of VEGF-E (NZ-7 VEGF) with No Affinity to Heparin
To examine the biological and biochemical features of VEGF-E, we synthesized its cDNA as described under "Experimental Procedures," and prepared its gene product on a large scale using Baculovirus system. Detection of the proteins was carried out using a rabbit antiserum against the carboxylterminal peptide of VEGF-E (Fig. 1B).
VEGF-E is predicted to be a 149-amino acid protein including a signal peptide of about 20 amino acids, and its overall structure without the basic amino acid cluster region is similar to the shortest subtype of VEGF, VEGF 121 (Fig. 1A). As expected from the structural similarity to VEGF 121 , VEGF-E did not show any affinity for the heparin column (data not shown). In addition, VEGF-E had only a weak affinity for the cation exchange column, thus, we purified it by using S-P column under acidic (pH 6.0) conditions as shown in Fig. 2. The elution pattern of VEGF-E showed a double band of 22 and 24 kDa, the former was 2-3-fold more intense than the latter based on immunoblotting. The difference of these two forms could be due to a minor difference of glycosylation. The silver staining for the 24-kDa VEGF-E was very faint. Repeated experiments suggest that this form is rather resistant to this staining.
The VEGF family belongs to the VEGF-PDGF supergene family, all the members of which have a dimer structure under natural conditions. Using reducing and nonreducing conditions, we found that VEGF-E exists as a homodimer of 44 kDa in the absence of reducing reagent, while it is separated to the 22-24-kDa monomer in the presence of a high concentration of dithiothreitol (Fig. 1B). These results indicate that the basic structure of VEGF-E is consistent with that of a member of the VEGF-PDGF supergene family.

Biological Activity of VEGF-E on Endothelial Cells
Stimulation of Endothelial Cell Growth-Since an important characteristic of VEGF is endothelial cell-specific growth stimulatory activity, we examined the effect of VEGF-E on the proliferation of HUVEC as well as rat liver sinusoidal endothelial cells (25). Although the structural similarity between VEGF-E and VEGF 121 is only 25% at the amino acid level, unexpectedly, VEGF-E showed a potent mitotic activity and stimulated the growth of these two types of endothelial cells to almost the same degree as VEGF 165 (Fig. 3). Cell numbers at 1 ng/ml VEGF-E corresponded to those at 0.8 ng/ml VEGF 165 and at 10 ng/ml VEGF 121 . The maximum number of cells stimulated with VEGF 121 was one-half that with VEGF 165 and VEGF-E. Furthermore, VEGF-E induced an elongated morphology of rat sinusoidal endothelial cells to a degree comparable to that with VEGF 165 (Fig. 4).
Enhancement of Vascular Permeability-To examine the effect of VEGF-E upon vascular permeability, we carried out the Miles assay using guinea pigs and Evans Blue dye as a marker. As shown in Fig. 5, 20 ng of VEGF 165 was sufficient to saturate the release of dye from the capillary. The same concentration of VEGF-E exerted comparable vascular permeability factor activity as VEGF 165 . The margin of the blue spots generated with VEGF-E was more diffuse than those with VEGF 165 , probably due to little, if any, affinity of VEGF-E to the heparin or heparan sulfate of matrix or mesenchymal cell surface. VEGF 121 (20 ng/0.2 ml) gave only a weak spot (data not shown). Control experiments using the conditioned medium of insect cells or PBS did not show any vascular permeability factor activity.

VEGF-E Competes for the Binding of VEGF to KDR/Flk-1 Receptor but Not to Flt-1 Receptor
To examine why VEGF-E has comparable biological activities to VEGF 165 , we examined the receptors specific to VEGF-E. Since VEGF binds both the Flt-1 tyrosine kinase and a related kinase KDR/Flk-1 at high affinity (17-19, 24, 28, 31), we carried out competition experiments using 125 I-labeled VEGF and unlabeled VEGF-E.
As indicated in Fig. 6A, VEGF-E competed the binding of 125 I-VEGF to NIH3T3-KDR/Flk-1. Competition of VEGF-E for binding of 125 I-VEGF to KDR/Flk-1 showed a kinetics very similar to that of unlabeled VEGF. Surprisingly, however, even a 1,000-fold excess of VEGF-E could not inhibit the binding of 125 I-VEGF to the Flt-1 receptor (Fig. 6B). As a positive control, unlabeled VEGF competed the binding of 125 I-VEGF to NIH3T3-Flt-1 and NIH3T3-KDR/Flk-1 (Fig. 6, A and B). These results strongly suggest that VEGF-E can bind to KDR/Flk-1 but not to Flt-1.

VEGF-E Binds to KDR/Flk-1 but Not to Flt-1
To more directly examine the receptor molecule(s) for VEGF-E, an aliquot of the purified VEGF-E was labeled with 125 I and used for the binding assay. As expected, the 125 Ilabeled VEGF-E bound NIH3T3-KDR/Flk-1 cells but neither NIH3T3-Flt-1 nor NIH3T3-neo cells (Fig. 6, C and D). The binding of 125 I-VEGF-E to KDR/Flk-1 was competed by unlabeled VEGF-E to the same degree as that of unlabeled VEGF (Fig. 6D).
A Scatchard analysis indicated that the K d for the binding of VEGF-E to KDR/Flk-1 was about 330 pM (Fig. 6E). This K d value indicates that the affinity of VEGF-E for KDR/Flk-1 is almost the same as that of VEGF 165 for KDR/Flk-1 under our experimental conditions (our VEGF 165 K d is 300 pM, and other groups have reported a VEGF 165 K d of 400 -800 pM) (24,32). These results indicate that VEGF-E binds KDR/Flk-1 but not Flt-1 as the receptor on the cell surface.

VEGF-E Activates KDRFlk-1 but Not Flt-1 Tyrosine Kinase
To further examine KDR/Flk-1 as the receptor for VEGF-E, we tested whether VEGF-E could stimulate intracellular signaling from KDR/Flk-1. The first event for the signal transduction from receptor tyrosine kinase is the autophosphorylation of the receptor molecules on tyrosine. NIH3T3-KDR/Flk-1 cells were serum-starved overnight and then stimulated with VEGF 165 , VEGF-E, or fetal bovine serum. As shown in Fig. 7A, both VEGF 165 and VEGF-E clearly stimulated autophosphorylation of KDR/Flk-1 based on the immunoprecipitation with anti-KDR/Flk-1 antiserum, followed by Western blotting using an anti-phosphotyrosine antibody. Addition of serum to the culture medium to 5% did not stimulate any KDR/Flk-1 autophosphorylation (data not shown), indicating that the serum does not contain VEGF, VEGF-E, or related growth factors at sufficient levels for activation of KDR/Flk-1.
On the other hand, VEGF-E could not activate Flt-1 overexpressed on NIH3T3 cells, although VEGF induced autophosphorylation of Flt-1 (Fig. 7B). In both cases of VEGF and VEGF-E, the autophosphorylation of KDR/Flk-1 occurred only on the upper molecule of the two major bands of KDR/Flk-1 (Fig. 7A). We have recently shown that the largest KDR/Flk-1,

FIG. 2. Purification of VEGF-E using S-P column.
VEGF-E was purified on a cation exchange S-P column, eluted with 10 mM NaClcontaining 20 mM sodium phosphate buffer (pH 6.0) (see "Experimental Procedures"). Total proteins and VEGF-E were detected by silver staining and by Western blotting using anti-VEGF-E antiserum, respectively. Tube fraction numbers 1 and 2 contained most of the bulk proteins, and numbers 3-6 carried purified VEGF-E.

FIG. 3. Growth stimulatory activity of VEGF-E on HUVEC.
HUVEC were cultured in HUVE culture medium with various amounts of VEGF 121 , VEGF 165 , or VEGF-E for 4 days, then the cell growth was monitored by MTT assay. Results represent the mean of triplicate experiments with standard deviation. 230 kDa in size, is the mature form expressed on the cell surface (26).
To further confirm no detectable binding of VEGF-E to Flt-1, we tested whether the soluble form of Flt-1 could inhibit the biological activity of VEGF-E on endothelial cell culture. We  (27). About 20 ng per 0.2 ml of VEGF 165 was sufficient to saturate the release of Evans Blue dye from the capillary. VEGF-E stimulated vascular permeability in a dose-dependent manner the same as VEGF 165 . Control experiments using PBS did not show any vascular permeability activity. lial cell growth-stimulatory activity of VEGF-E (data not shown). Thus, the affinity of VEGF-E for Flt-1 was undetectable in these experimental conditions.
In addition to Flt-1 and KDR/Flk-1, the VEGF receptor family includes Flt-4 as a third member which specifically binds VEGF-C and -D but not VEGF (8,36,37). However, VEGF-E did not induce any autophosphorylation of the processed 125-kDa form of Flt-4 in the Flt-4-overexpressing NIH3T3 cells (data not shown).
Furthermore, we examined the autophosphorylation of other possible receptors such as PDGF receptor and FGF receptor by VEGF-E, since VEGF-E shows 15% amino acid identity with PDGF. However, these receptors endogenously expressed on NIH3T3 cells were not autophosphorylated with VEGF-E, although the control PDGF and FGF stimulated the autophosphorylation of their receptors (data not shown) and induced cell proliferation (Fig. 8A), as well as morphological change (Fig.  8B). Therefore, from these results we concluded that VEGF-E binds and activates only one of the VEGF receptors, KDR/Flk-1.

Signal Transduction of VEGF-E
Recently we found that the major signal transduction pathway from KDR/Flk-1 overexpressed in NIH3T3 cells and in primary endothelial cells after stimulation with VEGF is through the PLC␥-protein kinase C-mitogen-activated protein kinase pathway (26). 2 Therefore, we examined the signal transduction of VEGF-E on rat liver primary sinusoidal endothelial cells. As shown in Fig. 7C, VEGF-E rapidly induced tyrosine phosphorylation of PLC␥ followed by the activation of mitogenactivated protein kinase. The time course of the phosphorylation of PLC␥ was quite similar to that of autophosphorylation of KDR/Flk-1 receptor, and the degree of the phosphorylation was essentially the same as that in the presence of VEGF 165 (Fig. 7, A and C) (26). The highest level of autophosphorylation of KDR/Flk-1 was observed 5 min after stimulation with VEGF-E. Based on these results, we conclude that the signal transduction of VEGF-E via KDR/Flk-1 is very similar to that of VEGF 165 .

Histological Analysis of Angiogenesis Induced by VEGF-E in Vivo
Various samples were mixed with Matrigel and implanted subcutaneously into the flanks of the mice. Four days after injection the animals were sacrificed and the gels were obtained for analysis. Two strains of mice, BALB/c nude mice (Fig. 9, A-G) and ICR mice (Fig. 9, H and I) were used in these experiments. Macroscopically, Matrigels containing VEGF 165 or VEGF-E were hypervascularized, compared with the control Matrigel with PBS alone (Fig. 9, A, B, and C). Under stereoscopic microscope, Matrigel containing VEGF-E showed many newly formed microvessels invading into Matrigel from preexisting dilated vessels (Fig. 9G). Sections of gel were stained with hematoxylin and eosin and examined under a light microscope (Fig. 9, D, E, F, and H). The control Matrigel without growth factor was not vascularized. On the other hand, several dilated vessels, newly formed microvessels, and hemorrhages were detected within the Matrigels mixed with VEGF 165 or VEGF-E. Microvessels close to the ligand-containing Matrigels were frequently congested with red blood cells. Essentially the same histological changes were observed in these two strains of mice except for inflammatory cells surrounding vessels which were detected only in ICR mice.
Immunostaining of VEGF-E-containing Matrigel with antivon Willebrand factor antibody confirmed that the inner layer of the enlarged and microvessel-like structures consisted of vascular endothelial cells (Fig. 9I) Matrigel was not detected. The pathological characteristics induced with VEGF-E were quite similar to those reported in Orf virus-infected lesions. These results indicate that VEGF-E bears a potent angiogenic activity similar to VEGF 165 not only in in vitro cell culture but also in in vivo experimental conditions. DISCUSSION In this study we have examined the characteristics of VEGF-E (NZ-7 VEGF) which was originally identified as an open reading frame in the genome of the Orf virus NZ-7 strain (9), and demonstrated that VEGF-E is a novel type of VEGF family in several ways. First, although VEGF-E is only 19 -25% identical at the amino acid level to VEGF (Fig. 1), it binds KDR/Flk-1 (VEGFR-2) at a high affinity similar to VEGF 165 . Second, unlike any of the VEGF subtypes, VEGF-E cannot bind Flt-1 nor transduce signals from Flt-1. Third, although VEGF-E does not bear a heparin-binding basic region, its biological activities for endothelial cell growth and vascular permeability are almost equal to those of the potent angiogenesis and vascular permeability factor, VEGF 165 . Therefore, VEGF-E is unique in that it carries a simple structure and utilizes only one of two VEGF receptors but shows significant biological activities on vascular endothelial cells.
The region on Flt-1 for binding to VEGF has been shown to be localized on the second Ig domain in the extracellular domain, however, a portion of the third Ig domain downstream is required for high affinity binding (35,38,39). A similar region of the second Ig domain on KDR was also shown to be important for binding with VEGF, but the binding affinity is still low, suggesting the surrounding portion is also important. The region for binding VEGF-E on KDR/Flk-1 has yet to be studied, however, the efficient suppression of VEGF binding to KDR/ Flk-1 by VEGF-E and vice versa strongly suggests that the binding regions for both ligands on KDR/Flk-1 overlap at least in part. A series of point mutational analysis of VEGF 165 by Keyt et al. (12) have revealed that 3 basic amino acid residues, Arg-82, Lys-84, and His-86, on VEGF play a role in the interaction of VEGF with KDR/Flk-1. Interestingly, a region on VEGF-E corresponding to these 3 amino acids on VEGF does not contain any basic amino acid residues. Therefore, although a hydrophilic interaction between this basic 82-86 stretch on VEGF and the binding site on the second and third Ig domains of KDR/Flk-1 is critical for high affinity binding of VEGF to KDR/Flk-1, this interaction model may not be generalized to other ligands such as VEGF-E. Since VEGF-E can bind KDR/ Flk-1 at almost the same affinity as VEGF 165 without the FIG. 7. Stimulation of the autophosphorylation of KDR/Flk-1 but not Flt-1 by VEGF-E. A, NIH3T3-KDR cells were starved in 0.5% serum overnight, then stimulated with 30 ng/ml VEGF 165 or VEGF-E for 5 min. The cell lysates were immunoprecipitated with anti-human KDR/Flk-1 antiserum, and immunoblotted with anti-phosphotyrosine antibody (lanes 1-3), or with anti-KDR antiserum (lanes 4 -6). B, NIH3T3-Flt-1 cells were starved in 0.1% serum overnight, then stimulated with 30 ng/ml VEGF 165 or VEGF-E for 5 min. The cell lysates were immunoprecipitated with anti-Flt-1, and immunoblotted with antiphosphotyrosine antibody (lanes 1-3), or with anti-Flt-1 antiserum (lanes 4 -6). C, signal transduction of VEGF-E. Rat liver sinusoidal endothelial cells cultured with VEGF 165 were starved in VEGF-free medium overnight, then stimulated with 30 ng/ml VEGF-E. The cell lysates were analyzed with anti-KDR, anti-PLC␥, or antiactive (phosphorylated) mitogen-activated protein kinase antiserum as indicated.
82-86 basic stretch, a different binding motif with rather hydrophobic residues may exist on VEGF-E. Furthermore, since VEGF-E induces a strong activation of KDR/Flk-1, the interaction of this putative motif on VEGF-E with KDR/Flk-1 is thought to be sufficient for inducing dimerization of the receptor. It seems important to elucidate the motif critical for interaction between KDR/Flk-1 and this new type of ligand, VEGF-E.
Heparin is known to enhance the biological activity of VEGF at lower concentrations, but is inhibitory at higher concentrations (14). Heparin interacts not only with the basic region of VEGF 165 but also with two VEGF receptors, although its interaction with KDR/Flk-1 is not very strong. Therefore, the effects of heparin or heparan sulfate-containing molecules on the endothelial cell surface appear complicated and might differ with biological conditions. However, that VEGF 165 has 10 -100-fold more biological activity than VEGF 121 clearly indicates that the interaction of VEGF 165 with heparin-like molecules through the basic stretch or the existence of this basic stretch itself is important for a higher affinity binding of VEGF 165 to KDR/Flk-1 and for the generation of strong cytoplasmic signals. Recently, a 130-kDa membrane-associated molecule named neuropilin-1 was reported to bind the basic stretch on VEGF 165 (20,40). This molecule might also be involved in VEGF-KDR/Flk-1 signaling. Interestingly, however, VEGF-E does not carry the basic stretch corresponding to exon 7 on VEGF 165 nor show any affinity to heparin column at detectable levels. Furthermore, in a cross-linking study using 125 I-VEGF 165 and HUVEC, VEGF-E strongly competed the binding of VEGF 165 with KDR but not the binding of VEGF 165 with 130-kDa molecule (data not shown). Therefore, the interaction of VEGF-E with heparin-like molecules or with the 130-kDa molecule may not be necessary for generation of a strong mitotic and permeability signal via KDR/Flk-1. A possible explanation for the strong biological activity of VEGF-E is that high affinity of a ligand is sufficient for a strong activation of KDR/Flk-1 and that the basic stretch of VEGF 165 is simply functional for the increase in the affinity of VEGF 165 for KDR/ Flk-1. In addition, the diffuse margin of the blue spots generated with VEGF-E might be due to no affinity of VEGF-E to Flt-1 or neuropilin-1. Further studies are required to understand the molecular mechanism of VEGF-E activity and the function of exon 7-derived basic stretch on VEGF 165 . VEGF binds both KDR/Flk-1 and Flt-1, whereas VEGF-E binds and stimulates only KDR/Flk-1. Flt-1 as well as KDR/ Flk-1 is expressed at higher levels in vascular endothelial cells during embryogenesis and most of the pathological angiogenesis. Flt-1 carries about a 10-fold higher binding affinity for VEGF but much weaker tyrosine kinase activity than KDR/ Flk-1. Furthermore, Flt-1 knockout mice studies have shown that flt-1(Ϫ/Ϫ) homozygous mice are embyonic lethal at embryonic day 8.5-9.0 due to an overgrowth of endothelial-like cells and disorganization of a variety of blood vessels (41). These results suggest that Flt-1 functions as a negative regulator of VEGF signaling at least in embryogenesis. Since VEGF utilizes both receptors, VEGF might control a balance between positive and negative signals (42). On the other hand, VEGF-E utilizes only KDR/Flk-1 which is a positive regulator of endothelial proliferation. This feature of VEGF-E might be sufficient to generate pathological angiogenesis as shown in Fig. 9, but not be appropriate for regulating the fine architecture of the physiological blood vessel network in embryogenesis.
An interesting possibility is that the cellular VEGF-E gene exists in the vertebrate genome similar to the case of parapoxvirus OV IL-10 gene (43), and is utilized at certain periods or in a specific tissue in vivo at the embryonic or postnatal/adult stage. The KDR/flk-1 gene was shown to be essential for the establishment of the blood vessel network and for hematopoiesis at early embryogenesis by gene targeting (44). Thus, VEGF-E gene may be a candidate gene for regulating a part of these processes in cooperation with the classical VEGF.