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Antagonistic Effect of NK4, a Novel Hepatocyte Growth Factor Variant, on in Vitro Angiogenesis of Human Vascular Endothelial Cells¹

Metastasis Research Group, University Department of Surgery, University of Wales College of Medicine, Cardiff CF14 4XN, United Kingdom [W. G. J., S. E. H., C. P., T. A. M., R. E. M.], and Biomedical Research Centre, Osaka University Medical School, Suita, Osaka 565, Japan [K. M., T. N.]

	ABSTRACT

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Hepatocyte growth factor (HGF), also known as scatter factor (SF), is known to act on cancer cells as well as endothelial cells and stimulate angiogenesis, thus playing an unwanted role in the development and progression of cancer. The current study examined the effects of a newly discovered HGF variant, NK4, on angiogenesis in vitro. Chemically generated NK4 (from recombinant human HGF/SF) was found to be able to inhibit HGF-induced activation (tyrosine phosphorylation) of the HGF/SF receptor cMET but was itself unable to activate cMET. Furthermore, NK4 was demonstrated to inhibit tubule formation from human umbilical vein endothelial cells that was induced by both HGF/SF and a HGF/SF-producing fibroblast (MRC5). Under the same settings, NK4 failed to increase tubular formation. NK4 had no effects on interleukin 8- and vascular endothelial growth factor-induced tubule formation. Using computer-assisted motion analysis, it was further shown that NK4 inhibited HGF-induced migration of human umbilical vein endothelial cells in a migration assay and in an endothelial wounding assay. These data show that NK4 is a complete antagonist to HGF. It inhibits HGF-induced endothelial movement and tubule formation. Thus, NK4 may have an important bearing on the control of cancer progression through its role in angiogenesis. Additional in vivo studies are warranted.

	INTRODUCTION

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HGF,³ which is also known as SF (HGF/SF; 1, 2), has been demonstrated over the past decade as a cytokine playing multiple and diverse roles in cancer. The factor is mainly produced by fibroblasts in vivo and acts via its specific receptor, the cMET proto-oncogene.

HGF/SF is a known angiogenic factor that stimulates angiogenesis both in vitro and in vivo (3, 4, 5, 6) . The effects of HGF/SF on angiogenesis are achieved by increasing the motility, migration, and proliferation and enhancing the morphogenesis of vascular endothelial cells. This function of HGF/SF is particularly important in cancer. Many solid tumors overexpress HGF/SF receptor, and some tumor cells also express and secrete bioactive HGF/SF. Furthermore, stromal fibroblasts in tumor tissues also overproduce HGF/SF. Thus, both tumor cells and stromal cells contribute to increased levels of HGF/SF in tumor-bearing hosts. HGF/SF acts directly on cancer cells by increasing their migration, invasion, and adhesion to the matrix. Suppression of apoptosis also occurs (for a recent review, see Ref. 7 ). Given the key role of angiogenesis in the development and progression of cancer, HGF/SF has been strongly implicated as a key player in cancer progression. Indeed, HGF/SF and its receptor have been demonstrated to be important prognostic factors in patients with cancer (8, 9, 10, 11) . A few agents have been demonstrated to have an inhibitory role on HGF/SF in cancer, such as retinoic acid and -linolenic acid, IL-4, and invasion-inhibiting factor 2 (12, 13, 14, 15, 16, 17) .

Mature HGF/SF is a heterodimeric protein that consists of a M_r 69,000 chain and a M_r 34,000 chain (1) . The chain is of particular interest, because it possesses a receptor binding domain and four kringle domains. The chain is therefore the key subunit for the biological function of HGF/SF. The chain of HGF/SF has no receptor binding domain and does not elicit any biological activity on its own (1) . Alternative spliced variants of the chain that contain different parts of the subunit have been discovered. The variant that contains the hairpin and the first kringle domain (K1) is known as HGF/NK1 and is naturally occurring. The one that contains the hairpin and the first and the second kringle domains (K2) is known as HGF/NK2. Both variants are able to bind HGF/SF receptor and have partial agonist and antagonist activity (18, 19, 20, 21, 22, 23) . For example, NK2 has no mitogenic effect on hepatocytes but retains its ability to induce cellular motility. Both variants have very limited therapeutic value in term of cancer intervention, due to their induction of cancer cellular motility.

Recently, another variant called NK4 has been obtained, which contains the hairpin and all four kringle domains (24) . NK4 exhibits a completely different biological spectrum than NK1 and NK2. NK4 is generated through enzymatic cleavage of mature human HGF/SF, whose receptor binding hairpin domain and four kringle domains have been retained, but the chain and amino acids responsible for dimerization on the COOH terminus of the chain have been removed. The new variant is a protein of M_r 50,000. Thus, NK4 has retained a receptor binding domain and is able to specifically bind to HGF/SF receptor. Furthermore, NK4 is unable to activate the HGF/SF receptor but is able to compete with HGF/SF in the receptor binding, thus deactivating HGF/SF-induced receptor activation in MDCK cells. Using hepatocytes and MDCK cells, NK4 has been found to have no mitogenic effect and abolishes the mitogenic effect induced by HGF/SF in hepatocytes. It has no motogenic effect and abolishes the motogenic effects of HGF/SF in MDCK cells (24 , 25) .

This study examined the effect of NK4 on endothelial cells and HGF/SF-induced tubule formation with human vascular endothelial cells. Here we report that NK4 had no agonistic effect on human vascular endothelial cells at concentrations of up to 500 ng/ml. Furthermore, NK4 was able to suppress HGF/SF-induced activation of its receptor. Consequently, NK4 inhibited HGF/SF tubule formation and endothelial cell migration.

	MATERIALS AND METHODS

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Cell Culture
HUVECs were obtained from umbilical veins using a method described previously (26) . Briefly, fresh umbilical cords were obtained immediately after birth. After washing debris with a balanced salt solution (BSS) buffer, the vein was irrigated with a buffer containing 0.1% collagenase (Clostridium histoliyticum; Worthington Biochemical Corp., Freehold, NJ) for 20 min at 37°C. Cells were then washed out and washed with PBS. They were cultured in Medium 199 (Sigma, Poole, United Kingdom) with 20% FCS, 50 µg/ml endothelial cell growth supplement (ECG; First Link Ltd., Milton Keynes, United Kingdom), 50 units/ml penicillin, and 50 units/ml streptomycin. After a 6–10-day culture at 37°C in 5% CO₂, these primary cultured cells were maintained in Medium 199 supplemented with 10% FCS and were always used within three passages. A human vascular endothelial cell line, ECV304, and MDCK cells were obtained from the European Collection of Animal Cell Culture (Porton Down, United Kingdom). Cells were routinely cultured with Medium 199 supplemented with 10% FCS. Human fibroblast MRC5 cells (known to produce a large quantity of bioactive HGF) were purchased from the European Collection of Animal Cell Culture.

Matrigel (reconstituted basement membrane) was purchased from Collaborative Research Products (Bedford, MA). A transwell plate equipped with a porous insert (pore size, 0.45 µm) was obtained from Becton Dickinson Labware (Oxford, United Kingdom) and used for the coculture study. Goat antihuman HGF/SF, antihuman cMET antibodies, and a mouse anti-phosphotyrosine antibody (PY99) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). rhHGF/SF was prepared from human HGF cDNA-transfected Chinese hamster ovary cells, as reported previously (15) . Human recombinant IL-8 and VEGF were from the National Biological Standard Bureau (NBSB) (Hertfordshire, United Kingdom) and Sigma, respectively. Neutralizing antibody to human IL-8 was from Sigma, anti-HGF/SF and VEGF were from R&D Systems (Abingdon, United Kingdom). Peroxidase-conjugated goat antimouse IgG for Western blotting was from Santa Cruz Biotechnology. Protein A/G-agarose was from Santa Cruz Biotechnology. All other materials were purchased from Sigma-Aldrich Co., Ltd. (Poole, United Kingdom) unless otherwise stated.

Preparation of NK4 from rhHGF
The preparation of NK4 from rhHGF was based on that reported previously (24) . Briefly, purified rhHGF/SF was first digested with pancreatic elastase and then applied to a µBondapak C4 reverse phase high-performance liquid chromatography column. NK4 was obtained and purified after elution with a gradient of acetonitrile containing 0.05% trifluoroacetic acid. NK4 obtained in this fashion retains the NH₂-terminal hairpin and four kringle domains of chain HGF but has 16 amino acids deleted from the COOH terminus of the chain (4) . NK4 retained the capacity to bind to HGF/SF receptor without activating it.

Tubule Formation
Tubule formation assays of endothelial cells were performed as described previously (27 , 28) . Twenty-four multiwell plates were first coated with Matrigel (200 µg/well) and allowed to form a thin gel layer. HUVECs (5 x 10⁴) in 0.5 ml of DMEM with 10% FCS were then added over Matrigel for 24 h. The medium was aspirated, and an additional 0.5 ml of Matrigel was overlaid with an additional 0.5 ml of medium that contained either medium, HGF, NK4, or NK4 and HGF in combination. Cell cultures were observed under a phase-contrast microscope after 24 h. Each well was photographed four times at random, and tubule length was measured using image analysis software (Optimas 6; Optimas UK).

Coculturing Technique
This was based on a method described previously (29) . Transwell chambers (Costar, Cambridge, MA) equipped with a 6.5-mm-diameter polycarbonate membrane (pore size, 0.45 µm) were used. MRC5 cells were plated in the top chamber, and HUVECs (prepared in between Matrigel layers) were placed in the lower chamber. This system allowed cells to be physically separated from each other but allowed molecules to communicate freely. Tubule length was similarly measured after 24 h. Supernatant was also collected for HGF analysis.

Cell Motion Analysis
The migration of endothelial cells was carried out using a motion analysis package. Two separate methods were used.

Migration of Individual Endothelial Cells.
Endothelial cells were seeded at 5000 cells/well in a 96-well plate. After being firmly stuck to the surface, they were washed, and HEPES-buffered DMEM (10% FCS) was added. The cells in culture medium were then placed on a heated stage (37°C) and then overlaid with light mineral oil to prevent evaporation. Medium or medium containing HGF, NK4, or NK4 and HGF in combination was applied to the cells. Cells were then immediately placed under a Hoffmann inverted microscope equipped with a stage heater to allow a constant temperature of 37°C (Leica, Cambridge, United Kingdom). Images were recorded using a Panasonic Digital camera and a time lapse recorder. Cells were identified using motion analysis function (Optimas 6). The distance the cells traveled was calculated and given in µm. A total of 6–10 cells in each frame were analyzed.

Endothelial Wounding Assay.
Endothelial cells were seeded in a glass chamber slide to reach confluence. They were then similarly prepared with HEPES buffer and light oil. The monolayer was wounded with an 82-gauge sterile needle to produce a wound of approximately 200 µm in width in the presence of either medium, HGF, NK4, or NK4 and HGF in combination. Images were similarly recorded, and the migration distance of the wound front was analyzed using the motion analysis package. Over 10 points were measured per frame. The distance that the cells had migrated from the initial wound edge was given in µm. Data were analyzed with Exel software, and statistical analyses were performed using the Mann-Whitney U test.

	Immunoprecipitation and Western Blotting

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Cells were pelleted and lysed in HCMF buffer containing 1% Triton X-100, 0.1% SDS, 2 mM CaCl₂, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin for 30 min. They were then boiled at 100°C for 5 min before clarification at 13,000 x g for 10 min. Protein concentrations were measured using fluorescamine and quantified by using a multifluoroscanner (Denly, Sussex, United Kingdom). Equal amounts of protein from each cell sample (30 µg/lane; controls and treated cells) were added to an 8% polyacrylamide gel. After electrophoresis, proteins were blotted on to nitrocellulose sheets and blocked in 10% skimmed milk for 60 min before probing with the HGF/SF or cMET antibody (1:800 and 1:1,000, respectively) and a peroxidase-conjugated secondary antibody (1:2,000). A molecular weight marker mixture (SDS-6H; Sigma) was used to determine the protein size. Protein bands were visualized with an enhanced chemiluminescence system (Amersham). Protein band densities were measured with a laser densitometer, and band volumes were analyzed with Molecular Analyst (Bio-Rad).

In the case of immunoprecipitation, cells were treated with HGF/SF for a given period of time and then lysed in the same lysis buffer as described above. Cells treated with sodium orthorvanadate were used as positive control. After the removal of cell debris by centrifugation at 13,000 x g for 10 min, anti-cMET antibody was added to each sample, each of which had the same quantity of proteins. Protein A/G-agarose was added after 1 h and incubated at 4°C for overnight. The precipitates were washed three times and lysed in the sample buffer. After Western blotting, the membrane was probed with anti-phosphotyrosine antibody or anti-cMET antibody.

	RT-PCR

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Cellular mRNA was extracted using a mRNA extraction kit (Sigma). cDNA was synthesized using a first-strand synthesis kit from Promega. PCR primers used were as follows (30) : (a) for HGF, 5'-ATAAAGGAAAGTTGGGTTCTT-3' and 5'-TCCACGGCCGGGAACAAT; and (b) for cMET, 5'-GTCCAGGCAGTGCAGCATGTA-3' and 5'-ACTATAGTATTCTTTATCATACATGTC-3'. PCR was performed using these primers under the following conditions using a Perkin-Elmer thermocycler and Pharmacia PCR beads: (a) 5 min at 95°C; (b) 30 s at 94°C, 60 s at 64°C (60°C for cMET), and 60 s at 72°C for 40 cycles; and (c) 72°C for 7 min. PCR products were then separated on a 0.8% agarose gel and visualized under UV light.

	HGF/SF ELISA and Bioassay

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The colony scattering assay was based on a method established previously (1 , 31) . Briefly, MDCK cells were seeded to a 96-well plate at 4000 cells/well. Serially diluted supernatant from MRC5 cells at the end of coculture or rhHGF/SF (as an internal standard) was added. Anti-HGF/SF was included in selected wells. After 24 h, cells were fixed and stained with crystal violet, and scattering was assessed. The quantity of HGF/SF was given in units/ml, in which a unit was defined as the highest dilution of supernatant that caused scattering. In this study, 1 unit/ml was equal to 0.25 ng/ml rhHGF/SF. An ELISA kit for human HGF/SF was obtained from Immunology Institute (Osaka, Japan), and the assay was carried out according to the manufacturer’s instructions, with rhHGF/SF as the internal standard. The detection limit of the ELISA was 0.5 ng/ml.

Statistical analysis was performed using Minitab software and the Mann-Whitney U test.

	RESULTS

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HGF Increased Tubule Formation of Endothelial Cells but Was Inhibited by NK4.
HGF/SF increased tubule formation in a concentration-dependent manner (Fig. 1) . NK4 was then included in the system over a range of concentrations (1–500 ng/ml; Fig. 2 ). The effect of HGF/SF at 5 and 10 ng/ml was significantly reduced by NK4 at concentrations over 50 ng/ml, and the effect of HGF/SF at 50 ng/ml was reduced by NK4 at concentrations over 100 ng/ml. The inhibition was significant but only partial because NK4 was unable to reduce the tubule length to control levels. When HGF/SF was used at 100 ng/ml, a small but significant degree of inhibition was seen at 500 ng/ml NK4. This has given a concentration ratio of NK4:HGF of approximately 2–5:1 in which significant inhibition of tubule formation was achieved. NK4 alone had no effect on the tubule formation.

View larger version (16K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Induction of tubule formation by HGF/SF from the vascular endothelial cells. HUVECs were embedded in Matrigel and treated with HGF/SF at different concentrations for 24 h. The length of the tubule formed was analyzed using digitized images and is measured in µm/frame.

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. NK4 suppressed tubule formation induced by HGF. Endothelial cells were treated with NK4 alone or in combination with HGF/SF at 1 ng/ml (A), 5 ng/ml (B), 10 ng/ml (C), 50 ng/ml (D), or 100 ng/ml (E). A partial inhibition of tubule formation was seen. NK4 alone had no effect on the tubule formation. *, P < 0.05 when comparing cells treated with HGF/SF and cells treated with a combination of HGF/SF and NK4 by Mann-Whitney U test.

View larger version (43K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. The effects of NK4 on human fibroblast (MRC5)-induced tubule formation. Matrigel-embedded endothelial cells were cocultured with MRC5 at a ratio of 1:5 (endothelial cell:MRC5) in the presence or absence of NK4. MRC5 cells significantly increased tubule formation, an effect that was significantly reduced by NK4 at concentrations higher than 50 ng/ml. HGF/SF concentration and activity from MRC5 cells are given in "Results." *, P < 0.05 when comparing HUVECs treated with MRC5 and control HUVEC cells; **, P < 0.05 when comparing HUVECs treated with MRC5 and HUVECs treated with a combination of MRC5 and NK4.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. tection of HGF/SF receptor (cMET; A) and HGF/SF (B) mRNA by RT-PCR (top) and protein by Western blotting (bottom) in MRC5 and endothelial cells. HGF/SF mRNA and protein were detected in MRC5 cells, but not in endothelial cells (B). HGF/SF receptor was detected in both endothelial cells and MRC5 cells (A).

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. GF/SF and NK4 and endothelial cell migration using motion analysis. Endothelial cells placed on a plastic surface were treated with either medium as a control, NK4 alone, HGF/SF, or a combination of NK4 and HGF/SF. The migration of cells was recorded using a time-lapse recorder on a Hoffmann microscope. The movement of the cells was analyzed using a motion analysis package, and the distance the cell moved is given here in µm. HGF/SF was seen to increase the migration after 60 min, but this effect was reduced by NK4. NK4 did not exhibit an effect on migration. *, P < 0.05 when comparing cells treated with HGF/SF and cells treated with a combination of HGF/SF and NK4.

View larger version (25K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Effects of NK4 on the movement of endothelial cells using a wounding assay. Fully confluent endothelial cell monolayer was wounded using a sharp needle in the presence of either medium as a control, NK4 alone, HGF/SF, or a combination of NK4 and HGF/SF. The migration of the leading fronts was recorded and similarly analyzed using a motion analysis package. HGF/SF significantly increased the migration of the leading fronts and was significantly reduced by NK4. No effect was seen with NK4 alone. *, P < 0.05 when comparing cells treated with HGF/SF and cells treated with a combination of HGF/SF and NK4.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. NK4 had no effects on IL-8 and VEGF-induced tubule formation. Recombinant HGF/SF, IL-8, and VEGF were used at a final concentration of 10 ng/ml. NK4 was used at a concentration of 50 ng/ml. All of the antibodies were used at a concentration of 100 ng/ml. All three cytokines increased tubule formation and were blocked by their specific antibodies. However, NK4 had no effect on IL-8- and VEGF-induced tubule formation. *, P < 0.05 versus control; **, P < 0.05 versus treatment with cytokine alone by the Mann-Whitney U test.

View larger version (38K): [in this window] [in a new window] [Download PPT slide]   Fig. 8. NK4 suppressed HGF/SF-induced cMET activation. Endothelial cells were treated with medium (Lane A, control), 10 ng/ml HGF/SF alone (Lane B), 10 ng/ml HGF/SF in combination with NK4 (Lane C, 10 ng/ml NK4; Lane D, 100 ng/ml NK4; Lane E, 500 ng/ml NK4), or NK4 alone (Lane F, 10 ng/ml NK4; Lane G, 100 ng/ml NK4; Lane H, 500 ng/ml NK4) for 10 min. The cells were then lysed, and cMET was precipitated with anti-cMET antibody. Precipitates were separated on an 8% SDS-PAGE gel and probed with PY20 anti-phosphotyrosine (top) or anti-cMET antibody (middle). The density of the phosphorylated protein bands (from the top) is given in the bar graph (bottom). Sodium orthovanadate was used as a positive control (LaneI). NK4 suppressed cMET activation by HGF/SF, but NK4 alone had no effect on the receptor.

	DISCUSSION

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Here we provide in vitro evidence that a HGF/SF variant, NK4, exerted an inhibitory effect on HGF/SF-induced angiogenesis. Firstly, HGF/SF, as reported previously, was able to increase tubule formation from HUVECs. We further demonstrated that the inclusion of NK4 significantly suppressed the formation of tubules from the HUVECs. In the current study, the concentration ratio of NK4:HGF/SF required to suppress the action of HGF/SF began at 2:1 and reached approximately a 50% inhibition at 5:1. This inhibitory effect of NK4 may be the consequence of suppression of HGF/SF-induced endothelial migration, as indicated by the motion analysis and wounding assay in this study.

The other key feature of NK4 was its complete lack of agonist activity on endothelial cells. Our study on tubule formation and migration assays clearly demonstrated that NK4, at concentrations of up to 500 ng/ml, did not induce any detectable change in endothelial cells, whereas HGF/SF exerted effects at a concentration as low as 1 ng/ml. Furthermore, NK4 at these concentrations had no effect on the phosphorylation of the HGF/SF receptor and antagonized HGF/SF-induced activation. The specificity of NK4 on the action of HGF/SF has been further demonstrated by the fact that NK4 has no inhibitory effects on IL-8- and VEGF-induced tubule formation (Fig. 7) .

Thus, NK4 exerted a different profile of functions on endothelial cells when compared with previously reported effects of other HGF/SF variants such as NK1 and NK2, which exert dual action (partial agonistic and partial antagonistic effects on target cells; Refs. 18, 19, 20, 21, 22, 23) . This suggests that NK4 holds promise as an antiangiogenesis agent. Furthermore, recent work on NK4 and cancer cells showed that NK4 inhibited HGF/SF-induced motility and invasion of bladder cancer cells (25) without inducing the activation of HGF/SF receptor on these cells. The promising nature of the effect of NK4 on angiogenesis and cancer cell motility should now be tested under in vivo conditions. We are currently carrying out animal studies to test this theme.

A few agents have been demonstrated to have an opposite or inhibitory effect on the activity of HGF/SF, such as certain cytokines, retinoids, polyunsaturated fatty acids, and invasion-inhibiting factor 2 (12, 13, 14, 15, 16, 17) . However, the exact mechanism by which these agents act on HGF/SF is less clear, although the effect on cell adhesion molecules such as E-cadherin complex, suppression of cell motility, and intracellular signaling have been demonstrated (33 , 34) . HGF/SF enhances angiogenesis via several mechanisms, including migration/motility, adhesion to the matrix, intra- and intercellular communications, and morphogenesis of endothelial cell tubules (3, 4, 5 , 35, 36, 37) . All of these effects are specifically elicited by the HGF/SF receptor. The effect of NK4 in blocking receptor activation is central to all of these activities of HGF/SF, thus presenting a new class of specific HGF/SF antagonist.

The fundamental importance of angiogenesis in cancer development and metastasis has prompted the discovery of a large number of angiogenesis inhibitors, including those agents specifically designed as antiangiogenesis agents (such as anti-VEGF antibody, anti-basic fibroblast growth factor antibody, and fumagillin) and those discovered unintentionally (such as IFN-, tamoxifen, and IL-4 and IL-12; Refs. 37, 38, 39) . NK4 may belong to the former group by virtue of being a specific antagonist to a known angiogenic factor, i.e., HGF/SF. Given the strong effects of HGF/SF on cancer cells and endothelial cells, it is anticipated that NK4 may be very useful in treating the following types of tumors: (a) tumors that have a high stromal cell content (because these stromal cells often produce bioactive HGF/SF); (b) tumors that overexpress the HGF/SF receptor c-MET; (c) tumors that express and produce bioactive HGF/SF; and (d) tumors that are rich in angiogenesis.

In summary, this study demonstrated that a new HGF/SF variant, NK4, was a potent inhibitor of HGF/SF-induced angiogenesis in vitro. The inhibitory effect was achieved by blocking phosphorylation of the HGF/SF receptor. The data presented here may bear implications for future in vivo and clinical testing.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by the World Cancer Research Fund.

² To whom requests for reprints should be addressed, at Metastasis Research Group, University Department of Surgery, University of Wales College of Medicine, Cardiff CF14 4XN, United Kingdom. Phone: 44-29-2074-2895; Fax: 44-29-2076-1623; E-mail: jiangw{at}cf.ac.uk

³ The abbreviations used are: HGF, hepatocyte growth factor; SF, scatter factor; rhHGF/SF, recombinant human HGF/SF; HUVEC, human umbilical vein endothelial cell; IL, interleukin; VEGF, vascular endothelial growth factor; MDCK, Madin-Darby canine kidney; RT-PCR, reverse transcription-PCR.

Received 5/28/99; revised 8/24/99; accepted 8/27/99.

	REFERENCES

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