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[Cancer Research 64, 2062-2069, March 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Evidence of Antiangiogenic and Antimetastatic Activities of the Recombinant Disintegrin Domain of Metargidin

Véronique Trochon-Joseph1,2, Dominique Martel-Renoir3, Lluis M. Mir3, Annick Thomaïdis1, Paule Opolon3, Elisabeth Connault3, Hong Li3,4, Christophe Grenet1, Françoise Fauvel-Lafève1, Jeannette Soria5, Chantal Legrand1, Claudine Soria1,4, Michel Perricaudet3 and He Lu1

1 Inserm U553, Hôpital Saint-Louis, Paris; 2 Bioalliance Pharma SA, Paris; 3 Unité Mixte de Recherche 8121, Centre National de la Recherche Scientifique, Institut Gustave-Roussy, Villejuif; 4 DIFEMA, Faculté de Médecine et Pharmacie de Rouen, Rouen; and 5 Laboratoires Sainte-Marie and EMI 353 INSERM, Hôtel-Dieu, Paris, France


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metargidin, a transmembrane protein of the adamalysin family, and integrins, e.g., {alpha}5ß1 and {alpha}v, are preferentially expressed on endothelial cells on angiogenesis. Furthermore, metargidin interacts with these integrins via its disintegrin domain. In this study, recombinant human disintegrin domain (RDD) was produced in Escherichia coli by subcloning its cDNA into the pGEX-2T vector, and the effect of purified RDD on different steps of angiogenesis was evaluated. At concentrations of 2–10 µg/ml, RDD exhibited inhibitory activities in a variety of in vitro functional assays, including endothelial cell proliferation and adhesion on the integrin substrates fibronectin, vitronectin, and fibrinogen. RDD (10 µg/ml) totally abrogated endothelial cell migration and blocked most capillary formation in a three-dimensional fibrin gel. To test RDD efficacy in vivo, the RDD gene inserted into pBi vector containing a tetracycline-inducible promoter was electrotransferred into nude mouse muscle. RDD was successfully synthesized by muscle cells in vivo as shown by immunolabeling and Western blotting. In addition, 78% less MDA-MB-231 tumor growth, associated with strong inhibition of tumor angiogenesis, was observed in athymic mice bearing electrotransferred RDD. Moreover, in the presence of RDD, 74% fewer B16F10 melanoma lung metastases were found in C57BL/6 mice. Taken together, these results identified this RDD as a potent intrinsic inhibitor of angiogenesis, tumor growth, and metastasis, making it a promising tool for use in anticancer treatment.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Angiogenesis, the development of new capillaries from preexisting blood vessels, is essential for the growth and progression of primary solid tumors. A strategy aimed at inhibiting such neovascularization within tumors has thus been proposed (1) . To date, various proteolytic fragments of extracellular matrix (ECM) components, e.g., angiostatin, endostatin, canstatin, and tumstatin, have been found to inhibit tumor angiogenesis and subsequent tumor growth (2, 3, 4, 5, 6, 7) . Antiangiogenic therapy presents at least two obvious advantages to contain cancer: (a), the vast variety of cancer phenotypes and genotypes becomes unimportant because angiogenesis is a phenomenon common to all malignancies; and (b), restriction and regression of tumor neovascularization should prevent the passage of tumor cells into the circulation and, therefore, lower the metastatic risk.

The family of adamalysin proteins, also referred to as A disintegrin and metalloproteinase proteins (ADAMs) or metalloproteinase-disintegrin cysteine-rich proteins (MDCs), has a particular primary structure containing disintegrin domain located on the COOH-terminal side of metalloproteinase domain. The disintegrin region contains an integrin-binding sequence in a disintegrin loop that interacts with integrins and may mediate cell–cell interactions (8) . All family members potentially possess cell-adhesion and protease activities; however, only half of them have been reported to have an active metalloproteinase site to mediate proteolytic cleavage of ECM components (9) . Adamalysins are implicated in diverse biological processes, such as fertilization, myogenesis, neurogenesis, and cytokine release (10) .

Metargidin (metalloprotease-RGD-disintegrin protein), also called human MDC-15 or ADAM-15, is a transmembrane adamalysin expressed by smooth muscle cells, mesangial cells, and at a much higher level, activated endothelial cells. To date, its function has remained poorly understood (11, 12, 13) . However, recently reported data implicated metargidin in mesangial cell migration associated with the gelatinase activity of its metalloproteinase domain (13) . Metargidin has also been shown to colocalize with a cell-adhesion molecule, vascular endothelial cadherin, which mediates endothelial cell adherent junction formation (14) . Furthermore, metargidin is the only known adamalysin that possesses a RGD motif at the tip of its disintegrin loop (CRPTRGDCD), which binds integrins {alpha}vß3 and {alpha}5ß1 (11 , 15 , 16) . More recently, mouse ADAM-15, a homolog of human metargidin, has been shown to bind integrin {alpha}9ß1 in an RGD-independent manner (17) . Integrins are heterodimeric transmembrane cell-surface molecules that mediate adhesion to ECM and cell–cell interactions (18) . In particular, {alpha}v integrins are involved in angiogenic processes; monoclonal antibodies, e.g., vitaxin, and cyclic RGD peptides targeting these integrins inhibited angiogenesis, leading to tumor regression (18, 19, 20, 21, 22) . However, the mechanisms of how {alpha}v integrins modulate angiogenesis remain unclear (23, 24, 25, 26, 27, 28, 29, 30) because the deletion of ß3, ß5, or {alpha}v integrins in knockout mice failed to block angiogenesis but rather showed extensive angiogenesis (27 , 28) .

In a previous study using a synthetic adamalysin inhibitor (GL129471) to block the metalloproteinase domain, we provided evidence implicating adamalysin in angiogenesis in vitro (31) . Because angiogenesis is thought to require the interaction between metargidin and integrin, we postulated that the disintegrin domain of metargidin, expressed as soluble recombinant protein, might prevent this interaction. To test this hypothesis, we produced this recombinant disintegrin domain (RDD) and evaluated its effect on angiogenesis in vitro. Moreover, the potential therapeutic efficacy of RDD against tumor angiogenesis, growth, and metastasis was evaluated after plasmid DNA electrotransfer using a previously described tetracycline-inducible system (32) in two experimental animal models: human MDA-MB-231 tumors grafted into nude mice, and the dissemination of B16F10 melanoma cells to the lungs of syngeneic mice.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
A peptide (KGWQCRPTRGDC) from the disintegrin domain of human metargidin (MDC-15) was synthesized by Neosystem (Strasbourg, France) and used to immunize rabbits as described by Herren et al. (12) . The resulting antiserum was used to detect purified Escherichia coli-produced RDD by Western blotting.

RDD Production in E. coli.
Glutathione S-transferase (GST)–RDD fusion protein was prepared by inserting cDNA that encodes the entire disintegrin domain of metargidin (MAAFCGNMFVEPGEQCDCGFLDDCVDPCCDSLTCQLRPGAQCASDGPCCQNCQLRPSGWQCRPTRGDCDLPEFCPGDSSQCPPDVSLGDGE) into the BamHI site of the pGEX-2T vector, yielding pGEX-2T-RDD, a generous gift from Y. Takada (The Scripps Research Institute, La Jolla, CA). This fusion protein has a thrombin cleavage site between GST and RDD. E. coli DH5{alpha} synthesis of GST–RDD was induced by incubating the bacteria in 1 mM isopropyl-1-thio-ß-D-galactopyranoside (Life Technologies, Inc., Paisley, United Kingdom) for 4 h. After addition of 1% Triton X-100, the bacterial suspension was sonicated, and the fusion protein was extracted. The fusion protein was purified by glutathione-agarose (Sigma-Aldrich, Saint Quentin Fallavier, France) affinity chromatography and eluted by competition with free glutathione (Sigma-Aldrich), as described by Smith and Johnson (33) . RDD was cleaved from the fusion protein by use of bovine thrombin (Amersham Pharmacia; 1 unit/100 µg of fusion protein) for 16 h at room temperature. The GST fragment was then removed by glutathione-agarose affinity chromatography, and the final product was dialyzed against PBS for 3 h. Purified RDD (20 µl) was analyzed by use of a fast-performance liquid chromatography system (P-500 pump; Amersham Biosciences Europe, Orsay, France) with a Superdex 75 High Resolution 10/30 column (Amersham) connected to a Beckman Gold 168 spectrophotometer (detection at 230 nm; Beckman Instruments, Fullerton, CA) with PBS as the elution buffer at a flow rate of 0.5 ml/min and pressure of 0.7 MPa. The protein concentration was determined with the BCA assay (Pierce; Rockford, IL). Vehicle-control cultures (without RDD) for in vitro experiments contained a final bovine thrombin concentration never exceeding 1.6 units/ml (used for the highest RDD dose).

Plasmid Preparation for Electric Pulse Delivery to Muscle.
The cDNA containing the disintegrin domain sequence from pGEX-2T–RDD was amplified by PCR with the forward primer 5'-ATC CGA GCT CTT ATG GCT GCT TTC TGC G-3' and the reverse primer 5'-GCA TGC GGA TCC TTA CTC GCC A-3' and subcloned into SacI–BamHI sites of the p1519 vector, which harbors the sequence of mouse urokinase signal peptide (34) . The EcoRV–BamHI restriction fragment encoding this signal peptide-fused RDD sequence was then subcloned into EcoRV–BglII sites of the pCO5 vector (Clontech, Palo Alto, CA). The NaeI–EcoRV restriction fragment was finally subcloned into the EcoRV site of the pBi vector (Clontech). This vector (pBi–RDD) carries the gene of interest from a tetracycline-responsive promoter in the Tet-On eukaryotic gene-expression system. The cDNA fragment of RDD fused to the urokinase signal peptide, with Flag-tag (DYKDDDDK) placed at the COOH terminus, was amplified by PCR from pBi–RDD by use of the forward primer 5'-AAT ACT AGC TAG CAT GAA AGT CTG GCT G-3' and the reverse primer 5'-TTG ATA TCT CACTTG TCA TCG TCG TCC TTG TAG TCC TCG CCA TCC CCT AG-3'. The NheI–EcoRV restriction fragment was then subcloned into NheI/EcoRV sites of pBi to generate pBi–RDD–Flag. The tag enables detection of muscle cell-synthesized RDD by Western blotting and immunolabeling of muscle sections. The Tet-On vector expressing the transactivator rtTA (reverse tetracycline transcriptional activator) and the Tet-tTS vector expressing the silencer tTS (tetracycline transcriptional silencer) were purchased from Clontech. Plasmids were prepared by use of the Endo-Free Plasmid Maxi kit (Qiagen, Courtaboeuf, France). Purified plasmid DNA was solubilized in endotoxin-free 0.9% NaCl at the working concentration.

Intramuscular Electrotransfer of Plasmid DNA.
Female nude (Janvier, Le Genest-sur-Isle, France) and C57BL/6 (Harlan, Gannat, France) mice, 8 weeks of age, were acclimated for 7 days and caged in groups of five or fewer. The hindlegs of C57BL/6 mice were shaved with an electric razor on the day before electrotransfer. Animals were anesthetized by intraperitoneal injection of ketamine (100 mg/kg of body weight; Ketalar; Panpharma, Fougères, France) and xylazine (40 mg/kg; Rompun; Bayer, Sens, France) before all procedures. Experiments were conducted in accordance with the recommendations of the NIH for animal experimentation. We injected 20 µg each of pBi (control), pBi–RDD (experimental treatment), or pBi–RDD–Flag (to visualize RDD secreted by muscle cells), together with 10 µg of the Tet-tTS and 20 µg of the Tet-On plasmids, in sterile 0.9% NaCl (final volume, 30 µl) into the tibialis cranialis muscle, and electrotransfer was conducted as described previously (32 , 35) . Briefly, eight transcutaneous square electric pulses (200 V/cm) were applied for 20 ms by use of two stainless steel plate electrodes placed ~5.7 mm apart on the leg at a frequency of 1 Hz by use of a PS-15 electropulsator (Jouan, St. Herblain, France) to obtain muscle cell electroporation that allowed passage of plasmids into the cells. The entire procedure was repeated for the second leg.

Cell Lines and Culture.
Human microvascular endothelial cells (HMEC-1) were provided by Dr. E. W. Ades (CDC, Atlanta, GA), who established this line by transfecting human dermal endothelial cells with SV40 Large T antigen (36) . HMEC-1 cells were grown as monolayers in MCDB 131 medium (Sigma-Aldrich) supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 10% FCS (Life Technologies, Inc.), 10 ng/ml epidermal growth factor (R&D Systems, Abingdon, United Kingdom), and 1 µg/ml hydrocortisone (Sigma-Aldrich). Human umbilical vein endothelial cells (HUVECs), obtained as described by Jaffe et al. (37) , were grown in M199 culture medium supplemented with 20% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, 75 mM HEPES, 3.7 mM sodium bicarbonate (pH 7.5), and 5 µg/ml fungizone (Life Technologies, Inc.). Human dermal microvascular endothelial cells (HMVEC-d; Biowhittaker Europe, Verviers, Belgium) were grown in EGM-2MV medium (Biowhittaker Europe) containing 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin and used at passages 4–6. Calf pulmonary artery endothelial (CPAE) cells provided by Dr. J. Badet (Laboratoire de Biotechnologie des Cellules Eucaryotes, Université de Créteil, Créteil, France), were grown as monolayers in MEM supplemented with 20% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin and used at passages 12–20. Human mammary adenocarcinoma cell line MDA-MB-231 and murine skeletal muscle cell line C2C12 (American Type Culture Collection, Rockville, MD) were maintained in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Murine melanoma B16F10 (ATCC) was grown in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin, adjusted to contain 1.5 g/L of sodium bicarbonate (Invitrogen, Cergy-Pontoise, France). The six media supplemented as stated above for the seven corresponding cell lines are hereafter referred to as the appropriate complete media.

Assessment of Capillary Formation by Use of Microcarrier Beads in a Fibrin Gel.
HMEC-1-coated microcarrier beads were cultured according to the method of Nehls and Drenckhahn (38) , and details of the experiment were described by Trochon et al. (31) . Briefly, HMEC-1-covered beads were embedded in a fibrin matrix containing RDD (5 µg/ml) or bovine thrombin (0.8 units/ml) as the control. Sprouting of capillary-like tubes from the periphery of microcarrier beads was observed as of the fourth day of culture. This neovascularization was photographed with an Olympus OM-2 camera on an inverted microscope, and capillary lengths were measured on the photos.

Cell Proliferation Assay.
Cells were cultured in 96-well plates in the appropriate complete medium (2000 cells/well). After 24 h, the serum concentration in the same culture medium was halved to induce the cells to accumulate at the G0-G1 phase during the next 24 h. The cells were then cultured for 30 h in fresh medium with the full concentration in the presence of RDD at the indicated concentrations or 1.6 units/ml bovine thrombin. [3H]Thymidine (1 µCi/well) was then added to the cells and allowed to incorporate for 16 h. The incorporated [3H]thymidine was adsorbed on a filter paper with a Skatron (Lier, Norway) harvester, and the radioactivity was counted in a liquid scintillation counter (Beckman).

Assessment of Apoptosis by Flow Cytometry Analysis.
To detect and quantify apoptosis, we used the AnnexinV-FITC Kit (R&D Systems). Briefly, CPAE (1 x 106) cells were cultured for 24 h in the presence of RDD (5 µg/ml) or bovine thrombin (0.8 units/ml). Pellets of 1 x 106 cells were resuspended in 100 µl of the kit reaction buffer containing propidium iodide (5 µg/ml) and 1:100 AnnexinV-FITC, according the manufacturer’s instructions. After mixing, cells were incubated for 15 min in the dark at room temperature. Flow cytometry was performed on a FACS flow cytometer (EPICS XL-MCL; Coulter, Hialeah, FL). Experiments were run in triplicate.

Cell Attachment Assay.
We coated 96-well plates overnight at 4°C with 100 µl of fibrinogen (40 µg/ml; Sigma-Aldrich), vitronectin (10 µg/ml; Sigma-Aldrich), or fibronectin (30 µg/ml; Sigma-Aldrich) in PBS. Negative controls consisted of wells coated with 2% BSA. CPAE cells (1 x 106) were cultured with 10 µg/ml RDD or bovine thrombin (1.6 units/ml) for 48 h. Cells were then detached from culture flasks by incubation with 1.5 mM EDTA and resuspended to a final concentration of 5 x 105 cells/ml in adhesion buffer [140 mM NaCl, 10 mM HEPES, 5.56 mM glucose, 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2 (pH 7.4)]]. The cell suspension (100 µl) was incubated for 20 min at 37°C on coated plates. Nonadherent cells were removed by washing the wells three times with 200 µl of adhesion buffer containing 1% BSA. Adherent cells were quantified by measuring cell phosphatase activity. Briefly, 100 µl of p-nitrophenylphosphate (3 mg/ml; Sigma-Aldrich) in sodium acetate buffer (pH 5.5) containing 0.1% Triton X-100 were added to the wells and incubated for 2 h at 37°C. The reaction was then stopped by the addition of 65 µl of 1 N NaOH. Released p-nitrophenol, which indicates the number of adherent cells, was measured by reading the absorbance at 405 nm in an ELISA reader (Labsystem, Cergy Pontoise, France). Nonspecific binding on BSA-coated plates was subtracted from the raw data. All experiments were run in triplicate.

Endothelial Cell Migration Assay.
The migration assay was performed in 24-well culture plates, as described previously (39) . Briefly, the wells were filled with 1.2% agarose (Sigma-Aldrich) dissolved in the culture medium, and the gel was allowed to set. The agarose cylinders were removed and cutoff in half, and each half was placed in a separate well into which 6.0 x 104 CPAE cells were deposited on the empty side and allowed to grow to confluence. The demi-agarose gel was then removed, RDD at the indicated concentrations or bovine thrombin (1.6 units/ml) was added, and the cells were allowed to migrate toward and through the newly created free space. Cell migration into free space could be measured by use of transparent graph paper stuck to the bottom of the culture plates.

Transient Transfection of C2C12 Skeletal Muscle Cells.
C2C12 murine muscle cells were routinely grown in the appropriate complete medium. C2C12 cells maintained in tetracycline-free serum (Clontech) for at least 1 week were transfected with equimolar amounts (3 µg of total DNA) of each plasmid (pBi, pBi–RDD-Flag and both Tet-On and Tet-tTS) at 50% saturation in 6-well plates with Lipofectamine plus reagent (Life Technologies, Inc.), according to the protocol recommended by the manufacturer. Doxycycline (1 µg/ml final concentration; Sigma-Aldrich) was added daily to the culture medium starting on the day of transfection, and supernatants were collected 48 h later.

Subcellular Fractionation and Western Blotting.
The two tibialis cranialis muscles per mouse were washed in ice-cold PBS, minced with scissors, and homogenized in 800 µl of iced-cold PBS in a glass Potter Elvejem homogenizer (VWR International, Strasbourg, France). The homogenate was then centrifuged at 700 x g for 10 min at 4°C, and the supernatant was collected. To detect RDD–Flag synthesized after electrotransfer of plasmids, 100 µg of total protein, determined with the BCA assay, were loaded on 10–20% tricine gels from Novex (Invitrogen, Cergy-Pontoise, France). Proteins were separated electrophoretically and transferred to nitrocellulose membranes (Sartorius, Göttingen, Germany). Membranes were blocked for 1 h in 10% nonfat dry milk in Tris-buffered saline [10 mM Tris (pH 7.5), 200 mM NaCl] containing 0.05% Tween 20. Membranes were then either incubated with polyclonal rabbit anti-RDD serum (Neosystem, Strasbourg, France), diluted 1:1000, for 1 h to detect E. coli-produced RDD or rabbit anti-Flag antibody (5 µg/ml; Sigma) to visualize RDD–Flag synthesized by skeletal muscle cells. After five washes with Tris-buffered saline–Tween, filters were incubated with an appropriate horseradish peroxidase-linked secondary antibody (Dako, Trappes, France), diluted 1:2000, for 1 h. Membranes were washed five times in Tris-buffered saline–Tween, and immunolabeling was detected by the enhanced chemiluminescence protocol (Amersham Pharmacia Biotech, Orsay, France).

Tumor Growth in Nude Mice.
The regulatory plasmids Tet-On and Tet-tTS, together with 20 µg of either pBi–RDD or pBi (control), were electrotransferred into female nude mice. Cultured log-phase MDA-MB-231 cells were harvested with EDTA (0.2 g/L), washed, and resuspended in sterile PBS to a final concentration of 2 x 107 cells/ml. Cell suspension (200 µl) was injected s.c. into the backs of mice. Tumor size was monitored by measuring two perpendicular diameters with a dial caliper, and tumor volume was calculated as: (length x width/2)3 x {pi}/6. When tumors reached ~18 mm3 in volume, RDD synthesis was induced in plasmid-electrotransferred muscle by the addition of doxycycline (200 µg/ml) to drinking water supplemented with 5% sucrose. Tumor size was monitored until day 14 after initiation of doxycycline stimulation.

Melanoma Pulmonary Metastases in Syngeneic Mice.
The regulatory plasmids Tet-On and Tet-tTS together with 20 µg of either pBi–RDD or pBi (control) were electrotransferred into female C57BL/6 mice, and doxycycline RDD induction (see above) was started 3 days before log-phase cultured B16F10 melanoma cells were detached with 0.02% EDTA and resuspended to the final concentration of 2 x 106/ml in sterile 0.9% NaCl. We injected 100 µl of the suspension i.v. into the retro-orbital sinus of the mice. Ten days later, the mice were sacrificed, the lungs were excised, and metastatic nodules were counted under a dissecting microscope.

Immunohistochemistry.
Paraffin sections (5-µm thick) of MDA-MB-231 tumor and tibialis cranialis muscles were cut, treated with xylene, rehydrated, and stained with H&E–saffranin. For immunohistochemistry, endogenous peroxidase activity was quenched by incubation with 3% H2O2 for 10 min. The sections were then washed in distilled water, incubated with blocking serum in Optimax wash buffer 1/10 (BioGenex, San Ramon, CA) for 10 min, and incubated with monoclonal rat anti-CD31 antibody (volume/volume mixture of MEC13.1 and 390; Becton Dickinson Europe, Le Pont De Claix, France), diluted 1:50, or polyclonal rabbit anti-Flag antibodies (Sigma), diluted 1:400, for 1 h. After two washes with Optimax, slides were incubated with peroxidase-conjugated goat polyclonal antirat or peroxidase-conjugated goat polyclonal antirabbit antibodies diluted 1:50, followed by two washes with Optimax. Slides were exposed to diaminobenzidine chromogenic substrate (PowerVision Histostaining Kit; ImmunoVision Technologies, Dady City, CA) for 10 min, washed with distilled water, counterstained with Mayer’s hematoxylin, and mounted in permanent medium (Pertex). All slides were immunolabeled the same day, thus assuring standardized intensities of immunochemical signals and counterstaining.

Image Analysis.
For each nude mouse, a representative histological sample, CD31-immunolabeled, was subjected to image analysis with a Zeiss Axiophot microscope coupled with a Sony 3 charge-coupled device camera (resolution, 768 x 576 pixels). Only viable tumor tissue was considered, excluding necrotic and fibrotic areas. For each specimen, the whole surface or eight contiguous fields were digitized when specimens were too large. Images were then analyzed with a specifically developed Linux-based program, as described by Martel-Renoir et al. (32) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RDD Purification.
Human RDD was synthesized in E. coli as a GST fusion protein. Soluble GST–RDD (36 kDa) was purified by glutathione-agarose affinity chromatography and detected by Western blotting with rabbit polyclonal antiserum directed against a RDD peptide (Fig. 1ACitation , Lane 1). GST–RDD was cleaved by bovine thrombin and further purified by glutathione-agarose chromatography to remove the fused GST. Purified RDD (10 kDa) migrated as a single band on tricine gel, and no residual GST–RDD was detected by immunoblotting (Fig. 1ACitation , Lane 2). Size-exclusion fast-performance liquid chromatography gave a single RDD peak at the expected position, demonstrating a purity >95% (Fig. 1B)Citation .



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  		Fig. 1. Western blotting and fast-performance liquid chromatography analysis of purified recombinant human disintegrin domain (RDD). Recombinant glutathione S-transferase (GST)–RDD (36 kDa) was synthesized in E. coli. RDD (10 kDa) was obtained after thrombin cleavage of GST–RDD and further purified by glutathione-agarose affinity chromatography. GST–RDD (Lane 1) and RDD (Lane 2) were visualized by immunoblotting with rabbit antiserum (1:1000) to an RDD peptide (A). Purified RDD was analyzed by fast-performance liquid chromatography with Sephadex, and a single peak corresponding to RDD was observed (B).

 
RDD Inhibition of Capillary-Like Structure Formation.
To study the effect of RDD on angiogenesis, we induced HMEC-1 cells, adapted for tube-like structure formation (38) , to form anastomosing networks of capillary-like tubes in a fibrin gel, as described previously (40) . Cells were cultured on microcarrier beads until confluence and were then embedded a in three-dimensional fibrin gel. As illustrated in Fig. 2Citation , after 3 days of incubation, a capillary-like tube sprouting from HMEC-1 cells was visible (Fig. 2A)Citation , and RDD (5 µg/ml) inhibition of this proliferation could be observed (Fig. 2C)Citation . This inhibition was more obvious after 10 days of incubation (Fig. 2D)Citation . The mean length was reduced by 90.3 ± 1.5% after 10 days compared with control (Fig. 2BCitation ; P < 0.05, Mann–Whitney U test; data from 10 microcarrier beads for each condition).



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  		Fig. 2. Recombinant human disintegrin domain (RDD) inhibition of in vitro angiogenesis. The effect on capillary-like structure formation was investigated after 3 and 10 days of RDD treatment. Microcarrier beads entirely covered with confluent HMEC-1 cells were embedded in three-dimensional fibrin matrix in the absence (A and B) or the presence (C and D) of RDD (5 µg/ml). Aggregates were photographed under an inverted microscope.

 
Effect of RDD on Endothelial Cell Proliferation and Apoptosis.
The ability of RDD (5 µg/ml) to inhibit proliferation of several endothelial cell lines (microvascular HMEC-1 and HMVEC-d cells, and macrovascular CPAE cells and HUVECs) was assessed in thymidine incorporation assays. The respective mean (±SE) percentages of inhibition were 33.1 ± 3.9, 53.6 ± 0.8, 57.2 ± 13.1 and 52.7 ± 3.0%. CPAE inhibition was dose-dependent with a maximum inhibition of 59.3% (P < 0.01, Student’s t test; n = 3; Fig. 3ACitation ) at the RDD concentration of 10 µg/ml. We also studied the effect of RDD on CPAE apoptosis. The Annexin V method was used to determine the percentage of apoptotic cells after 24 h of treatment with RDD. Although cells entering apoptosis represented only a minor cell population (mean ± SE, 12.8 ± 2.5%), there were 3-fold more of them compared with the control (4.6 ± 0.9%; n = 3), suggesting that RDD did not intervene strongly in CPAE apoptosis.



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  		Fig. 3. Recombinant human disintegrin domain (RDD) inhibition of calf pulmonary artery endothelial (CPAE) cell proliferation, migration, and adhesion. CPAE proliferation (A) and migration (B) were estimated in the presence of the indicated RDD concentrations. To evaluate CPAE adhesion (C), cells preincubated for 48 h in the absence or presence of RDD (10 µg/ml) were detached and then incubated for 20 min in wells precoated with vitronectin (10 µg/ml), fibronectin (30 µg/ml), or fibrinogen (40 µg/ml). Nonadherent cells were removed by washing and adherent cells were quantified by measurement of cell phosphatase activity. Results are expressed as percentages of the controls. Results are the means ± SE (error bars) of three determinations. *, P < 0.05, Student’s t test.

 
RDD Inhibition of Endothelial Cell Migration.
Because angiogenesis is highly dependent on endothelial cell motility, we examined the effect of RDD on CPAE migration in an in vitro assay, as we described previously (39) . During this study, we observed morphological changes in the features of CPAE cells incubated with 5 µg/ml RDD: they formed long pseudopodia, and cell–cell interactions were altered (not shown). RDD inhibited CPAE migration in a concentration-dependent fashion between 2 and 10 µg/ml (Fig. 3B)Citation , with migration totally stopped at 10 µg/ml.

Effect of RDD on Endothelial Cell Adhesion.
Endothelial cell adhesion to ECM essentially implicates integrins {alpha}vß3 and {alpha}5ß1, which have been reported to interact with metargidin (15 , 16) . We therefore analyzed the RDD effect on CPAE adhesion to immobilized fibrinogen, vitronectin, or fibronectin. Cells were cultured for 2 days in the presence of RDD (2, 5, or 10 µg/ml), and their adhesion to each substrate was evaluated. RDD at 10 µg/ml inhibited CPAE adhesion by ~30% (Fig. 3C)Citation , compared with the control, for the three adhesive proteins tested, indicating that RDD may interfere with cell adhesion but not block integrin-mediated cell adhesion. Lower concentrations showed no significant effect (results not shown).

RDD Synthesis by Skeletal Muscle Cells in Vitro and in Vivo.
We evaluated RDD secretion by C2C12 muscle cells in culture, after transient transfection with the plasmids Tet-On and Tet-tTS together with pBi–RDD–Flag or control pBi. Starting the day of transfection, doxycycline (1 µg/ml) was added daily to the cell culture medium. RDD–Flag was detected in the supernatants of day 2 cultures by Western blotting with an anti-Flag antibody (Fig. 4ACitation , Lane 2). A 10-kDa band was observed, corresponding to the RDD–Flag molecular mass, whereas no band appeared in control conditioned medium (Fig. 4ACitation , Lane 1). In vivo RDD synthesis was evaluated after muscle-targeted gene electrotransfer of pBi–RDD–Flag or pBi together with Tet-On and Tet-tTS into nude mouse skeletal muscle (tibialis cranialis). After 1 week of doxycycline stimulation, the presence of RDD–Flag in muscle extracts was evaluated by Western blotting with anti-Flag antibodies. The expected 10-kDa band was detected (Fig. 4ACitation , Lane 3) in RDD–Flag-treated animals. This band migrated just like the one produced in vitro by the pBi–RDD–Flag plasmid transfected into C2C12 muscle cells. No band was detected in the control animals (Fig. 4ACitation , Lane 4). The presence of RDD–Flag in the muscle sections was visualized by immunochemical labeling (Fig. 4, B and C)Citation . This demonstration of transgene expression in the myofibers validated our use of this expression system in this animal model.



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  		Fig. 4. Recombinant human disintegrin domain (RDD)–Flag secretion by skeletal muscle cells detected by Western blotting and immunolabeling on muscle sections. A, Western blot of RDD–Flag identified with anti-Flag antibody and visualized by chemiluminescence. Supernatants of muscle C2C12 cells transfected with the regulatory plasmids Tet-On and Tet-tTS plus empty pBi tetracycline-inducible plasmid (Lane 1) or plus pBi–RDD–Flag plasmid (Lane 2) were collected after 48 h of incubation in the presence of doxycycline (1 µg/ml). Nude mice were subjected to electrotransfer into muscle of the same plasmid system. Transgene expression was turned on for 1 week by the addition of doxycycline (200 µg/ml) to the drinking water. Muscle extracts were prepared, and 100 µg of total protein were loaded onto the gel. Lane 3 contains the muscle extract from pBi–RDD–Flag-treated mice; Lane 4 contains the pBi control plasmid. The molecular markers are indicated on the left. Sections of paraffin-embedded tibialis cranialis muscle from nude mice that had received the pBi control (B) or pBi–RDD–Flag plasmids (C) were immmunolabeled with anti-Flag antibodies then revealed by peroxidase-conjugated antibodies and diaminobenzidine chromogen.

 
RDD Inhibition of MDA-MB-231 Tumor Growth in Nude Mice.
We then examined the effect of skeletal muscle-synthesized and -secreted RDD on the growth of established MDA-MB-231 breast tumors. As above, mice received Tet-On, Tet-tTS, and pBi–RDD or pBi by electrotransfer before being inoculated s.c. with 4 x 106 MDA-MB-231 cells/animal. When tumors reached 18 mm3, RDD expression was turned on by doxycycline. Tumor growth in the RDD-treated group was significantly inhibited compared with the controls (Fig. 5A)Citation . This inhibition became detectably by day 7 post-RDD induction. By day 14 postinduction, the mean tumor volume in RDD-treated mice was 98.1 mm3, whereas in the control group, it had reached 451.2 mm3, which corresponded to 78.2% inhibition of tumor growth. The tumors were removed on day 14, and intratumoral vascularization was visualized by immunolabeling of CD31 in tissue sections (Fig. 5, B and C)Citation and quantified of the staining by digitized color imaging; there were 53.4 ± 0.2% (P < 0.01, Student’s t test; n = 10) fewer vessels within RDD-treated tumors than in control tumors. This observation suggests that RDD inhibition of tumor growth corresponds to the inhibition of blood vessel development.



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  		Fig. 5. Recombinant human disintegrin domain (RDD) inhibition of MDA-MB-231 tumor growth and angiogenesis. Equimolar concentrations of the Tet-On and Tet-tTS regulatory plasmids and pBi–RDD 20 µg or pBi (control) plasmids in 0.9% NaCl were injected into the tibialis cranialis of female nude mice and subjected to electrotransfer. MDA-MB-231 cells (4 x 106) were then injected s.c. into the flank. When tumor volumes reached 18 mm3, muscle RDD production was turned on by the addition of doxycycline (200 µg/ml) to the drinking water. A, {triangleup} represent individual tumor volumes; the histograms are the mean values of each group of five mice. ***, P < 0.01, Mann–Whitney U test. The tumors were removed 14 days after transgene induction. Paraffin-embedded MDA-MB-231 sections were immunolabeled with an anti-CD31 antibody. B and C, photographs show control (B) and RDD-treated (C) tumors.

 
RDD Inhibition of Experimental Metastatic Spread.
The same gene delivery system as that described above for MDA-MB-231 was used to investigate the effect of RDD on an experimental model of lung metastasis formation, using the melanoma cells line B16F10. The plasmids were electrotransferred into syngeneic C57BL/6 mice, and doxycycline induction was started immediately. Three days later, these mice received an i.v. injection of B16F10 cells. One week after inoculation, mice were sacrificed, their lungs were excised, and lung metastases were counted. In the presence of RDD, 74.2% fewer metastatic nodules were detected in the experimental group than the controls (Fig. 6)Citation . The experiment was repeat twice with similar results. This observation demonstrates that, in the presence of RDD, fewer metastatic nodules developed in the lungs.



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  		Fig. 6. Effect of recombinant human disintegrin domain (RDD) on B16F10 melanoma dissemination. C57BL/6 mice received pBi–RDD or pBi (control), Tet-tTS and Tet-On plasmids; doxycycline (200 µg/ml) was added to the drinking water from the day of electrotransfer. After 3 days, B16F10 cells (2 x 105) were injected into the retro-orbital sinuses of these animals. Lungs were removed from the mice 10 days after induction of RDD expression. {triangleup} represent the number of the pulmonary metastatic nodules from one mouse, and histograms are the mean values for each group of 15 mice. ***, P < 0.01, Mann–Whitney U test.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
During angiogenesis, activated endothelial cells undergo phenotypic changes and express a variety of functionally important molecules on their surfaces (41) . Metargidin expression has been reported to be up-regulated on angiogenic endothelial cell surfaces (12) . Because the soluble disintegrin domain of metargidin binds integrins, such as {alpha}5ß1, a proangiogenic adhesion integrin, and {alpha}vß3, an integrin preferentially expressed on angiogenic blood vessel (15 , 16) , it can be suggested that the functions of these integrins and metargidin may be mutually dependent. This hypothesis led us to explore the action of the metargidin disintegrin domain, RDD, on angiogenesis. RDD synthesized and secreted by E. coli was tested in vitro on endothelial cells derived from large vessels (HUVECs and CPAE cells) or from microcapillaries (HMVEC-d and HMEC-1 cells). RDD antiangiogenic activity was clearly demonstrated in the three-dimensional fibrin gel model. RDD (5 µg/ml) potently inhibited capillary tube formation by 90%. Because capillary formation represents an end result of endothelial cell proliferation, adhesion, and migration, we further examined the effect of RDD on each of these processes. We noticed that RDD strongly affected endothelial cell migration. This assay, previously developed in our laboratory (39) , enabled the accurate measurement of the CPAE cell front edge migration. RDD inhibited endothelial cell migration in a dose-dependent manner within the range of 2–10 µg/ml, with cell mobility being completely stopped at a RDD concentration of 10 µg/ml. In contrast, cell adhesion was only moderately affected, regardless of the integrin ligand substrate tested (vitronectin, fibronectin, or fibrinogen), suggesting that RDD action on cell migration is only partially dependent on inhibition of cell adhesion via integrin molecules.

RDD also inhibited endothelial cell proliferation in a concentration-dependent manner. An inhibition of ~50% at 5 µg/ml RDD was constantly observed in our experiments using HUVEC, CPAE, or HMVEC-d cell lines. Apparently RDD affects both endothelial cell migration and proliferation, and these dual actions have been reported for other antiangiogenic molecules, such as angiostatin and endostatin (2 , 3 , 42 , 43) . In addition, we found weak proapoptotic activity for RDD. RDD inhibition of angiogenesis therefore probably results from combined actions on endothelial cell migration, proliferation, and apoptosis.

One of the key mechanisms involved in RDD inhibition was first thought to be related to its integrin-binding domain. On the basis of the critical role of integrins in vascular development and cell survival (25 , 44 , 45) , therapeutic agents blocking those integrins, expressed by angiogenic endothelial cells, have been tested in several laboratories. Specific reagents targeting {alpha}v integrins, such as vitaxin or RGD-based peptides, inhibited tumor-associated angiogenesis in various animal models (18, 19, 20) and induced endothelial cell apoptosis (25) . At present, the exact roles of those integrins expressed on endothelial cells during angiogenesis is still unknown, because {alpha}v- and ß3-integrin-null mice were found to exhibit enhanced neovascularization (28, 29, 30) . Recently, Hynes (30) has suggested that these integrins are negative regulators of angiogenesis and that the antiangiogenic effect of drugs targeting them results from an agonistic rather than antagonistic action. In contrast to {alpha}vß3, {alpha}5ß1 integrin is unambiguously angiogenic, and an antagonist ligating this integrin was shown to successfully inhibit angiogenesis (46) . Thus, RDD may exert its action by neutralizing integrin proangiogenic actions and/or increasing integrin inhibitory actions. Moreover, a recent observation showing the colocalization of metargidin with vascular endothelial cadherin (14) suggests that the antiangiogenic activity of RDD might also be associated with a defect in the formation of cell junctions between endothelial cells. This possibility could represent another mechanism involved in the RDD effect.

The observed multiple inhibitory activities of RDD were not surprising because metargidin is a multifunctional molecule, as suggested by its structure. Metargidin is composed of an active metalloproteinase domain and an intracellular signaling tail separated by a disintegrin domain (13 , 47 , 48) . The interaction of metargidin with integrins expressed on cell membranes suggests that metargidin functions may be dictated by localization or distribution on the cell membrane. This situation would imply that its functions might be regulated by integrins or other, as yet unidentified, metargidin-binding proteins. On other hand, it has been postulated that RDD may most likely affect metargidin functions, e.g., the proteolytic activity of its metalloproteinase domain, by preventing its association with binding proteins. Thus, by blocking metargidin degradation of ECM components, RDD may prevent endothelial cell migration. This hypothesis is in good agreement with the potent inhibitory action of RDD on cell migration. Moreover, RDD may inhibit endothelial cell proliferation by interfering with metargidin intracellular signaling via binding partners of its cytoplasmic domain and/or a related integrin (23 , 47, 48, 49) .

We further evaluated the effect of RDD on angiogenesis-mediated tumor growth and metastasis. The RDD gene inserted in plasmids was electrotransferred into mouse skeletal muscles in a tetracycline-inducible system. This system has been used successfully in many in vivo studies (32 , 35 , 50, 51, 52, 53) . We recently demonstrated that the in vivo expression of the transgene in this model was rapid, stable, and tightly regulated (32) . Moreover, protein was detected in the blood (~50 ng/ml) and could last more than 2 months (32) . Our present results show that RDD production in vivo inhibited s.c. MDA-MB-231 tumor growth by 78% at day 14 after transgene induction and that this was associated with significantly less tumor vascularization. In addition, in the presence of RDD, 74% fewer B16F10 melanoma metastatic nodules formed in mouse lungs after 1 week of treatment.

Although the precise mechanisms involved in these effects require further investigation, the results of this study demonstrate the antiangiogenic and anticancer activities of RDD. These results are in good agreement with recent data showing the involvement of ADAM-15 in pathological neovascularization in knockout mice (54) . It is reasonable to postulate that the human RDD used in this study might affect angiogenesis and tumor metastasis through {alpha}vß3 and {alpha}5ß1 in vivo. Nevertheless, the disintegrin domain of mouse metargidin differs from that of human metargidin because it has a TDD sequence instead of RGD (55) . Thus, mouse metargidin interacts with {alpha}9ß1 instead of {alpha}vß3 and {alpha}5ß1 (17) . Therefore, it will be very interesting to investigate the effect of mouse RDD in vivo in comparison with human RDD.

Disintegrins derived from snake venom, such as rhodostomin, accutin, and salmosin, have already been shown to have important inhibitory effects on angiogenesis (56, 57, 58) and on metastatic properties of melanoma cells (59 , 60) . Compared with those disintegrin, RDD has the singular advantage of being the first endogenous disintegrin molecule of human origin and, hence, might avoid immunogenic side effects from the point of the view of future clinical settings. We believe that further investigations will support the potential clinical use of RDD as a potent and promising anticancer agent.


   ACKNOWLEDGMENTS
 
We are grateful to Monique Stanciu (Institut Gustave Roussy, Villejuif, France) for excellent technical contributions to this study. We thank Dr. E. W. Ades for the gift of the HMEC-1 cell line, Dr. J. Badet for the CPAE cell line, and Dr. Y. Takada for the pGEX-2T vector.


   FOOTNOTES
 
Grant support: Grant 8364 from Association Française contre la Myopathie (AFM); the Ligue National Contre le Cancer; Grants 7593 and 9061 from the Association pour la Recherche contre le Cancer (ARC); Grant 1999 001858 from Fondation de France and the Institut National de la Santé et de la Recherche Médicale (INSERM); le Centre National pour la Recherche Scientifique (CNRS); Institut Gustave Roussy; Institut Universitaire d’Hématologie of Paris VII; and Bioalliance Pharma SA.

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.

Note: V. Trochon-Joseph and D. Martel-Renoir contributed equally to the manuscript.

Requests for reprints: He Lu, U553 INSERM, Hôpital Saint-Louis, 1, av. Claude Vellefaux, 75475 Paris Cedex 10, France. Phone: (33) 1 53 72 40 26; Fax: (33) 1 53 72 40 27; E-mail: helu{at}chu-stlouis.fr

Received 10/27/03. Revised 12/12/03. Accepted 1/ 9/04.


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