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• W2135925543 abstract "Vascular endothelial growth factor (VEGF), a potent angiogenic mitogen, plays a crucial role in angiogenesis under various pathophysiological conditions. We have recently demonstrated that VEGF165, one of the VEGF isoforms, binds connective tissue growth factor (CTGF) and that its angiogenic activity is inhibited in the VEGF165·CTGF complex form (Inoki, I., Shiomi, T., Hashimoto, G., Enomoto, H., Nakamura, H., Makino, K., Ikeda, E., Takata, S., Kobayashi, K. and Okada, Y. (2002) FASEB J. 16, 219–221). In the present study, we further examined the susceptibility of the VEGF165·CTGF complex to matrix metalloproteinases (MMP-1, -2, -3, -7, -9, and -13), ADAMTS4 (aggrecanase-1), and serine proteinases, and evaluated the recovery of the angiogenic activity of VEGF165 after the treatment. Among the MMPs, MMP-1, -3, -7, and -13 processed CTGF of the complex into the major NH2- and COOH-terminal fragments, whereas VEGF165 was completely resistant to the MMPs. On the other hand, elastase and plasmin cleaved both CTGF and VEGF165 of the complex, but they were completely resistant to ADAMTS4. By digestion of the immobilized VEGF165·CTGF complex with MMP-3 or MMP-7, both NH2- and COOH-terminal fragments of CTGF were dissociated and released from the complex into the liquid phase. The in vitro angiogenic activity of VEGF165 blocked in the VEGF165·CTGF complex was reactivated to original levels after CTGF digestion of the complex with MMP-1, -3, and -13. Recovery of angiogenic activity was further confirmed by in vivoangiogenesis assay using a Matrigel injection model in mice. These results demonstrate for the first time that CTGF is a substrate of MMPs and that the angiogenic activity of VEGF165 suppressed by the complex formation with CTGF is recovered through the selective degradation of CTGF by MMPs. MMPs may play a novel role through CTGF degradation in VEGF-induced angiogenesis during embryonic development, tissue maintenance, and/or pathological processes of various diseases. Vascular endothelial growth factor (VEGF), a potent angiogenic mitogen, plays a crucial role in angiogenesis under various pathophysiological conditions. We have recently demonstrated that VEGF165, one of the VEGF isoforms, binds connective tissue growth factor (CTGF) and that its angiogenic activity is inhibited in the VEGF165·CTGF complex form (Inoki, I., Shiomi, T., Hashimoto, G., Enomoto, H., Nakamura, H., Makino, K., Ikeda, E., Takata, S., Kobayashi, K. and Okada, Y. (2002) FASEB J. 16, 219–221). In the present study, we further examined the susceptibility of the VEGF165·CTGF complex to matrix metalloproteinases (MMP-1, -2, -3, -7, -9, and -13), ADAMTS4 (aggrecanase-1), and serine proteinases, and evaluated the recovery of the angiogenic activity of VEGF165 after the treatment. Among the MMPs, MMP-1, -3, -7, and -13 processed CTGF of the complex into the major NH2- and COOH-terminal fragments, whereas VEGF165 was completely resistant to the MMPs. On the other hand, elastase and plasmin cleaved both CTGF and VEGF165 of the complex, but they were completely resistant to ADAMTS4. By digestion of the immobilized VEGF165·CTGF complex with MMP-3 or MMP-7, both NH2- and COOH-terminal fragments of CTGF were dissociated and released from the complex into the liquid phase. The in vitro angiogenic activity of VEGF165 blocked in the VEGF165·CTGF complex was reactivated to original levels after CTGF digestion of the complex with MMP-1, -3, and -13. Recovery of angiogenic activity was further confirmed by in vivoangiogenesis assay using a Matrigel injection model in mice. These results demonstrate for the first time that CTGF is a substrate of MMPs and that the angiogenic activity of VEGF165 suppressed by the complex formation with CTGF is recovered through the selective degradation of CTGF by MMPs. MMPs may play a novel role through CTGF degradation in VEGF-induced angiogenesis during embryonic development, tissue maintenance, and/or pathological processes of various diseases. vascular endothelial growth factor a disintegrin and metalloproteinase with thrombospondin motifs bovine aortic endothelial cells bovine serum albumin COOH-terminal connective tissue growth factor extracellular matrix fms-like tyrosine kinase-1 insulin-like growth factor binding protein monocyte chemoattractant protein-3 matrix metalloproteinase phosphate-buffered saline thrombospondin type 1 repeat von Willebrand (vWF) factor type C repeat Vascular endothelial growth factor (VEGF)1 has been reported to play a key role in angiogenic processes under various pathophysiological conditions such as embryonic development, inflammatory diseases, diabetic retinopathy, and tumor growth (1Plate K.H. Breier G. Weich H.A. Risau W. Nature. 1992; 359: 845-848Crossref PubMed Scopus (2126) Google Scholar, 2Carmeliet P. Ferreira V. Breier G. Pollefeyt S. Kieckens L. Gertsenstein M. Fahrig M. Vandenhoeck A. Harpal K. Eberhardt C. Declercq C. Pawling J. Moons L. Collen D. Risau W. Nagy A. Nature. 1996; 380: 435-439Crossref PubMed Scopus (3475) Google Scholar, 3Ferrara N. Carver-Moore K. Chen H. Dowd M., Lu, L. O'Shea K.S. Powell-Braxton L. Hillan K.J. Moore M.W. Nature. 1996; 380: 439-442Crossref PubMed Scopus (3062) Google Scholar, 4Koch A.E. Harlow L.A. Haines G.K. Amento E.P. Unemori E.N. Wong W.L. Pope R.M. Ferrara N. J. Immunol. 1994; 152: 4149-4156PubMed Google Scholar, 5Ikeda M. Hosoda Y. Hirose S. Okada Y. Ikeda E. J. Pathol. 2000; 191: 426-433Crossref PubMed Scopus (112) Google Scholar, 6Ishida S. Shinoda K. Kawashima S. Oguchi Y. Okada Y. Ikeda E. Investig. Ophthalmol. Vis. Sci. 2000; 41: 1649-1656PubMed Google Scholar). Five different splicing variants of VEGF (VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206) have been identified so far (7Ferrara N. J. Mol. Med. 1999; 77: 527-543Crossref PubMed Scopus (1087) Google Scholar). The angiogenic activity of VEGF in vivo may be regulated by the gene expression of the VEGF isoforms and their receptors, fms-like tyrosine kinase-1 (Flt-1 = VEGFR-1) (8de Vries C. Escobedo J.A. Ueno H. Houck K. Ferrara N. Williams L.T. Science. 1992; 255: 989-991Crossref PubMed Scopus (1896) Google Scholar) and kinase insert domain-containing receptor (KDR = VEGFR-2) (9Terman B.I. Dougher-Vermazen M. Carrion M.E. Dimitrov D. Armellino D.C. Gospodarowicz D. Bohlen P. Biochem. Biophys. Res. Commun. 1992; 187: 1579-1586Crossref PubMed Scopus (1405) Google Scholar). However, another important regulation mechanism is extracellular inhibition of activity through complex formation with proteins, which include platelet factor-4 and soluble forms of VEGF receptors (10Maione T.E. Gray G.S. Petro J. Hunt A.J. Donner A.L. Bauer S.I. Carson H.F. Sharpe R.J. Science. 1990; 247: 77-79Crossref PubMed Scopus (627) Google Scholar, 11Gengrinovitch S. Greenberg S.M. Cohen T. Gitay-Goren H. Rockwell P. Maione T.E. Levi B.Z. Neufeld G. J. Biol. Chem. 1995; 270: 15059-15065Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 12Goldman C.K. Kendall R.L. Cabrera G. Soroceanu L. Heike Y. Gillespie G.Y. Siegal G.P. Mao X. Bett A.J. Huckle W.R. Thomas K.A. Curiel D.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8795-8800Crossref PubMed Scopus (417) Google Scholar). By screening a human chondrocyte cDNA library using a yeast two-hybrid system, we have recently demonstrated that connective tissue growth factor (CTGF) inhibits VEGF165-induced in vitroand in vivo angiogenesis through binding to VEGF165 (13Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K.I. Ikeda E. Takata S. Kobayashi K.I. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (329) Google Scholar). CTGF is a member of the CCN (CTGF/cysteine-rich 61/nephroblastoma-overexpressed gene) family and consists of 349 amino acids with four distinct domains, i.e.insulin-like growth factor-binding protein (IGFBP), von Willebrand factor type C repeat (vWFC), thrombospondin type 1 repeat (TSP-1), and COOH-terminal (CT) domains (14Bradham D.M. Igarashi A. Potter R.L. Grotendorst G.R. J. Cell Biol. 1991; 114: 1285-1294Crossref PubMed Scopus (811) Google Scholar, 15Lau L.F. Lam S.C. Exp. Cell Res. 1999; 248: 44-57Crossref PubMed Scopus (582) Google Scholar). Because both VEGF and CTGF are expressed in physiological conditions including growth plate morphogenesis and wound healing and pathological fibrosis such as hepatic fibrosis and myocardial fibrosis, the angiogenic activity of VEGF under pathophysiological conditions may be controlled by interaction with CTGF. However, this regulation mechanism can be affected by proteinases, since VEGF and CTGF are susceptible to proteolytic degradation. Plasmin is known to digest VEGF165and inactivate its angiogenic activity (16Keyt B.A. Berleau L.T. Nguyen H.V. Chen H. Heinsohn H. Vandlen R. Ferrara N. J. Biol. Chem. 1996; 271: 7788-7795Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar). Several fragments of CTGF have been detected in culture media of various cells (17Yang D.H. Kim H.S. Wilson E.M. Rosenfeld R.G. Oh Y. J. Clin. Endocrinol. Metab. 1998; 83: 2593-2596Crossref PubMed Scopus (98) Google Scholar), biological fluids such as sera (17Yang D.H. Kim H.S. Wilson E.M. Rosenfeld R.G. Oh Y. J. Clin. Endocrinol. Metab. 1998; 83: 2593-2596Crossref PubMed Scopus (98) Google Scholar) and uterine luminal flushing (18Brigstock D.R. Steffen C.L. Kim G.Y. Vegunta R.K. Diehl J.R. Harding P.A. J. Biol. Chem. 1997; 272: 20275-20282Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). However, no information is available for the proteinases that digest CTGF and modulate the angiogenic activity of the VEGF165·CTGF complex. Matrix metalloproteinases (MMPs) are a gene family of structurally and functionally related zinc endopeptidases that consist of 23 different members in human (19Sternlicht M.D. Werb Z. Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3256) Google Scholar). MMPs play an essential role in physiological turnover and pathological degradation of extracellular matrix (ECM) macromolecules such as proteoglycans and collagens (20Okada Y. Ruddy S. Harris Jr., E.D. Sledge C.B. Kelly's Textbook of Rheumatology. 6th Ed. W. B. Saunders Co., Philadelphia2001: 55-72Google Scholar). However, recent studies indicate that MMPs can also digest molecules other than ECM components. Mcquibban et al. (21McQuibban G.A. Gong J.H. Tam E.M. McCulloch C.A. Clark-Lewis I. Overall C.M. Science. 2000; 289: 1202-1206Crossref PubMed Scopus (646) Google Scholar) demonstrate that monocyte chemoattractant protein-3 (MCP-3) is clipped by MMP-2 and that the cleaved chemokine becomes an antagonist of chemoattractive activity. Interleukin-1β, Fas ligand, and IGFBP-3 are also susceptible to MMPs including MMP-1, -2, -3, -7, and/or -9 (22Ito A. Mukaiyama A. Itoh Y. Nagase H. Thogersen I.B. Enghild J.J. Sasaguri Y. Mori Y. J. Biol. Chem. 1996; 271: 14657-14660Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 23Mitsiades N., Yu, W.H. Poulaki V. Tsokos M. Stamenkovic I. Cancer Res. 2001; 61: 577-581PubMed Google Scholar, 24Fowlkes J.L. Suzuki K. Nagase H. Thrailkill K.M. Endocrinology. 1994; 135: 2810-2813Crossref PubMed Scopus (95) Google Scholar, 25Manes S. Llorente M. Lacalle R.A. Gomez-Mouton C. Kremer L. Mira E. Martinez A.C. J. Biol. Chem. 1999; 274: 6935-6945Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Thus, it might be possible that MMPs are capable of degrading the VEGF165·CTGF complex to affect biological activity. In the present study, we examined the susceptibility of the VEGF165·CTGF complex to six different MMPs including MMP-1 (tissue collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin-1), MMP-7 (matrilysin), MMP-9 (gelatinase B), and MMP-13 (collagenase-3) and assayed the angiogenic activity of the complex after MMP treatment. The data demonstrate for the first time that MMP-1, -3, -7, and -13 selectively degrade CTGF bound to VEGF165, which itself is resistant to MMPs, and that the angiogenic activity of VEGF165 is recovered by dissociation of CTGF fragments from VEGF165 after digestion. Recombinant CTGF was prepared as described previously (13Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K.I. Ikeda E. Takata S. Kobayashi K.I. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (329) Google Scholar). Briefly, CTGF cDNA amplified by PCR from a chondrocyte cDNA library (CLONTECH, Palo Alto, CA) was cloned into pCMVtag4a (Stratagene, La Jolla, CA) and expressed as FLAG-tagged protein. Culture media were harvested 3 days after transfection of the CTGF expression vector into COS-7 cells, concentrated by ultrafiltration, and subjected to anti-FLAG M2-agarose affinity column chromatography (Sigma-Aldrich). Recombinant CTGF was eluted with 6 m urea, washed with 50 mm Tris-HCl (pH 7.5), 3 m NaCl, 10 mm CaCl2, 0.05% Brij 35 and dialyzed against 50 mm Tris-HCl (pH 7.5), 0.15 m NaCl, 10 mm CaCl2, 0.05% Brij 35 (TNCB buffer) immediately after elution (13Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K.I. Ikeda E. Takata S. Kobayashi K.I. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (329) Google Scholar). Purified CTGF showed two bands of 38 and 35 kDa on silver-stained gels after SDS-PAGE, which correspond to the glycosylated and non-glycosylated forms (13Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K.I. Ikeda E. Takata S. Kobayashi K.I. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (329) Google Scholar). The biological activity of recombinant CTGF was confirmed by tube formation assay (13Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K.I. Ikeda E. Takata S. Kobayashi K.I. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (329) Google Scholar). Protein concentration was determined using BCA protein assay reagents (Pierce). Aliquots of purified CTGF were radioiodinated according to the method of Fraker and Speck (26Fraker P.J. Speck Jr., J.C. Biochem. Biophys. Res. Commun. 1978; 80: 849-857Crossref PubMed Scopus (3626) Google Scholar) and used for binding assay. The zymogens of MMP-1, -2, -3, -7, -9, and -13 were purified and activated by incubation with p-aminophenylmercuric acetate (Sigma-Aldrich) according to previous methods (27Okada Y. Harris Jr., E.D. Nagase H. Biochem. J. 1988; 254: 731-741Crossref PubMed Scopus (133) Google Scholar, 28Okada Y. Morodomi T. Enghild J.J. Suzuki K. Yasui A. Nakanishi I. Salvesen G. Nagase H. Eur J Biochem. 1990; 194: 721-730Crossref PubMed Scopus (385) Google Scholar, 29Imai K. Yokohama Y. Nakanishi I. Ohuchi E. Fujii Y. Nakai N. Okada Y. J. Biol. Chem. 1995; 270: 6691-6697Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 30Okada Y. Gonoji Y. Naka K. Tomita K. Nakanishi I. Iwata K. Yamashita K. Hayakawa T. J. Biol. Chem. 1992; 267: 21712-21719Abstract Full Text PDF PubMed Google Scholar, 31Knauper V. Lopez-Otin C. Smith B. Knight G. Murphy G. J. Biol. Chem. 1996; 271: 1544-1550Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar). Concentrations of MMPs were determined by titration with tissue inhibitors of metalloproteinases as described previously (32Shimada T. Nakamura H. Ohuchi E. Fujii Y. Murakami Y. Sato H. Seiki M. Okada Y. Eur. J. Biochem. 1999; 262: 907-914Crossref PubMed Scopus (88) Google Scholar, 33Nakamura H. Fujii Y. Inoki I. Sugimoto K. Tanzawa K. Matsuki H. Miura R. Yamaguchi Y. Okada Y. J. Biol. Chem. 2000; 275: 38885-38890Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Recombinant human ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4, which is aggrecanase-1) was purified according to our method (33Nakamura H. Fujii Y. Inoki I. Sugimoto K. Tanzawa K. Matsuki H. Miura R. Yamaguchi Y. Okada Y. J. Biol. Chem. 2000; 275: 38885-38890Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), and activity was verified in an assay using aggrecan as a substrate (34Hashimoto G. Aoki T. Nakamura H. Tanzawa K. Okada Y. FEBS Lett. 2001; 494: 192-195Crossref PubMed Scopus (169) Google Scholar). Concentration of ADAMTS4 was determined using BCA protein assay reagents (Pierce). Plasminogen (Sigma-Aldrich) and pro-elastase (Calbiochem) were activated with streptokinase (Calbiochem-Novabiochem) and trypsin (Sigma-Aldrich), respectively. Polyclonal antibodies against the IGFBP and CT domains of CTGF (anti-IGFBP and anti-CT antibodies, respectively) were raised in rabbits by injecting bovine serum albumin (BSA)-conjugated oligopeptides corresponding to the amino acid sequences of the IGFBP domain (residues 83–95, DFGSPANRKIGVC) and CT domain (residues 257–273, IRTPKISKPIKFELSGC), respectively. IgG was isolated from the antisera by DEAE-Sephacel column chromatography. Specific reaction to CTGF was demonstrated by immunoblotting of culture media of 293T cells (an immortalized human embryonic kidney cell line) transfected with pCMVtag4a/CTGF plasmid as described previously (13Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K.I. Ikeda E. Takata S. Kobayashi K.I. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (329) Google Scholar). Non-glycosylated recombinant VEGF165 produced in Escherichia coli was purchased from Pepro Tech EC Ltd. (London, UK). VEGF165·CTGF complex was prepared by incubation of VEGF165 with CTGF at a molar ratio of 1:1 at 4 °C for 24 h as described previously (13Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K.I. Ikeda E. Takata S. Kobayashi K.I. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (329) Google Scholar). Digestion of the complex was initially examined by incubation of the substrate at 37 °C for 24 h with MMPs, ADAMTS4, elastase, or plasmin in an enzyme-to-substrate ratio of 1:30 in TNCB buffer. Because CTGF in the complex was efficiently digested with MMP-1, -3, -7, and -13, time course digestion was performed by incubation of the complex with these MMPs for different periods ranging from 0 to 24 h in the same enzyme-to-substrate ratio. The reactions were terminated with 20 mm EDTA, and the digestion products were analyzed on silver-stained gels after SDS-PAGE and by immunoblotting using anti-IGFBP (1 μg/ml), anti-CT (1 μg/ml), or anti-VEGF antibodies (10 μg/ml; Santa Cruz Biotechnology Inc., Santa Cruz, CA) according to our method (30Okada Y. Gonoji Y. Naka K. Tomita K. Nakanishi I. Iwata K. Yamashita K. Hayakawa T. J. Biol. Chem. 1992; 267: 21712-21719Abstract Full Text PDF PubMed Google Scholar). CTGF alone was also digested with MMP-1, -3, -7, and -13 at 37 °C for 24 h to confirm the digestion of CTGF without complex formation. CTGF (10 μg) was incubated with MMP-1, -3, -7 or -13 at an enzyme-to-substrate ratio of 1:30 at 37 °C for 24 h for MMP-1, -3, and -7 and for 4 h for MMP-13 in a total reaction volume of 100 μl in TNCB buffer. Reactions were stopped by treatment with 20 mm EDTA, and the digestion products were subjected to SDS-PAGE. Proteins in the gels were transferred to polyvinylidene difluoride membranes and located by staining with 0.1% Coomassie Brilliant Blue R-250. The bands of interest were excised and sequenced by Edman degradation using Procise 491 Protein Sequencer (PerkinElmer Life Sciences) (33Nakamura H. Fujii Y. Inoki I. Sugimoto K. Tanzawa K. Matsuki H. Miura R. Yamaguchi Y. Okada Y. J. Biol. Chem. 2000; 275: 38885-38890Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Microtiter plates with 96 wells (Immunomodule, NalgeNunc, Rochester, NY) were coated with 50 μl of VEGF165 (500 ng/well) for 16 h at 4 °C. Then the plates were washed twice with phosphate-buffered saline (PBS) containing 0.05% Brij 35 and subsequently blocked with 1% BSA in PBS for 2 h at room temperature. 125I-Labeled CTGF (1∼3 × 105 cpm, 10 ng/well) was bound to each well by incubation for 24 h at 4 °C. After washing carefully twice with PBS containing 0.05% Brij 35, the bound proteins were digested with MMP-3 (1 ng) or MMP-7 (1 ng) in TNCB buffer or buffer alone for 0.5, 4, or 24 h at 37 °C. Reaction mixtures were collected, and CTGF fragments were immunoprecipitated with 50 μl of anti-IGFBP antibody or anti-CT antibody, which were then trapped onto protein G-Sepharose beads (Amersham Biosciences). CTGF remaining on the wells was dissociated by treatment with 1 n NaOH. Radioactivity of the precipitates and NaOH-dissociated fractions was counted by γ-counter ARC-600 (Aloka, Tokyo, Japan). Tube formation assay using bovine aortic endothelial cells (BAEC) in type I collagen gel was carried out as described previously (13Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K.I. Ikeda E. Takata S. Kobayashi K.I. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (329) Google Scholar). Briefly, type I collagen (3 mg/ml) (Vitrogen, Cohesion, Palo Alto, CA) was mixed with 10-fold concentrated M199 (Invitrogen) and 0.1 n NaOH at a ratio of 8:1:1, respectively. The mixture (500 μl) was dispensed to 24-well culture plates and incubated at 37 °C for 1 h to form collagen fibrils. BAEC were trypsinized and plated on the collagen gel at a density of 5 × 104 cells/well. They were cultured in M199 medium supplemented with 10% fetal bovine serum overnight. After removal of the media, 500 μl of the collagen mixture was overlaid on the cells and incubated for 1 h at 37 °C. The cells were stimulated for 3 days with VEGF165, CTGF, VEGF165·CTGF complex, VEGF165·CTGF complex digested with MMPs or MMPs by adding media containing these molecules onto the top of the collagen gel layers. For VEGF165·CTGF complex formation, VEGF165 (40 ng) was incubated for 24 h at 4 °C with increasing concentrations of CTGF (40 and 200 ng and 1 μg) in M199 medium containing 1% fetal bovine serum. Digestion mixtures of the complex, which were made by incubation of VEGF165 (40 ng) with CTGF (100 ng) for 24 h at 4 °C, were prepared by digestion of the complex with MMP-1, -3, and -13 (3 ng each) for 24 h at 37 °C. To demonstrate that recovered angiogenic activity after digestion of the VEGF165·CTGF complex with MMP-1, -3, or -13 is derived from VEGF165, the MMP digestion mixtures prepared as mentioned above were incubated with 40 μg of mouse monoclonal anti-VEGF IgG (Upstate Biotechnology, Lake Placid, NY) or non-immune mouse IgG (DAKO, Glostrup, Denmark) for 1 h at 37 °C and then used for tube formation assay. As for a control, the cells were also stimulated with CTGF (100 ng) or CTGF (100 ng) digested with MMP-1, -3, or -13 (3 ng each) for 24 h at 37 °C. After 3 days of culture, each well was photographed, and the tubular length of the cells was measured in five different areas in 0.25 mm2 using NIH Image 1.62 as described previously (13Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K.I. Ikeda E. Takata S. Kobayashi K.I. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (329) Google Scholar). The length was expressed as mm/mm2. Experiments were repeated three times, and similar results were obtained. In vivo angiogenesis assay using a Matrigel injection model was performed as previously described (13Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K.I. Ikeda E. Takata S. Kobayashi K.I. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (329) Google Scholar). Before injection, 500 μl of Matrigel (Collaborative Biomedical Products, Bedford, MA) was mixed with 50 μl of PBS, VEGF165 (50 ng), VEGF165 (50 ng)-CTGF (100 ng) complex, complex digested with MMP-3 (3 ng) for 24 h at 37 °C, MMP-3 digestion mixture incubated with anti-VEGF IgG (50 μg; Upstate Biotechnology) or non-immune mouse IgG (50 μg; DAKO) for 1 h at 37 °C, CTGF (100 ng), CTGF (100 ng) digested with MMP-3 (3 ng) for 24 h at 37 °C, or MMP-3 (3 ng). MMP-3 activity was inactivated before mixing with Matrigel by incubation with sheep polyclonal anti-human MMP-3 IgG (0.5 μg) (35Okada Y. Takeuchi N. Tomita K. Nakanishi I. Nagase H. Ann. Rheum. Dis. 1989; 48: 645-653Crossref PubMed Scopus (174) Google Scholar). Matrigel containing these factors was injected subcutaneously near the abdominal midline of 4-week-old male C57BL/6J mice. Cutaneous tissues with Matrigel plugs were removed 5 days after injection, fixed in periodate-lysine-paraformaldehyde, and embedded in paraffin. The serial sections were stained with hematoxylin and eosin and immunostained with rabbit anti-vWF antibody (1:200; DAKO) or non-immune rabbit IgG. The degree of angiogenesis was determined by counting vWF-positive cells and blood vessels with an apparent luminal area/mm2 area using NIH Image 1.62 according to our previous method (13Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K.I. Ikeda E. Takata S. Kobayashi K.I. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (329) Google Scholar). Measured values were expressed as mean ± S.D. In the tube formation assay, the difference of tubular length treated with VEGF165 and VEGF165·CTGF complex was analyzed by Bonferroni/Dunn test. In the in vivo angiogenesis assay, the differences of spindle cells and blood vessels between two independent groups were analyzed by the Mann-Whitney test. These tests were performed using StatView 5.0. p values of less than 0.05 were considered significant. Our previous studies (13Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K.I. Ikeda E. Takata S. Kobayashi K.I. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (329) Google Scholar) have demonstrated that the angiogenic activity of glycosylated VEGF165 is inhibited with CTGF by in vitro and in vivo assays. To further confirm the inhibitory effect of CTGF on the angiogenic activity of non-glycosylated recombinant VEGF165 used in the present study, the activity was assayed by tube formation assay. As shown in Fig. 1, A andB, VEGF165 stimulated the tubular extension 5.5-fold more than the untreated control. In contrast, VEGF165-induced tube formation was remarkably reduced in the presence of CTGF. Increasing concentrations of CTGF inhibited the activity with a peak at 200 ng/ml. These data are consistent with our previous data of CTGF inhibition of angiogenesis using glycosylated VEGF165 (13Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K.I. Ikeda E. Takata S. Kobayashi K.I. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (329) Google Scholar), indicating that glycosylated chains of VEGF165 are not involved in the inhibitory effect of CTGF. VEGF165 and CTGF were complexed by incubating them at a molar ratio of 1:1 for 16 h at 4 °C, and then the complex was digested at 37 °C for 24 h with 6 different MMPs (MMP-1, -2, -3, -7, -9, and -13), ADAMTS4, elastase, or plasmin. When the reaction products of the VEGF165·CTGF complex with MMPs were analyzed on silver-stained gels, CTGF of 35 and 38 kDa was cleaved by MMP-1, -3, -7, and -13 into a few fragments with lower molecular weights ranging from 19 to 23 kDa (data not shown). However, because VEGF165 of 19 kDa co-migrated with these fragments, some digestion fragments could not be differentiated from VEGF165. On the other hand, when the reaction mixtures were analyzed by immunoblotting with two different anti-CTGF antibodies specific to the IGFBP domain or CT domain, intact CTGF disappeared, and several 19∼23-kDa bands immunoreactive with these antibodies were observed in the digestion products with MMP-1, -3, -7, and -13 (Fig.2, A and B). In addition, weak immunoreactive bands were also detected by digestion with MMP-2 and MMP-9 (Fig. 2, A and B), although they were not detectable on silver-stained gels (data not shown). CTGF was also digested by elastase and plasmin into small peptides, most of which were not observed by immunoblotting with antibodies (Fig. 2,A and B), or on a silver-stained gel (data not shown). However, CTGF was resistant to ADAMTS4 (Fig. 2, Aand B). In contrast to the susceptibility of CTGF to MMPs, VEGF165 was completely resistant to digestion with MMPs and ADAMTS4 under these conditions, whereas both elastase and plasmin cleaved VEGF165 of 19 kDa into 13-kDa fragments (Fig.2 C). To compare the susceptibility of the VEGF165·CTGF complex to degradation with MMP-1, -3, -7, and -13, all of which digested CTGF in a 24-h incubation, time course digestion was carried out and monitored by immunoblotting with anti-CT antibody. Among the four MMPs examined, MMP-13 appeared to most efficiently digest CTGF in the complex, since intact CTGF was degraded fastest. A complete digestion of CTGF was observed after a 4-h incubation with MMP-13 (Fig.3 D) and after an 8-h incubation with MMP-3 (Fig. 3 B). However, MMP-1 and MMP-7 required a 24-h incubation (Fig. 3, A and C). When CTGF alone was digested with MMP-1, -3, -7, and -13 and the digestion patterns of CTGF were compared with those obtained by VEGF165·CTGF complex digestion with MMPs, the fragments detected with anti-IGFBP and anti-CT antibodies were identical between the samples of CTGF alone and VEGF165·CTGF complex (data not shown). Thus, NH2-terminal sequence analyses of the CTGF fragments generated by digestion with MMP-1, -3, -7, and -13 were performed by incubation of CTGF alone with MMPs. As shown in TableI, the NH2-terminal sequences of the COOH-terminal fragments of CTGF were successfully determined. However, the NH2-terminal sequence of intact CTGF was not obtained, indicating that its NH2 terminus is blocked. Besides intact CTGF, the NH2-terminal sequences of several 19∼23-kDa CTGF fragments recognized with anti-IGFBP antibody could not be obtained, suggesting that these fragments contain the NH2 terminus of CTGF. By CTGF digestion with these MMPs, CTGF was cleaved at several peptide bonds including the Ala181-Tyr182 (MMP-3 and -7), Arg183-Leu184 (MMP-7 and -13), Met194-Ile195 (MMP-1, -3, -7, and -13), or Cys199-Leu200 bond (MMP-1, -7, and -13) (TableI), all of which are located between the vWFC and TSP domains. Among them, the Met194-Ile195 bond wa" @default.
• W2135925543 created "2016-06-24" @default.
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• W2135925543 date "2002-09-01" @default.
• W2135925543 modified "2023-09-30" @default.
• W2135925543 title "Matrix Metalloproteinases Cleave Connective Tissue Growth Factor and Reactivate Angiogenic Activity of Vascular Endothelial Growth Factor 165" @default.
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• W2135925543 doi "https://doi.org/10.1074/jbc.m201674200" @default.
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