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• W2017759961 abstract "ADAMTS1 is a metalloprotease previously shown to inhibit angiogenesis in a variety of in vitro and in vivo assays. In the present study, we demonstrate that ADAMTS1 significantly blocks VEGFR2 phosphorylation with consequent suppression of endothelial cell proliferation. The effect on VEGFR2 function was due to direct binding and sequestration of VEGF165 by ADAMTS1. Binding was confirmed by co-immunoprecipitation and cross-linking analysis. Inhibition of VEGF function was reversible, as active VEGF could be recovered from the complex. The interaction required the heparin-binding domain of the growth factor, because VEGF121 failed to bind to ADAMTS1. Structure/function analysis with independent ADAMTS1 domains indicated that binding to VEGF165 was mediated by the carboxyl-terminal (CT) region. ADAMTS1 and VEGF165 were also found in association in tumor extracts. These findings provide a mechanism for the anti-angiogenic activity of ADAMTS1 and describe a novel modulator of VEGF bioavailability. ADAMTS1 is a metalloprotease previously shown to inhibit angiogenesis in a variety of in vitro and in vivo assays. In the present study, we demonstrate that ADAMTS1 significantly blocks VEGFR2 phosphorylation with consequent suppression of endothelial cell proliferation. The effect on VEGFR2 function was due to direct binding and sequestration of VEGF165 by ADAMTS1. Binding was confirmed by co-immunoprecipitation and cross-linking analysis. Inhibition of VEGF function was reversible, as active VEGF could be recovered from the complex. The interaction required the heparin-binding domain of the growth factor, because VEGF121 failed to bind to ADAMTS1. Structure/function analysis with independent ADAMTS1 domains indicated that binding to VEGF165 was mediated by the carboxyl-terminal (CT) region. ADAMTS1 and VEGF165 were also found in association in tumor extracts. These findings provide a mechanism for the anti-angiogenic activity of ADAMTS1 and describe a novel modulator of VEGF bioavailability. Extracellular matrix proteins can significantly modulate growth factor signaling. This occurs to a large extent, but not exclusively, from direct non-covalent interactions that mediate selective anchorage of growth factors to the extracellular milieu (1Vlodavsky I. Folkman J. Sullivan R. Fridman R. Ishai-Michaeli R. Sasse J. Klagsbrun M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2292-2296Google Scholar, 2Taipale J. Keski-Oja J. FASEB J. 1997; 11: 51-59Google Scholar, 3Damsky C. Ilic D. Curr. Opin. Cell Biol. 2002; 14: 594Google Scholar). Several extracellular matrix molecules have been shown to bind and sequester growth factors, as well as to enhance signaling by altering presentation to receptor binding sites (4Gengrinovitch S. Berman B. David G. Witte L. Neufeld G. Ron D. J. Biol. Chem. 1999; 274: 10816-10822Google Scholar, 5Miralem T. Steinberg R. Price D. Avraham H. Oncogene. 2001; 20: 5511-5524Google Scholar, 6Brekken R.A. Sage E.H. Matrix Biol. 2001; 19: 816-827Google Scholar, 7Kim Y.M. Hwang S. Kim Y.M. Pyun B.J. Kim T.Y. Lee S.T. Gho Y.S. Kwon Y.G. J. Biol. Chem. 2002; 277: 27872-27879Google Scholar, 8Barillari G. Albonici L. Franzese O. Modesti A. Liberati F. Barillari P. Ensoli B. Manzari V. Santeusanio G. Am. J. Pathol. 1998; 152: 1161-1166Google Scholar, 9Sahni A. Francis C.W. Blood. 2000; 96: 3772-3778Google Scholar, 10Ikuta T. Ariga H. Matsumoto K. Genes Cells. 2000; : 913-927Google Scholar). Angiogenesis is particularly sensitive to this type of regulation due to the critical role of paracrine growth factors in endothelial cell migration and proliferation. The vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial growth factor; ADAMTS1, a disintegrin and metalloproteinase with thrombospondin motifs; BAEC, bovine aortic endothelial cells; BSA, bovine serum albumin; CT, carboxyl-terminal region of ADAMTS1; DSS, decanesulfonic acid sodium salt; HAEC, human aortic endothelial cells; HPLC, high pressure liquid chromatography; mAb, monoclonal antibody; METH1, methalloprotease and thrombospondin; Met, metalloproteinase construct; Met-Dis, metalloproteinase and disintegrin-truncated construct; MMP, matrix metalloproteinase; PAE, porcine aortic endothelial cells; TGF-β, transforming growth factor β; TSP, thrombospondin; VEGFR, VEGF receptor; FGF, fibroblast growth factor; ELISA, enzyme-linked immunosorbent assay. gene produces several splice variants critical for capillary morphogenesis and tumor angiogenesis (11Veikkola T. Karkkainen M. Claesson-Welsh L. Alitalo K. Cancer Res. 2000; 60: 203-212Google Scholar, 12Ferrara N. Am. J. Physiol. Cell Physiol. 2001; 280: 1358-1366Google Scholar, 13Ferrara N. Nat. Rev. Cancer. 2002; 2: 795-803Google Scholar). Haploinsufficiency of this gene is incompatible with development due to major vascular abnormalities, as demonstrated by inactivation of the gene through homologous recombination (14Carmeliet 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: 435-439Google Scholar, 15Ferrara 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: 439-442Google Scholar). Isoforms of VEGF are secreted by diverse cell types including smooth muscle, fibroblasts, and epithelial cells. These proteins function by activation of two tyrosine kinase receptors, VEGFR1 and VEGFR2, as well as by binding to non-receptor tyrosine kinase coreceptors such as neuropilin 1 and 2 on endothelial cells (16Shibuya M. Cell Struct. Funct. 2001; 26: 25-35Google Scholar, 17Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Google Scholar). ADAMTS1 was the first member of a growing family of ADAMTS extracellular proteases characterized by the presence of disintegrin-like metalloprotease and a variable number of thrombospondin-like domains (18Vazquez F. Hastings G. Ortega M.A. Lane T.F. Oikemus S. Lombardo M. Iruela-Arispe M.L. J. Biol. Chem. 1999; 274: 23349-23357Google Scholar, 19Kuno K. Kanada N. Nakashima E. Fujiki F. Ichimura F. Matsushima K. J. Biol. Chem. 1997; 272: 556-562Google Scholar). Once secreted, ADAMTS1 activation requires furin cleavage and removal of the pro-domain. The active protease can undergo a secondary processing event that separates the catalytic subunit from the thrombospondin (TSP) repeats (20Kuno K. Matsushima K. J. Biol. Chem. 1998; 273: 13912-13917Google Scholar, 21Rodriguez-Manzaneque J.C. Milchanowski A.B. Dufour E.K. Leduc R. Iruela-Arispe M.L. J. Biol. Chem. 2000; 275: 33471-33479Google Scholar). These TSP motifs in TSP1 and TSP2 have been shown to block angiogenesis by several, and likely not independent, mechanisms (22Taraboletti G. Belotti D. Borsotti P. Vergani V. Rusnati M. Presta M. Giavazzi R. Cell Growth Differ. 1997; 8: 471-479Google Scholar, 23Rodriguez-Manzaneque J.C. Lane T.F. Ortega M.A. Hynes R.O. Lawler J. Iruela-Arispe M.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12485-12490Google Scholar, 24Jimenez B. Volpert O.V. Crawford S.E. Febbraio M. Silverstein R.L. Bouck N. Nat. Med. 2000; 6: 41-48Google Scholar). We demonstrated previously that ADAMTS1 is able to suppress capillary growth using multiple in vivo and in vitro assays (18Vazquez F. Hastings G. Ortega M.A. Lane T.F. Oikemus S. Lombardo M. Iruela-Arispe M.L. J. Biol. Chem. 1999; 274: 23349-23357Google Scholar). Interestingly, the ability of ADAMTS1 to inhibit neovascularization in vivo was greater than that of endostatin and TSP1 at the same molar ratio. The findings are intriguing and appear in marked contrast to the current paradigm that MMPs are pro-migratory and pro-angiogenic (25Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Google Scholar, 26Coussens L.M. Fingleton B. Matrisian L.M. Science. 2002; 295: 2387-2392Google Scholar, 27Vihinen P. Kahari V.M. Int. J. Cancer. 2002; 99: 157-166Google Scholar, 28Jiang Y. Goldberg I.D. Shi Y.E. Oncogene. 2002; 21: 2245-2252Google Scholar). In an effort to determine the mechanism(s) responsible for the angiostatic properties of ADAMTS1, we investigated its effects on endothelial cell growth and found that ADAMTS1 was able to drastically decrease VEGFR2 phosphorylation by a mechanism that involved direct binding and sequestration of VEGF165. The interaction was also verified in vivo using xenograft assays engineered to express ADAMTS1. Our results demonstrate that ADAMTS1 binds to VEGF165, and that this impacts the bioavailability of VEGF with consequences to receptor phosphorylation, endothelial proliferation, and angiogenesis. Cells—Bovine aortic endothelial cells (BAEC), human embryonic kidney 293T cells expressing full-length human ADAMTS1 or vector alone (18Vazquez F. Hastings G. Ortega M.A. Lane T.F. Oikemus S. Lombardo M. Iruela-Arispe M.L. J. Biol. Chem. 1999; 274: 23349-23357Google Scholar), and breast tumor-derived T47D cells expressing deletion ADAMTS1 constructs were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. Human aortic endothelial cells (HAEC, provided by Dr. Judith Berliner, Department of Pathology, UCLA) were grown in medium 199 containing 20% fetal calf serum, endothelial cell growth supplement (20 μg/ml), and heparin (90 μg/ml) (Sigma). Porcine aortic endothelial cells (PAE) transfected with VEGFR2 or vector alone (provided by Dr. Gera Neufeld, Technicon, Israel) were grown in Ham's F-12 medium supplemented with 10% fetal calf serum. Generation of Monoclonal Antibodies against ADAMTS1—Balb/c mice were immunized by intraperitoneal injections of purified ADAMTS1 (10 μg) (21Rodriguez-Manzaneque J.C. Milchanowski A.B. Dufour E.K. Leduc R. Iruela-Arispe M.L. J. Biol. Chem. 2000; 275: 33471-33479Google Scholar) and complete Freud coadjuvant (300 μl). Additional immunizations were done with incomplete Freud coadjuvant at days 15 and 33 (intraperitoneal injection) and intravenously at day 45. At day 48, spleenocytes were obtained from immunized mice and fused with SP2 myeloma cells at a 4:1 ratio following established techniques (29Luque A. Gomez M. Puzon W. Takada Y. Sanchez-Madrid F. Cabanas C. J. Biol. Chem. 1996; 271: 11067-11075Google Scholar). Conditioned media from hybridoma cultures were screened by ELISA against purified ADAMTS1 coated to plastic. Further characterization of positive clones was performed by immunoprecipitation, Western blot analysis, and immunohistochemistry. From 101 positive wells, twelve hybridomas were cloned by repeated limiting dilution based on epitopes and specific properties. Table I summarizes the selected monoclonal antibodies (mAbs).Table ICharacterization of mAbs against ADAMTSmAbs against ADAMTS1No.mAbsTiter by ELISAI.P.W.B.I.F.N.R.C.R.C.FormaldehydeMethanol11H12G80.868110/87/65110/87/65NEG+++22G7F100.72687/65NEGNEG++-33B12F60.624110/8787NEG+++43C8F80.878110/87110/87NEG++++53E4C6B41.025110/87/65110/87/65- 65++++63E7G100.861110/87/65110/87/65NEG+++/-73G3A6B110.947110/87/65NEGNEG+++/-84A11C70.726110/87/65110/87/65NEG++++95C6D50.899110/87110/87NEG++++105D4E11B50.470NEG110/87/65110/87/65+-115D3H30.980110/87/65110/87/65NEGNDND124C2C41.358110/8787NEGNDND+Mouse scrum1.755110/87/65110/87/65110/87/65NDND-Irrelevant IgG0.093NEGNEGNEGNEGNEG Open table in a new tab Endothelial Cell Proliferation—Quiescent BAEC were trypsinized and plated onto 24-well plates in Dulbecco's modified Eagle's medium supplemented with VEGF165 (R&D Systems) (25 ng/ml), basic fibroblast growth factor (FGF-2) (2 ng/ml) (provided by Dr. Gera Neufeld) or a combination of both, in the presence or absence of recombinant ADAMTS1/METH-1 protein (5 μg/ml). A pulse of 1 μCi/well of [3H]thymidine (Amersham Biosciences) was applied over the last 8 h prior to harvesting. Cells were washed and fixed in 10% trichloroacetic acid. Incorporation of [3H]thymidine was determined by scintillation counting, as described previously (30Iruela-Arispe M.L. Sage E.H. J. Cell. Biochem. 1993; 52: 414-430Google Scholar). Phosphorylation Assays—Subconfluent cells were incubated overnight in serum-free medium and subsequently preincubated for 5 min with 0.1 mm Na3VO4 to inhibit phosphatase activity. Cultures were then washed once and pretreated with 1.5 ml of conditioned media from 293T cells expressing ADAMTS1 or vector alone for 15 min at 37 °C. The concentration of ADAMTS1 in the conditioned media ranges between 5.85 μg/ml (66 nm) and 16.7 μg/ml (191 nm). These values were obtained by ELISA against a standard curve of purified ADAMTS1 (data not shown). Cells were stimulated for 6 min at 37 °C with specified concentration of VEGF165. The incubation was terminated by removal of the medium and washes with cold phosphate-buffered saline/0.2 mm Na3VO4. Cells were solubilized in lysis buffer (1% Triton X-100, 10 mm Tris-HCl, pH 7.6, 150 mm NaCl, 30 mm sodium pyrophosphate, 50 mm sodium fluoride, 2.1 mm sodium orthovanadate, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 2 μg/ml of aprotinin) at 4 °C for 15 min. Insoluble material was removed by centrifugation at 4 °C for 30 min at 14,000 × g. Equal amounts of cell lysate were separated by SDS-PAGE and transferred to nitrocellulose membranes. Phosphorylated proteins were detected by immunoblotting using antiphosphotyrosine antibodies (polyclonal or mAb PY20, BD Transduction Laboratories) followed by secondary antibodies coupled with horseradish peroxidase and visualized by chemiluminescence (ECL kit, Pierce). Protein-loading control was assessed by Western blot using anti-VEGFR2 (A-3, Santa Cruz Biotechnology) and or anti-enolase antibodies. Immunoprecipitation—Cell lysates were precleared with 40 μl of protein G-agarose (Roche Applied Science) for 1 h at 4 °C. Beads were discarded by centrifugation, and the supernatant was incubated with 1 μg/ml of the antiphosphotyrosine antibody (PY20) overnight at 4 °C followed by addition of protein G-agarose for 1 h under continuous agitation. Immunoprecipitates were washed three times with lysis buffer and extracted in 2× SDS-PAGE sample buffer by boiling 5 min, fractionated by one-dimensional SDS-PAGE, and further analyzed by Western blot with antiphosphotyrosine antibodies. ADAMTS1 was immunoprecipitated from tumor lysates (450 μg) as described above using the mAb 5C6D5. The presence of coimmunoprecipitated VEGF was assessed by Western blot using polyclonal anti-VEGF, 375 (generous gift from Don Senger, Beth Israel Deaconess Medical Center, Boston). Levels of ADAMTS1 were determined by Western analysis (mAb 5C6D5). Radiolabeling of VEGF—VEGF165 was labeled with 125I-Na using iodogen as coupling agent. Briefly, VEGF165 (2 μg) was incubated in 200 μl of borate buffer (0.01 m Na2B4O7, 0.14 m NaCl, pH 8.2) with 0.3 mCi of 125I-Na (Amersham Biosciences) in IODO-GEN-precoated iodination tubes (Pierce) 5 min at room temperature. The reaction was stopped by transfer to a fresh tube containing 40 μl of 0.4 mg/ml tyrosine in borate buffer for 1 min at room temperature and adding 200 μl of 1 mg/ml IK in phosphate-buffered saline/1% BSA. 125I-VEGF165 was separated from free iodine using size exclusion chromatography on Sephadex-G25 columns (Amersham Biosciences). The specific radioactivity of the purified iodinated VEGF165 was 18,886 cpm/ng. Quality and integrity of the labeled VEGF was assessed by SDS-PAGE followed by autoradiography of the dried gel. Binding Assays—Subconfluent cells were incubated at low serum (2% serum for HAEC and 0.1% for PAE and PAE-VEGFR2 cells) for 5 h at 37 °C, rinsed in binding buffer (media containing 20 mm HEPES, 0.2% gelatin) and equilibrated at 4 °C for 15 min. Indicated concentrations of 125I-VEGF165 were added to the wells in a final volume of 350 μl of binding buffer in the presence or absence of ADAMTS1. After 1 h of incubation at 4 °C, cells were washed four times in binding buffer and solubilized in 500 μl of lysis buffer (2% Triton X-100, 10% glycerol, 1 mg/ml BSA). Bound protein was assessed on a γ-counter. Nonspecific binding was calculated in the presence of 20-fold excess of cold VEGF165. Specific binding was determined by subtracting nonspecific binding from total binding. Purification of ADAMTS1 and Evaluation of VEGF Levels—Recombinant ADAMTS1 protein was purified from conditioned media of stable 293T cells expressing ADAMTS1 by heparin and Zn2+-chelate affinity chromatography (21Rodriguez-Manzaneque J.C. Milchanowski A.B. Dufour E.K. Leduc R. Iruela-Arispe M.L. J. Biol. Chem. 2000; 275: 33471-33479Google Scholar). Conditioned media from 293T cells transfected with the vector alone was used as control. Presence of VEGF in the ADAMTS1 fractions was assessed by Western blot (Chemicon). Levels of ADAMTS1 were tested using the mAb 5D4E11B5 after stripping of the same membrane. Gel Permeation Chromatography—ADAMTS-VEGF complexes purified by heparin chromatography were concentrated to 1 ml and dialyzed into 10 mm HEPES, 150 mm NaCl, 25 mm 1-decanesulfonic acid sodium salt (DSS). Sample was loaded onto a Superdex 75 HR 10/30, previously equilibrated in 10 mm HEPES, 150 mm NaCl, and 0.3% Zwittergent 3–12. Chromatography was carried out at 4 °C with a flow rate of 0.3 ml/min (at 1100 psi). Fractions of 300 μl were collected and evaluated by Western blots. Binding of VEGF to Immobilized ADAMTS1—ADAMTS1 was immunoprecipitated from conditioned media of stably transfected 293T cells using the mAb 5C6D5. Protein A beads were washed three times with phosphate-buffered saline, 1% Triton X-100, 1% BSA, 1 mm CaCl2,1mm MgCl2, and equilibrated in binding buffer (50 mm HEPES, 150 mm NaCl, 0.5% Nonidet P-40, 0.5% BSA, 1 mm CaCl2,1mm MgCl2). Soluble ligands: VEGF165 (50 ng) (R&D Systems) and/or heparin (50 ng) (Sigma), were added to the pellets in a final volume of 500 μl of binding buffer, and incubated for 30 min at 4 °C. The beads were subsequently washed three times with binding buffer. Proteins bound to the immunoconjugates were subjected to SDS-PAGE, and the presence of coimmunoprecipitated VEGF was analyzed by Western blots. Immunoprecipitated ADAMTS1 was tested reproving the same membrane with the mAb 5D4E11B5. Cross-linking Experiments Using Purified Proteins—Purified ADAMTS1 (1.25 μg) was incubated with VEGF165 (25 ng) in the presence or absence of heparin (50 ng) in 75 μl of binding buffer (50 mm HEPES, 100 mm NaCl, pH 7.2) for 2 h at room temperature. Bound proteins were cross-linked with disuccinimydil suberate (1.5 mm) (Pierce) for 30 min at room temperature. The reaction was stopped by adding 20 mm Tris, 50 mm glycine, 2 mm EDTA (pH7.4) for 15 min at room temperature. The samples were processed by SDS-PAGE, and protein complexes, indicating protein-protein interaction, were visualized by Western blots. Cross-linking Experiments Using Conditioned Media—Subconfluent 293T or T47D cells expressing full-length or different deletion constructs of ADAMTS1 or pCDNA (negative control) grown on 24-well plates were washed twice with serum-free Dulbecco's modified Eagle's medium and incubated in 300 μl of serum-free media for 18 h in the presence or absence of VEGF165 (150 ng) or VEGF121 (125 ng) (provided by Dr. Gera Neufeld) with or without heparin (1.5 μg). Conditioned media was collected, and the interacting proteins were cross-linked and analyzed as described above. Xenograft Tumor Assays and Tumor Protein Extraction—Control and ADAMTS1 tumors were generated in 6-week-old male nude mice (Charles River Laboratories) by subcutaneous flank injection of both T47D control (empty vector) or ADAMTS1 (5 × 106 cells/injection). When tumors reached 1,500 mm3, mice were euthanized, and dissected tumors were diced, sieved, and solubilized in lysis buffer (1% Triton X-100, 10 mm Tris-HCl, pH 7.6, 150 mm NaCl, 30 mm sodium pyrophosphate, 50 mm sodium fluoride, 2.1 mm sodium orthovanadate, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 2 μg/ml aprotinin) at 4 °C for 1 h. Insoluble material was removed by centrifugation at 4 °C for 1 h at 14,000 × g. Expression of the Carboxyl-terminal (CT) Region of ADAMTS1 in T47D Cells—The construct for expression of the CT region of ADAMTS1 corresponding to Pro549–Ser951 was obtained by PCR of the full-length cDNA. A KpnI site was introduced at the 5′-end by site-directed mutagenesis using the following forward oligo: 5′TTTTCATGGTACCTGGGGAATGTGGG-3′. The reverse oligo used was 5′-ACTGCATTCTGCCTTTGTGCAAAAGTC-3′. The resulting PCR product was cloned into the pSecTag2/Hygro B vector (Invitrogen) using KpnI/EcoRV. Stable transfectant clones were generated using this plasmid in T47D cells by calcium phosphate transfection and selected with hygromycin-B (300 μg/ml). ADAMTS1 Inhibits the Mitogenic Effect of VEGF165Affecting VEGFR2 Phosphorylation—In a previous report we showed that ADAMTS1 antagonizes endothelial cell proliferation induced by a combination of FGF-2 and VEGF165 (18Vazquez F. Hastings G. Ortega M.A. Lane T.F. Oikemus S. Lombardo M. Iruela-Arispe M.L. J. Biol. Chem. 1999; 274: 23349-23357Google Scholar). To further dissect the mechanism of action of ADAMTS1, we repeated these experiments using each growth factor independently and in combination. Addition of recombinant ADAMTS1 decreased cell proliferation induced by FGF2 (49% ± 16%) and FGF-2 + VEGF165 (78% ± 18%). Proliferative signals mediated by VEGF165 were completely blocked in the presence of ADAMTS1 (Fig. 1A). The mitogenic action of all VEGF isoforms is mediated through binding to the 205-kDa receptor tyrosine kinase VEGFR2 (31Bernatchez P.N. Soker S. Sirois M.G. J. Biol. Chem. 1999; 274: 31047-31054Google Scholar). Therefore, we tested whether activation of VEGFR2 was affected by ADAMTS1. VEGFR2 phosphorylation was strongly inhibited by ADAMTS1 in several endothelial cell cultures including PAE-VEGFR2 (Fig. 1B), BAEC (Fig. 1C) and human aortic endothelial cells (data not shown). Stimulation of BAEC with increasing concentrations of VEGF165 resulted in a dose-dependent phosphorylation of VEGFR2 and ADAMTS1 reduced the levels of phospho-VEGFR2 in all cases (Fig. 1D). Together these results imply a direct link between VEGF165 signaling and the anti-angiogenic effects mediated by ADAMTS1. As a possible explanation for the ADAMTS1 inhibition of VEGFR2 phosphorylation, we tested whether binding of VEGF165 to the endothelial cell surface was affected by ADAMTS1. 125I-VEGF165 bound to HAEC (Fig. 2A) and PAEVEGFR2 cells (Fig. 2B) in a concentration-dependent manner; however it did not bind to VEGFR2-deficient cells (PAE) (Fig. 2B). It has been documented that saturation binding occurs at 10 ng/ml with VEGF165 (32Bikfalvi A. Sauzeau C. Moukadiri H. Maclouf J. Busso N. Bryckaert M. Plouet J. Tobelem G. J. Cell. Physiol. 1991; 149: 50-59Google Scholar). The specific binding of 125I VEGF165 (16 ng/ml) to HAEC (Fig. 2A) was 14.7%, while saturation binding in PAE-VEGFR2 was lower, 8.9% after incubation with 12 ng/ml of radioiodinated VEGF (Fig. 2B). Presence of purified ADAMTS1 reduced 125I-VEGF165 specific binding by 69% (Fig. 2C). VEGF Co-purifies with ADAMTS1—It has been reported that TSP repeats in thrombospondin 1 and in connective tissue growth factor bind to VEGF165 and modulate its activity on endothelial cells (33Gupta K. Gupta P. Wild R. Ramakrishnan S. Hebbel P. Angiogenesis. 1999; 3: 147-158Google Scholar, 34Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K. Ikeda E. Takata S. Kobayashi K. Okada Y. FASEB J. 2002; 16: 219-221Google Scholar, 35Criscuolo G.R. Merrill M.J. Oldfield E.H. J. Neurosurg. 1988; 69: 254-262Google Scholar). The CT of ADAMTS1 contains three domains that share significant homology to the TSP repeats of the TSP1 molecule (18Vazquez F. Hastings G. Ortega M.A. Lane T.F. Oikemus S. Lombardo M. Iruela-Arispe M.L. J. Biol. Chem. 1999; 274: 23349-23357Google Scholar). Therefore, we investigated the possibility of an interaction between ADAMTS1 and VEGF165 as a regulatory mechanism to explain the effect of ADAMTS1 on VEGFR2 phosphorylation and the binding to the endothelial cell surface. We found that indeed VEGF (Fig. 3A, lanes 1–3) was present in the samples containing ADAMTS1 (Fig. 3A, lanes 5–7) after purification from heparin affinity chromatography (Fig. 3A, lanes 1 and 5) and from chromatography on a Zn2+-chelate affinity column (Fig. 3A, lanes 2 and 6, 3 and 7). Although detection of VEGF in the first purification step was not surprising, since VEGF interacts avidly with heparin (34Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K. Ikeda E. Takata S. Kobayashi K. Okada Y. FASEB J. 2002; 16: 219-221Google Scholar, 36Ferrara N. Henzel W.J. Biochem. Biophys. Res. Commun. 1989; 161: 851-858Google Scholar); its presence after Zn2+ chromatography was unexpected since this growth factor does not bind to Zn2+. To ascertain the degree of purification of VEGF from these two chromatography procedures in the absence of ADAMTS1, conditioned media from parental cell lines transfected with vector alone was subjected to the same chromatography purification. As expected, VEGF was eluted from heparin column at 1 m (Fig. 3B), whereas no binding was detected on Zn2+-chelate affinity chromatography in the absence of ADAMTS1 (Fig. 3C). 293T cells have been shown previously to secrete VEGF; our results concur with those findings (37Mukhopadhyay D. Akbarali H.I. Biochem. Biophys. Res. Commun. 1996; 229: 733-738Google Scholar, 38Tian X. Song S. Wu J. Meng L. Dong Z. Shou C. Biochem. Biophys. Res. Commun. 2001; 286: 505-512Google Scholar). These results provide evidence that VEGF binds to ADAMTS1. The two molecules can be dissociated and purified from one another by gel filtration chromatography (Fig. 4A). When bound to ADAMTS1, VEGF165 was unable to phosphorylate VEGFR2 (Fig. 4B, sample A). However, VEGF165 regained its activity when dissociated from the protease (Fig. 4B, sample 13). ADAMTS1 Binds to VEGF165—The interaction of ADAMTS1 with VEGF165 was also evaluated by co-immunoprecipitation and cross-linking assays. Conditioned media was collected from cells that were stably transfected with either vector alone (pCDNA) or ADAMTS1 and immunoprecipitated with anti-ADAMTS1 antibodies. The immunocomplexes were subsequently incubated with VEGF165, and binding was evaluated by Western blots. VEGF165 was detected in the ADAMTS1 immunocomplexes and addition of exogenous heparin increased binding of ADAMTS1 to VEGF165 (Fig. 5A). No VEGF165 was bound to immunoprecipitated complexes when media conditioned from cells expressing vector alone was used (Fig. 5A). Cross-linking experiments were conducted with disuccinimydil suberate to evaluate physical proximity of ADAMTS1 and VEGF165. Heterodimeric complexes of about 250 and 130 kDa were detected with VEGF antibodies in the presence of ADAMTS1 (Fig. 5B). VEGF antibodies also recognized 130-kDa species in the ADAMTS1 preparations, indicating presence of VEGF in ADAMTS1 preparations as demonstrated previously (Fig. 3). We predict that the 130-kDa form corresponds to the sum of ADAMTS1 (87 kDa) and VEGF (43 kDa). Addition of heparin enhanced the formation of the high molecular weight complexes; however, the presence of heparin was not a requirement (Fig. 5B). To ascertain whether ADAMTS1-VEGF165 complexes were formed in vivo, we incubated 293T cells transfected with ADAMTS1 or empty vector with purified VEGF165 and heparin. Evaluation of VEGF after cross-linking revealed a shifted band of about 130 kDa only in the ADAMTS1-conditioned media (Fig. 5C). Under these experimental conditions, formation of the complex ADAMTS1-VEGF165 required heparin. To ensure the presence of ADAMTS1 in the 130-kDa band, the same blot was re-probed with anti-ADAMTS1 antibodies (Fig. 5C, blot to the right). Indeed a 130-kDa band was found only in the presence of VEGF165 and overlapped with the band identified by the VEGF antisera. Cross-linking experiments were also performed with VEGF121; however, no interaction was found (Fig. 5D). These results indicated that the carboxyl-terminal heparin-binding domain of VEGF participates in an interaction with ADAMTS1. ADAMTS1 and VEGF Form Complexes in Vivo—The in vitro data implied that one possible mechanism for ADAMTS1 activity is binding and inactivation of VEGF165 function. Therefore, we investigated whether ADAMTS1 and VEGF formed complexes in vivo. The experiments were performed using xenograft tumor lysates from T47D mammary carcinoma cells expressing either ADAMTS1 or vector alone (pCDNA). As previously described, we found that biochemical evaluation of VEGF from tissues/tumors reveals multiple bands immunoreactive with several independent VEGF antibodies (Fig. 6A) (39Goldbrunner R.H. Bernstein J.J. Plate K.H. Vince G.H. Roosen K. Tonn J.C. J. Neurosci. Res. 1999; 55: 486-495Google Scholar, 40Sasaki H. Ray P.S. Zhu L. Galang N. Maulik N. Toxicology. 2000; 155: 27-35Google Scholar), representing different VEGF forms (17Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Google Scholar, 41Robinson C.J. Stringer S.E. J. Cell Sci. 2001; 114: 853-865Google Scholar) as well as possible complexes formed with other proteins (42Soker S. Svahn C.M. Neufeld G. J. Biol. Chem. 1993; 268: 7685-7691Google Scholar, 43Anthony F.W. Evans P.W. Wheeler T. Wood P.J. Ann. Clin. Biochem. 1997; 34: 276-280Google Scholar, 44Vuorela-Vepsalainen P. Alfthan H. Orpana A. Alitalo K. Stenman U.H. Halmesmaki E. Hum. Reprod. 1999; 14: 1346-1351Google Scholar, 45Wijelath E.S. Murray J. Rahman S. Patel Y. Ishida A. Strand K. Aziz S. Cardona C. Hammond W.P. Savidge G.F. Rafii S. Sobel M. Circ. Res. 2002; 91: 25-31Google Scholar). Comparison between control and ADAMTS1 expressing tumors revealed a novel VEGF immunoreactive species of ∼130 kDa present exclusively in the ADAMTS1 tumor lysates (Fig. 6A). The 130-kDa band co-migrated and overlapped with a species recog" @default.
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• W2017759961 title "ADAMTS1/METH1 Inhibits Endothelial Cell Proliferation by Direct Binding and Sequestration of VEGF165" @default.
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