Structural and Functional Uncoupling of the Enzymatic and Angiogenic Inhibitory Activities of Tissue Inhibitor of Metalloproteinase-2 (TIMP-2)
2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês
10.1074/jbc.m306176200
ISSN1083-351X
AutoresCecilia A. Fernández, Catherine Butterfield, Geraldine Jackson, Marsha A. Moses,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoTissue inhibitors of metalloproteinases (TIMPs) regulate tumor growth, progression, and angiogenesis in a variety of experimental cancer models and in human malignancies. Results from numerous studies have revealed important differences between TIMP family members in their ability to inhibit angiogenic processes in vitro and angiogenesis in vivo despite their universal ability to inhibit matrix metalloproteinase (MMP) activity. To address these differences, a series of structure-function studies were conducted to identify and to characterize the anti-angiogenic domains of TIMP-2, the endogenous MMP inhibitor that uniquely inhibits capillary endothelial cell (EC) proliferation as well as angiogenesis in vivo. We demonstrate that the COOH-terminal domain of TIMP-2 (T2C) inhibits the proliferation of capillary EC at molar concentrations comparable with those previously reported for intact TIMP-2, while the NH2-terminal domain (T2N), which inhibits MMP activity, has no significant anti-proliferative effect. Interestingly, although both T2N and T2C inhibited embryonic angiogenesis, only T2C resulted in the potent inhibition of angiogenesis driven by the exogenous addition of angiogenic mitogen, suggesting that MMP inhibition alone may not be sufficient to inhibit the aggressive neovascularization characteristic of aberrant angiogenesis. We further mapped the anti-proliferative activity of T2C to a 24-amino acid peptide corresponding to Loop 6 of TIMP-2 and show that Loop 6 is a potent inhibitor of both embryonic and mitogen-stimulated angiogenesis in vivo. These findings demonstrate that TIMP-2 possesses two distinct types of anti-angiogenic activities which can be uncoupled from each other, the first represented by its MMP-dependent inhibitory activity which can inhibit only embryonic neovascularization and the second represented by an MMP-independent activity which inhibits both normal angiogenesis and mitogen-driven angiogenesis in vivo. In addition, we report, for the first time, the discovery of Loop 6 as a novel and potent inhibitor of angiogenesis. Tissue inhibitors of metalloproteinases (TIMPs) regulate tumor growth, progression, and angiogenesis in a variety of experimental cancer models and in human malignancies. Results from numerous studies have revealed important differences between TIMP family members in their ability to inhibit angiogenic processes in vitro and angiogenesis in vivo despite their universal ability to inhibit matrix metalloproteinase (MMP) activity. To address these differences, a series of structure-function studies were conducted to identify and to characterize the anti-angiogenic domains of TIMP-2, the endogenous MMP inhibitor that uniquely inhibits capillary endothelial cell (EC) proliferation as well as angiogenesis in vivo. We demonstrate that the COOH-terminal domain of TIMP-2 (T2C) inhibits the proliferation of capillary EC at molar concentrations comparable with those previously reported for intact TIMP-2, while the NH2-terminal domain (T2N), which inhibits MMP activity, has no significant anti-proliferative effect. Interestingly, although both T2N and T2C inhibited embryonic angiogenesis, only T2C resulted in the potent inhibition of angiogenesis driven by the exogenous addition of angiogenic mitogen, suggesting that MMP inhibition alone may not be sufficient to inhibit the aggressive neovascularization characteristic of aberrant angiogenesis. We further mapped the anti-proliferative activity of T2C to a 24-amino acid peptide corresponding to Loop 6 of TIMP-2 and show that Loop 6 is a potent inhibitor of both embryonic and mitogen-stimulated angiogenesis in vivo. These findings demonstrate that TIMP-2 possesses two distinct types of anti-angiogenic activities which can be uncoupled from each other, the first represented by its MMP-dependent inhibitory activity which can inhibit only embryonic neovascularization and the second represented by an MMP-independent activity which inhibits both normal angiogenesis and mitogen-driven angiogenesis in vivo. In addition, we report, for the first time, the discovery of Loop 6 as a novel and potent inhibitor of angiogenesis. Matrix metalloproteinases (MMPs), 1The abbreviations used are: MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; EC, endothelial cell(s); CAM, chorioallantoic membrane; T2N, NH2-terminal domain of TIMP-2; T2C, COOH-terminal domain of TIMP-2; EA-T2N, MMP inihibition-deficient mutant of T2N; HPLC, high performance liquid chromatography; bFGF, basic fibroblast growth factor. the multigene family of zinc-dependent endopeptidases, have been implicated in a number of important physiological events, both normal and pathological (1Brew K. Dinakarpandian D. Nagase H. Biochim. Biophys. Acta. 2000; 1477: 267-283Crossref PubMed Scopus (1628) Google Scholar). Among these is angiogenesis, the formation of new capillaries from the pre-existing vasculature. Regulation of MMP activity is achieved at the transcriptional level (1Brew K. Dinakarpandian D. Nagase H. Biochim. Biophys. Acta. 2000; 1477: 267-283Crossref PubMed Scopus (1628) Google Scholar) as well as by a family of endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Shifts in the proteolytic balance in favor of MMP activity have been shown to play a crucial role in regulating angiogenesis as well as tumor growth and metastasis (2Moses M.A. Sudhalter J. Langer R. Science. 1990; 248: 1408-1410Crossref PubMed Scopus (435) Google Scholar, 3Stetler-Stevenson W.G. Hewitt R. Corcoran M. Semin. Cancer Biol. 1996; 7: 147-154Crossref PubMed Scopus (343) Google Scholar, 4Moses M.A. Stem Cells. 1997; 15: 180-189Crossref PubMed Scopus (264) Google Scholar, 5Vu T.H. Shipley J.M. Bergers G. Berger J.E. Helms J.A. Hanahan D. Shapiro S.D. Senior R.M. Werb Z. Cell. 1998; 93: 411-422Abstract Full Text Full Text PDF PubMed Scopus (1524) Google Scholar, 6O'Reilly M.S. Wiederschain D. Stetler-Stevenson W.G. Folkman J. Moses M.A. J. Biol. Chem. 1999; 274: 29568-29571Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 7Fang J. Shing Y. Wiederschain D. Yan L. Butterfield C. Jackson G. Harper J. Tamvakopoulos G. Moses M.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3884-3889Crossref PubMed Scopus (353) Google Scholar, 8Bergers G. Brekken R. McMahon G. Vu T.H. Itoh T. Tamaki K. Tanzawa K. Thorpe P. Itohara S. Werb Z. Hanahan D. Nat. Cell Biol. 2000; 2: 737-744Crossref PubMed Scopus (2320) Google Scholar, 9Yan L. Borregaard N. Kjeldsen L. Moses M.A. J. Biol. Chem. 2001; 276: 37258-37265Abstract Full Text Full Text PDF PubMed Scopus (591) Google Scholar). Historically, the potential therapeutic value of endogenous, as well as of synthetic inhibitors of metalloproteinases, has been predicated on their ability to inhibit MMP activity. More recently, attention has been focused on the ability of TIMPs to regulate angiogenesis. Since it was first demonstrated that TIMPs could inhibit angiogenesis, numerous studies have sought to characterize the specific angiogenic processes effected by MMP inhibitors (2Moses M.A. Sudhalter J. Langer R. Science. 1990; 248: 1408-1410Crossref PubMed Scopus (435) Google Scholar, 10Moses M.A. Langer R. Biotechnology. 1991; 9: 630-634Crossref PubMed Scopus (86) Google Scholar, 11Murphy A.N. Unsworth E.J. Stetler-Stevenson W.G. J. Cell Physiol. 1993; 157: 351-358Crossref PubMed Scopus (316) Google Scholar, 12Johnson M.D. Kim H.R. Chesler L. Tsao-Wu G. Bouck N. Polverini P.J. J. Cell. Physiol. 1994; 160: 194-202Crossref PubMed Scopus (269) Google Scholar, 13Anand-Apte B. Pepper M.S. Voest E. Montesano R. Olsen B. Murphy G. Apte S.S. Zetter B. Invest. Ophthalmol. Vis. Sci. 1997; 38: 817-823PubMed Google Scholar). It is now widely appreciated that although all three TIMPs (TIMP-1, -2, and -3) tested to date inhibit MMP activity and the migration of endothelial cells stimulated by angiogenic mitogens, these inhibitors differ in their ability to regulate other angiogenic processes (4Moses M.A. Stem Cells. 1997; 15: 180-189Crossref PubMed Scopus (264) Google Scholar, 11Murphy A.N. Unsworth E.J. Stetler-Stevenson W.G. J. Cell Physiol. 1993; 157: 351-358Crossref PubMed Scopus (316) Google Scholar, 13Anand-Apte B. Pepper M.S. Voest E. Montesano R. Olsen B. Murphy G. Apte S.S. Zetter B. Invest. Ophthalmol. Vis. Sci. 1997; 38: 817-823PubMed Google Scholar). For example, TIMP-2 has the ability to inhibit the proliferation of capillary endothelial cells driven by angiogenic mitogens, while TIMP-1 has been reported to be a modest stimulator of capillary EC growth (11Murphy A.N. Unsworth E.J. Stetler-Stevenson W.G. J. Cell Physiol. 1993; 157: 351-358Crossref PubMed Scopus (316) Google Scholar, 14Hayakawa T. Yamashita K. Tanzawa K. Uchijima E. Iwata K. FEBS Lett. 1992; 298: 29-32Crossref PubMed Scopus (652) Google Scholar). Although TIMP-3 has recently been reported to inhibit the proliferation of genetically modified aortic endothelial cells that overexpress the vascular endothelial growth factor receptor KDR (15Qi J.H. Ebrahem Q. Moore N. Murphy G. Claesson-Welsh L. Bond M. Baker A. Anand-Apte B. Nat. Med. 2003; 9: 407-415Crossref PubMed Scopus (559) Google Scholar), previous studies have reported that TIMP-3 actually had no effect on capillary endothelial cell proliferation (13Anand-Apte B. Pepper M.S. Voest E. Montesano R. Olsen B. Murphy G. Apte S.S. Zetter B. Invest. Ophthalmol. Vis. Sci. 1997; 38: 817-823PubMed Google Scholar). TIMP-4, the latest member of the TIMP family to be cloned, has yet to be rigorously tested. Moreover, neither Batimastat (BB-94), a synthetic MMP inhibitor with potent MMP-inhibitory activity, nor immunoneutralizing antibodies to MMP-2, inhibit capillary EC proliferation (11Murphy A.N. Unsworth E.J. Stetler-Stevenson W.G. J. Cell Physiol. 1993; 157: 351-358Crossref PubMed Scopus (316) Google Scholar). In fact, Batimastat has been shown to stimulate the outgrowth of capillary vessels, another process required for successful angiogenesis (16Koolwijk P. Sidenius N. Peters E. Sier C.F. Hanemaaijer R. Blasi F. van Hinsbergh V.W. Blood. 2001; 97: 3123-3131Crossref PubMed Scopus (98) Google Scholar). It has been suggested that the pleiotropic effects that MMP inhibitors have on specific angiogenic processes, such as the inhibition of endothelial cell proliferation by TIMP-2, may be independent of their metalloproteinase inhibitory activity. The goal of the current study was to identify and characterize the structural domains of TIMP-2 responsible for its endothelial cell growth-modulating activity and its anti-angiogenic activity in vivo. Previous reports have shown that the NH2-terminal domain of TIMPs house the MMP inhibitory activity and that the amino acid sequence of this inhibitory site is highly conserved throughout the TIMP family (17Murphy G. Houbrechts A. Cockett M.I. Williamson R.A. O'Shea M. Docherty A.J. Biochemistry. 1991; 30: 8097-8102Crossref PubMed Scopus (294) Google Scholar, 18O'Shea M. Willenbrock F. Williamson R.A. Cockett M.I. Freedman R.B. Reynolds J.J. Docherty A.J. Murphy G. Biochemistry. 1992; 31: 10146-10152Crossref PubMed Scopus (74) Google Scholar, 19Bodden M.K. Harber G.J. Birkedal-Hansen B. Windsor L.J. Caterina N.C. Engler J.A. Birkedal-Hansen H. J. Biol. Chem. 1994; 269: 18943-18952Abstract Full Text PDF PubMed Google Scholar, 20Gomis-Ruth F.X. Maskos K. Betz M. Bergner A. Huber R. 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We have independently expressed and purified both the NH2-terminal (T2N) and the COOH-terminal (T2C) domains of TIMP-2 as defined by the biochemical and three-dimensional structural information available (17Murphy G. Houbrechts A. Cockett M.I. Williamson R.A. O'Shea M. Docherty A.J. Biochemistry. 1991; 30: 8097-8102Crossref PubMed Scopus (294) Google Scholar, 18O'Shea M. Willenbrock F. Williamson R.A. Cockett M.I. Freedman R.B. Reynolds J.J. Docherty A.J. Murphy G. Biochemistry. 1992; 31: 10146-10152Crossref PubMed Scopus (74) Google Scholar, 19Bodden M.K. Harber G.J. Birkedal-Hansen B. Windsor L.J. Caterina N.C. Engler J.A. Birkedal-Hansen H. J. Biol. Chem. 1994; 269: 18943-18952Abstract Full Text PDF PubMed Google Scholar, 22Muskett F.W. Frenkiel T.A. Feeney J. Freedman R.B. Carr M.D. Williamson R.A. J. Biol. Chem. 1998; 273: 21736-21743Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 23Tolley S. Murphy G. O'Shea M. Ward R. Docherty A. Cockett M. Rawas A. Davies G. J. Mol. Biol. 1993; 229: 1163-1164Crossref PubMed Scopus (6) Google Scholar, 24Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (317) Google Scholar, 25Butler G.S. Hutton M. Wattam B.A. Williamson R.A. Knauper V. Willenbrock F. Murphy G. J. Biol. Chem. 1999; 274: 20391-20396Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) and have systematically analyzed these domains in a variety of in vitro and in vivo angiogenesis assays as well as in MMP activity assays (2Moses M.A. Sudhalter J. Langer R. Science. 1990; 248: 1408-1410Crossref PubMed Scopus (435) Google Scholar, 7Fang J. Shing Y. Wiederschain D. Yan L. Butterfield C. Jackson G. Harper J. Tamvakopoulos G. Moses M.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3884-3889Crossref PubMed Scopus (353) Google Scholar, 26Moses M.A. Sudhalter J. Langer R. J. Cell Biol. 1992; 119: 475-482Crossref PubMed Scopus (92) Google Scholar, 27O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3196) Google Scholar, 28Moses M.A. Wiederschain D. Wu I. Fernandez C.A. Ghazizadeh V. Lane W.S. Flynn E. Sytkowski A. Tao T. Langer R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2645-2650Crossref PubMed Scopus (194) Google Scholar). In this report, we demonstrate, for the first time, that TIMP-2 possesses two distinct anti-angiogenic activities which can be dissociated from each other, both in terms of their structure and their angiogenesis-modulating activities in vivo. The NH2-terminal domain, T2N, which inhibits MMP activity but not capillary EC growth, suppresses embryonic neovascularization in vivo but not the neovascularization driven by an angiogenic mitogen. These results suggest that inhibition of MMP activity alone may not be sufficient to inhibit the mitogen-stimulated neovascularization that is characteristic of pathologic angiogenesis. In contrast, the COOH-terminal domain of TIMP-2, T2C, which is deficient in MMP-inhibitory activity but which inhibits capillary EC proliferation, is a potent inhibitor of both embryonic and mitogen-stimulated angiogenesis in vivo. Further structural mapping of this activity using synthetic peptides demonstrates that the anti-angiogenic activity of T2C can be found in Loop 6. In summary, we have uncoupled the MMP-dependent and MMP-independent angiogenesis-modulating activities of TIMP-2, and in doing so, have identified a novel, potent, small molecular weight inhibitor of angiogenesis. Cloning and Expression of hTIMP-2 and hTIMP-2 Domains—Human TIMP-2 was cloned via PCR of a human fetal heart cDNA library (Clontech, Palo Alto, CA) using primers specific for the mature form of TIMP-2. Two separate TIMP-2 domains were produced using PCR primers designed to yield two fragments of TIMP-2 which encode for either the three NH2-terminal loops (Cys1–Glu127), designated T2N, or the three COOH-terminal loops (Cys128–Pro194), designated T2C. A fourth construct, designated EA-T2N, was designed to produce an inactive mutant of T2N using PCR to add two amino acid residues, glutamic acid and alanine, to the NH2 terminus of T2N. The full-length TIMP-2 PCR product, as well as the two TIMP-2 fragments and the mutant EA-T2N, were subcloned into the yeast expression vector pPICZαA (Invitrogen) and their sequences verified. COOH-terminal His tags were designed into each of the constructs to aid in the purification of expressed proteins. Linearized vectors were electroporated into the methylotrophic yeast Pichia pastoris for expression (Invitrogen), and integrants were selected by culturing on YPDS (2% peptone, 1% yeast extract, 2% glucose, 1 m sorbitol, 2% agar) plates with 100 μg/ml zeocin (Invitrogen) for 3 days. Successful insertion of the genes of interest into the Pichia genome was verified by PCR using Pichia-specific primers, which also verified that recombination occurred at the proper site such that expression of the gene of interest is under the control of the methanol-inducible AOX1 promoter. Four Pichia clones for each gene of interest were tested for expression levels, and the clone expressing the highest amount of each protein was chosen for subsequent studies. Expression conditions were as follows: 25-ml overnight cultures were grown at 30 °C in BMGY medium (2% peptone, 1% yeast extract, 100 mm potassium phosphate pH 6.0, 1.34% yeast nitrogenous base, 1% glucose) containing 100 μg/ml zeocin, and cell pellets were collected the next day by centrifugation at 1500 × g. Cultures were induced by resuspending the cell pellets in 250 ml of methanol-containing medium (BMMY: 2% peptone, 1% yeast extract, 100 mm potassium phosphate, pH 6.0, 1.34% yeast nitrogenous base, 1% methanol), and allowed to grow for 24 h. Medium containing the secreted expressed protein was cleared of cell content by centrifugation at 3000 × g. Purification of Recombinant TIMP-2 and TIMP-2 Domains—Expressed proteins were initially purified from the yeast media using histidine affinity binding to a nickel-nitrilotriacetic acid-agarose resin (Qiagen, Valencia, CA) under native conditions. Briefly, expressed protein in 250 ml of cleared medium was allowed to bind to 5 ml of resin by nutating for 1 h at 4 °C and then centrifuged at low speed to collect the resin. Resin carrying the expressed protein was then loaded into a 12-ml Bio-Rad glass column by gravity, and the resin was washed with 15 ml of buffer containing 10 mm imidazole (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 10 mm imidazole) to reduce nonspecific binding. Protein was then eluted using 10 ml of elution buffer containing 100 mm imidazole (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 100 mm imidazole) and concentrated by centrifugation using membrane concentrators with 3-kDa molecular mass cutoff (Centriprep, Amicon, Beverly, MA). Concentrated protein was further purified to homogeneity by C4 reverse phase HPLC. Separation was carried out over a gradient, from 100% Buffer A (0.05% trifluoroacetic acid in water) to 60% Buffer B (0.05% trifluoroacetic acid in acetonitrile) in 60 min at a flow rate of 1 ml/min. Purity was confirmed by silver staining of SDS-PAGE gels and amino acid composition. SDS-PAGE Electrophoresis and Protein Sequencing—Proteins were resolved on 12% NuPage gels (Invitrogen) run at 200 V for 1 h and visualized either by silver or Coomassie Blue staining. Once purified to homogeneity, protein identity was verified via NH2-terminal amino acid sequencing. Briefly, proteins to be sequenced were blotted onto polyvinylidene difluoride using a Bio-Rad Transblot apparatus for 1 h at 100 V, stained with Amido Black, and excised from the membrane. NH2-terminal sequence was determined by Edman degradation using an Applied Biosystems 477A protein sequencer (Dana Farber Microsequencing Facility, Boston, MA). Peptide Synthesis and Purification—Peptide sequences were designed to represent various smaller structural domains of the carboxyl terminus of TIMP-2. These include: a 10-amino acid peptide corresponding to Loop 5 with sequence TRCPMIPCYI, a 24-amino acid peptide corresponding to Loop 6 with sequence ECLWMDWVTEKNINGHQAKFFACI, and a 19-amino acid peptide corresponding to the carboxyl-terminal tail with sequence AWYRGAAPPKQEFLDIEDP. A fourth peptide of sequence VIRAK corresponding to a conserved sequence in the NH2-terminal domain of all TIMPs was also synthesized for use as a control peptide. All four peptides were synthesized via Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid phase synthesis on Advanced ChemTech 396-5000 multiple peptide synthesizers (Advanced ChemTech, Louisville, KY) to yield peptides as a trifluoroacetic acid salt (ResGen, Invitrogen). Synthetic peptides were further purified by us using C18 reverse phase HPLC to remove any truncation products. Briefly, 1 mg of lyophilized peptide was resuspended in 1 ml of Buffer A (0.05% trifluoroacetic acid in water) and loaded onto the column. Separation was carried out over a gradient, from 20% Buffer B to 60% Buffer B (0.05% trifluoroacetic acid in acetonitrile) in 60 min at a flow rate of 1 ml/min. Fractions containing the peak of interest were collected by hand and subjected to mass spectroscopy to confirm identity and purity of the peptides. Yield was determined by amino acid composition. MMP Inhibitory Activity—MMP inhibitory activity was assessed using a quantitative [14C]collagen film assay, as described previously by us (2Moses M.A. Sudhalter J. Langer R. Science. 1990; 248: 1408-1410Crossref PubMed Scopus (435) Google Scholar). Briefly, 14C-labeled collagen was added to 96-well plates and allowed to polymerize. To determine inhibitory activity, wells were treated with a known amount of activated type I collagenase plus test sample or with collagenase alone and the plates incubated at 37 °C for 2.5 h to allow for release of 14C by the enzyme. Supernantants were then analyzed in a Wallac scintillation counter, and percent inhibition of collagenolytic activity was calculated. An IC50 unit was defined as the amount of protein necessary to inhibit the proteolytic activity of collagenase by 50%. Cell Culture and Capillary Endothelial Cell Proliferation—Capillary EC, isolated from bovine adrenal cortex, were a kind gift of Dr. Judah Folkman (Children's Hospital, Boston) and were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% calf serum (HyClone) and 3 ng/ml basic fibroblast growth factor (bFGF), and grown at 37 °C in 10% CO2. Capillary EC proliferation was measured as reported previously by us (2Moses M.A. Sudhalter J. Langer R. Science. 1990; 248: 1408-1410Crossref PubMed Scopus (435) Google Scholar, 26Moses M.A. Sudhalter J. Langer R. J. Cell Biol. 1992; 119: 475-482Crossref PubMed Scopus (92) Google Scholar, 27O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3196) Google Scholar, 28Moses M.A. Wiederschain D. Wu I. Fernandez C.A. Ghazizadeh V. Lane W.S. Flynn E. Sytkowski A. Tao T. Langer R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2645-2650Crossref PubMed Scopus (194) Google Scholar, 29Braunhut S.J. Moses M.A. J. Biol. Chem. 1994; 269: 13472-13479Abstract Full Text PDF PubMed Google Scholar). Briefly, capillary EC were plated on pregelatinized 96-well plates at a density of 2000 cells per well in Dulbecco's modified Eagle's medium supplemented with 5% calf serum and allowed to attach for 24 h. The next day, cells were treated with fresh medium with or without 1 ng/ml bFGF and challenged with the test proteins at various concentrations. All samples were tested in duplicate. Control wells contained cell treated with medium alone or medium with bFGF. After 72 h, the medium was removed, and the cells were lysed in buffer containing Triton X-100 and the phosphatase substrate p-nitrophenyl phosphate. After a 2-h incubation at 37 °C, NaOH was added to each well to terminate the reaction and cell density was determined by colorimetric analysis using a SpectraMax 190 multiwell plate reader (Molecular Devices, Sunnyvale, CA). All samples were tested in duplicate in at least three independent experiments. Chick Chorioallantoic Membrane (CAM) Assay—The chick CAM assay was conducted as reported previously by us (2Moses M.A. Sudhalter J. Langer R. Science. 1990; 248: 1408-1410Crossref PubMed Scopus (435) Google Scholar, 7Fang J. Shing Y. Wiederschain D. Yan L. Butterfield C. Jackson G. Harper J. Tamvakopoulos G. Moses M.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3884-3889Crossref PubMed Scopus (353) Google Scholar, 26Moses M.A. Sudhalter J. Langer R. J. Cell Biol. 1992; 119: 475-482Crossref PubMed Scopus (92) Google Scholar, 27O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3196) Google Scholar, 28Moses M.A. Wiederschain D. Wu I. Fernandez C.A. Ghazizadeh V. Lane W.S. Flynn E. Sytkowski A. Tao T. Langer R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2645-2650Crossref PubMed Scopus (194) Google Scholar). Briefly, 3-day-old chick embryos were removed from their shells and incubated in plastic Petri dishes for 3 days. On embryonic day 6, samples and controls mixed into methylcellulose discs were applied to the surfaces of developing CAMs, above the dense subectodermal plexus. After 48 h of incubation, the eggs were examined for vascular reactions under a dissecting scope (×60) and photographed. All samples were tested in triplicate for each dose tested and determinations were made by three independent members of the laboratory, in a double-blinded fashion. Mouse Corneal Pocket Assay—In vivo inhibition of angiogenesis was also tested using the mouse corneal pocket assay as described previously (27O'Reilly M.S. Holmgren L. Shing Y. Chen C. Rosenthal R.A. Moses M. Lane W.S. Cao Y. Sage E.H. Folkman J. Cell. 1994; 79: 315-328Abstract Full Text PDF PubMed Scopus (3196) Google Scholar, 28Moses M.A. Wiederschain D. Wu I. Fernandez C.A. Ghazizadeh V. Lane W.S. Flynn E. Sytkowski A. Tao T. Langer R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2645-2650Crossref PubMed Scopus (194) Google Scholar). Hydron pellets containing sucrose octasulfate and either test sample (5 μg) plus bFGF (40 ng) or bFGF (40 ng) alone were implanted into corneal micropockets of C57Bl/6. Each animal carried a pellet containing the test sample plus bFGF in one eye, and a control bFGF pellet in the contralateral eye. After 6 days, angiogenesis was evaluated using a slit lamp microscope, and each eye was photographed. The area of neovascularization for each cornea was calculated from the length of the vessels (VL) invading the cornea as well as the clock hours (CH) covered as described by the formula VL × CH × 0.0628. Cloning, Expression, and Purification of TIMP-2 and TIMP-2 Domains—Human TIMP-2 was cloned from a human heart cDNA library using high fidelity PCR and primers designed to produce full-length TIMP-2, a 3′-deletion fragment and a 5′-deletion fragment (Fig. 1B). Cloning of these gene fragments result in the expression of two deletion mutants of TIMP-2 as distinct peptides. The first is composed of the three NH2-terminal disulfide-bonded loops (Cys1–Glu127) designated T2N (∼15 kDa), and the second is composed of the three COOH-terminal disulfide-bonded loops (Cys128–Pro194) designated T2C (∼8.5 kDa). In addition, following recent reports suggesting that the addition of amino acid residues to the NH2-terminal Cys1 results in the abrogation of MMP-inhibitory activity (30Wingfield P.T. Sax J.K. Stahl S.J. Kaufman J. Palmer I. Chung V. Corcoran M.L. Kleiner D.E. Stetler-Stevenson W.G. J. Biol. Chem. 1999; 274: 21362-21368Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 31Hoegy S.E. Oh H.R. Corcoran M.L. Stetler-Stevenson W.G. J. Biol. Chem. 2001; 276: 3203-3214Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), a mutant form of T2N that encodes for a form of T2N with additional glutamic acid and alanine residues at the NH2 terminus of the protein, and designated EA-T2N, was designed, expressed, and purified for use as a control for MMP inhibition. All expressed proteins were NH2-terminally sequenced to verify either native or mutant forms. All proteins, as well as intact human TIMP-2, were expressed using the Pichia pastoris yeast expression system, which has been shown to successfully produce other disulfide-bonded proteins (32Ikegaya K. Hirose M. Ohmura T. Nokihara K. Anal. Chem. 1997; 69: 1986-1991Crossref PubMed Scopus (24) Google Scholar, 33Sun J. Bottomley S.P. Kumar S. Bird P.I. Biochem. Biophys. Res. Commun. 1997; 238: 920-924Crossref PubMed Scopus (31) Google Scholar). Expressed proteins with incorporated COOH-terminal His-tags are secreted into the growth media and purified to homogeneity by histidine affinity chromatography followed by C4 reverse phase HPLC. A sample chromatogram of the purification of T2C by reverse phase HPLC is shown in Fig. 1C. Sample purity was monitored by SDS-PAGE run under reducing conditions followed by silver-staining as previously reported (2Moses M.A. Sudhalter J. Langer R. Science. 1990; 248: 1408-1410Crossref PubMed Scopus (435) Google Scholar, 28Moses M.A. Wiederschain D. Wu I. Fernandez C.A. Ghazizadeh V. Lane W.S. Flynn E. Sytkowski A. Tao T. Langer R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2645-2650Crossref PubMed Scopus (194) Google Scholar). Fig. 1D shows a representative example of a silver-stained gel of both the HPLC starting material (lane 1) and purified T2C (lane 2). An exa
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