Structural and Functional Uncoupling of the Enzymatic and Angiogenic Inhibitory Activities of Tissue Inhibitor of Metalloproteinase-2 (TIMP-2)

Abstract

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. 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). is that although TIMPs and to inhibit MMP activity and the of endothelial by angiogenic these inhibitors in their ability to regulate angiogenic processes 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). TIMP-2 has the ability to inhibit the proliferation of capillary endothelial driven by angiogenic while has been reported to be a of capillary EC growth A.N. Unsworth E.J. Stetler-Stevenson W.G. J. Cell Physiol. 1993; 157: 351-358Crossref PubMed Scopus (316) Google Scholar, T. K. Tanzawa K. E. K. PubMed Scopus Google Scholar). has been reported to inhibit the proliferation of endothelial that the endothelial growth N. Murphy G. L. M. A. B. Nat. 9: PubMed Scopus Google studies have reported that no on capillary endothelial cell proliferation 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). the of the TIMP family to be has to be a synthetic MMP inhibitor with potent activity, to inhibit capillary EC proliferation A.N. Unsworth E.J. Stetler-Stevenson W.G. J. Cell Physiol. 1993; 157: 351-358Crossref PubMed Scopus (316) Google Scholar). In has been shown to the of capillary for angiogenesis P. N. E. R. 2001; 97: PubMed Scopus Google Scholar). has been that the that MMP inhibitors have on specific angiogenic as the inhibition of endothelial cell proliferation by TIMP-2, may be of their metalloproteinase inhibitory activity. of the was to identify and characterize the domains of TIMP-2 for its endothelial cell activity and its anti-angiogenic activity in vivo. have shown that the NH2-terminal domain of TIMPs the MMP inhibitory activity and that the acid of inhibitory is the TIMP family G. A. M. 1991; PubMed Scopus Google Scholar, M. Murphy G. PubMed Scopus Google Scholar, B. J.A. H. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, K. M. A. R. K. N. Nagase H. K. H. 1997; PubMed Scopus Google Scholar, S. R. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. M.D. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). We that the anti-proliferative activity of TIMP-2 be in the COOH-terminal domain To we have conducted a series of structure-function studies to identify the of the anti-angiogenic activity of We have and both the NH2-terminal and the COOH-terminal (T2C) domains of TIMP-2 as by the and G. A. M. 1991; PubMed Scopus Google Scholar, M. Murphy G. PubMed Scopus Google Scholar, B. J.A. H. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, J. M.D. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Murphy G. M. R. A. M. A. G. J. Biol. 1993; PubMed Scopus Google Scholar, C. R. D. A. H. K. J. 1998; PubMed Scopus Google Scholar, M. Murphy G. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) and have these domains in a variety of in vitro and in vivo angiogenesis as well as in MMP activity (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, M.A. Sudhalter J. Langer R. J. Cell Biol. PubMed Scopus Google Scholar, M.S. L. Shing Y. C. Moses M. Y. Folkman J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, M.A. Wiederschain D. E. A. T. Langer R. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). In report, we for the first time, that TIMP-2 possesses two distinct anti-angiogenic activities which can be from each other, both in of their and their activities in vivo. NH2-terminal T2N, which inhibits MMP activity not capillary EC growth, embryonic neovascularization in vivo not the neovascularization driven by an angiogenic These that inhibition of MMP activity alone may not be sufficient to inhibit the mitogen-stimulated neovascularization that is characteristic of angiogenesis. In the COOH-terminal domain of TIMP-2, T2C, which is in activity which inhibits capillary EC is a potent inhibitor of both embryonic and mitogen-stimulated angiogenesis in vivo. of activity synthetic that the anti-angiogenic activity of T2C can be in Loop In we have uncoupled the MMP-dependent and MMP-independent activities of TIMP-2, and in have a inhibitor of angiogenesis. and of and TIMP-2 was of a human specific for the of TIMP-2 domains were to two of TIMP-2 which for the NH2-terminal T2N, the COOH-terminal EA-T2N, was to an mutant of T2N to two acid acid and to the of TIMP-2 as well as the two TIMP-2 and the mutant EA-T2N, were the and their COOH-terminal were each of the to in the of were the for and were by on with for of the of the was by which that at the that of the of is the of the for each of were for and the the of each was for were as were at in and cell were the by at were by the cell in of and to for the was of cell by at of TIMP-2 and TIMP-2 were from the to a in of was to to of by for at and at to the the was a by and the was with of to was of and by with was further to by was a from acid in to acid in in at a of was by of and acid and were on at for and by to was NH2-terminal acid to be were a for at with and from the NH2-terminal was by an and were to domains of the of These a acid peptide corresponding to Loop with a 24-amino acid peptide corresponding to Loop 6 with and a acid peptide corresponding to the with peptide of corresponding to a in the NH2-terminal domain of TIMPs was for as a were on peptide to as a acid were further by to of peptide was in of acid in and the was a from to acid in in at a of the of were by and to to and of the was by acid MMP inhibitory activity was a as previously by (2Moses M.A. Sudhalter J. Langer R. Science. 1990; 248: 1408-1410Crossref PubMed Scopus (435) Google Scholar). was to and to To inhibitory activity, were with a of with alone and the at for to for of by the were in a and inhibition of activity was was as the of to inhibit the proteolytic activity of by Cell and Cell EC, from were a of Folkman and were in with and basic fibroblast growth and at in EC proliferation was as reported previously by (2Moses M.A. Sudhalter J. Langer R. Science. 1990; 248: 1408-1410Crossref PubMed Scopus (435) Google Scholar, M.A. Sudhalter J. Langer R. J. Cell Biol. PubMed Scopus Google Scholar, M.S. L. Shing Y. C. Moses M. Y. Folkman J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, M.A. Wiederschain D. E. A. T. Langer R. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, Moses M.A. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). capillary EC were on at a of well in with and to for were with with and with the at were in cell with alone with the was and the were in and the a at was to each well to the and cell was by a were in in at was conducted as reported previously by (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, M.A. Sudhalter J. Langer R. J. Cell Biol. PubMed Scopus Google Scholar, M.S. L. Shing Y. C. Moses M. Y. Folkman J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, M.A. Wiederschain D. E. A. T. Langer R. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). were from their and in for embryonic and were to the of the of the were for a and were in for each and were by members of the in a vivo inhibition of angiogenesis was the as previously M.S. L. Shing Y. C. Moses M. Y. Folkman J. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, M.A. Wiederschain D. E. A. T. Langer R. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). and alone were of a the in and a in the 6 angiogenesis was a and each was of neovascularization for each was from the of the the as well as the as by the and of TIMP-2 and TIMP-2 TIMP-2 was from a human high and to TIMP-2, a and a of these in the of two of TIMP-2 as distinct first is of the NH2-terminal T2N and the second is of the COOH-terminal T2C In addition, suggesting that the addition of acid to the NH2-terminal in the of activity J. Corcoran Stetler-Stevenson W.G. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, H.R. Corcoran Stetler-Stevenson W.G. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google a mutant of T2N that for a of T2N with acid and at the of the and EA-T2N, was and for as a for MMP were to mutant as well as intact human TIMP-2, were the which has been shown to K. M. T. K. Chem. 1997; PubMed Scopus Google Scholar, J. S. Biophys. 1997; PubMed Scopus Google Scholar). with COOH-terminal the growth and to by by of the of T2C by is shown in was by by as previously reported (2Moses M.A. Sudhalter J. Langer R. Science. 1990; 248: 1408-1410Crossref PubMed Scopus (435) Google Scholar, M.A. Wiederschain D. E. A. T. Langer R. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). a of a of both the and T2C of a of each of the TIMP-2 and TIMP-2 domains is shown in of each were as for T2N, for T2C, for EA-T2N, and for intact T2N, T2C, demonstrate that the NH2-terminal domain of TIMP-2, T2N, activity as reported previously G. A. M. 1991; PubMed Scopus Google Scholar, M. Murphy G. PubMed Scopus Google Scholar, J. M.D. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. Murphy G. M. R. A. M. A. G. J. Biol. 1993; PubMed Scopus Google Scholar, C. R. D. A. H. K. J. 1998; PubMed Scopus Google Scholar, M. Murphy G. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) and to the ability of to we TIMP-2, the TIMP-2 and an MMP of T2N, EA-T2N, for their ability to inhibit MMP activity a MMP (2Moses M.A. Sudhalter J. Langer R. Science. 1990; 248: 1408-1410Crossref PubMed Scopus (435) Google Scholar). T2N inhibited MMP activity at concentrations comparable to intact TIMP-2, with an of significant inhibition of MMP activity was for T2C EA-T2N, at concentrations that of the of T2N T2C, T2N, the of the TIMP-2 domains for their ability to capillary EC proliferation in vitro and that T2C alone inhibited capillary EC proliferation driven by the angiogenic with an of These with those previously reported for TIMP-2 A.N. Unsworth E.J. Stetler-Stevenson W.G. J. Cell Physiol. 1993; 157: 351-358Crossref PubMed Scopus (316) Google Scholar). T2C inhibited endothelial growth proliferation at the not T2N is for inhibition of MMP activity, it no significant on capillary EC These demonstrate that the inhibitory of TIMP-2 on capillary EC proliferation is of MMP inhibition and that these can be from each anti-proliferative activity of T2C a second anti-angiogenic domain T2C and T2N in in the that MMP inhibitors have been shown to inhibit angiogenesis and that T2C inhibits capillary endothelial cell we both T2C and T2N for their ability to inhibit angiogenesis in vivo in the were in these shown in were with of each of the at the in which inhibition be achieved for addition of T2C T2N to the chorioallantoic resulted in a in neovascularization and T2C at as as while T2N inhibited at EA-T2N, which is in MMP and capillary EC inhibitory activity, not inhibit neovascularization TIMP-2 was used as a T2C in the the ability of T2C to inhibit angiogenesis in a second and in vivo the the neovascularization is by the addition of an exogenous angiogenic T2C resulted in an of neovascularization with the with only and of the with T2N resulted in only inhibition of angiogenesis and not from the inhibition were with the MMP of T2N, and In intact TIMP-2 resulted in potent inhibition of neovascularization which was the as that with T2C inhibition of neovascularization from of is shown in Loop a of T2C, EC further the anti-proliferative activity of TIMP-2 T2C, corresponding to domains of T2C were and for their ability to inhibit capillary EC proliferation in These domains and their acid in corresponding to Loop 6 of intact TIMP-2 with acid inhibited capillary EC while a peptide corresponding to the no effect. peptide corresponding to Loop of intact TIMP-2, the proliferation of capillary EC peptide corresponding to the TIMP of Loop of intact TIMPs acid was used as a peptide and resulted in no inhibition of capillary EC proliferation not Loop 6 inhibited capillary EC proliferation at comparable with those of intact TIMP-2, suggesting that Loop 6 may the anti-proliferative activity of Interestingly, Loop 6 not inhibit the proliferation of as suggesting that the inhibitory of Loop 6 may be specific to endothelial not of and of Loop 6 no in the suggesting that Loop 6 as a and that both Loop 6 and in the in vivo anti-angiogenic activity of T2C is Loop we Loop 6 in the chorioallantoic synthetic to Loop and to were used as Loop 6 resulted in at comparable with those for T2C Loop the significant on neovascularization not Interestingly, although Loop the proliferation of capillary EC in not the of neovascularization in vivo in We Loop 6 in the to peptide alone could inhibit in vivo angiogenesis driven by the exogenous addition of an angiogenic with Loop 6 a significant of neovascularization with the These comparable with those for intact TIMP-2 and T2C and demonstrate that Loop 6 is a inhibitor of angiogenesis in vivo. Loop a inhibitor of angiogenesis. is that TIMPs with to cell growth, angiogenesis, and (1Brew K. Dinakarpandian D. Nagase H. Biochim. Biophys. Acta. 2000; 1477: 267-283Crossref PubMed Scopus (1628) Google Scholar, Murphy G. J. Cell Sci. PubMed Scopus Google Scholar). of these differences in TIMP activities can be to differences in their for their role in MMP to be both and TIMP-2 have been shown to activity R.M. PubMed Scopus Google Scholar, W.G. N. PubMed Scopus Google Scholar). has been reported to inhibit tumor A. A. M. C. M. Murphy G. 1998; PubMed Scopus Google Scholar) as well as H. Y. 1997; 9: PubMed Scopus Google Scholar, Murphy G. J. 1999; PubMed Scopus Google Scholar, M. Murphy G. A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google while can inhibit L. Stetler-Stevenson W.G. L. J. P. A. Stetler-Stevenson M. J. Invest. 1998; PubMed Scopus Google Scholar, L. L. Stetler-Stevenson M. 1998; PubMed Google Scholar, G. R. Kim H.R. Cancer 1999; Google Scholar). TIMP-2 has been shown to in M.S. L. Stetler-Stevenson W.G. Stetler-Stevenson M. N. Y. Acad. Sci. 1999; PubMed Scopus Google Scholar, K. A. A. M. M. Cancer 2000; Google Scholar) to have no in M. Murphy G. A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). is the that only TIMP-2 has been shown to inhibit mitogen-driven capillary EC proliferation 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). that TIMPs can inhibit MMP activity, the ability of TIMP-2 to inhibit capillary EC proliferation has been to a second of TIMP-2, of its activity A.N. Unsworth E.J. Stetler-Stevenson W.G. J. Cell Physiol. 1993; 157: 351-358Crossref PubMed Scopus (316) Google Scholar, H.R. Corcoran Stetler-Stevenson W.G. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). on these we that be a to TIMP-2 that be for the inhibition of mitogen-driven capillary EC a second anti-angiogenic the no of TIMP for growth inhibition has been demonstrated A.N. Unsworth E.J. Stetler-Stevenson W.G. J. Cell Physiol. 1993; 157: 351-358Crossref PubMed Scopus (316) Google Scholar, N. Murphy G. L. M. A. B. Nat. 9: PubMed Scopus Google Scholar, H.R. Corcoran Stetler-Stevenson W.G. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, T. K. E. A. J. Cell Sci. 1994; PubMed Google Scholar). In structure-function we and characterize the MMP-dependent and MMP-independent anti-angiogenic of TIMP-2 and for the first time, that these activities In we have a novel inhibitor of angiogenesis, Loop first series of demonstrated that TIMP-2 possesses two anti-angiogenic that is with MMP inhibition and that is not These in vitro studies that the ability of TIMP-2 to inhibit capillary EC proliferation is in the MMP In addition, T2C inhibited angiogenesis in both the chorioallantoic and in the at a level comparable to that with intact TIMP-2 We further mapped the anti-proliferative activity of T2C to the 24-amino acid of Loop 6 as with T2C, Loop 6 inhibited angiogenesis in two in vivo models is to that the of the in the with T2C Loop 6 was from in those with with T2C Loop 6 and the a of Jackson Cancer PubMed Scopus Google Scholar). inhibition of neovascularization in the of the T2N, could be by a of that numerous studies have demonstrated the for MMP activity at the of angiogenesis T. Y. Stetler-Stevenson W.G. Cancer 1993; Google Scholar, G. J. Stetler-Stevenson W.G. Science. 1997; PubMed Scopus Google Scholar, Z. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google the of the with T2N is with the inhibition of that T2C Loop 6 inhibit MMP activity, demonstrate that TIMP-2 has an anti-angiogenic domain that is of MMP Interestingly, a has that T2C can inhibit the of by from the J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). further that activity is by the of the of TIMP-2 to the domain of an that is for cell of These findings with to with that demonstrate that has no anti-proliferative anti-angiogenic activity that the ability of TIMP-2 to inhibit capillary EC which we have to Loop is not only of its ability to inhibit MMP activity, is to be of its in to be not the anti-proliferative activities of T2C its Loop in vivo alone as of intact of was to not domains in TIMP-2 that be of activity. in that is in the and that TIMP-2 two activities that inhibit angiogenesis in vivo in two models with in vivo angiogenesis studies conducted in important differences in the types of angiogenesis inhibition effected by domains of the MMP inhibitory T2N, embryonic activity, domain not the mitogen-stimulated neovascularization that aberrant angiogenesis in vivo. To the role of MMP inhibition alone on these two types of in vivo angiogenesis, we T2N studies by an MMP in the of on that the addition of to the of TIMP-2 resulted in the of MMP inhibitory activity J. Corcoran Stetler-Stevenson W.G. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). not inhibit MMP activity and not inhibit embryonic angiogenesis in the In although T2N resulted in only inhibition of angiogenesis in the these not from those with the MMP EA-T2N, suggesting that MMP inhibition alone may not be sufficient to inhibit of mitogen-stimulated In both the anti-angiogenic domain T2C and its Loop to be inhibitors of both normal and mitogen-stimulated angiogenesis in vivo. These that the inhibition of MMP activity may be sufficient to inhibit angiogenesis as represented by the embryonic of the chorioallantoic alone may not be sufficient to inhibit the neovascularization which is characteristic of pathological may the of of synthetic MMP inhibitors B. Science. PubMed Scopus Google activity is on In we identify Loop 6 as a new inhibitor of angiogenesis the of is that inhibitors as Loop 6 may therapeutic potential both in of of and as well as Loop as we have demonstrated in is to by synthetic as a of its acid and these from may the of J. T. J. 2001; PubMed Scopus Google Scholar). a number of with to the TIMPs and their important (1Brew K. Dinakarpandian D. Nagase H. Biochim. Biophys. Acta. 2000; 1477: 267-283Crossref PubMed Scopus (1628) Google Scholar, Murphy G. J. Cell Sci. PubMed Scopus Google Scholar). Among these Nagase and (1Brew K. Dinakarpandian D. Nagase H. Biochim. Biophys. Acta. 2000; 1477: 267-283Crossref PubMed Scopus (1628) Google Scholar) have the for a of the between MMP cell and and the inhibition of angiogenesis. these and the for capillary endothelial cell and the inhibition of angiogenesis in vivo by these activities from the inhibition of MMP activity. In we have Loop a potent inhibitor of angiogenesis, which as a of its may a role in the of by angiogenesis. We to Harper for of the and for