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. 1992 Oct 2;119(2):475–482. doi: 10.1083/jcb.119.2.475

Isolation and characterization of an inhibitor of neovascularization from scapular chondrocytes

PMCID: PMC2289656  PMID: 1383232

Abstract

An inhibitor of neovascularization from the conditioned media of scapular chondrocytes established and maintained in serum-free culture has been isolated and characterized. To determine whether this chondrocyte-derived inhibitor (ChDI) was capable of inhibiting neovascularization in vivo, this protein was assayed in the chick chorioallantoic membrane assay. ChDI was a potent inhibitor of angiogenesis in vivo (4 micrograms = 87% avascular zones). This inhibitor is also an inhibitor of fibroblast growth factor-stimulated capillary endothelial cell (EC) proliferation and migration, as well as being an inhibitor of mammalian collagenase. ChDI significantly suppressed capillary EC proliferation in a dose-dependent, reversible manner with an IC50 (the inhibitory concentration at which 50% inhibition is achieved) of 2.025 micrograms/ml. Inhibition by ChDI of growth factor-stimulated capillary EC migration was also observed using a modified Boyden chamber assay (IC50 = 255 ng/ml). SDS-PAGE analysis followed by silver staining of ChDI purified to apparent homogeneity revealed a single band having an M(r) of 35,550. Gel elution experiments demonstrated that only protein eluting at this molecular weight was anti-angiogenic. These studies are the first demonstration that chondrocytes in culture can produce a highly enriched, potent inhibitor of neovascularization which also inhibits collagenase.

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Selected References

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  1. Ausprunk D. H., Folkman J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res. 1977 Jul;14(1):53–65. doi: 10.1016/0026-2862(77)90141-8. [DOI] [PubMed] [Google Scholar]
  2. Block J. A., Inerot S. E., Gitelis S., Kimura J. H. Synthesis of chondrocytic keratan sulphate-containing proteoglycans by human chondrosarcoma cells in long-term cell culture. J Bone Joint Surg Am. 1991 Jun;73(5):647–658. [PubMed] [Google Scholar]
  3. Braunhut S. J., Palomares M. Modulation of endothelial cell shape and growth by retinoids. Microvasc Res. 1991 Jan;41(1):47–62. doi: 10.1016/0026-2862(91)90007-x. [DOI] [PubMed] [Google Scholar]
  4. Brem H., Folkman J. Inhibition of tumor angiogenesis mediated by cartilage. J Exp Med. 1975 Feb 1;141(2):427–439. doi: 10.1084/jem.141.2.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bunning R. A., Murphy G., Kumar S., Phillips P., Reynolds J. J. Metalloproteinase inhibitors from bovine cartilage and body fluids. Eur J Biochem. 1984 Feb 15;139(1):75–80. doi: 10.1111/j.1432-1033.1984.tb07978.x. [DOI] [PubMed] [Google Scholar]
  6. Cambray G. J., Murphy G., Page-Thomas D. P., Reynolds J. J. The production in culture of metalloproteinases and an inhibitor by joint tissues from normal rabbits, and from rabbits with a model arthritis. I. Synovium. Rheumatol Int. 1981;1(1):11–16. doi: 10.1007/BF00541217. [DOI] [PubMed] [Google Scholar]
  7. Connolly D. T., Knight M. B., Harakas N. K., Wittwer A. J., Feder J. Determination of the number of endothelial cells in culture using an acid phosphatase assay. Anal Biochem. 1986 Jan;152(1):136–140. doi: 10.1016/0003-2697(86)90131-4. [DOI] [PubMed] [Google Scholar]
  8. Doctrow S. R., Folkman J. Protein kinase C activators suppress stimulation of capillary endothelial cell growth by angiogenic endothelial mitogens. J Cell Biol. 1987 Mar;104(3):679–687. doi: 10.1083/jcb.104.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eisenstein R., Kuettner K. E., Neapolitan C., Soble L. W., Sorgente N. The resistance of certain tissues to invasion. III. Cartilage extracts inhibit the growth of fibroblasts and endothelial cells in culture. Am J Pathol. 1975 Nov;81(2):337–348. [PMC free article] [PubMed] [Google Scholar]
  10. Falk W., Goodwin R. H., Jr, Leonard E. J. A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration. J Immunol Methods. 1980;33(3):239–247. doi: 10.1016/0022-1759(80)90211-2. [DOI] [PubMed] [Google Scholar]
  11. Folkman J. Angiogenesis and its inhibitors. Important Adv Oncol. 1985:42–62. [PubMed] [Google Scholar]
  12. Folkman J., Haudenschild C. C., Zetter B. R. Long-term culture of capillary endothelial cells. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5217–5221. doi: 10.1073/pnas.76.10.5217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Folkman J., Klagsbrun M. Angiogenic factors. Science. 1987 Jan 23;235(4787):442–447. doi: 10.1126/science.2432664. [DOI] [PubMed] [Google Scholar]
  14. Gabrielides C., Barreau F. A vertebrate interstitial collagenase inhibitor from bovine scapular cartilage: purification and characterization. Biochim Biophys Acta. 1987 Apr 16;924(1):238–247. doi: 10.1016/0304-4165(87)90092-4. [DOI] [PubMed] [Google Scholar]
  15. Gitlin J. D., D'Amore P. A. Culture of retinal capillary cells using selective growth media. Microvasc Res. 1983 Jul;26(1):74–80. doi: 10.1016/0026-2862(83)90056-0. [DOI] [PubMed] [Google Scholar]
  16. Harris E. D., Jr Recent insights into the pathogenesis of the proliferative lesion in rheumatoid arthritis. Arthritis Rheum. 1976 Jan-Feb;19(1):68–72. doi: 10.1002/art.1780190111. [DOI] [PubMed] [Google Scholar]
  17. Hoyer L. W., De los Santos R. P., Hoyer J. R. Antihemophilic factor antigen. Localization in endothelial cells by immunofluorescent microscopy. J Clin Invest. 1973 Nov;52(11):2737–2744. doi: 10.1172/JCI107469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Johnson-Wint B. A quantitative collagen film collagenase assay for large numbers of samples. Anal Biochem. 1980 May 1;104(1):175–181. doi: 10.1016/0003-2697(80)90295-x. [DOI] [PubMed] [Google Scholar]
  19. Klagsbrun M. Large-scale preparation of chondrocytes. Methods Enzymol. 1979;58:560–564. doi: 10.1016/s0076-6879(79)58171-3. [DOI] [PubMed] [Google Scholar]
  20. Kuettner K. E., Hiti J., Eisenstein R., Harper E. Collagenase inhibition by cationic proteins derived from cartilage and aorta. Biochem Biophys Res Commun. 1976 Sep 7;72(1):40–46. doi: 10.1016/0006-291x(76)90957-8. [DOI] [PubMed] [Google Scholar]
  21. Kuettner K. E., Pauli B. U., Soble L. Morphological studies on the resistance of cartilage to invasion by osteosarcoma cells in vitro and in vivo. Cancer Res. 1978 Feb;38(2):277–287. [PubMed] [Google Scholar]
  22. Kusaka M., Sudo K., Fujita T., Marui S., Itoh F., Ingber D., Folkman J. Potent anti-angiogenic action of AGM-1470: comparison to the fumagillin parent. Biochem Biophys Res Commun. 1991 Feb 14;174(3):1070–1076. doi: 10.1016/0006-291x(91)91529-l. [DOI] [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Langer R., Brem H., Falterman K., Klein M., Folkman J. Isolations of a cartilage factor that inhibits tumor neovascularization. Science. 1976 Jul 2;193(4247):70–72. doi: 10.1126/science.935859. [DOI] [PubMed] [Google Scholar]
  25. Langer R., Conn H., Vacanti J., Haudenschild C., Folkman J. Control of tumor growth in animals by infusion of an angiogenesis inhibitor. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4331–4335. doi: 10.1073/pnas.77.7.4331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Langer R., Murray J. Angiogenesis inhibitors and their delivery systems. Appl Biochem Biotechnol. 1983 Feb;8(1):9–24. doi: 10.1007/BF02798344. [DOI] [PubMed] [Google Scholar]
  27. Lutty G. A., Thompson D. C., Gallup J. Y., Mello R. J., Patz A., Fenselau A. Vitreous: an inhibitor of retinal extract-induced neovascularization. Invest Ophthalmol Vis Sci. 1983 Jan;24(1):52–56. [PubMed] [Google Scholar]
  28. Maione T. E., Gray G. S., Petro J., Hunt A. J., Donner A. L., Bauer S. I., Carson H. F., Sharpe R. J. Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides. Science. 1990 Jan 5;247(4938):77–79. doi: 10.1126/science.1688470. [DOI] [PubMed] [Google Scholar]
  29. Morales T. I., Kuettner K. E., Howell D. S., Woessner J. F. Characterization of the metalloproteinase inhibitor produced by bovine articular chondrocyte cultures. Biochim Biophys Acta. 1983 Oct 18;760(2):221–229. doi: 10.1016/0304-4165(83)90167-8. [DOI] [PubMed] [Google Scholar]
  30. Morris G. M. A high molecular weight collagenase inhibitor made by rabbit chondrocytes in cell culture. Matrix. 1989 Mar;9(2):127–134. doi: 10.1016/s0934-8832(89)80030-7. [DOI] [PubMed] [Google Scholar]
  31. Moses M. A., Langer R. A metalloproteinase inhibitor as an inhibitor of neovascularization. J Cell Biochem. 1991 Nov;47(3):230–235. doi: 10.1002/jcb.240470308. [DOI] [PubMed] [Google Scholar]
  32. Moses M. A., Langer R. Inhibitors of angiogenesis. Biotechnology (N Y) 1991 Jul;9(7):630–634. doi: 10.1038/nbt0791-630. [DOI] [PubMed] [Google Scholar]
  33. Moses M. A., Sudhalter J., Langer R. Identification of an inhibitor of neovascularization from cartilage. Science. 1990 Jun 15;248(4961):1408–1410. doi: 10.1126/science.1694043. [DOI] [PubMed] [Google Scholar]
  34. Murphy G., McGuire M. B., Russell R. G., Reynolds J. J. Characterization of collagenase, other metallo-proteinases and an inhibitor (TIMP) produced by human synovium and cartilage in culture. Clin Sci (Lond) 1981 Dec;61(6):711–716. doi: 10.1042/cs0610711. [DOI] [PubMed] [Google Scholar]
  35. Murray J. B., Allison K., Sudhalter J., Langer R. Purification and partial amino acid sequence of a bovine cartilage-derived collagenase inhibitor. J Biol Chem. 1986 Mar 25;261(9):4154–4159. [PubMed] [Google Scholar]
  36. Pauli B. U., Memoli V. A., Kuettner K. E. Regulation of tumor invasion by cartilage-derived anti-invasion factor in vitro. J Natl Cancer Inst. 1981 Jul;67(1):65–73. [PubMed] [Google Scholar]
  37. Pepper M. S., Montesano R., Vassalli J. D., Orci L. Chondrocytes inhibit endothelial sprout formation in vitro: evidence for involvement of a transforming growth factor-beta. J Cell Physiol. 1991 Jan;146(1):170–179. doi: 10.1002/jcp.1041460122. [DOI] [PubMed] [Google Scholar]
  38. Presta M., Rifkin D. B. New aspects of blood vessel growth: tumor and tissue-derived angiogenesis factors. Haemostasis. 1988;18(1):6–17. doi: 10.1159/000215778. [DOI] [PubMed] [Google Scholar]
  39. Roughley P. J., Murphy G., Barrett A. J. Proteinase inhibitors of bovine nasal cartilage. Biochem J. 1978 Mar 1;169(3):721–724. doi: 10.1042/bj1690721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sano H., Forough R., Maier J. A., Case J. P., Jackson A., Engleka K., Maciag T., Wilder R. L. Detection of high levels of heparin binding growth factor-1 (acidic fibroblast growth factor) in inflammatory arthritic joints. J Cell Biol. 1990 Apr;110(4):1417–1426. doi: 10.1083/jcb.110.4.1417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shing Y. Heparin-copper biaffinity chromatography of fibroblast growth factors. J Biol Chem. 1988 Jun 25;263(18):9059–9062. [PubMed] [Google Scholar]
  42. Shirai E. [Production of an anti-angiogenesis factor by cultured chondrocytes and establishment of a cell line which produces the factor]. Osaka Daigaku Shigaku Zasshi. 1987 Jun;32(1):163–180. [PubMed] [Google Scholar]
  43. Sorgente N., Kuettner K. E., Soble L. W., Eisenstein R. The resistance of certain tissues to invasion. II. Evidence for extractable factors in cartilage which inhibit invasion by vascularized mesenchyme. Lab Invest. 1975 Feb;32(2):217–222. [PubMed] [Google Scholar]
  44. Staskus P. W., Masiarz F. R., Pallanck L. J., Hawkes S. P. The 21-kDa protein is a transformation-sensitive metalloproteinase inhibitor of chicken fibroblasts. J Biol Chem. 1991 Jan 5;266(1):449–454. [PubMed] [Google Scholar]
  45. Takigawa M., Shirai E., Enomoto M., Hiraki Y., Fukuya M., Suzuki F., Shiio T., Yugari Y. Cartilage-derived anti-tumor factor (CATF) inhibits the proliferation of endothelial cells in culture. Cell Biol Int Rep. 1985 Jul;9(7):619–625. doi: 10.1016/0309-1651(85)90054-2. [DOI] [PubMed] [Google Scholar]
  46. Taylor S., Folkman J. Protamine is an inhibitor of angiogenesis. Nature. 1982 May 27;297(5864):307–312. doi: 10.1038/297307a0. [DOI] [PubMed] [Google Scholar]
  47. Thorgeirsson U. P., Liotta L. A., Kalebic T., Margulies I. M., Thomas K., Rios-Candelore M., Russo R. G. Effect of natural protease inhibitors and a chemoattractant on tumor cell invasion in vitro. J Natl Cancer Inst. 1982 Nov;69(5):1049–1054. [PubMed] [Google Scholar]
  48. Treadwell B. V., Pavia M., Towle C. A., Cooley V. J., Mankin H. J. Cartilage synthesizes the serine protease inhibitor PAI-1: support for the involvement of serine proteases in cartilage remodeling. J Orthop Res. 1991 May;9(3):309–316. doi: 10.1002/jor.1100090302. [DOI] [PubMed] [Google Scholar]
  49. Voyta J. C., Via D. P., Butterfield C. E., Zetter B. R. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol. 1984 Dec;99(6):2034–2040. doi: 10.1083/jcb.99.6.2034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]

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