WO1997044059A2 - Collagene de type ii de cartilage utilise en tant que facteur angiogenique - Google Patents

Collagene de type ii de cartilage utilise en tant que facteur angiogenique Download PDF

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WO1997044059A2
WO1997044059A2 PCT/US1997/009861 US9709861W WO9744059A2 WO 1997044059 A2 WO1997044059 A2 WO 1997044059A2 US 9709861 W US9709861 W US 9709861W WO 9744059 A2 WO9744059 A2 WO 9744059A2
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collagen
type
cartilage
cells
angiogenesis
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PCT/US1997/009861
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WO1997044059A3 (fr
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A. Robin Poole
Anne Marriott
Mauro Alini
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Shriner's Hospitals For Children
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • Angiogenesis is a fundamental biological process whereby new capillaries are formed (Folkman, J. [1991] In "Biologic Therapy of Cancer” (V. DeVita, S. Hellman, and S.A. Rosenberg, Eds.), Lippmcott, Philadelphia, pp. 743-753; Folkman, J., Y. Shing [1992] J. Bwl Chem. 267 : 10931 - 10934).
  • angiogenesis can contribute to the pathogenesis of a number of diseases such as retinopathy, tumor growth and metastasis, rheumatoid arthritis, osteoarth ⁇ tis, retrolental fibroplasia, neovascular glaucoma, psoriasis, angiofibromas, immune and non- immune inflammation (including rheumatoid arthritis), capillary proliferation within atherosclerotic plaques, hemangiomas, and Kaposi's Sarcoma.
  • diseases such as retinopathy, tumor growth and metastasis, rheumatoid arthritis, osteoarth ⁇ tis, retrolental fibroplasia, neovascular glaucoma, psoriasis, angiofibromas, immune and non- immune inflammation (including rheumatoid arthritis), capillary proliferation within atherosclerotic plaques, hemangiomas, and Kaposi's Sarcoma.
  • PF4 platelet factor 4
  • major basic protein has demonstrated hepa ⁇ n-binding activity but has toxicity.
  • Platelet factor 4 is a well-known protein which has been completely sequenced (Deuel, T.F., P.S. Keim, M. Farmer, R.L. Hemrikson [1977] Proc Natl. Acad. Sci USA 74(6):2256-2258).
  • Ada Anal. 68:1-17 is closely associated with the destruction of the largely uncalcified longitudinal septa.
  • the remaining calcified trabeculae serve as a scaffold onto which osteoblasts can then settle and form woven bone.
  • Cartilage is a predominantly avascular tissue except during development when endochondral bone formation occurs.
  • capillaries from the developing bone invade the uncalcified and calcified cartilage of the physis which results in the subsequent removal of the cartilage and its replacement by bone and a low-molecular weight angiogenic factor known as endothehal cell- stimulating factor (ESAF) (Brown, R.A., C. Taylor, B. McLaughlin, CD. McFarland, J.B Weiss, Y.S. Ah [1987] Bone Mineral. 3: 143-158), have been shown to be present in growth- plate cartilage. These molecules could act as potential chemoattractants for endothehal cells
  • a cascade of events that characte ⁇ ze angiogenesis occurs. These include degradation and remodeling of the matrix, migration and proliferation of endothehal cells, invasion of the uncalcified transverse septum cartilage, and vessel maturation. These processes continue during development until growth-plate closure occurs at the end of puberty, again due to mechanisms that remain unknown.
  • Type II collagen (tropocollagen) and type I collagen (tropocollagen) form the primary organic components of the skeletal tissues cartilage and bone, respectively
  • the molecules form collagen fibrils that are destroyed as part of a process of controlled cartilage and bone turnover in healthy persons. In diseases such as arth ⁇ tis and osteoporosis, there is increased cleavage of these molecules leading to the release of degradation products into serum
  • the collagens are each composed of three ⁇ -chains that are chemically characteristic of each collagen. They combine together to form the triple helix, of which each molecule is primarily composed.
  • This triple helix can only be cleaved by collagenase, particularly MMP-1 , known as interstitial collagenase.
  • MMP-1 collagenase
  • MMP- 13 tissue collagenase
  • a ncutrophil collagenase (MMP-8) may also be important in arth ⁇ tis.
  • MMP-8, and MMP- 13 are identical
  • Vitamin-D deficiency affects endochondral ossification resulting in rickets. Characte ⁇ stics of this condition are the impairment of calcification and a lengthened hypertrophic zone of the growth plate. Vitamin D has been shown to exert a direct effect on the metabolism of growth-plate chondrocytes. l,25-(OH) 2 vitamin D, promotes expression of the hypertrophic phenotype in chick sternal prehypertrophic cells and stimulates mRNA expression and synthesis of types II, IX, and X collagen, the core protein of aggrecan, and fibronectm (Gersternfeld, L.C., C M. Kelly, M. von Deck, J.B.
  • vitamin D 3 can maximally stimulate matrix calcification m healthy rat growth-plate chondrocyte cultures (Hinek, A., R.A. Poole [1988] J Bone Miner Res. 3:421-429). However, in rachitic (vitamin-D deficient) rats, only 24,25-(OH) 2 vitamin D 3 is required to stimulate calcification Receptors for l,25-(OFI) 2 vitamin D 3 are present on growth-plate chondrocytes, with receptor density being highest in hypertrophic cells (Iwamoto, M., K. Sato, K. Nakashima, A. Shimazu, Y. Kato [1989] Dev Biol 136:500-507)
  • cartilage type II collagen is an angiogenic factor.
  • Cartilage type II collagen is a well known structural molecule which has never before been known to promote, or otherwise modulate, angiogenesis.
  • angiogenesis it has now been discovered that the release of cartilage type II collagen from cartilage can induce angiogenesis, thereby playing a c ⁇ tical role m erosive joint destruction such as that which occurs m rheumatoid arth ⁇ tis.
  • cartilage type II collagen promotes angiogenesis makes it possible for the first time to treat angiogenic diseases including, but not limited to, erosive joint diseases by specifically inhibiting the release and/or biological activity of cartilage type II collagen.
  • This inhibition may be achieved, for example, by the use of compounds which inhibit the production and/or release of cartilage type II collagen or which inhibit its effect on endothehal cells which form new blood vessels.
  • antibodies may be used to block the activity of this molecule.
  • proteases may be used to eliminate the biological activity of this molecule
  • a further aspect of the subject invention pertains to diagnostic assays which detect the presence of cartilage type II collagen as a means for detecting the existence of joint destruction and/or monitoring the progression of joint destruction
  • Methods for detecting the presence of cartilage type II collagen would be readily evident to a person skilled in this art having the benefit of the instant disclosure.
  • the presence of this molecule could be detected, for example, by using antibodies to this molecule or by a biological assay such as the rabbit cornea assay which tests for angiogenic activity.
  • a further aspect of the subject invention pertains to the use of cartilage type II collagen to promote angiogenesis This can be useful, for example, in wound repair situations.
  • the use of cartilage type II collagen in such applications is particularly advantageous because this molecule has been determined to be non-mitogenic.
  • cartilage type II collagen is in assays, such as the rabbit cornea assay, which are used to test the efficacy of va ⁇ ous angiogenesis-inhibitmg molecules.
  • cartilage type II collagen can be used to induce angiogenesis so that test compounds can then be administered in order to evaluate the ability of these compounds to inhibit angiogenesis
  • the subject invention relates to the identification of cartilage type II collagen as an angiogenic factor.
  • the subject invention provides therapies useful for the amelioration of destructive jomt conditions. These therapies involve the blocking of cartilage type II collagen activity This blocking can be achieved by inhibiting or preventing the release of this molecule or by inhibiting its angiogenic activity after release.
  • the subject invention also provides diagnostic procedures and procedures useful for certain in vitro assays
  • Angiogenesis is a pivotal event in endochondral ossification. Vessels grow into the hypertrophic cartilage and erode it to produce a scaffold on which osteoblasts settle to produce woven bone.
  • a new culture system was used to identify an angiogenic molecule produced by growth plate chondrocytes. Chondrocytes from p ⁇ mary growth plates of bovine fetuses were separated into maturationally distinct subpopulations. When cultured, these cells produce an extensive extracellular matrix, and the prehypertrophic cells mature to express the hypertrophic phenotype defined by the synthesis of type X collagen and mat ⁇ x calcification.
  • the culture medium collected from the hypertrophic cells contains a chemoattractant, nonmitogenic molecule for bovine endothehal cells which can induce angiogenesis in vivo in the rabbit cornea model
  • This molecule has an A/, of approximately 120 x lO 3 .
  • vitamin D 3 regulation by vitamin D 3 is complex, is maturation-dependent, and requires further careful studies. However, the establishment of this culture system which separates growth plate chondrocytes into distinct developmental stages (prehypertrophic, early hypertrophic, and advance hypertrophic), enabled us to isolate this molecule and investigate its regulation.
  • angiogenic molecules such as bFGF, aFGF, TGF- ⁇ , and platelet-derived endothehal cell growth factor are mitogenic, whereas angiogemn is not (Folkman and Shing, 1992, supra)
  • angiogemn is not (Folkman and Shing, 1992, supra)
  • these molecules are much smaller than the molecule we describe here
  • the molecule has a potency (nanogram amounts) in the rabbit cornea assay similar to that of other angiogenic molecules such as basic and acid FGF and TGF- ⁇ (Folkman and Shing, 1992, supra).
  • the angiogenic molecule desc ⁇ bed herein was found only m conditioned medium from chondrocytes that were expressing the hypertrophic phenotype The synthesis of this molecule may therefore be switched on when chondrocytes undergo hypertrophy. Alternatively, it may have been synthesized at an earlier stage of growth-plate chondrocyte differentiation and stored within the extracellular matrix.
  • Cartilage type II collagen is a well known and extensively described molecule. Sec, for example, Poole, A.R. (1993) "Cartilage in Health and Disease” (Chapter 15) in Arthritis and Allied Conditions. A Textbook of Rheumatology, 12th Ed , Lea & Febiger, Philadelphia, pp 279, and McCarty, D J , W.J. Koopman, eds., in Scientific Basis for the Study of the Rheumatic Diseases, p. 284
  • cartilage type II collagen includes its various forms including those resulting from alterative splicing and allehc variation, so long as the compound possesses angiogenic activity.
  • Recombmant human type II collagen is also chemotactic and can be substituted for natural human type II collagen
  • the 120 kDa protein referred to herein refers to the apparent molecular weight of the type II collagen as it migrates on an SDS gel as desc ⁇ bed herein
  • the actual molecular weight of one ⁇ -cham of type II collagen is approximately 100 kDa This is known by those skilled in the art.
  • Intact and pepsin-extracted type II collagen is chemotactic for endothehal cells
  • the chemotactic activity is related to the structure of the collagen and concentration-dependent
  • chemotactic activity for fibroblasts was demonstrated for collagenase-de ⁇ ved peptides of type II collagen (Postlethwaite, et al , 1978)
  • chemotactic activity of type II collagen for endothehal cells is not observed following a single or multiple proteolytic cleavage of the molecule.
  • one method for reducing the biological activity of type II collagen is to treat with a protease.
  • Enzymic treatments of the collagen with pepsin, bacte ⁇ al collagenase, and rMMP-1 demonstrate that the chemotactic activity of type II collagen is dependent on the region of the ⁇ -cham that forms a triple helix
  • chemotactic activity does not necessarily require an intact helix.
  • the activity of enzymes can be used to reduce the amount of chemotactic type II collagen molecules released from the tissue Enhanced angiogenesis will result from inhibition of collagenase
  • the activity of type II collagen is mitigated by preventing the release of the compound from cartilage. This can be achieved by enhancing incorporation of newly-synthesized type II collagen and/or by preventing cartilage breakdown
  • the prevention of cartilage breakdown can be achieved by, for example, the administration of metalloprotemase inhibitor or other inhibitors of cartilage matrix degradation.
  • Endothehal cells were obtained from bovine umbilical veins as previously desc ⁇ bed (Jaffe, E.A., R.L. Machman, C.G. Becker, C.R Mimck [1973] J Chn Invest 52:2745-2756). Briefly, umbilical veins were washed several times with DMEM and infused with 1% collagenase (type 1A, Sigma Chemical Co.) in DMEM containing 10% FCS for 15 minutes at room temperature (by clamping the ends of the veins) The collagenase solution containing detached EC was removed and the cells were washed several times with
  • DMEM containing 0.25 mg/ml fungizone (Gibco BRL, Grand Island, NY). Endothehal cells were cultured on gelatin-coated 25-cm 3 flasks at 37 °C with DMEM supplemented with 10% FCS. After 18-24 hours, nonadherent cells were removed from the flask and fresh medium was added Cells from passages 3-6 were used for the Boyden chamber assays. The endothehal nature of the cells was confirmed by staining with an antiserum to factor Vlll-related antigen
  • Type X collagen synthesis was measured during culture under serum-free conditions as previously desc ⁇ bed (Ahni et al, 1994, supra). Twenty-five ⁇ Ci/ml of [ 3 H]prohne (Amersham Canada, Inc) and 70 ⁇ g/ml p-ammo-propionit ⁇ le were added for 48 hours to the culture medium as indicated.
  • Radiolabeled culture media (shown previously to be representative of cell layer biosynthesis of type X collage) (Ahni et al, 1994, supra) were precipitated by ammonium sulfate (33% saturation) overnight at 4°C Pellets were washed twice with 70% ethanol and analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmh, U.K. [1970] Nature 227:680-685) using 7.5% gels followed by fluorography (Laskey, R.A., A.D. Hills [1975] Eur J. Biochem. 56:335-341).
  • Vitamm-D, metabolites Either l,25-(OH) 2 D 3 or 24,25-(OH) 2 D 2 or both metabolites (at 10 8 , 10 " l0 , or 10 " 12 M) were added for 48 hours in serum-free medium as indicated above Control wells received the highest amounts (less than 1%) of vehicle (ethanol). Serum-free media were stored at -20°C prior to assay for endothehal cell chemotactic activity.
  • Endothehal cell chemotaxis assay Endothehal cell chemotaxis assay. EC migration was measured using a modified Boyden chamber assay (Neuroprobe, Inc., Bethesda, MD) as described by Falk et al. (Falk, W.K., R.H Goodwin, Jr., E.R. Leonard [1980] J Immunol Methods 33:239-247). Polycarbonate membranes with 8- ⁇ m pores (Nucleopore Corp., Pleasanton, CA) were precoated by the manufacturer with gelatin. Culture media were assayed at the end of the 48-hour serum- free culture.
  • Migration was quantified by counting the number of EC that had migrated to the lower surface of the polycarbonate membrane using a Photomicroscope III (Carl Zeiss Inc., Montreal). Between 7 and 10 fields (7.1 mm 2 ) were counted. These determinations were performed in triplicate. To determine whether the migration of EC was due to movement along a concentration gradient (chemotaxis) or random migration (chemokinesis), checkerboard analysis was performed (Zigmond, S.H., J.G. Hirsh [1973] J. Exp Med 137:387-410) by adding various concentrations of the chondrocyte serum- free medium to the upper wells together with the EC.
  • chemotaxis chemotaxis
  • chemokinesis chemokinesis
  • Chemotactic-positive chondrocyte-conditions serum-free media up to 500 ml were concentrated using a YM-1 membrane (molecular weight cutoff 1000; Amicon, Beverly, MA).
  • the retentate (reconstituted to its original volume with DMEM) and the filtrate were stored at -20°C until they were examined for chemotactic activity in the Boyden chamber assay. Chemotactic activity was found to be totally bound to the membrane. The activity was eluted with 2 M NaCl in 10 mM T ⁇ s-HCl, pH 7 4, overnight at 4°C, lyophihzed, and desalted by washing with 70% ethanol.
  • the residue was dissolved in water and examined for chemotactic activity
  • the positive residue was adjusted to 0.1 % t ⁇ fluoroacetic acid (TFA) (maximal volume of 1 ml) and chromatographed with high-performance liquid chromatography (HPLC) using a C18 ⁇ Bondapak column (3.9 x 300 mm) (Waters).
  • the column was developed with 100% solvent A (0.1% TFA) for 10 minutes, followed by a linear gradient (from 0 to 100%) of solvent B (0.1% TFA in 80% acetonit ⁇ le) over a 60-mmute pe ⁇ od.
  • the eluate was monitored at 214 and 280 nm.
  • Fraction (1.5 ml) were pooled (as indicated in the Examples, below), lyophihzed, washed with 70% ethanol, redissolved m water, and evaluated for endothehal cell chemotactic activity in vitro and angiogenesis in vivo.
  • Bovine fetal growth-plate chondrocytes were isolated and fractionated into subpopulations, as previously described (Lee et al, 1990, supra; Ahni et al , 1994, supra), except that reduced concentrations of enzymes were used, namely 800 ⁇ g/ml hyaluronidase (bovine testicular type V, Sigma Chemical Co.), and 50 ⁇ g/ml DNAse 1 (from bovine pancreas, Sigma Chemical Co.), m order to increase the recovery and viability (over 80%) of subpopulation A (the least dense and the largest cells).
  • chondrocyte subpopulation (A, B, C, and D/E) were seeded on gelatin-coated 96-well flat-bottom microtiter plates (Falcon, Becton Dickinson, NJ) at a density of 2 x 10 6 cells m 200 ⁇ l medium per well.
  • the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), containing 50 ⁇ g/ml ascorbic acid and a 5 mM sodium ⁇ -giycerophosphate (both additives were freshly prepared at each medium change).
  • DMEM Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • cells were also cultured for 48-hour periods (Days 0-2, 2-4, 4-6, 6-8) in serum-free DMEM, containing 5 ⁇ g/ml insulin, 5 ⁇ g/ml transfer ⁇ n, 5 ng/ml sodium selenite (ITS), 1 mg/ml fatty acid-free bovine serum albumin (BSA), ascorbic acid, and sodium ⁇ -glycerophosphate (as above) with or without v ⁇ tam ⁇ n-D 3 metabolites as indicated below. Only serum-free media were examined for chemotactic activity.
  • serum-free DMEM containing 5 ⁇ g/ml insulin, 5 ⁇ g/ml transfer ⁇ n, 5 ng/ml sodium selenite (ITS), 1 mg/ml fatty acid-free bovine serum albumin (BSA), ascorbic acid, and sodium ⁇ -glycerophosphate (as above) with or without v ⁇ tam ⁇ n-D 3 metabolites as indicated below. Only serum-free media were examined
  • chondrocyte subpopulations adhered to the gelatin-coated wells after 406 hours of culture, losing their rounded shape and assuming a polygonal appearance They demonstrated a characte ⁇ stic "cobblestone" morphology.
  • the cells rapidly synthesized an extensive extracellular mat ⁇ x ⁇ ch in collagen and proteoglycan.
  • Type X collagen a definitive marker of the hypertrophic phenotype, was detected in the serum- free medium (DMEM-ITS) of subpopulation A within 2-4 days of isolation. At this stage, these cells are, by definition, hypertrophic chondrocytes.
  • type X collagen synthesis increased m the A subpopulation.
  • Type X collagen was first observed in the B subpopulation at 4-6 days and later m the C population at 8-10 days and the D/E subpopulation sat 12-14 days. The time of appearance of type X collagen was dependent on fetal age The synthesis of type X collagen corresponded to an increase in cell size as the cells underwent hypertrophic changes revealed by light and electron microscopy (Ahni et al, 1994, supra). Matrix calcification occurred in the same sequential order as observed for type X collagen synthesis but always 1 to 3 days after the synthesis of this molecule (Ahni et al, 1994, supra).
  • chondrocyte serum-free conditioned media were analyzed using the Boyden chamber assay after different periods of exposure to FCS.
  • Chemotactic activity was detected only m cultures synthesizing type X collagen (Table 1). Using chondrocytes from five different aged fetuses, chemotactic activity was first observed at 0-2 days m the A subpopulation and then at 2-4 days in the B subpopulation accompanying the synthesis of type X collagen. Cultures of subpopulations C (6-8 days) and D/E (8-10 days) took longer before type X collagen was synthesized. In the C and D/E subpopulations, chemotactic activity was detected after type X collagen synthesis was first detected at 8- 10 and 10-12 days, respectively (Table 1).
  • Table 1 The presence (+) or absence (-) of chemotactic activity and type X collagen (X) in the culture media
  • Bio-Gel P-30 chemotactic-positive fractions indicated that the molecule was of an apparent low molecular mass (M, below 5 x 10 3 ) and/or was weakly bound to the gel.
  • the HPLC fractions that contained chemotactic activity were pooled and tested for the ability to induce EC proliferation. There was no effect on EC proliferation
  • Chemotactic-positive HPLC fractions were pooled, lyophihzed, washed with 70% ethanol, and analyzed under reducing conditions using SDS-PAGE. Chemotactic-positive HPLC fractions were pooled, lyophihzed, washed twice with 70% ethanol, and analyzed by SDS-PAGE using 4-20% gradient gels. Following electrophoresis, proteins were either stained directly with Coomassie blue or transferred to PVDF membrane in the presence of 10 mM 3-
  • the rabbit cornea assay was used. After partial purification using HPLC (as desc ⁇ bed above), chemotactic-positive fractions were mixed in a hydroxyethyl-methacrylate polymer (Polyscience, Inc., War ⁇ ngton, Hampshire, UK) in 70% ethanol at room temperature as descnbed (Langer, R, J Folkman [1976] Nature 263:797-800) The molecule trapped within the polymer matrix was implanted as a pellet of 1 mm 3 in the rabbit cornea stroma (New Zealand white, female, 3 4 kg) 2 mm away from the corneal-scleral junction Basic FGF was used as positive control.
  • a hydroxyethyl-methacrylate polymer Polyscience, Inc., War ⁇ ngton, Hampshire, UK
  • 70% ethanol 70% ethanol at room temperature as descnbed
  • the molecule trapped within the polymer matrix was implanted as a pellet of 1 mm 3 in the rabbit cornea stroma (New Zealand white, female, 3 4 kg)
  • the corneas were examined every 2 days to monitor for infection and capillary growth
  • the rabbits were sacnficed 10 days after implantation Just prior to euthanasia, some of the rabbits were perfused from the carotid artery with colloidal carbon to improve definition of new corneal vessels
  • Paraffin wax-embedded sections (6 ⁇ m) were stained with hematoxyhn and eosin and examined using light microscopy
  • Induction of corneal neovascula ⁇ zation occurred after 10 days implantation of 300 ng of the chemotactic peptide in a methacrylate pellet This effect was also observed when 200 or 100 ng samples were implanted in the rabbit cornea This observation was reproduced m six rabbits on different occasions using two different preparations of the chemotactic molecule
  • rabbit corneas implanted with control pellets did not exhibit angiogenic responses Histological studies revealed an absence
  • the B and C chondrocyte subpopulations were cultured for 48-hour penods with vanous concentrations of l,25-(OH) 2 D unten 24,25-(OH) 2 D 3 , or both at 10 ⁇ 10 ,0 , and 10 12 M under serum- free conditions following different penods of culture in the presence of 10% FCS Media were compared for their ability to induce EC migration using the Boyden chamber assay
  • This expenmental protocol allowed us to investigate the effect of the v ⁇ tamm-D 3 metabolites on the production of the angiogenic molecule at different maturational stages, namely prehypertrophic (no type X collagen synthesis), early hypertrophic (type X collagen synthesis but no calcification), and advanced hypertrophic states (type X collagen production and matrix calcification)
  • An increase in EC migration was observed with both v ⁇ tam ⁇ n-D 3 metabolites (alone or in combination) at Day 5 (early hypertrophic stage) at all of the concentrations tested
  • TCA degradation products can both be detected by the anti-carboxy-termini polyclonal antibody, whereas the anti-amino-termini TCB polyclonal antibody and monoclonal only recognize the MMP- 1 product.
  • These antibodies can be used m evaluating and monito ⁇ ng the degradation of these collagens m bone and cartilage.
  • TCA directed antibodies we can selectively detect only type II collagen collagenase cleavage products, whereas, if we use the antibodies to TCB products, we can detect both type I and type II degradation products.
  • the antibodies provide the potential to examine either cartilage or bone resorption in patients with arthritis or osteoporosis, for example.

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Abstract

La présente invention se rapporte à des matières et à des procédés permettant d'inhiber ou de favoriser l'angiogénèse. On a établit que le collagène de type II de cartilage est un promoteur d'angiogénèse. L'invention se rapporte également à des procédés visant à réduire l'angiogénèse non désirée ainsi qu'à des procédés visant à favoriser une angiogénèse appropriée, notamment pour la cicatrisation.
PCT/US1997/009861 1996-05-23 1997-05-23 Collagene de type ii de cartilage utilise en tant que facteur angiogenique WO1997044059A2 (fr)

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Cited By (6)

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EP1149111A1 (fr) * 1999-01-06 2001-10-31 University Of Southern California Technique et composition pour l'inhibition de l'angiogenese
GB2433508A (en) * 2005-12-20 2007-06-27 Pentax Corp Agents obtainable by culturing chondrocytes capable of hypertrophication
US7365167B2 (en) 2001-11-26 2008-04-29 Cell Matrix, Inc. Humanized collagen antibodies and related methods
US7390885B2 (en) 2001-11-26 2008-06-24 Cell Matrix, Inc. Humanized collagen antibodies and related methods
US7488792B2 (en) 2002-08-28 2009-02-10 Burnham Institute For Medical Research Collagen-binding molecules that selectively home to tumor vasculature and methods of using same
US7566449B2 (en) 1999-07-13 2009-07-28 University Of Southern California Method and composition for inhibition of angiogenesis using antagonists based on MMP-9 and β1 integrins

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WO1994007520A1 (fr) * 1992-09-25 1994-04-14 Autoimmune, Inc. Procede de traitement de la polyarthrite rhumatoide avec du collagene de type ii
WO1994018563A1 (fr) * 1993-02-12 1994-08-18 Rhode Island Hospital Detection de collagene de type ii et de ses peptides

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Publication number Priority date Publication date Assignee Title
WO1994007520A1 (fr) * 1992-09-25 1994-04-14 Autoimmune, Inc. Procede de traitement de la polyarthrite rhumatoide avec du collagene de type ii
WO1994018563A1 (fr) * 1993-02-12 1994-08-18 Rhode Island Hospital Detection de collagene de type ii et de ses peptides

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EP1149111A4 (fr) * 1999-01-06 2004-08-11 Univ Southern California Technique et composition pour l'inhibition de l'angiogenese
US7122635B2 (en) 1999-01-06 2006-10-17 University Of Southern California Method and composition for angiogenesis inhibition
US8025883B2 (en) 1999-01-06 2011-09-27 University Of Southern California Antagonists and methods for inhibiting angiogenesis
US7345151B2 (en) 1999-01-06 2008-03-18 University Of Southern California Antagonists specific for denatured collagen and methods of using same
US7588760B2 (en) 1999-01-06 2009-09-15 University Of Southern California Antagonists specific for denatured collagen and methods of using same
EP1149111A1 (fr) * 1999-01-06 2001-10-31 University Of Southern California Technique et composition pour l'inhibition de l'angiogenese
US7566449B2 (en) 1999-07-13 2009-07-28 University Of Southern California Method and composition for inhibition of angiogenesis using antagonists based on MMP-9 and β1 integrins
US7566770B2 (en) 2001-11-26 2009-07-28 Cell-Matrix, Inc. Humanized collagen antibodies and related methods
US7390885B2 (en) 2001-11-26 2008-06-24 Cell Matrix, Inc. Humanized collagen antibodies and related methods
US7365167B2 (en) 2001-11-26 2008-04-29 Cell Matrix, Inc. Humanized collagen antibodies and related methods
US7763248B2 (en) 2001-11-26 2010-07-27 Cell Matrix, Inc. Humanized collagen antibodies and related methods
US7763247B2 (en) 2001-11-26 2010-07-27 Cell Matrix, Inc. Humanized collagen antibodies and related methods
US7488792B2 (en) 2002-08-28 2009-02-10 Burnham Institute For Medical Research Collagen-binding molecules that selectively home to tumor vasculature and methods of using same
GB2433508A (en) * 2005-12-20 2007-06-27 Pentax Corp Agents obtainable by culturing chondrocytes capable of hypertrophication

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WO1997044059A3 (fr) 1997-12-24

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