Abstract
Many studies in arthritis research require an evaluation of the cellular responses within the joint and the ensuing matrix degradation in articular cartilage. The early histochemical/histological scale of Mankin (1) has been widely used but recently challenged as insufficient (2). Imaging techniques such as microscopic magnetic resonance imaging (MRI) (3), polarized light microscopy (3), atomic force microscopy (4), and infrared spectral analysis (5) have opened new approaches to evaluating cartilage structure. Histological methods now include in situ hybridization for cell-specific gene expression and immunohistochemistry for the spatial organization of cartilage proteins and their processed forms.
This chapter details of a method for immunohistochemical analysis of aggrecan degradation in articular cartilage samples which have been prepared by standard methods of formalin fixation and paraffin embedding. The procedure focuses on the application of antibodies (e.g., anti-ADAMTS4, anti-MT4MMP) which detect some of the proteinases most likely involved, and anti-NITEGE which detects the terminal product of the aggrecanase-mediated cleavage of aggrecan at Glu392-Ala393 (bovine, human, dog, rat, pig, sheep, horse, mouse) or Glu393-Ala394 (chick).
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mankin, H. J., Dorfman, H., Lippiello, L., and Zarins, A. (1971) Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. II. Correlation of morphology with biochemical and metabolic data. J. Bone Joint Surg. Am. 53, 523–537.
Ostergaard, K., Petersen, J., Andersen, C. B., Bendtzen, K., and Salter, D. M. (1997) Histologic/histochemical grading system for osteoarthritic articular cartilage: reproducibility and validity. Arthritis Rheum. 40, 1766–1771.
Alhadlaq, H. A., Xia, Y., Moody, J. B., and Matyas, J. R. (2004) Detecting structural changes in early experimental osteoarthritis of tibial cartilage by microscopic magnetic resonance imaging and polarised light microscopy. Ann. Rheum. Dis. 63, 709–717.
Stolz, M., Raiteri, R., Daniels, A.U., VanLandingham, M. R., Baschong, W., and Aebi, U. (2004) Dynamic elastic modulus of porcine articular cartilage determined at two different levels of tissue organization by indentation-type atomic force microscopy. Biophys. J. 86, 3269–3283.
West, P. A., Bostrom, M.P., Torzilli, P. A., and Camacho, N.P. (2004) Fourier transform infrared spectral analysis of degenerative cartilage: an infrared fiber optic probe and imaging study. Appl. Spectrosc. 58, 376–381.
Koshy, P. J., Lundy, C. J., Rowan, A. D., et al. (2002) The modulation of matrix metalloproteinase and ADAM gene expression in human chondrocytes by interleukin-1 and oncostatin M: a time-course study using real-time quantitative reverse transcription-polymerase chain reaction. Arthritis Rheum. 46, 961–967.
Ng, L., Grodzinsky, A. J., Patwari, P., Sandy, J., Plaas, A., and Ortiz, C. (2003) Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy. J. Struct. Biol. 143, 242–257.
Lohmander, L. S., Neame, P. J., and Sandy, J. D. (1993) The structure of aggrecan fragments in human synovial fluid. Evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis. Arthritis Rheum. 36, 1214–1222.
Sandy, J. D. and Verscharen, C. (2001) Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activity is required for C-terminal processing in vivo. Biochem. J. 358, 615–626.
Oshita, H., Sandy, J. D., Suzuki, K., et al. (2004) Mature bovine articular cartilage contains abundant aggrecan that is C-terminally truncated at Ala719-Ala720, a site which is readily cleaved by m-calpain. Biochem. J. 382, 253–259.
Sandy, J. D., Flannery, C. R., Neame, P. J., and Lohmander, L. S. (1992) The structure of aggrecan fragments in human synovial fluid. Evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain. J. Clin. Invest. 89, 1512–1516.
Malfait, A. M., Liu, R. Q., Ijiri, K., Komiya, S., and Tortorella, M. D. (2002) Inhibition of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in osteoarthritic cartilage. J. Biol. Chem. 277, 22,201–22,208.
Collins-Racie, L.A., Flannery, C.R., Zeng, W., et al. (2004) ADAMTS-8 exhibits aggrecanase activity and is expressed in human articular cartilage. Matrix Biol. 23, 219–230.
Arner, E. C., Pratta, M. A., Trzaskos, J. M., Decicco, C. P., and Tortorella, M. D. (1999) Generation and characterization of aggrecanase. A soluble, cartilage-derived aggrecan-degrading activity. J. Biol. Chem. 274, 6594–6601.
Pratta, M. A., Scherle, P. A., Yang, G., Liu, R. Q., and Newton, R. C. (2003) Induction of aggrecanase 1 (ADAM-TS4) by interleukin-1 occurs through activation of constitutively produced protein. Arthritis Rheum. 48, 119–133.
Patwari, P., Gao, G., Lee, J. H., Grodzinsky, A. G., and Sandy, J. D. (2005) Analysis of ADAMTS4 and MT4-MMP indicates that both are involved in aggrecanolysis in interleukin-1-treated bovine cartilage. Osteoarth. Cartilage 13, 269–277.
Stanton, H., East, C., Golub, S., et al. (2005) ADAMTS-5 is the major aggrecanase in cartilage: in vitryo studies with ADAMTS-4 and ADAMTS-5 deficient mice. Orthopedic Research Society, Washington, DC, 206.
Gao, G., Plaas, A., Thompson, V. P., Jin, S., Zuo, F., and Sandy, J. D. ADAMTS4 (aggrecanase-1) activation on the cell surface involves C-terminal cleavage by glycosylphosphatidyl inositol-anchored membrane type 4-matrix metalloproteinase and binding of the activated proteinase to chondroitin sulfate and heparan sulfate on syndecan-1. J. Biol. Chem. 279, 10,042–10,051.
Gao, G., Westling, J., Thompson, V. P., Howell, T. D., Gottschall, P. E., and Sandy, J. D. Activation of the proteolytic activity of ADAMTS4 (aggrecanase-1) by C-terminal truncation. J. Biol. Chem. 277, 11,034–11,041.
Kashiwagi, M., Enghild, J.J., Gendron, C et al. (2004) Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing. J. Biol. Chem. 279, 10,109–10,119.
Singer, II, Scott, S., Kawka, D.W., et al. (1997) Aggrecanase and metalloproteinase-specific aggrecan neo-epitopes are induced in the articular cartilage of mice with collagen II-induced arthritis. Osteoarthritis Cartilage 5, 407–418.
Lark, M.W., Bayne, E.K., Flanagan, J., et al. (1997) Aggrecan degradation in human cartilage. Evidence for both matrix metalloproteinase and aggrecanase-activity in normal, osteoarthritic, and rheumatoid joints. J. Clin. Invest. 100, 93–106.
van Meurs, J. B., van Lent, P. L., Holthuysen, A. E., Singer, II, Bayne, E. K., and van den Berg, W. B. (1999) Kinetics of aggrecanase-and metalloproteinase-induced neoepitopes in various stages of cartilage destruction in murine arthritis. Arthritis Rheum. 42, 1128–1139.
McLean, I. W. and Nakane, P. K. (1974) Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077–1083.
Chambers, M. G., Cox, L., Chong, L., et al. (2001) Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice. Arthritis Rheum. 44, 1455–1465.
Bayliss, M. T., Hutton, S., Hayward, J., and Maciewicz, R. A. (2001) Distribution of aggrecanase (ADAMts 4/5) cleavage products in normal and osteoarthritic human articular cartilage: the influence of age, topography and zone of tissue. Osteoarthritis Cartilage 9, 553–560.
Clements, K. M., Price, J. S., Chambers, M. G., Visco, D. M., Poole, A. R., and Mason, R. M. (2003) Gene deletion of either interleukin-1beta, interleukin-1beta-converting enzyme, inducible nitric oxide synthase, or stromelysin 1 accelerates the development of knee osteoarthritis in mice after surgical transection of the medial collateral ligament and partial medial meniscectomy. Arthritis Rheum. 48, 3452–3463.
Lee, E. R., Lamplugh, L., Leblond, C. P., Mordier, S., Magny, M. C., and Mort, J. S. (1998) Immunolocalization of the cleavage of the aggrecan core protein at the Asn341-Phe342 bond, as an indicator of the location of the metalloproteinases active in the lysis of the rat growth plate. Anat. Rec. 252, 117–132.
Lee, E. R., Lamplugh, L., Davoli, M. A., et al. (2001) Enzymes active in the areas undergoing cartilage resorption during the development of the secondary ossification center in the tibiae of rats ages 0–21 days: I. Two groups of proteinases cleave the core protein of aggrecan. Dev. Dyn. 222, 52–70.
Wang, P., Tortorella, M., England, K., et al. (2004) JBCA-EJ. Proprotein convertase furin interacts with and cleaves pro-ADAMTS4 (Aggrecanase-1) in the trans-Golgi network. J. Biol. Chem. 279, 15,434–15,440.
Itoh, Y., Kajita, M., Kinoh, H., Mori, H., Okada, A., and Seiki, M. (1999) Membrane type 4 matrix metalloproteinase (MT4-MMP, MMP-17) is a glycosylphosphatidylinositol-anchored proteinase. J. Biol. Chem. 274, 34,260–34,266.
Hunziker, E. B., Michel, M., and Studer, D. (1997) Ultrastructure of adult human articular cartilage matrix after cryotechnical processing. Microsc. Res. Tech. 37, 271–284.
Sandy, J. D., Gamett, D., Thompson, V., and Verscharen, C. (1998) Chondrocyte-mediated catabolism of aggrecan: aggrecanase-dependent cleavage induced by interleukin-1 or retinoic acid can be inhibited by glucosamine. Biochem. J. 335, 59–66.
Patwari, P., Kurz, B., Sandy, J. D., and Grodzinsky, A.J. (2000) Mannosamine inhibits aggrecanase-mediated changes in the physical properties and biochemical composition of articular cartilage. Arch. Biochem. Biophys. 374, 79–85.
Sandy, J. D., Neame, P. J., Boynton, R. E., and Flannery, C.R. (1991) Catabolism of aggrecan in cartilage explants. Identification of a major cleavage site within the interglobular domain. J. Biol. Chem. 266, 8683–8685.
Tortorella, M. D., Burn, T. C., Pratta, M. A., and Abbaszade, I., (1999) Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins. Science 284, 1664–1666.
Abbaszade, I., Liu, R. Q., Yang, F., et al. (1999) Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family. J. Biol. Chem. 274, 23,443–23,450.
Roberts, C. R., Roughley, P. J., and Mort, J. S. (1989) Degradation of human proteoglycan aggregate induced by hydrogen peroxide. Protein fragmentation, amino acid modification and hyaluronic acid cleavage. Biochem. J. 259, 805–811.
Sandy, J. D., Thompson, V., Verscharen, C., and Gamett, D. (1999) Chondrocyte-mediated catabolism of aggrecan: evidence for a glycosylphosphatidylinositol-linked protein in the aggrecanase response to interleukin-1 or retinoic acid. Arch. Biochem. Biophys. 367, 258–264.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Osborn, B., Bai, Y., Plaas, A.H.K., Sandy, J.D. (2007). Image Analysis of Aggrecan Degradation in Articular Cartilage With Formalin-Fixed Samples. In: Cope, A.P. (eds) Arthritis Research. Methods in Molecular Medicine, vol 135. Humana Press. https://doi.org/10.1007/978-1-59745-401-8_10
Download citation
DOI: https://doi.org/10.1007/978-1-59745-401-8_10
Publisher Name: Humana Press
Print ISBN: 978-1-58829-344-2
Online ISBN: 978-1-59745-401-8
eBook Packages: Springer Protocols