The major clinical features of osteoarthritis are focal loss of articular cartilage and osteophytic outgrowth of subchondral bone. Loss of articular cartilage is preceded by changes in the content and composition of cartilage matrix proteoglycan aggregates. Mature chondrocytes, although biosynthetically active, appear unable to maintain or repair matrix aggregates so that cartilage degeneration is progressive. Proteoglycan aggregates are composed of monomers, link protein and hyaluronate in ternary complex. Monomers bind to hyaluronate (and link protein) via a specific binding region and this process immobilizes the molecules and possibly protects them from proteolytic attack. An understanding of how proteoglycans acquire hyaluronate binding properties and what occurs during loss of binding is therefore central to the question of matrix maintenance in normal and diseased cartilages. Three closelyrelated systems will be studied (1) rabbit chondrocytes in monolayer culture which secrete a """"""""precursor"""""""" form of proteoglycan with a low affinity for hyaluronate, which can be markedly enhanced by treatment in vitro with mild alkali. We plan to study conditions of culture which modify precursor affinity and the molecular basis of the alkaliinduced change in binding activity. (2) a cartilage explant system in which the hyaluronate binding activity of proteoglycans is lost. This appears to be due to limited proteoysis of the binding region domain and we plan to investigate the change in binding region structure. (3) Purified stromelysin, a neutral metalloendopeptidase, will be incubated with binding region to determine its effect on the binding properties and structure of this protein. Essential methodologies are established in our laboratory (culture systems, proteoglycan isolation, immunoassay, binding affinity assays, electrophoresis and blotting) and we are developing reversed- phase HPLC for mapping of peptides in proteoglycan core protein. The long-term objective of these studies is to increase our understanding of the extracellular processing of cartilage proteoglycans, particularly in relation to modification of hyaluronate binding activity. Such knowledge might suggest methods for the useful intervention in joint disease at the level of improving matrix repair by mature chondrocytes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
1R01AR038580-01
Application #
3158639
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1987-08-01
Project End
1992-07-31
Budget Start
1987-08-01
Budget End
1988-07-31
Support Year
1
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Rhode Island Hospital (Providence, RI)
Department
Type
DUNS #
161202122
City
Providence
State
RI
Country
United States
Zip Code
02903
Flannery, C R; Sandy, J D (1994) Effects of proteinase inhibitors on aggrecan catabolism in chondrocyte cultures. Ann N Y Acad Sci 732:380-3
Plaas, A H; Sandy, J D (1993) A cartilage explant system for studies on aggrecan structure, biosynthesis and catabolism in discrete zones of the mammalian growth plate. Matrix 13:135-47
Lohmander, L S; Neame, P J; 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 36:1214-22
Sandy, J D; Flannery, C R; Neame, P J et al. (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 89:1512-6
Flannery, C; Stanescu, V; Morgelin, M et al. (1992) Variability in the G3 domain content of bovine aggrecan from cartilage extracts and chondrocyte cultures. Arch Biochem Biophys 297:52-60
Flannery, C R; Lark, M W; Sandy, J D (1992) Identification of a stromelysin cleavage site within the interglobular domain of human aggrecan. Evidence for proteolysis at this site in vivo in human articular cartilage. J Biol Chem 267:1008-14
Sandy, J D; Neame, P J; Boynton, R E et al. (1991) Catabolism of aggrecan in cartilage explants. Identification of a major cleavage site within the interglobular domain. J Biol Chem 266:8683-5
Sandy, J D; Boynton, R E; Flannery, C R (1991) Analysis of the catabolism of aggrecan in cartilage explants by quantitation of peptides from the three globular domains. J Biol Chem 266:8198-205
Flannery, C R; Urbanek, P J; Sandy, J D (1990) The effect of maturation and aging on the structure and content of link proteins in rabbit articular cartilage. J Orthop Res 8:78-85
Sandy, J D; Flannery, C R; Boynton, R E et al. (1990) Isolation and characterization of disulfide-bonded peptides from the three globular domains of aggregating cartilage proteoglycan. J Biol Chem 265:21108-13

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