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Products resulting from cleavage of the interglobular domain of aggrecan in samples of synovial fluid collected from dogs with early- and late-stage osteoarthritis

John F. Innes BVSc, PhD1, Chris B. Little BVMS, PhD2,3, Clare E. Hughes PhD4, and Bruce Caterson PhD5
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  • 1 Small Animal Hospital, Department of Veterinary Clinical Science, Faculty of Veterinary Science, University of Liverpool, Liverpool L7 7EX, UK.
  • | 2 Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, Cardiff CF1 3US, UK.
  • | 3 Present address is the Raymond Purves Bone and Joint Research Laboratories, Institute of Bone and Joint Research and Department of Orthopaedic and Traumatic Surgery, University of Sydney, Royal North Shore Hospital, St Leonards, NSW 2065, Australia.
  • | 4 Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, Cardiff CF1 3US, UK.
  • | 5 Connective Tissue Biology Laboratories, School of Biosciences, Cardiff University, Cardiff CF1 3US, UK.

Abstract

Objective—To investigate interglobular domain (IGD) cleavage of aggrecan in dogs with naturally developing osteoarthritis (OA).

Sample Population—Samples of synovial fluid (SF) obtained from 3 cubital (elbow) joints and 3 stifle joints of 4 clinically normal dogs, 24 elbow joints of 12 dogs with early-stage OA, 8 stifle joints of 5 dogs with early-stage OA, and 10 stifle joints of 9 dogs with latestage OA.

Procedure—Fractions of SF were assayed for total glycosaminoglycan (GAG) content and also subjected to western blot analysis by use of monoclonal antibodies against neoepitopes generated by cleavage of the IGD of the aggrecan protein core by matrix metalloproteinase (MMP; BC-14) and aggrecanase (BC-3).

Results—Total GAG content of SF from joints of clinically normal dogs did not differ from that of dogs with early-stage OA. The GAG content of SF from joints of dogs with late-stage OA was significantly lower, compared with GAG content for other SF samples. Aggrecanase-generated fragments were detected in SF from all groups but not in all samples. Matrix metalloproteinase– generated fragments were not detected in any SF samples. In early-stage OA, high-molecularweight aggrecanase-generated aggrecan catabolites were evident.

Conclusions and Clinical Relevance—GAG content of SF obtained from dogs with late-stage OA is significantly decreased, suggesting proteoglycan depletion of cartilage. Aggrecanases, but not MMPs, are the major proteolytic enzymes responsible for IGD cleavage of aggrecan in canine joints. Analyses of SF samples to detect aggrecanase-generated catabolites may provide an early biomarker for discriminating early- and latestage OA in dogs. (Am J Vet Res 2005;66:1679–1685)

Abstract

Objective—To investigate interglobular domain (IGD) cleavage of aggrecan in dogs with naturally developing osteoarthritis (OA).

Sample Population—Samples of synovial fluid (SF) obtained from 3 cubital (elbow) joints and 3 stifle joints of 4 clinically normal dogs, 24 elbow joints of 12 dogs with early-stage OA, 8 stifle joints of 5 dogs with early-stage OA, and 10 stifle joints of 9 dogs with latestage OA.

Procedure—Fractions of SF were assayed for total glycosaminoglycan (GAG) content and also subjected to western blot analysis by use of monoclonal antibodies against neoepitopes generated by cleavage of the IGD of the aggrecan protein core by matrix metalloproteinase (MMP; BC-14) and aggrecanase (BC-3).

Results—Total GAG content of SF from joints of clinically normal dogs did not differ from that of dogs with early-stage OA. The GAG content of SF from joints of dogs with late-stage OA was significantly lower, compared with GAG content for other SF samples. Aggrecanase-generated fragments were detected in SF from all groups but not in all samples. Matrix metalloproteinase– generated fragments were not detected in any SF samples. In early-stage OA, high-molecularweight aggrecanase-generated aggrecan catabolites were evident.

Conclusions and Clinical Relevance—GAG content of SF obtained from dogs with late-stage OA is significantly decreased, suggesting proteoglycan depletion of cartilage. Aggrecanases, but not MMPs, are the major proteolytic enzymes responsible for IGD cleavage of aggrecan in canine joints. Analyses of SF samples to detect aggrecanase-generated catabolites may provide an early biomarker for discriminating early- and latestage OA in dogs. (Am J Vet Res 2005;66:1679–1685)