Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: The research carried out in this project focused on determining the role of ADAMTS aggrecanases in equine osteoarthritis. The hypothesis addressed by the proposal is as follows: Aggrecan degradation in equine osteoarthritis is mediated by ADAMTS 'aggrecanase' activity (as opposed to MMP-mediated proteolysis). The data indicate that the interglobular domain NITEGE 'aggrecanase' cleavage motif is the predominant neo-epitope present in arthritic equine cartilage, consistent with the hypothesis under investigation. Further, equine articular chondrocytes isolated from arthritic cartilage rapidly cleave newly secreted aggrecan at the NITEGE site when maintained as three-dimensional aggregate cultures in defined, serum-free serum. Finally, known catabolic agents, such as interkeukin-1b, endotoxin, lipoteichoic acid and peptidoglycan stimulate aggrecan degradation predominantly by ADAMTS-mediated NITEGE cleavage. In contrast, the MMP-generated aggrecan core fragment (the VDIPEN
peptide fragment) was not present at high levels in these analyses. As expected, ADAMTS-1, -4 and -5 mRNA transcripts were identified in both normal and osteoarthritic articular cartilage collected from a number of horses. The current scientific literature on this subject indicates that regulation of ADAMTS enzymatic activity largely effects post-transcriptional targets, so the QPCR data does not necessarily reflect relative enzymatic activities of these ADAMTS gene products. In the context of these experiments, we have fully sequenced and characterized equine aggrecan, and have confirmed that the ADAMTS and MMP cleavage sites identified in the core protein sequence are fully conserved in the horse. Further, we have identified a previously unreported polymorphism in the repetitive elements of the chondroitin sulfate binding domain of equine aggrecan. This polymorphism directly impacts the extent of glycosaminoglycan substitution of the core protein and affects the net fixed charge
density the aggrecan:GAG complex can impart on the cartilage extracellular matrix. We are currently continuing this aspect of the study to determine whether this polymorphism is linked to the incidence of equine osteoarthritis and/or breed morphological differences. In related studies, we have found that the epigenetic regulatory agents 5 azacytidine and trichostatin A inhibit aggrecanase activity and result in an increase in secretion of full-length aggrecan, with a concomitant reduction in aggrecan fragment levels.
PARTICIPANTS: Matt Stewart - Principle Investigator. Evelyn Caporali - Graduate Student. Trina Kuykendall - Research Specialist. John Sandy - Collaborator.
TARGET AUDIENCES: The results of this research are intended to be of interest to veterinarians with a particular interest in articular disease and arthritis in horses, and to biomedical researchers focused on aggrecan dynamics in articular cartilage.
PROJECT MODIFICATIONS: The experiments proposed in the grant document constituted the primary focus of the completed work. We also carried out a considerable amount of work sequencing and characterizing equine aggrecan in the context of this work and also included aspects of a related research project currently in the lab, addressing the influence of epigenetic regulatory pathways and modulatory agents on articular chondrocytes.
Impacts
The results of this research indicate that ADAMTS-mediated aggrecan cleavage is a critical activity in equine osteoarthritis and is a promising target for therapeutic intervention. Our ongoing research in this area is focused on identifying agents that inhibit ADAMTS enzymatic activity in chondrocytes.
Publications
- Caporali, E., Stewart, M., Kuykendall, T., Gawriluk, T. and Jones, P.L. 2007. Sequencing and characterization of equine aggrecan. American Journal of Veterinary Research (Submitted).
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