MMP and non-MMP-mediated release of aggrecan and its fragments from articular cartilage: a comparative study of three different aggrecan and glycosaminoglycan assays.

OBJECTIVE: Aggrecan is the major proteoglycan in articular cartilage and is known to be degraded by various proteases, including matrix metalloproteinases (MMPs). The present study was undertaken to develop immunoassays detecting aggrecan and its fragments generated by MMP and non-MMP-mediated proteolysis. METHODS: Two immunoassays were developed: (1) the G1/G2 sandwich assay employing a monoclonal antibody (F-78) both as a capturing and a detecting antibody, and (2) the 342-G2 sandwich assay substituting the capturing antibody in the G1/G2 test with a monoclonal antibody, AF-28 recognizing the 342FFGVG neo-epitope generated by MMP cleavage. These assays were compared to the commercially available glycosaminoglycan (GAG) assay. RESULTS: In supernatants of Oncostatin M and Tumor Necrosis Factor alpha (OSM/TNFalpha) stimulated explants, high levels of G1/G2 fragments and GAGs were released in the initial phase (days 2-5), followed by low levels in the intermediate (days 9-12) and late phase (days 12-21). MMP-generated fragments were detected in the late phase only. In the presence of the general MMP inhibitor GM6001, 342-G2 was not detected, whereas the G1/G2 profile remained virtually unchanged. In patients with rheumatoid arthritis (RA), the release of G1/G2 molecules was decreased (27.3%), and that of the 342-G2 fragments increased compared to healthy controls (33.3%). CONCLUSION: The stimulation of bovine articular cartilage explants with OSM/TNFalpha released aggrecan fragments both in an MMP and non-MMP-mediated route. These immunoassays carry a potential as diagnostic tools for the quantitative assessment of the cartilage turnover in RA patients in addition to their utility in ex vivo explant cultures.

Application of biomarkers in the clinical development of new drugs for chondroprotection in destructive joint diseases: a review.

Emerging evidence supports the concept that biochemical markers are clinically useful non-invasive diagnostic tools for the monitoring of changes in cartilage turnover in patients with destructive joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). Epidemiological studies demonstrated that measurements of different degradation products of proteins in the extracellular matrix of hyaline cartilage in urine or serum samples are (1) increased in OA or RA patients compared with healthy individuals, (2) correlate with disease activity, and (3) are predictive for the rate of changes in radiographic measures of cartilage loss. The present review provides an updated list of available biomarkers and summarize the research data arguing for their clinical utility. In addition, it addresses the question whether or not the monitoring of biomarkers during different treatment modalities could be a useful approach to characterize the chondro-protective effects of approved and candidate drugs. Finally, it briefly reviews the in vitro/ex vivo experimental settings - isolated chondrocyte cultures and articular cartilage explants - that can assist in the verification of novel markers, but also studies assessing direct effects of drug candidates on chondrocytes. Collectively, biomarkers may acquire a function as established efficacy parameters in the clinical development of novel chondro-protective agents.

In vitro, ex vivo, and in vivo methodological approaches for studying therapeutic targets of osteoporosis and degenerative joint diseases: how biomarkers can assist?

Although our approach to the clinical management of osteoporosis (OP) and degenerative joint diseases (DJD)-major causes of disability and morbidity in the elderly-has greatly advanced in the past decades, curative treatments that could bring ultimate solutions have yet to be found or developed. Effective and timely development of candidate drugs is a critical function of the availability of sensitive and accurate methodological arsenal enabling the recognition and quantification of pharmacodynamic effects. The established concept that both OP and DJD arise from an imbalance in processes of tissue formation and degradation draws attention to need of establishing in vitro, ex vivo, and in vivo experimental settings, which allow obtaining insights into the mechanisms driving increased bone and cartilage degradation at cellular, organ, and organism levels. When addressing changes in bone or cartilage turnover at the organ or organism level, monitoring tools adequately reflecting the outcome of tissue homeostasis become particularly critical. In this context, bioassays targeting the quantification of various degradation and formation products of bone and cartilage matrix elements represent a useful approach. In this review, a comprehensive overview of widely used and recently established in vitro, ex vivo, and in vivo set-ups is provided, which in many cases effectively take advantage of the potentials of biomarkers. In addition to describing and discussing the advantages and limitations of each assay and their methods of evaluation, we added experimental and clinical data illustrating the utility of biomarkers for these methodological approaches.