Background Collagen isomerization is a non-enzymatical process associated with tissue aging, and changes in levels of isomerized peptide fragments may reflect unique changes during pathological conditions. The D-G sequence in C2M is likely to undergo beta-isomerization over time, and may be altered in tissue turnover diseases affecting the joint. We therefore hypothesize that pathological conditions, such as ankylosing spondylitis (AS) or rheumatoid arthritis (RA), which affects the general turnover of cartilage may result in altered levels of isomerized collagen type II.
Objectives The aims of this investigation were to develop a novel assay measuring an isomerized form of MMP degraded collagen II and to investigate whether it can be used as a clinical marker of joint deterioration in inflammatory joint diseases.
Methods A solid phase competitive ELISA was developed applying a specific monoclonal antibody targeting an isomerized type II collagen fragment, C2Mβ. The biomarker was characterized by measuring its generation in vitro by cleavage of bovine cartilage with a range of proteases. Furthermore, the release of C2Mβ into the supernatant of bovine explants cultured ex vivo were measured under normal (without cytokines) and catabolic (TNF-α and oncostatin M) conditions. Serum samples from AS patients (n=28, age 25–76) or RA patients (n=149, age 15–86) were collected and C2Mβ measured. The change in C2Mβ after TNF-alpha treatment for 52 weeks was analyzed in AS patients. ROC curves were generated and the area under the curve calculated (AUC) to assess the assays capacity to distinguish between the diseases. Multivariable logistic regression was employed to correct for age and gender.
Results The antibody against C2Mβ was specific towards the neo-epitope and did not recognize the non-isomerized form. C2Mβ was released from bovine cartilage when cleaved with MMP2 or MMP9 but not Cathepsin S. Levels of C2Mβ was increased in the supernatant of ex vivo cultured bovine cartilage explants in response to the catabolic cytokines TNF-α and Oncostatin M (p<0.001), compared to unstimulated explants which did not release C2Mβ. The addition of the MMP inhibitor GM6001 to catabolically activated explants completely abrogated the release of C2Mβ. C2Mβ was slightly decreased in AS patients treated with TNF-α inhibitors (10%), although the effect was not significant. C2Mβ was able to distinguish AS from RA with an AUC of 0.785, which increased to 0.905 when including age and gender
Conclusions The C2Mβ neoepitope is released from catabolically activated cartilage and is measurable in human serum. C2Mβ is elevated in AS patients compared to RA patients and the marker can discriminate AS from RA with high diagnostic accuracy. Anti-inflammatory treatment in AS patients resulted in a trend towards a decrease in the marker compared to baseline. These findings suggest that systemic levels of C2Mβ may potentially serve as a joint specific biomarker for monitoring joint turnover and destruction as well as treatment efficacy in rheumatic diseases
Disclosure of Interest C. Thudium Employee of: Nordic Bioscience A/S, Y. He Employee of: Nordic Bioscience A/S, N. Gudmann Employee of: Nordic Bioscience A/S, K. De Vlam: None declared, R. Lories: None declared, S. Hirata: None declared, Y. Tanaka: None declared, M. Karsdal Shareholder of: Nordic Bioscience A/S, Employee of: Nordic Bioscience A/S, A.-C. Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Nordic Bioscience A/S
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