Background Synovitis (synovial inflammation) is the main characteristics of rheumatoid arthritis (RA); a chronic inflammatory disease leading to progressive joint destruction. Synovitis is characterised by thickening of the synovial membrane due increased synoviocyte proliferation, leukocyte infiltration, innervations, vascularization and fibrosis. Fibrosis is described as increased extracellular matrix turnover, which is a result of the increased amount of proteolytic enzymes (MMPs and aggrecanases) and cell numbers. Especially, MMP-1, 3, and 9 is up regulated in RA. In this study, we investigated type I and III collagen, as these collagens are pronounced in the synovial membrane. Furthermore, we investigated type II collagen as this is the main collagen in cartilage and it is adjacent to the synovial membrane.
Objectives We investigated the turnover of collagens related to synovitis by utilising a panel of extracellular turnover biomarkers in a RA animal model (collagen induced arthritis (CIA)). Such panel of biomarkers reflecting the turnover of the affected tissues in RA may be a better approach in diagnosis differentiation of early and severe disease.
Methods CIA was induced in 10 Wistar rats by immunizations with 450 μl 2 mg/ml porcrine type II collagen dissolved in 0.05M acetic acid and emulsified 1:1 in incomplete Freunds Adjuvant in day 0 and 7. 10 Wistar rats were used as controls with injection of 0.05M acetic acid. On day 16 all 10 CIA rats showed signs of disease (paw swelling). All rats were sacrificed on day 26 and blood samples were taken throughout the experiment.
Six different ELISA’s were used to quantify the level of collagen turnover. Two unpublished assays P2NP and P3NP and four published assays PINP, C1M, C2M and C3M were used. P1NP, P2NP, and P3NP utilize neo-epitopes in the pro-peptide of type I, II and III collagen, respectively. C1M, C2M, and C3M are MMP-dependent degradation markers of the same collagens.
Results Increased degradation of type I (C1M) and III (C3M) collagen was detected from disease onset until termination (57.5±8.8% (P<0.01) and 41.8±5.2% compared to baseline (P<0.001) respectively), when compared to controls. However, there was no significant difference in the formation of type I (P1NP) and III (P3NP) collagen between CIA rats and control. A significant decrease (-19.4±5.6% compared to baseline, P<0.01) in type II collagen formation (P2NP) was detected in the CIA rats at day 16. A 31.9±10.5% increase in degradation of type II collagen (C2M) was detected in CIA rats compared to baseline at day 22.
The diagnostic utility of the biomarkers was investigated by ROC curves. None of the markers could discriminate between groups at the early time point (day 7). At the later time point (day 22), the formation markers all had an AUC of 0.53-0.67 (p>0.05). C1M had an AUC of 0.88 (P=0.0041), C3M had an AUC of 0.97 (p=0.00038) and C2M had an AUC of 0.85 (P=0.0082), suggesting that the three degradation markers were diagnostic for RA.
Conclusions In summary, a profile of tissue balance in RA was investigated. The results indicate that cartilage, synovium, and connective tissue turnover are important events in RA. We speculate that a combination of these biological relevant collagen turnover markers might be utilized in a panel as diagnostic and prognostic markers of RA.
Disclosure of Interest None Declared
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