Objective and noninvasive biochemical markers in rheumatoid arthritis: where are we and where are we going?

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic autoimmune disease that affects approximately 1% of the adult population. RA is multi-factorial, and as such our understanding of the molecular pathways involved in the disease is currently limited. An increasing number of studies have suggested that several molecular phenotypes (i.e. endotypes) of RA exist, and that different endotypes respond differently to various treatments. Biochemical markers may be an attractive means for achieving precision medicine, as they are objective and easily obtainable. AREAS COVERED: We searched recent publications on biochemical markers in RA as either diagnostic or prognostic markers, or as markers of disease activity. Here, we provide a narrative overview of different classes of markers, such as autoantibodies, citrulline products, markers of tissue turnover and cytokines, that have been tested in clinical cohorts or trials including RA patients. EXPERT OPINION: Although many biochemical markers have been identified and tested, few are currently being used in clinical practice. As more treatment options are becoming available, the need for precision medicine tools that can aid physicians and patients in choosing the right treatment is growing.

Effect of n-3 PUFA on extracellular matrix protein turnover in patients with psoriatic arthritis: a randomized, double-blind, placebo-controlled trial.

Psoriatic arthritis (PsA) is a chronic inflammatory disease characterized by involvement of skin, axial and peripheral skeleton. An altered balance between extracellular matrix (ECM) formation and breakdown is a key event in PsA, and changes in ECM protein metabolites may provide insight to tissue changes. Dietary fish oils (n-3 PUFA) might affect the inflammation driven tissue turnover. The aim was to evaluate ECM metabolites in patients with PsA compared to healthy individuals and investigate the effects of n-3 PUFA. The 24-week randomized, double-blind, placebo-controlled trial of PUFA included 142 patients with PsA. Fifty-seven healthy individuals were included for comparison. This study is a sub-study investigating biomarkers of tissue remodelling as secondary outcomes. Serum samples at baseline and 24 weeks and healthy individuals were obtained, while a panel of ECM metabolites reflecting bone and soft tissue turnover were measured by ELISAs: PRO-C1, PRO-C3, PRO-C4, C1M, C3M, C4M, CTX-I and Osteocalcin (OC). C1M, PRO-C3, PRO-C4 and C4M was found to be elevated in PsA patients compared to the healthy individuals (from 56 to 792%, all p < 0.0001), where no differences were found for OC, CTX-I, PRO-C1 and C3M. PRO-C3 was increased by 7% in patients receiving n-3 PUFA after 24 weeks compared to baseline levels (p = 0.002). None of the other biomarkers was changed with n-3 PUFA treatment. This indicates that tissue turnover is increased in PsA patients compared to healthy individuals, while n-3 PUFA treatment for 24 weeks did not have an effect on tissue turnover. Trial registration NCT01818804. Registered 27 March 2013-Completed 18 February 2016. https://clinicaltrials.gov/ct2/show/NCT01818804?term=NCT01818804&rank=1.

The Anti-ADAMTS-5 Nanobody® M6495 Protects Cartilage Degradation Ex Vivo.

Osteoarthritis (OA) is associated with cartilage breakdown, brought about by ADAMTS-5 mediated aggrecan degradation followed by MMP-derived aggrecan and type II collagen degradation. We investigated a novel anti-ADAMTS-5 inhibiting Nanobody® (M6495) on cartilage turnover ex vivo. Bovine cartilage (BEX, n = 4), human osteoarthritic - (HEX, n = 8) and healthy-cartilage (hHEX, n = 1) explants and bovine synovium and cartilage were cultured up to 21 days in medium alone (w/o), with pro-inflammatory cytokines (oncostatin M (10 ng/mL) + TNFα (20 ng/mL) (O + T), IL-1α (10 ng/mL) or oncostatin M (50 ng/mL) + IL-1ß (10 ng/mL)) with or without M6495 (1000-0.46 nM). Cartilage turnover was assessed in conditioned medium by GAG (glycosaminoglycan) and biomarkers of ADAMTS-5 driven aggrecan degradation (huARGS and exAGNxI) and type II collagen degradation (C2M) and formation (PRO-C2). HuARGS, exAGNxI and GAG peaked within the first culture week in pro-inflammatory stimulated explants. C2M peaked from day 14 by O + T and day 21 in co-culture experiments. M6495 dose dependently decreased huARGS, exAGNxI and GAG after pro-inflammatory stimulation. In HEX C2M was dose-dependently reduced by M6495. M6495 showed no effect on PRO-C2. M6495 showed cartilage protective effects by dose-dependently inhibiting ADAMTS-5 mediated cartilage degradation and inhibiting overall cartilage deterioration in ex vivo cartilage cultures.

Association of metabolites reflecting type III and VI collagen formation with modified Rodnan skin score in systemic sclerosis - a cross-sectional study.

Objective: The objective was to investigate blood-based biomarkers of type I (PRO-C1), III (PRO-C3) and VI (PRO-C6) collagen formation in systemic sclerosis (SSc) patients and examine their correlation to modified Rodnan skin score (mRSS). Methods: Limited (lSSc, n = 76) and diffuse SSc (dSSc, n = 41) fulfilling the ACR/EULAR 1980 and 2013 classification criteria for SSc and asymptomatic controls (n = 9) were included. PRO-C1, PRO-C3 and PRO-C6 were measured in serum. Results: LSSc compared to dSSc were significantly older, had longer disease duration and lower mRSS. PRO-C3 was higher in early dSSc compared to early lSSc (mean [95 percentile], 27.4 [13.1-39.1] ng/mL vs 14.9 [8.2-28.8] ng/mL, p = 0.006). PRO-C6 levels were higher in early dSSc compared to early lSSc and late dSSc (early dSSc: 28.2 [10.4-92.3] ng/ml vs early lSSc: 11.0 [6.9-28.5] ng/ml; p = 0.006 and late dSSc: 12.6 [6.5-25.3] ng/mL, p = 0.04). No difference was observed with PRO-C1. PRO-C3 and PRO-C6 were moderately correlated with mRSS with R-partials of 0.36 (p < 0.001) and 0.29 (p = 0.002), respectively Conclusion: Measures of type III and VI collagen formation are potential objective biomarkers of fibrosis in systemic sclerosis. These biomarkers could be useful in monitoring the disease and efficacy of treatment.

Citrullinated vimentin and biglycan protein fingerprints as candidate serological biomarkers for disease activity in systemic sclerosis: a pilot study.

Purpose: Extracellular matrix (ECM) deposition and remodelling in skin and lungs of systemic sclerosis (SSc) subjects lead to release of metabolites/biomarkers into circulation. We investigated if biomarkers of ECM degradation (biglycan and elastin) and macrophage activation (citrullinated vimentin) could identify diffuse SSc (dSSc) subjects from controls and the biomarkers discriminative power. Methods: DSSc subjects (n = 40) fulfilling the 2013 EULAR/ACR classification criteria were divided in early (<2years of symptoms) and late (≥10 years of symptoms). Early were subdivided into intermediate and rapid skin thickness progression rate (STPR). Twenty controls were included. Citrullinated and matrix metalloproteinase (MMP)-2/8-degraded vimentin (VICM), MMP-9/12-degraded biglycan (BGM) and MMP-7-degraded elastin (ELM-7) were assessed in serum. Analysis between groups was by Kruskal-Wallis and ROC AUC for discriminative power. Results: VICM and BGM levels were increased in early compared with late dSSc (p< =0.023). VICM was increased in rapid and intermediate STPR compared with controls (p< =0.025). No differences in ELM-7 levels were observed. AUC of VICM was 0.71 for early versus late dSSc and BGM had an AUC of 0.79 for dSSc versus controls. Conclusion: This pilot study found differences in biomarker levels between early and late dSSc. This study offers new perspectives of ECM metabolites as potential biomarkers of dSSc.

Metabolites of C-reactive protein and vimentin are associated with disease activity of axial spondyloarthritis.

OBJECTIVES: Non-radiographic (nr-axSpA) and radiographic (AS) forms of axial spondyloarthritis (axSpA) share clinical features, but have different radiographic patterns. Radiographic progression is not associated with the current disease activity biomarkers. We investigated a matrix metalloproteinase mediated metabolite of C-reactive protein (CRPM) and two biomarkers of citrullinated vimentin (VICM and anti-MCV) as novel biomarkers of disease activity. METHODS: AxSpA patients (n=121 nr-axSpA and n=72 AS) were characterised by activity (AS disease activity score with CRP [ASDAS-CRP], Bath AS disease activity index [BASDAI] and functional index [BASFI]), radiographic scores and quality of life questionnaires. CRPM, VICM and anti-MCV levels were analysed by ELISA in serum. Asymptomatic controls (n=100) were used as reference. Multiple regression investigated association with disease activity and diagnostic potential. RESULTS: CRPM and VICM levels were increased in AS compared to nr-axSpA (11.9nM vs. 10.2nM, p<0.001 and 4.92nM vs. 3.77nM, p=0.0025). Anti-MCV was similar in both axSpA subgroups, but lowered in controls. In nr-axSpA, CRPM correlated with CRP (ρ=0.33, p<0.001) and VICM (ρ=0.29, p=0.001); in AS, VICM correlated with CRP (ρ=0.34, p=0.0032) and ESR (ρ=0.38, p<0.001). ASDAS-CRP correlated with CRPM and anti-MCV, but when adjusting for CRP the correlation only remained with CRPM. CRPM and VICM separated the subgroups with odds ratios of 1.19 and 1.10 adjusted for age, gender, BMI, and disease duration. VICM lost significance when adjusting for CRP. CONCLUSIONS: CRPM was associated with disease activity in axSpA, and CRPM and VICM separated the axSpA groups. This study indicates that serological biomarkers may be novel biomarkers in axSpA.

Suppression of active, but not total MMP-3, is associated with treatment response in a phase III clinical study of rheumatoid arthritis.

OBJECTIVE: Biologics for rheumatoid arthritis (RA) patients with moderate to severe disease may preserve joint function. Matrix metalloproteinase 3 (MMP-3), a key tissue degrading protease, is highly elevated in RA. MMP-3, which measures the total pool of circulating MMP-3 species (cMMP3), is a commonly measured biomarker in rheumatology. The aim was to investigate the association of activated MMP-3 (actMMP3) species with treatment response compared to cMMP-3. METHODS: The LITHE biomarker study (n=741) was a 1-year phase III, double-blind, placebo-controlled, parallel group study of TCZ in RA patients on stable methotrexate. cMMP-3 and actMMP-3 were assessed in fasting serum at baseline, week 4, 16, 24 and 52. Patients not achieving ACR20 remission at week 16 or 28 received rescue treatment (escapers). Spearman's correlation was analysed between biomarker baseline level or biomarker delta and clinical measures. Changes in biomarker levels were studied as a function of time and treatment. RESULTS: ActMMP-3 16-week change in treatment groups was predictive of 1-year radiographic progression; a small change in actMMP3 was equal to worsening radiographics. Baseline cMMP-3 was associated with 52-weeks' radiographic status and cMMP3 16-weeks' change was predictive of 1-year change in disease activity. ActMMP-3 was dose-dependently decreased by TCZ, and escapers decreased in actMMP-3 upon treatment. CONCLUSIONS: ActMMP-3 and cMMP-3 were found to be efficacy biomarkers of TCZ and actMMP-3 were able to differentiated doses. Moreover, the suppression of actMMP3, but not cMMP3 was associated with treatment response. This study illustrates that two biomarkers of the same protein may have different predictive capacities.

IL-6 receptor inhibition modulates type III collagen and C-reactive protein degradation in rheumatoid arthritis patients with an inadequate response to anti-tumour necrosis factor therapy: analysis of connective tissue turnover in the tocilizumab RADIATE study.

OBJECTIVES: The objective of this study was to examine the tissue degradation in response to anti-IL6 receptor treatment in rheumatoid arthritis (RA) patients which are anti-TNF-α inadequate responders. METHODS: RADIATE was a randomised, double-blinded, placebo-controlled, parallel-group, phase III trial. RA patients with previous inadequate response to anti-TNFα therapy (n=299) were randomly assigned to tocilizumab 4 or 8 mg/kg with methotrexate (10-25 mg weekly) or placebo with methotrexate. Type III collagen degradation (C3M) and CRP degradation (CRPM) were analysed in serum samples at baseline and 16 weeks. RESULTS: Treatment with 4 and 8 mg/kg tocilizumab significantly decreased C3M (p=0.0001 and p=0.0007) and CRPM (p<0.0001) levels after 16 weeks. Changes in C3M and CRPM levels after 16 weeks correlated well with the changes in disease activity score 28 (DAS28). Change in CRPM levels furthermore correlated moderately with the change in patient pain (VAS) (rpartial of 0.20) and Health assessment questionnaire disability index (HAQ-DI) (rpartial of 0.24). Changes in biomarker levels above median change led to an odds ratio of 1.95 (C3M) and 3.00 (CRPM) for achieving the American College of Rheumatology 20% improvement criteria (ACR20). CONCLUSIONS: This study shows that a decrease in inflammation leads to a decrease in excessive extracellular matrix degradation. It furthermore supports earlier shown evidence that tocilizumab works in the treatment of RA patients, although there is a clear need for identifying and selecting patients who are more likely to respond to treatment.

Type IV collagen metabolism is associated with disease activity, radiographic progression and response to tocilizumab in rheumatoid arthritis.

OBJECTIVES: The expanding spectrum of targeted therapies for rheumatoid arthritis (RA) implies a need for development of precision tools for disease assessment reflecting pathobiologic processes. Type IV collagen is an abundant protein of basement membranes, but is also present in the intercellular matrix of the synovial lining layer. We aimed to investigate the association of type IV collagen turnover with RA disease activity, response to IL-6 inhibition and radiographic progression. METHODS: C4M, a serologic marker of type IV collagen metabolism, was measured at baseline and at follow-up in serum samples of RA patients participating in the phase III studies LITHE (n=687) and RADIATE (n=217). Both were double-blinded, placebo-controlled clinical trials testing the safety and efficacy of 4 and 8 mg/kg tocilizumab (TCZ) in combination with methotrexate (MTX) vs. MTX plus placebo. Associations with disease activity, radiographic severity and ACR response were investigated. RESULTS: Baseline C4M correlated significantly with clinical disease parameters in both study populations, including DAS28, HAQ score and VASpain (all p<0.00001). C4M at baseline correlated significantly with change in JSN (p=0.001) and Sharp score (p=0.00002) at 52 weeks. TCZ lowered C4M by 11-40% in a dose dependent manner. The likelihood of achieving an ACR20 response by week 16 was associated with C4M suppression exceeding the median decrease at week 4 (p<0.0001). CONCLUSIONS: Type IV collagen remodelling was associated with disease activity and radiographic progression in RA and was persistently and dose-dependently suppressed by TCZ. These findings indicate that C4M may serve as a plausible biologic marker of destructive synovitis growth in RA.

Sprifermin (rhFGF18) modulates extracellular matrix turnover in cartilage explants ex vivo.

BACKGROUND: Sprifermin (recombinant human fibroblast growth factor 18) is in clinical development as a potential disease-modifying osteoarthritis drug (DMOAD). In vitro studies have shown that cartilage regenerative properties of sprifermin involve chondrocyte proliferation and extracellular matrix (ECM) production. To gain further insight into the process of sprifermin in the cartilage tissue, this study aimed at investigating the ECM turnover of articular cartilage explants in a longitudinal manner. METHODS: Bovine full-depth articular cartilage explants were stimulated with sprifermin or placebo at weekly intervals, similar to the dosing regimen used in clinical trials. Pre-culturing with oncostatin M and tumour necrosis factor-α, was also used to induce an inflammatory state before treatment. Metabolic activity was measured using AlamarBlue, and chondrocyte proliferation was visualized by immuno-histochemical detection of proliferating cell nuclear antigen. ECM turnover was quantified by biomarker ELISAs; ProC2 reflecting type II collagen formation, CS846 reflecting aggrecan formation, active MMP9, C2M and AGNx2 reflecting matrix metalloproteinase activity, and AGNx1 reflecting aggrecanase activity. RESULTS: Sprifermin was able to reach the chondrocytes through the extracellular matrix, as it increased cell proliferation and metabolic activity of explants. ProC2 and CS846 was dose-dependently increased (P < 0.05) by sprifermin compared to placebo, while C2M and AGNx2 were unaffected, active MMP9 was slightly decreased, and AGNx1 was slightly increased. Over the course of treatment, the temporal order of ECM turnover responses was AGNx1, then ProC2, followed by CS846 and MMP9. Pro-inflammatory activation of the explants diminished the ECM turnover responses otherwise observed under non-inflammatory conditions. CONCLUSIONS: The data suggest that sprifermin has chondrogenic effects on articular cartilage ex vivo, exerted through a sequential process of ECM turnover; aggrecan degradation seems to occur first, while type II collagen and aggrecan production increased at a later time point. In addition, it was observed that these chondrogenic effects are dependent on the inflammatory status of the cartilage prior to treatment.

Type I and III collagen turnover is increased in axial spondyloarthritis and psoriatic arthritis. Associations with disease activity and diagnostic capacity.

OBJECTIVES: To investigate the turnover of type I and III collagen by neo-epitope markers in patients with axial spondyloarthritis (axSpA) and psoriatic arthritis (PsA). METHODS: Patients with PsA (n=101) or axSpA (n=110) and healthy subjects (n=120) were included. Demographic and clinical data were recorded. Markers of type I and III collagen were quantified by RIA (ICTP) or ELISA (C1M and C3M). Non-parametric statistics were applied for intergroup comparisons and correlation studies. The diagnostic potential of these marker molecules was assessed by ROC analysis. RESULTS: C1M and C3M, which originate from soft connective tissues, were significantly higher in axSpA and PsA as compared with healthy control subjects. CIM and C3M correlated with ASDAS and DAS28. Overall, ICTP, which arises from bone degradation, did not differ between disease versus healthy. However, ICTP was lower in HLA-B27 positive than in HLA-B27 negative patients with axSpA. There was no association between bone and soft connective tissue collagen I markers (ICTP and C1M), while C1M and C3M were highly correlated (p<0.0001). C1M discriminated between healthy and diseased with AUCs of 0.83 for PsA and 0.79 for axSpA. C3M AUCs were 0.77 for PsA and 0.78 for axSpA. CONCLUSIONS: Type I and III collagen remodelling in soft connective tissue is increased in axSpA and PsA and associates with disease activity. Bone collagen degradation is lower in HLA-B27 positive compared with HLA-B27 negative axSpA, which may represent an aspect of enhanced enthesopathic bone proliferation in HLA-B27 carriers. C1M and C3M distinguish well between healthy and diseased individuals.

Intercritical circulating levels of neo-epitopes reflecting matrixmetalloprotease-driven degradation as markers of gout and frequent gout attacks.

OBJECTIVE: Recurrent flares constitute the main clinical burden of gout. Our aim was to assess whether biomarkers measuring MMP tissue degradation could be used as markers of frequent gout flares. METHODS: Fasting plasma samples from 112 men with gout and 170 controls, along with serum samples from 447 men with gout collected at baseline from an ongoing clinical trial, were analysed by ELISA for neo-epitopes from MMP degradation of collagens type I (C1M) and type III (C3M). The log10 levels of both markers were compared between cases and controls and between gout patients with three or more gout attacks in the past year and those with two or less attacks. RESULTS: The circulating levels of C1M and C3M correlated with gout status in the case-control study. Levels of both markers were associated with frequent gout flares (⩾3 attacks in the past year) in both cohorts (odds ratio, OR = 3.1; 95% CI: 1.4, 6.8; P = 0.0056 for log10C1M, and OR = 6.7; 95% CI: 2.3, 19.3; P = 0.0005 for log10C3M). The area under the curve in a receiver operating characteristic analysis of frequent flares increased from 0.68 to 0.74 in one cohort and from 0.60 to 0.66 in the other when log10C1M and log10C3M were added to clinical variables of the model. CONCLUSION: C1M and C3M, reflective of interstitial matrix destruction, are associated with gout status and with frequent gout flares in men, suggesting that increased MMP activity may contribute to gout flares. Further research is needed to find out whether this is independent of dietary and lifestyle risk factors for acute gout.

Cartilage collagen type II seromarker patterns in axial spondyloarthritis and psoriatic arthritis: associations with disease activity, smoking and HLA-B27.

The aim of the study was to assess the possible association between type II collagen turnover seromarkers and disease profile in patients with axial spondyloarthritis (SpA) and psoriatic arthritis (PsA). Outpatients with axial SpA (n = 110) or PsA (n = 101) underwent clinical examination including disease activity measures and HLA-B27 typing. The procollagen IIA N-terminal peptide (PIIANP) and a matrix metalloproteinase-generated type II collagen fragment (C2M) were quantified in serum by ELISA. C2M was higher in SpA than in controls, 0.41 versus 0.36 ng/ml (p = 0.004), while PIIANP did not differ between patients and healthy subjects, 2252 versus 2142 ng/ml (p = 0.13). However, DMARD-naïve SpA patients had higher PIIANP, 2461 ng/ml (p = 0.01) and C2M, 0.44 ng/ml (p = 0.0007) levels than controls, and PIIANP correlated with CRP (ρ = 0.34). C2M was lower in SpA smokers, 0.36 ng/ml versus non-smokers, 0.43 ng/ml (p = 0.02), while PIIANP was higher in HLA-B27 positive, 2312 ng/ml versus negative patients, 2021 ng/ml (p = 0.03). In PsA, PIIANP and C2M did not differ between patients and controls, but PIIANP was elevated in patients not receiving DMARDs, 2726 ng/ml. In PsA, PIIANP and C2M did not differ according to smoking and HLA-B27. Cartilage degradation assessed by C2M is increased in SpA irrespective of treatment but not in PsA. Cartilage synthesis reflected by PIIANP is increased in untreated SpA and PsA. PIIANP correlates with CRP in SpA while not in PsA. In DMARD-naïve SpA but not in PsA, HLA-B27 positivity and smoking are associated with a chondro-proliferative metabolic pattern.

Synovitis biomarkers: ex vivo characterization of three biomarkers for identification of inflammatory osteoarthritis.

OBJECTIVE: Characterize biomarkers measuring extracellular matrix turnover of inflamed osteoarthritis synovium. METHODS: Human primary fibroblast-like synoviocytes and synovial membrane explants (SMEs) treated with various cytokines and growth factors were assessed by C1M, C3M, and acMMP3 in the conditioned medium. RESULTS: TNFα significantly increased C1M up to seven-fold (p = 0.0002), C3M up to 24-fold (p = 0.0011), and acMMP3 up to 14-fold (p < 0.0001) in SMEs. IL-1ß also significantly increased C1M up to five-fold (p = 0.00094), C3M four-fold (p = 0.007), and acMMP3 18-fold (p < 0.0001) in SMEs. CONCLUSION: The biomarkers C1M, C3M, and acMMP-3 were synovitis biomarkers ex vivo and provide a translational tool together with the SME model.

The active form of MMP-3 is a marker of synovial inflammation and cartilage turnover in inflammatory joint diseases.

BACKGROUND: Matrix metalloproteinase-3 (MMP-3) plays an important role in the pathology of rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Measurement of active MMP-3 in clinical samples could provide information about progression of rheumatoid diseases, and potentially response to treatment. Hence, we aimed to develop a sensitive assay specifically measuring the active form of MMP-3 (act-MMP-3) both in ex vivo models and in human sera. METHODS: A monoclonal antibody against the first 6 amino acids of act-MMP-3 was developed, and the specificity was carefully tested by comparing total and active MMP-3. A technically robust act-MMP-3 ELISA was produced. For biological validation, human synovial membrane and human cartilage explant (HEX) culture models were measured and compared by ELISA and immunoblots. For clinical relevance, the serum levels of act-MMP-3 in AS and RA patients before and after anti-TNF-α treatment were evaluated. RESULTS: A highly specific and technically robust ELISA detecting act-MMP-3 in serum was developed. The lower limit of detection was 33.7 pg/mL. The dilution and spiking recovery of human serum was within 100 ± 20%. The average intra- and inter-assay variations were 3.1% and 13.5% respectively.High levels of act-MMP-3 expression were observed in human synovial membrane culture and oncostatin M and TNF-α stimulated human cartilage. In a cross-sectional study of both AS and RA patients, serum act-MMP-3 level was correlated with C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR). In addition, in patients receiving anti-TNF-α treatment, the serum level of act-MMP-3 was significantly reduced compared to baseline level reflecting the anti-inflammatory effects of the treatment. CONCLUSION: We have successfully developed an assay measuring act-MMP-3 in human serum showing correlation to inflammatory markers. Further studies are required to clarify, whether act-MMP-3 can serve as a predictive marker for outcome in chronic rheumatoid disorders.

Type X collagen levels are elevated in serum from human osteoarthritis patients and associated with biomarkers of cartilage degradation and inflammation.

BACKGROUND: Osteoarthritis (OA) is the most common degenerative joint disease, of which the pathogenesis is inadequately understood. Hypertrophy-like changes have been observed as part of the progression of OA. The aim of the study was to develop and characterize a novel biomarker of chondrocytes hypertrophy and investigate how this marker was associated with cartilage degradation and inflammation in patients with various degrees of OA. METHODS: A competitive ELISA, C-Col10, applying a well-characterized monoclonal antibody was developed as a biomarker of chondrocyte hypertrophy through measurement of type X collagen (ColX). The levels of C-Col10, C2M (matrix metalloproteinase-derived fragments of type II collagen) and hsCRP (high sensitive C-reactive protein) were quantified by ELISAs in serum of 271 OA patients stratified by Kellgren-Lawrence (KL) score 0-4. Associations between serum levels of the three biomarkers (log transformed) were analyzed by Pearson's correlation and differences in C-Col10 levels between patients with high and low levels of inflammation measured by hsCRP were analyzed by ANOVA. RESULTS: We developed a C-Col10 assay measuring the C-terminus of ColX. We found significantly higher levels of ColX in patients with KL score 2 compared to patients with no radiographic evidence of OA (KL0) (p = 0.04). Levels of ColX were significantly elevated in OA patients with above normal hsCRP levels (p < 0.0001), as well as significantly correlated with levels of C2M (r = 0.55, p < 0.0001), which suggested that chondrocyte hypertrophy was associated with inflammation and cartilage degradation. There was no correlation between C2M and hsCRP. Age and BMI adjustment didn't change the results. Immuno-staining revealed that ColX was predominately located around the hypertrophic chondrocytes and the clustered chondrocytes indicating that C-Col10 measures may be linked to cartilage hypertrophic changes. CONCLUSIONS: We developed a novel assay, C-Col10, for measurement of chondrocyte hypertrophy and found its levels significantly elevated in OA patients with KL score of 2, and also in OA patients with above normal hsCRP levels. Concentration of C-Col10 strongly correlated with levels of C2M, a marker of cartilage destruction. The data suggest that chondrocyte hypertrophy and subsequent collagen X fragmentation seem to be increased in a subset of patients with inflammatory OA.

Cartilage turnover reflected by metabolic processing of type II collagen: a novel marker of anabolic function in chondrocytes.

The aim of this study was to enable measurement of cartilage formation by a novel biomarker of type II collagen formation. The competitive enzyme-linked immunosorbent assay (ELISA) Pro-C2 was developed and characterized for assessment of the beta splice variant of type II procollagen (PIIBNP). This is expected to originate primarily from remodeling of hyaline cartilage. A mouse monoclonal antibody (Mab) was raised in mouse, targeting specifically PIIBNP (QDVRQPG) and used in development of the assay. The specificity, sensitivity, 4-parameter fit and stability of the assay were tested. Levels of PIIBNP were quantified in human serum (0.6-2.2 nM), human amniotic fluid (163-188 nM) and sera from different animal species, e.g., fetal bovine serum (851-901 nM) with general good linearity (100% (SD 7.6) recovery) and good intra- and inter-assay variation (CV% < 10). Dose (0.1 to 100 ng/mL) and time (7, 14 and 21 days) dependent release of PIIBNP were evaluated in the conditioned medium from bovine cartilage explants (BEX) and human cartilage explants (HEX) upon stimulation with insulin-like growth factor (IGF-1), transforming growth factor (TGF)-ß1 and fibroblastic growth factor-2 (FGF-2). TGF-ß1 and IGF-1 in concentrations of 10-100 ng/mL significantly (p < 0.05) induced release of PIIBNP in BEX compared to conditions without treatment (WO). In HEX, IGF-1 100 ng/mL was able to induce a significant increase of PIIBNP after one week compared to WO. FGF-2 did not induce a PIIBNP release in our models. To our knowledge this is the first assay, which is able to specifically evaluate PIIBNP excretion. The Pro-C2 assay seems to provide a promising and novel marker of type II collagen formation.

Dysregulation of circulating collagen turnover markers in very early systemic sclerosis.

OBJECTIVE: Clinical observation suggests that vascular activation and autoimmunity precede remodelling of the extracellular matrix (ECM) in systemic sclerosis (SSc). We challenge this paradigm by hypothesising that ECM biomarkers are already disturbed in patients with very early SSc (veSSc) when fibrosis is not yet clinically detectable. METHODS: 42 patients with veSSc, defined as the presence of Raynaud's phenomenon and at least one of puffy fingers, positive antinuclear antibodies or pathological nailfold capillaroscopy, not meeting the 2013 American College of Rheumatology/European Alliance of Associations for Rheumatology classification criteria for SSc, were compared with healthy controls (HCs, n=29). ECM degradation (BGM, C3M, C4M and C6M) and ECM formation biomarkers (PRO-C3, PRO-C4 and PRO-C5) were measured in serum using ELISAs. A cross-sectional analysis at baseline and a longitudinal analysis was performed. RESULTS: Compared with HC, veSSc patients showed a strongly dysregulated turnover of type III and IV collagens (higher C3M, C4M, both p<0.0001 and PRO-C3, p=0.004, lower turnover ratios PRO-C3/C3M and PRO-C4/C4M, both p<0.0001). The biglycan degradation biomarker BGM was higher in veSSc than in HC (p=0.006), whereas the degradation biomarker for type VI collagen, C6M, was lower (p=0.002). In an ROC analysis, biomarkers of type III and IV collagen excellently distinguished between veSSc and HC: C3M, AUC=0.95, p<0.0001; C4M, AUC=0.97, p<0.0001; turnover ratios PRO-C3/C3M, AUC=0.80, p<0.0001; PRO-C4/C4M, AUC=0.97; p<0.0001. CONCLUSION: These findings indicate ECM remodelling as a very early phenomenon of SSc occurring in parallel with microvascular and autoimmune changes. Biomarkers of type III and IV collagens distinguished between veSSc patients and HC, indicating them as potential biomarkers for the detection of veSSc.

Fibroblasts are not just fibroblasts: clear differences between dermal and pulmonary fibroblasts' response to fibrotic growth factors.

Systemic Sclerosis (SSc) hallmark is skin fibrosis, but up to 80% of the patients have fibrotic involvement in the pulmonary system. Antifibrotic drugs which have failed in a general SSc population have now been approved in patients with SSc-associated interstitial lung disease (ILD). This indicates that the fibrotic progression and regulation of fibroblasts likely depend on local factors specific to the tissue type. This study investigated the difference between dermal and pulmonary fibroblasts in a fibrotic setting, mimicking the extracellular matrix. Primary healthy fibroblasts were grown in a crowded environment and stimulated with TGF-ß1 and PDGF-AB. The viability, morphology, migration capacity, extracellular matrix formation, and gene expression were assessed: TGF-ß1 only increased the viability in the dermal fibroblasts. PDGF-AB increased the migration capacity of dermal fibroblasts while the pulmonary fibroblasts fully migrated. The morphology of the fibroblasts was different without stimulation. TGF-ß1 increased the formation of type III collagen in pulmonary fibroblasts, while PDGF-AB increased it in dermal fibroblasts. The gene expression trend of type VI collagen was the opposite after PDGF-AB stimulation. The fibroblasts exhibit different response profiles to TGF-ß1 and PDGF-AB; this suggests that drivers of fibrosis are tissue-dependent, which needs to be considered in drug development.

Matrix metalloproteinase-dependent turnover of cartilage, synovial membrane, and connective tissue is elevated in rats with collagen induced arthritis.

BACKGROUND: Rheumatoid arthritis is a disease affecting the extracellular matrix of especially synovial joints. The thickness of the synovial membrane increases and surrounding tissue degrades, leading to altered collagen balance in the tissues. In this study, we investigated the altered tissue balance of cartilage, synovial membrane, and connective tissue in collagen induced arthritis (CIA) in rats. METHODS: Six newly developed ELISAs quantifying MMP-derived collagen degradation (C1M, C2M, and C3M) and formation (P1NP, P2NP, and P3NP) was used to detect cartilage turnover in rats with CIA. Moreover, CTX-II was used to detect alternative type II collagen degradation and as control of the model. 10 Lewis rats were injected with porcrine type II collagen twice with a 7 day interval and 10 rats was injected with 0.05 M acetic acid as control. The experiment ran for 26 days. RESULTS: A significant increase in the degradation of type I, II, and III collagen (C1M, C2M, and C3M, respectively) was detected on day 22 (P = 0.0068, P = 0.0068, P < 0.0001, respectively), whereas no significant difference in formation (P1NP, P2NP, and P3NP) was detected at any time point (P=0.22, P=0.53, P=0.53, respectively). The CTX-II level increased strongly from disease onset and onwards. CONCLUSION: A nearly total separation between diseased and control animals was detected with C3M, making it a good diagnostic marker. The balance of type I, II, and III collagen was significantly altered with CIA in rats, with favour of degradation of the investigated collagens. This indicates unbalanced turnover of the surrounding tissues of the synovial joints, leading to increased pain and degeneration of the synovial joints.