Serologically assessed heat shock protein 47 is related to fibrosis stage in early compensated alcohol-related liver disease.

BACKGROUND AND AIMS: Heat shock protein (HSP)47 is a collagen-specific chaperone, essential for the correct formation of fibrillar procollagens. Collagen accumulation in the extracellular matrix (ECM) is a hallmark of fibrogenesis. The expression of HSP47 is proportional to the rate of collagen formation. Thus, HSP47 is a potential drug target for fibrotic diseases. We hypothesized that a C-terminal fragment of HSP47 (HSP47-C) could be quantified serologically and related to liver fibrosis stage. For this, a novel competitive enzyme-linked immunosorbent assay (ELISA) was developed. METHOD: An ELISA employing a monoclonal antibody targeting HSP47-C was developed and technically validated. The assay was evaluated in serum from a cross-sectional biopsy-controlled study of 281 patients with alcohol-related liver disease (ALD) and 50 gender, age and BMI matched healthy controls (HC). All liver biopsies from ALD patients were scored by one pathologist according to fibrosis stage (F0-4). RESULTS: The HSP47-C assay was technically robust and specific for the target sequence. HSP47-C was 39% higher in ALD patients (median 17.7 ng/mL, IQR 12.4-24.0 ng/mL) compared to HC (median 12.7 ng/mL, IQR 9.4-15.7 ng/mL, p < 0.0001). In addition, HSP47-C was elevated in patients with severe fibrosis (F3-4, median 22.8 ng/mL, IQR 17.5-33.3 ng/mL) compared to none-to-moderate fibrosis (F0-2, median 16.5 ng/mL, IQR 11.8-22.5 ng/mL) with an AUROC of 0.72 (p < 0.0001). HSP47-C also correlated with other liver disease parameters, albumin, bilirubin and aspartate transaminase. CONCLUSION: We developed a competitive ELISA for serological detection of HSP47-C. The study supports HSP47 as a potential marker of liver fibrosis in ALD.

PRO-C3 and ADAPT algorithm accurately identify patients with advanced fibrosis due to alcohol-related liver disease.

BACKGROUND: Alcohol is a main cause of preventable deaths and frequently leads to the development of alcohol-related liver disease. Due to the lack of diagnostics, patients are commonly diagnosed after developing clinical manifestations. Recently, the biomarker PRO-C3 was shown to accurately identify fibrosis due to non-alcoholic fatty liver disease. AIM: To assess the diagnostic accuracy of PRO-C3, the ADAPT score and best-performing non-patented serological test to detect advanced alcohol-related liver fibrosis. METHODS: We enrolled 426 patients with alcohol overuse in a prospective biopsy-controlled study. We evaluated the accuracy of PRO-C3 and the PRO-C3-based algorithm ADAPT to detect advanced liver fibrosis. RESULTS: The accuracy of PRO-C3 was good with an AUROC of 0.85 (95% CI 0.79-0.90). The best-performing non-patented test was the Forns index with an AUROC of 0.83 (95% CI 0.78-0.89). The ADAPT algorithm performed better as compared to both the Forns index and PRO-C3 alone with an AUROC = 0.88 (95% CI 0.83-0.93). CONCLUSION: PRO-C3 is a new marker with high accuracy to detect advanced alcohol-related liver fibrosis. The diagnostic accuracy of PRO-C3 can be further improved by using the ADAPT algorithm in which the test outperforms currently available non-patented serological fibrosis markers. The study is registered in the Odense Patient Data Exploratory Network (OPEN) under study identification numbers OP_040 (https://open.rsyd.dk/OpenProjects/da/openProject.jsp?openNo=40) and OP_239 (https://open.rsyd.dk/OpenProjects/openProject.jsp?openNo=239&lang=da).

Altered balance between collagen formation and degradation after successful direct-acting antiviral therapy of chronic hepatitis C.

The effect of direct-acting antiviral (DAA) therapy on extracellular matrix (ECM) turnover, a prominent feature of chronic hepatitis C (CHC), is unknown. ECM protein degradation and formation generate fragments reflecting the tissue turnover balance when quantified in the blood. PRO-C3 and PRO-C4 reflect type III and IV collagen formation; C3M and C4M are degradation markers of type III and IV. We aimed to assess the markers' dynamics with DAA therapy in CHC patients. Plasma PRO-C3, PRO-C4, C3M and C4M were assessed before, during and up till one year after 12-24 weeks of DAA therapy in 77 CHC patients with advanced fibrosis (n = 14) or cirrhosis (n = 63). Liver stiffness was evaluated using transient elastography. PRO-C3, C3M and C4M levels decreased significantly (P < .00001) while PRO-C4 was unchanged (P = .20) during the study period. There was a steep decrease in the PRO-C3/C3M ratio during DAA therapy and follow-up (P < .02). The PRO-C4/C4M ratio was unchanged (P > .27). The dynamics of the collagen markers behaved similarly between patients with advanced fibrosis and cirrhosis. However, the cirrhosis patients had >20% higher levels of C3M, PRO-C4 and C4M at all time points (P < .05). The collagen markers correlated with liver stiffness at baseline and follow-up.Markers of type III and IV collagen formation and degradation decreased during and after successful DAA therapy in CHC patients with advanced liver disease, and associated with disease severity. These results indicate an altered balance between collagen formation and degradation after viral clearance suggesting favourable effects on liver fibrosis.

An integrated multiomic and quantitative label-free microscopy-based approach to study pro-fibrotic signalling in ex vivo human precision-cut lung slices.

Fibrosis can affect any organ, resulting in the loss of tissue architecture and function with often life-threatening consequences. Pathologically, fibrosis is characterised by the expansion of connective tissue due to excessive deposition of extracellular matrix (ECM) proteins, including the fibrillar forms of collagen. A significant limitation for discovering cures for fibrosis is the availability of suitable human models and techniques to quantify mature fibrillar collagen deposition as close as possible to human physiological conditions.Here we have extensively characterised an ex vivo cultured human lung tissue-derived, precision-cut lung slices (hPCLS) model using label-free second harmonic generation (SHG) light microscopy to quantify fibrillar collagen deposition and mass spectrometry-based techniques to obtain a proteomic and metabolomic fingerprint of hPCLS in ex vivo culture.We demonstrate that hPCLS are viable and metabolically active, with mesenchymal, epithelial, endothelial and immune cell types surviving for at least 2 weeks in ex vivo culture. Analysis of hPCLS-conditioned supernatants showed a strong induction of pulmonary fibrosis-related ECM proteins upon transforming growth factor-ß1 (TGF-ß1) stimulation. This upregulation of ECM proteins was not translated into an increased deposition of fibrillar collagen. In support of this observation, we revealed the presence of a pro-ECM degradation activity in our ex vivo cultures of hPCLS, inhibition of which by a metalloproteinase inhibitor resulted in increased collagen deposition in response to TGF-ß1 stimulation.Together the data show that an integrated approach of measuring soluble pro-fibrotic markers alongside quantitative SHG-based analysis of fibrillar collagen is a valuable tool for studying pro-fibrotic signalling and testing anti-fibrotic agents.

Translational pharmacology of an inhaled small molecule αvß6 integrin inhibitor for idiopathic pulmonary fibrosis.

The αvß6 integrin plays a key role in the activation of transforming growth factor-ß (TGFß), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvß6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvß6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFß signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvß6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvß6, induces prolonged inhibition of TGFß signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.

Serum markers of type III and IV procollagen processing predict recurrence of fibrosis in liver transplanted patients.

Following liver transplantation (LT), 10-30% of patients develop recurrent cirrhosis (RC). There is an urgent need for predictive non-invasive markers for improved monitoring of these patients. Here we studied extracellular matrix biomarkers as predictors of RC after LT. Forty-seven LT patients were divided into groups of fast, intermediate or non-progressors towards RC (<1 year, 3-5 years or no advanced fibrosis >5 years after LT), assessed by follow-up liver biopsies. Markers of interstitial matrix type III and V collagen formation (PRO-C3 and PRO-C5), basement membrane type IV collagen formation (PRO-C4) and degradation (C4M) were assessed in serum samples collected 3, 6 and 12 months post-LT using specific ELISAs. PRO-C3, PRO-C4, and C4M were elevated in fast progressors compared to non-progressors 3 months after LT. C4M and PRO-C4 additionally differentiated between intermediate and fast progressors at 3 months. PRO-C3 was best predictor of survival, with LT patients in the highest PRO-C3 tertile having significantly shorter survival time. This shows that interstitial matrix and basement membrane remodeling in RC may be distinguishable. Markers originating from different sites in the extracellular matrix could be valuable tools for a more dynamic monitoring of patients at risk of RC. However, this needs validation in larger cohorts.

A Novel High Sensitivity Type II Collagen Blood-Based Biomarker, PRO-C2, for Assessment of Cartilage Formation.

N-terminal propeptide of type II collagen (PIINP) is a biomarker reflecting cartilage formation. PIINP exists in two main splice variants termed as type IIA and type IIB collagen NH2-propeptide (PIIANP, PIIBNP). PIIANP has been widely recognized as a cartilage formation biomarker. However, the utility of PIIBNP as a marker in preclinical and clinical settings has not been fully investigated yet. In this study, we aimed to characterize an antibody targeting human PIIBNP and to develop an immunoassay assessing type II collagen synthesis in human blood samples. A high sensitivity electrochemiluminescence immunoassay, hsPRO-C2, was developed using a well-characterized antibody against human PIIBNP. Human cartilage explants from replaced osteoarthritis knees were cultured for ten weeks in the presence of growth factors, insulin-like growth factor 1 (IGF-1) or recombinant human fibroblast growth factor 18 (rhFGF-18). The culture medium was changed every seven days, and levels of PIIBNP, PIIANP, and matrix metalloproteinase 9-mediated degradation of type II collagen (C2M) were analyzed herein. Serum samples from a cross-sectional knee osteoarthritis cohort, as well as pediatric and rheumatoid arthritis samples, were assayed for PIIBNP and PIIANP. Western blot showed that the antibody recognized PIIBNP either as a free fragment or attached to the main molecule. Immunohistochemistry demonstrated that PIIBNP was predominately located in the extracellular matrix of the superficial and deep zones and chondrocytes in both normal and osteoarthritic articular cartilage. In addition, the hsPRO-C2 immunoassay exhibits acceptable technical performances. In the human cartilage explants model, levels of PIIBNP, but not PIIANP and C2M, were increased (2 to 7-fold) time-dependently in response to IGF-1. Moreover, there was no significant correlation between PIIBNP and PIIANP levels when measured in knee osteoarthritis, rheumatoid arthritis, and pediatric serum samples. Serum PIIBNP was significantly higher in controls (KL0/1) compared to OA groups (KL2/3/4, p = 0.012). The hsPRO-C2 assay shows completely different biological and clinical patterns than PIIANP ELISA, suggesting that it may be a promising biomarker of cartilage formation.

No effect of rifaximin on soluble CD163, mannose receptor or type III and IV neoepitope collagen markers in decompensated cirrhosis: Results from a randomized, placebo controlled trial.

BACKGROUND AND AIMS: Macrophages play a significant role in chronic liver disease as reflected by elevated soluble (s)CD163 and mannose receptor (sMR) levels and associated with liver disease severity and prognosis. Extracellular matrix remodelling associated with fibrogenesis may be affected by systemic inflammation induced by bacterial translocation. Therefore, we aimed to investigate the effect of rifaximin-α, an antibiotic with effect on gut bacteria, on sCD163, sMR, and collagen metabolites. METHODS: Fifty-four clinically stable patients with decompensated cirrhosis were randomized to 4 weeks treatment with rifaximin-α (n = 36) or placebo (n = 18). Macrophage markers sCD163, sMR and markers of collagen fibrogenesis (C3M and C4M) and formation (PRO-C3 and P4NPS7) were analysed in plasma before and after treatment. RESULTS: sCD163 and sMR levels were associated with liver disease severity (MELD score, sCD163 rho = 0.47, p<0.001 and sMR rho = 0.37, p = 0.005). There was no effect of Rifaximin-α on sCD163 levels (median (range) sCD163 5.64(2.02 to 10.8) at baseline versus 4.42(1.98 to 8.92) at follow-up in the rifaximin-α group and 4.85 (2.29 to 12.1) at baseline versus 4.32 (1.98 to 12.4) at follow-up in the placebo-group), p = 0.34); nor sMR levels, p = 0.34. Also in patients with elevated lipopolysaccharide binding protein (> 5.9 µg/ml, 38 patients) there was no effect of rifaximin-α on sCD163 (p = 0.49) or sMR levels (p = 0.32). CONCLUSION: We confirmed that macrophage activation markers sCD163 and sMR are directly associated to liver disease severity (MELD score). However, rifaximin-α has no effect on sCD163, sMR or collagen markers in decompensated cirrhosis and does therefore not seem to interfere with macrophage activation or fibrogenesis.

Lung tissue destruction by proteinase 3 and cathepsin G mediated elastin degradation is elevated in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterized by high levels of protease activity leading to degradation of elastin followed by loss of elasticity of the lung and the development of emphysema. Elastin is an essential structural component of the lung parenchyma to support the expansion and recoil of the alveoli during breathing. The lung extracellular matrix is vulnerable to pathological structural changes upon upregulation of serine proteases, including cathepsin G (CG) and proteinase 3 (PR3). In this study, we explored the diagnostic features of elastin neo-epitopes generated by CG and PR3. Two novel competitive enzyme-linked immunosorbent assays (ELISA) measuring CG and PR3 generated elastin fragments (EL-CG and ELP-3 respectively) were developed for assessment in serum. Both assays were technically robust and biologically validated in serum from patients with COPD. Serological levels of both elastin fragments were significantly elevated in patients with COPD compared to healthy controls. These data suggest that EL-CG and ELP-3 may serve as plausible biologic markers of destructive changes in COPD.

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.

Increased remodelling of interstitial collagens and basement membrane is suppressed by treatment in patients with rheumatoid arthritis: serological evaluation of a one-year prospective study of 149 Japanese patients.

OBJECTIVES: This prospective study aimed to use serological biomarkers for evaluation of, connective tissue turnover, in a population of 149 Japanese patients with rheumatoid arthritis (RA). It was aimed to investigate how the connective tissue was affected by treatment at follow-up after 1 year (+/- 6 weeks) with either methotrexate (n=23) alone, or in combination with: adalimumab (n=49), tofacitinib (n=27) or tocilizumab (n=50). METHODS: Clinical characteristics were collected and connective tissue turnover, was evaluated by 4 serological biomarkers: C1M and C3M reflect degradation of types I and III collagen in interstitial tissue; C4M, reflecting degraded type IV collagen of the basement membranes; and CRPM, a marker of degraded C-reactive protein. Evaluated biomarker levels were measured at baseline and at follow-up. Levels were compared to the reference levels of healthy individuals. RESULTS: The four evaluated biomarkers were all elevated at baseline in patients with RA compared to healthy individuals. The biomarkers were higher in RA patients compared to healthy individuals at baseline and they were all significantly correlated with disease activity score of 28 joint (DAS28) (p<0.0001). The biomarker levels were all significantly decreased in all four patient groups at follow-up in all of the four treatment groups. CONCLUSIONS: Rheumatoid joint inflammation elicits enhanced turnover of major collagen constituents of the synovial membrane and this abnormal pathway may be implicated in erosive progression. Evaluations of the applied biomarkers: C1M, C3M, C4M and CRPM, indicate that the pathologically enhanced tissue turnover was attenuated, by all of the four studied treatments.

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.

Chondrocyte activity is increased in psoriatic arthritis and axial spondyloarthritis.

BACKGROUND: Psoriatic arthritis (PsA) and axial spondyloarthritis (axSpA) are chronic inflammatory rheumatic diseases with complex origins. Both are characterized by altered extracellular matrix remodeling in joints and entheses that results in destructive and osteochondral proliferative lesions. There is a need for biomarkers reflecting core disease pathways for diagnosis and disease mapping. Pro-C2 reflects mature cartilage collagen type IIB formation, while C-Col10 represents turnover of type X collagen, which is exclusively expressed by hypertrophic chondrocytes. The objectives of this study were to study cartilage metabolism in axSpA and PsA by assessing Pro-C2 and C-Col10 and to evaluate their diagnostic utility against a healthy reference population. METHODS: Patients with PsA (n = 101) or axSpA (n = 110) were recruited consecutively from three rheumatology outpatient clinics. Demographic and clinical disease measures were recorded. Pro-C2 and C-Col10 were quantified in serum by using newly developed and specific competitive enzyme-linked immunosorbent assays based on monoclonal antibodies. One-way analysis of variance and Tukey's multiple comparison tests were performed on log-transformed data. ROC curve analysis was carried out to evaluate their discriminative power. RESULTS: Pro-C2 levels in serum were significantly increased in both axSpA (median concentration 1.11 ng/ml, 0.67-1.64) and PsA (median concentration 1.03 ng/ml, 0.53-1.47) compared with healthy controls (median concentration 0.30 ng/ml, 0.16-0.41) (p < 0.0001). Pro-C2 did not differ according to treatment. C-Col10 was slightly but equally elevated in the PsA and axSpA groups vs. the control group, but it was significantly lower in patients with axSpA undergoing tumor necrosis factor-α inhibitor (TNFi) treatment. ROC curve analysis revealed AUCs of 0.85 (95 % CI 0.79-0.89) for axSpA and 0.81 (95 % CI 0.75-0.86) for PsA. CONCLUSIONS: These findings indicate that cartilage collagen metabolism was enhanced in the axSpA and PsA groups compared with the healthy control group. The lower C-Col10 level in patients with axSpA undergoing TNFi treatment may reflect that hypertrophic chondrocytes in axSpA are targeted by TNFi. ROC curve analysis showed a diagnostic potential for Pro-C2 in axSpA and PsA.

Neo-Epitopes--Fragments of Cartilage and Connective Tissue Degradation in Early Rheumatoid Arthritis and Unclassified Arthritis.

OBJECTIVE: Tissue destruction in rheumatoid arthritis (RA) is predominantly mediated by matrix metalloproteinases (MMPs), thereby generating protein fragments. Previous studies have revealed that these fragments include MMP-mediated collagen type I, II, and III degradation, citrullinated and MMP-degraded vimentin and MMP degraded C-reactive protein. We evaluated if biomarkers measuring serum levels of specific sequences of the mentioned fragments would provide further information of diagnostic and/or prognostic processes in early arthritis. METHODS: Ninety-two early arthritis patients (arthritis duration<1 year, DMARD naïve) were enrolled. Patients either fulfilled the ACR/EULAR2010 criteria for RA (n = 60) or had unclassified arthritis (UA) (n = 32). Patients fulfilling the RA criteria after 2 years follow-up were classified into non-erosive (n = 25), or erosive disease (n = 13). Concentrations of the biomarkers: C1M, C2M, C3M, VICM and CRPM were measured in baseline serum. RESULTS: C1M, C3M and CRPM were able to discriminate between the UA and RA baseline diagnosis in 92 patients with an AUROC of 0.64 (95%CI 0.517 to 0.762), 0.73 (95%CI 0.622 to 0.838) and 0.68 (95%CI 0.570 to 0.795). C2M showed a potential for discrimination between non-erosive and erosive disease in 38 patients with an AUROC of 0.75 (95%CI 0.597 to 0.910). All of the applied biomarkers correlated with one or more of the disease activity parameters: DAS28, ESR, CRP, SJC66, TJC68 and/or HAQ. CONCLUSION: This is the first study evaluating the applied biomarkers at this early stage of arthritis. C1M, C3M, CRPM might be the best diagnostic marker, whereas high levels of C2M indicated progression of disease at follow-up in early RA patients.

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.

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.

A comparison of osteoclast-rich and osteoclast-poor osteopetrosis in adult mice sheds light on the role of the osteoclast in coupling bone resorption and bone formation.

Osteopetrosis due to lack of acid secretion by osteoclasts is characterized by abolished bone resorption, increased osteoclast numbers, but normal or even increased bone formation. In contrast, osteoclast-poor osteopetrosis appears to have less osteoblasts and reduced bone formation, indicating that osteoclasts are important for regulating osteoblast activity. To illuminate the role of the osteoclast in controlling bone remodeling, we transplanted irradiated skeletally mature 3-month old wild-type mice with hematopoietic stem cells (HSCs) to generate either an osteoclast-rich or osteoclast-poor adult osteopetrosis model. We used fetal liver HSCs from (1) oc/oc mice, (2) RANK KO mice, and (3) compared these to wt control cells. TRAP5b activity, a marker of osteoclast number and size, was increased in the oc/oc recipients, while a significant reduction was seen in the RANK KO recipients. In contrast, the bone resorption marker CTX-I was similarly decreased in both groups. Both oc/oc and Rank KO recipients developed a mild osteopetrotic phenotype. However, the osteoclast-rich oc/oc recipients showed higher trabecular bone volume (40 %), increased bone strength (66 %), and increased bone formation rate (54 %) in trabecular bone, while RANK KO recipients showed only minor trends compared to control recipients. We here show that maintaining non-resorbing osteoclasts, as opposed to reducing the osteoclasts, leads to increased bone formation, bone volume, and ultimately higher bone strength in vivo, which indicates that osteoclasts are sources of anabolic molecules for the osteoblasts.

Osteoclasts are not crucial for hematopoietic stem cell maintenance in adult mice.

The osteoclast is vital for establishment of normal hematopoiesis in the developing animal. However, its role for maintenance of hematopoiesis in adulthood is more controversial. To shed more light on this process, we transplanted hematopoietic stem cells from two osteopetrotic mouse models, with lack of osteoclasts or defective osteoclast function, to normal adult mice and examined the bone phenotype and hematopoiesis in the recipients. B6SJL mice were lethally irradiated and subsequently transplanted with oc/oc, Receptor Activator of Nuclear Factor Kappa B knockout or control fetal liver cells. Osteoclasts derived from the recipient animals were tested in vitro for osteoclastogenesis and resorptive function. Bone remodeling changes were assessed using biomarkers of bone turnover and micro-CT. Hematopoiesis was assessed by flow cytometry and colony formation, and hematopoietic stem cell function by secondary competitive transplantations and cell cycle analysis. After transplantation, a donor chimerism of 97-98% was obtained, and by 15 weeks mild osteopetrosis had developed in recipients of cells from osteopetrotic mice. There were no alterations in the number of bone marrow cells. Colony formation was slightly reduced in Receptor Activator of Nuclear Factor Kappa B knockout recipients but unchanged in oc/oc recipients. Phenotypically, stem cells were marginally reduced in recipients of cells from osteopetrotic mice, but no significant difference was seen in cell cycle status and in competitive secondary transplantations all three groups performed equally well. Our results indicate that osteoclast function is not crucial for hematopoietic stem cell maintenance in adult mice.

Lentiviral gene transfer of TCIRG1 into peripheral blood CD34(+) cells restores osteoclast function in infantile malignant osteopetrosis.

Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by non-functional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. The aim of this study was to restore the resorptive function of IMO osteoclasts by lentiviral mediated gene transfer of the TCIRG1 cDNA. CD34(+) cells from peripheral blood of five IMO patients and from normal cord blood were transduced with lentiviral vectors expressing TCIRG1 and GFP under a SFFV promoter, expanded in culture and differentiated on bone slices to mature osteoclasts. qPCR analysis and western blot revealed increased mRNA and protein levels of TCIRG1, comparable to controls. Vector corrected IMO osteoclasts generated increased release of Ca(2+) and bone degradation product CTX-I into the media as well as increased formation of resorption pits in the bone slices, while non-corrected IMO osteoclasts failed to resorb bone. Resorption was approximately 70-80% of that of osteoclasts generated from cord blood. Furthermore, transduced CD34(+) cells successfully engrafted in NSG-mice. In conclusion we provide the first evidence of lentiviral-mediated correction of a human genetic disease affecting the osteoclastic lineage.