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Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study.

ABSTRACT: BACKGROUND: Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease that leads to progressive cartilage destruction. Advances in the treatment of RA-related destruction of cartilage require profound insights into the molecular mechanisms involved in cartilage degradation. Until now, comprehensive data about the molecular RA-related dysfunction of chondrocytes have been limited. Hence, the objective of this study was to establish a standardized in vitro model to profile the key regulatory molecules of RA-related destruction of cartilage that are expressed by human chondrocytes. METHODS: Human chondrocytes were cultured three-dimensionally for 14 days in alginate beads and subsequently stimulated for 48 hours with supernatants from SV40 T-antigen immortalized human synovial fibroblasts (SF) derived from a normal donor (NDSF) and from a patient with RA (RASF), respectively. To identify RA-related factors released from SF, supernatants of RASF and NDSF were analyzed with antibody-based protein membrane arrays. Stimulated cartilage-like cultures were used for subsequent gene expression profiling with oligonucleotide microarrays. Affymetrix GeneChip Operating Software and Robust Multi-array Analysis (RMA) were used to identify differentially expressed genes. Expression of selected genes was verified by real-time RT-PCR. RESULTS: Antibody-based protein membrane arrays of synovial fibroblast supernatants identified RA-related soluble mediators (IL-6, CCL2, CXCL1-3, CXCL8) released from RASF. Genome-wide microarray analysis of RASF-stimulated chondrocytes disclosed a distinct expression profile related to cartilage destruction involving marker genes of inflammation (adenosine A2A receptor, cyclooxygenase-2), the NF-kappaB signaling pathway (toll-like receptor 2, spermine synthase, receptor-interacting serine-threonine kinase 2), cytokines/chemokines and receptors (CXCL1-3, CXCL8, CCL20, CXCR4, IL-1beta, IL-6), cartilage degradation (matrix metalloproteinase (MMP)-10, MMP-12) and suppressed matrix synthesis (cartilage oligomeric matrix protein, chondroitin sulfate proteoglycan 2). CONCLUSION: Differential transcriptome profiling of stimulated human chondrocytes revealed a disturbed catabolic-anabolic homeostasis of chondrocyte function and disclosed relevant pharmacological target genes of cartilage destruction. This study provides comprehensive insight into molecular regulatory processes induced in human chondrocytes during RA-related destruction of cartilage. The established model may serve as a human in vitro disease model of RA-related destruction of cartilage and may help to elucidate the molecular effects of anti-rheumatic drugs on human chondrocyte gene expression.

SUBMITTER: Andreas K

PROVIDER: S-EPMC2374452 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study.

Andreas Kristin K Lübke Carsten C Häupl Thomas T Dehne Tilo T Morawietz Lars L Ringe Jochen J Kaps Christian C Sittinger Michael M

Arthritis research & therapy 20080118 1

<h4>Background</h4>Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease that leads to progressive cartilage destruction. Advances in the treatment of RA-related destruction of cartilage require profound insights into the molecular mechanisms involved in cartilage degradation. Until now, comprehensive data about the molecular RA-related dysfunction of chondrocytes have been limited. Hence, the objective of this study was to establish a standardized in vitro model t ...[more]

PMID: 18205922

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Project description:We investigated the expression and localization of the receptor activator nuclear factor κB ligand (RANKL) in cartilage from patients with rheumatoid arthritis (RA) of relevance to cartilage degeneration. We also examined the role of exogenous lymphotoxin (LT)-α on RANKL expression in human chondrocytes and its effect on in vitro osteoclast differentiation. Cartilage and synovial fluid samples were obtained from 45 patients undergoing total joint replacement surgery or joint puncture, including 24 patients with osteoarthritis (OA) and 21 patients with RA. RANKL expression in articular cartilage was examined by immunohistochemistry. LT-α concentrations in synovial fluid were measured using an enzyme-linked immunosorbent assay (ELISA). Normal human chondrocytes were stimulated with LT-α, and the relative mRNA levels of RANKL, osteoprotegerin (OPG), matrix metalloproteinase-9, and vascular endothelial growth factor were examined by real-time polymerase chain reaction. Soluble RANKL protein in culture media was measured using ELISA, and membrane-bound RANKL protein in cells was examined by western blotting. Co-cultures of human chondrocytes with peripheral blood mononuclear cells (PBMCs) were stimulated with macrophage-colony stimulating factor and LT-α, and osteoclast differentiation was evaluated by staining for tartrate-resistant acid phosphatase. LT-α concentrations were higher in RA synovial fluid than in OA samples. The population of RANKL-positive chondrocytes of RA cartilage was higher than that of OA cartilage, and correlated with cartilage degeneration. Stimulation of cultured human chondrocytes by LT-α increased RANKL expression, the RANKL/OPG ratio, and angiogenic factors. Membrane-bound RANKL in chondrocytes was up-regulated after stimulation of LT-α, whereas soluble RANKL in culture medium did not increase. Co-cultures of human chondrocytes and PBMCs demonstrated that LT-α stimulated human chondrocytes to produce RANKL and induced osteoclastic differentiation of PBMCs. RANKL produced by chondrocytes may contribute to cartilage destruction during RA and LT-α could promote the expression of RANKL in human chondrocytes.

Project description:ObjectiveRheumatoid arthritis (RA) is a chronic disease leading to joint destruction. Although many studies have addressed factors potentially correlated with the speed of joint destruction, less attention has been paid to the distribution of joint destruction in patients with RA. In this study, destruction of the hand bones in patients with RA was classified into 2 anatomic subgroups, the fingers and the non-fingers, with the aim of analyzing which factors are associated with destruction of the finger joints.MethodsA total of 1,215 Japanese patients with RA were recruited from 2 different populations. The degree of joint destruction was assessed using the total modified Sharp/van der Heijde score (SHS) of radiographic joint damage. The SHS score of joint damage in the finger joints was used as the dependent variable, and the SHS score in the non-finger joints was used as a covariate. Age, sex, disease duration, smoking, C-reactive protein level, treatment for RA, and positivity for and levels of anti-citrullinated protein antibodies and rheumatoid factor (RF) were evaluated as candidate correlates. Overall effect sizes were assessed in a meta-analysis. In addition, associations observed in the Japanese patients were compared to those in a cohort of 157 Dutch RA patients in the BeSt study (a randomized, controlled trial involving 4 different strictly specified treatment strategies for early RA).ResultsNot surprisingly, disease duration in Japanese patients with RA was associated with the finger SHS score (P ≤ 0.00037). Both positivity for and levels of RF showed significant associations with the finger SHS score after adjustment for covariates (P = 0.0022 and P = 8.1 × 10(-7) , respectively). These associations were also true in relation to the time-averaged finger SHS score. An association between RF positivity and the finger SHS score was also observed in Dutch patients with RA in the BeSt study (P = 0.049).ConclusionPositivity for and levels of RF are associated with finger joint destruction independent of non-finger joint destruction and other covariates. Our findings suggest that there are different mechanisms of joint destruction operating in the finger joints of patients with RA.

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Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study.

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Key regulatory molecules of cartilage destruction in rheumatoid arthritis: an in vitro study.

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