| Description: |
Chondrocytes were isolated from cadaver knees and grown in alginate suspension to mimic cartilage-like conditions. Then, chondrocytes were stimulated with conditioned medium from synovial fibroblasts (SFs) either from rheumatoid arthritis patients (RASFs) or from normal donors (NDSFs). SFs had been pre-treated with a drug from one of three classes: disease-modifying antirheumatic drugs (DMARDs: azathioprine, gold sodium thiomalate, chloroquine phosphate, or methotrexate), nonsteroidal anti-inflammatory drugs (NSAIDs: piroxicam or diclofenac), or steroidal anti-inflammatory drugs (SAIDs: methylprednisolone or prednisolone). [original description: Rheumatoid arthritis (RA) leads to progressive destruction of articular structures. Despite recent progress in controlling inflammation and pain, little cartilage repair has yet been observed. This in vitro study aims to determine the role of chondrocytes in RA-related cartilage destruction and antirheumatic drug-related regenerative processes. Human chondrocytes were three-dimensionally cultured in alginate beads. To determine the RA-induced gene expression pattern, human chondrocytes were stimulated with supernatant of RA synovial fibroblasts (RASF) and normal donor synovial fibroblasts (NDSF), respectively. To examine antirheumatic drug response signatures, human chondrocytes were stimulated with supernatant of RASF that have been treated with disease-modifying antirheumatic drugs (DMARD; azathioprine, sodium aurothiomalate, chloroquine phosphate, methotrexate), non-steroidal anti-inflammatory drugs (NSAID; piroxicam, diclofenac) or steroidal anti-inflammatory drugs (SAID; methylprednisolone, prednisolone). Genome-wide expression profiling with oligonucleotide microarrays was used to determine differentially expressed genes. Real-time RT-PCR and ELISA were performed for validation of microarray data. Following antirheumatic treatment, microarray analysis disclosed a reverted expression of 94 RA-induced chondrocyte genes involved in inflammation/NF-kappaB signaling, cytokine/chemokine activity, immune response, proliferation/differentiation and matrix remodeling. Hierarchical clustering analysis showed that treatment of RASF with the DMARD azathioprine, gold sodium thiomalate and methotrexate resulted in chondrocyte gene expression signatures that were closely related to the healthy?? pattern. Treatment with the SAID methylprednisolone and prednisolone strongly reverted the RA-related chondrocyte gene expression, in particular the expression of genes involved in inflammation/NF-kappaB and cytokine/chemokine activity. The NSAID piroxicam and diclofenac and the DMARD chloroquine phosphate had only moderate to marginal effects. Pathway analysis determined major mechanisms of drug action, for example pathways of cytokine-cytokine receptor interaction, TGF-beta/TLR/Jak-STAT signaling and ECM-receptor interaction were targeted. This in vitro study provides a comprehensive molecular insight into the antirheumatic drug response signatures in human chondrocytes, thereby revealing potential molecular targets, pathways and mechanisms of drug action involved in chondrocyte regeneration. Thus, the present study may contribute to the development of novel therapeutic chondro-protective compounds and strategies. Experiment Overall Design: Drug-related suppression of gene expression in activated chondrocytes was determined by genome-wide microarray analysis. Chondrocytes were stimulated with supernatant of RASF and NDSF. Effect of treatment with DMARDs, NSAIDs and glucocorticoids was tested by treating RASF prior to collection of supernatant. Two RNA pools were analyzed for each group (RASF-stimulated NDSF stimulated and RASF-treated), each pool consisting of equal amounts of RNA from three different donors.]
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| Publication(s): |
Andreas K, Häupl T, Lübke C, Ringe J, Morawietz L, Wachtel A, Sittinger M, Kaps C.
Antirheumatic drug response signatures in human chondrocytes: potential molecular targets to stimulate cartilage regeneration.
PubMed:19192274
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