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Background: Network inference of gene expression data is an important challenge in systems biology. Novel algorithms may provide more detailed gene regulatory networks (GRN) for complex, chronic inflammatory diseases such as rheumatoid arthritis (RA), in which activated synovial fibroblasts (SFBs) play a major role. Since the detailed mechanisms underlying this activation are still unclear, simultaneous investigation of multi-stimuli activation of SFBs offers the possibility to elucidate the regulatory effects of multiple mediators and to gain new insights into disease pathogenesis., Methods: A GRN was therefore inferred from RA-SFBs treated with 4 different stimuli (IL-1 β, TNF- α, TGF- β, and PDGF-D). Data from time series microarray experiments (0, 1, 2, 4, 12 h; Affymetrix HG-U133 Plus 2.0) were batch-corrected applying 'ComBat', analyzed for differentially expressed genes over time with 'Limma', and used for the inference of a robust GRN with NetGenerator V2.0, a heuristic ordinary differential equation-based method with soft integration of prior knowledge., Results: Using all genes differentially expressed over time in RA-SFBs for any stimulus, and selecting the genes belonging to the most significant gene ontology (GO) term, i.e., 'cartilage development', a dynamic, robust, moderately complex multi-stimuli GRN was generated with 24 genes and 57 edges in total, 31 of which were gene-to-gene edges. Prior literature-based knowledge derived from Pathway Studio or manual searches was reflected in the final network by 25/57 confirmed edges (44%). The model contained known network motifs crucial for dynamic cellular behavior, e.g., cross-talk among pathways, positive feed-back loops, and positive feed-forward motifs (including suppression of the transcriptional repressor OSR2 by all 4 stimuli., Conclusion: A multi-stimuli GRN highly concordant with literature data was successfully generated by network inference from the gene expression of stimulated RA-SFBs. The GRN showed high reliability, since 10 predicted edges were independently validated by literature findings post network inference. The selected GO term 'cartilage development' contained a number of differentiation markers, growth factors, and transcription factors with potential relevance for RA. Finally, the model provided new insight into the response of RA-SFBs to multiple stimuli implicated in the pathogenesis of RA, in particular to the 'novel' potent growth factor PDGF-D.

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory and destructive joint disease characterized by overexpression of pro-inflammatory/pro-destructive genes and other activating genes (for example, proto-oncogenes) in the synovial membrane (SM). The gene expression in disease is often characterized by significant inter-individual variances via specific synchronization/desynchronization of gene expression. To elucidate the contribution of the variance to the pathogenesis of disease, expression variances were tested in SM samples of RA patients, osteoarthritis (OA) patients, and normal controls (NCs)., Method: Analysis of gene expression in RA, OA, and NC samples was carried out using Affymetrix U133A/B oligonucleotide arrays, and the results were validated by real-time reverse transcription-polymerase chain reaction. For the comparison between RA and NC, 568 genes with significantly different variances in the two groups (P

Genome-wide gene expression was comparatively investigated in early-passage rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts (SFBs; n = 6 each) using oligonucleotide microarrays; mRNA/protein data were validated by quantitative PCR (qPCR) and western blotting and immunohistochemistry, respectively. Gene set enrichment analysis (GSEA) of the microarray data suggested constitutive upregulation of components of the transforming growth factor (TGF)-beta pathway in RA SFBs, with 2 hits in the top 30 regulated pathways. The growth factor TGF-beta1, its receptor TGFBR1, the TGF-beta binding proteins LTBP1/2, the TGF-beta-releasing thrombospondin 1 (THBS1), the negative effector SkiL, and the smad-associated molecule SARA were upregulated in RA SFBs compared to OA SFBs, whereas TGF-beta2 was downregulated. Upregulation of TGF-beta1 and THBS1 mRNA (both positively correlated with clinical markers of disease activity/severity) and downregulation of TGF-beta2 mRNA in RA SFBs were confirmed by qPCR. TGFBR1 mRNA (only numerically upregulated in RA SFBs) and SkiL mRNA were not differentially expressed. At the protein level, TGF-beta1 showed a slightly higher expression, and the signal-transducing TGFBR1 and the TGF-beta-activating THBS1 a significantly higher expression in RA SFBs than in OA SFBs. Consistent with the upregulated TGF-beta pathway in RA SFBs, stimulation with TGF-beta1 resulted in a significantly enhanced expression of matrix-metalloproteinase (MMP)-11 mRNA and protein in RA SFBs, but not in OA SFBs. In conclusion, RA SFBs show broad, constitutive alterations of the TGF-beta pathway. The abundance of TGF-beta, in conjunction with an augmented mRNA and/or protein expression of TGF-beta-releasing THBS1 and TGFBR1, suggests a pathogenetic role of TGF-beta-induced effects on SFBs in RA, for example, the augmentation of MMP-mediated matrix degradation/remodeling.

Rheumatoid arthritis is a chronic inflammatory disease with a high prevalence and substantial socioeconomic burden. Despite intense research efforts, its aetiology and pathogenesis remain poorly understood. To identify novel genes and/or cellular pathways involved in the pathogenesis of the disease, we utilized a well-recognized tumour necrosis factor-driven animal model of this disease and performed high-throughput expression profiling with subtractive cDNA libraries and oligonucleotide microarray hybridizations, coupled with independent statistical analysis. This twin approach was validated by a number of different methods in other animal models of arthritis as well as in human patient samples, thus creating a unique list of disease modifiers of potential therapeutic value. Importantly, and through the integration of genetic linkage analysis and Gene Ontology-assisted functional discovery, we identified the gelsolin-driven synovial fibroblast cytoskeletal rearrangements as a novel pathophysiological determinant of the disease., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

A better understanding of the initial mechanisms that lead to arthritic disease could facilitate development of improved therapeutic strategies. We characterized the synovial microcirculation of knee joints in susceptible mouse strains undergoing intradermal immunization with bovine collagen II in complete Freund's adjuvant to induce arthritis (i.e. collagen-induced arthritis [CIA]). Susceptible DBA1/J and collagen II T-cell receptor transgenic mice were compared with CIA-resistant FVB/NJ mice. Before onset of clinical symptoms of arthritis, in vivo fluorescence microscopy of knee joints revealed marked leucocyte activation and interaction with the endothelial lining of synovial microvessels. This initial inflammatory cell response correlated with the gene expression profile at this disease stage. The majority of the 655 differentially expressed genes belonged to classes of genes that are involved in cell movement and structure, cell cycle and signal transduction, as well as transcription, protein synthesis and metabolism. However, 24 adhesion molecules and chemokine/cytokine genes were identified, some of which are known to contribute to arthritis (e.g. CD44 and neutrophil cytosolic factor 1) and some of which are novel in this respect (e.g. CC chemokine ligand-27 and IL-13 receptor alpha1). Online in vivo data on synovial tissue microcirculation, together with gene expression profiling, emphasize the potential role played by early inflammatory events in the development of arthritis.

Global scale molecular profiling of diseased tissues is an important first step to unravel candidate target molecules that are involved in the pathogenesis of a disease. We have performed a comparative molecular characterization at the transcriptome (microarray with 12 526 gene specificities) and proteome level (multi-Western blot PowerBlot with 791 antibodies) of synovial tissue from rheumatoid arthritis (RA) compared to osteoarthritis (OA) patients. From the panel of 791 antibodies, 260 (33%) detected their corresponding protein. Out of 58 unambiguous changes at the protein level only 16 coincided at the transcript level (28%). Stat1, p47phox and manganese superoxide dismutase were shown to be reproducibly overexpressed in RA versus OA synovial tissue by Western blots with a panel of 8 RA versus 8 OA samples. Cathepsin D was among the most prominent proteins scored to be underexpressed in RA by the PowerBlot whereas no differences of the respective transcript were observed. The lower abundance of cathepsin D protein in RA compared to OA tissue was also reproduced in other patient samples. Immunohistochemistry assigned the Stat1 protein in RA synovial tissue mainly to macrophages and T lymphocytes and the p47phox protein in particular to macrophages. In conclusion, our approach provided us with new candidate molecules for further analysis of rheumatic diseases and stressed the importance of studies at the protein level.

Increasing attention has been directed towards identifying non-T-cell mechanisms as potential therapeutic targets in rheumatoid arthritis. Synovial fibroblast (SF) activation, a hallmark of rheumatoid arthritis, results in inappropriate production of chemokines and matrix components, which in turn lead to bone and cartilage destruction. We have demonstrated that SFs have an autonomous pathogenic role in the development of the disease, by showing that they have the capacity to migrate throughout the body and cause pathology specifically to the joints. In order to decipher the pathogenic mechanisms that govern SF activation and pathogenic potential, we used the two most prominent methods of differential gene expression analysis, differential display and DNA microarrays, in a search for deregulated cellular pathways in the arthritogenic SF. Functional clustering of differentially expressed genes, validated by dedicated in vitro functional assays, implicated a number of cellular pathways in SF activation. Among them, diminished adhesion to the extracellular matrix was shown to correlate with increased proliferation and migration to this matrix. Our findings support an aggressive role for the SF in the development of the disease and reinforce the perspective of a transformed-like character of the SF.

To improve our knowledge on the pathophysiology of rheumatoid arthritis (RA), we investigated gene expression patterns in synovial tissue from RA and osteoarthritis (OA) patients. DNA oligonucleotide microarray analysis was employed to identify differentially expressed genes in synovial tissue from pathologically classified tissue samples from RA (n = 20) and OA patients (n = 10). From 7131 gene sets displayed on the microarray chip, 101 genes were found to be upregulated and 300 genes to be downregulated in RA as compared with OA. Semiquantitative reverse-transcription polymerase chain reaction, Western blotting and immunohistochemistry were used to validate microarray expression levels. These experiments revealed that Cys-X-Cys receptor (CXCR)1, CXCR2 and CXCR3 mRNAs, as well as Cys-X-Cys ligand (CXCL)9 (monokine induced by IFN-gamma) and CXCL10 (IFN-gamma inducible protein 10) mRNAs, were significantly upregulated in RA as compared with OA disease. Elevated protein levels in RA synovial tissue were detected for CXCR1 and CXCR3 by Western blotting. Using immunohistochemistry, CXCR3 protein was found to be preferentially expressed on mast cells within synovial tissue from RA patients. These findings suggest that substantial expression of CXCR3 protein on mast cells within synovial tissue from RA patients plays a significant role in the pathophysiology of RA, accompanied by elevated levels of the chemokines CXCL9 and CXCL10. Mature mast cells are likely to contribute to and sustain the inflamed state in arthritic lesions (e.g. by production of inflammatory mediators such as histamine, proteinases, arachidonic acid metabolites and cytokines). Thus, the mast cell could become a potential target in therapeutic intervention.