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1.  The epigenome of synovial fibroblasts: an underestimated therapeutic target in rheumatoid arthritis 
Perturbed epigenetic landscape and deregulated microRNA networks are central to the permanent activation and aggressiveness of synovial fibroblasts in rheumatoid arthritis. Current anti-cytokine therapies, although effectively halting synovitis, cannot reverse the stably activated destructive phenotype of rheumatoid arthritis synovial fibroblasts, offering rather limited protection against ongoing joint destruction in rheumatoid arthritis. Targeting the deregulated epigenome of rheumatoid arthritis synovial fibroblasts is key to developing joint-protective strategies in rheumatoid arthritis. To date, different pathogenic mechanisms have been identified that can profoundly impact the epigenetic derangements in rheumatoid arthritis synovial fibroblasts, including increased consumption of S-adenosylmethionine, a principal methyl donor in DNA methylation reactions, together with deregulation of crucial DNA- and histone-modifying enzymes. Re-establishing globally disturbed DNA methylation patterns in rheumatoid arthritis synovial fibroblasts by supplementing S-adenosylmethionine while preventing its leakage into polyamine cycles may be a promising therapeutic strategy in rheumatoid arthritis and the first epigenetic treatment to target rheumatoid arthritis synovial fibroblasts at the scene of the crime. Given the dynamic nature and reversibility of epigenetic modifications, their involvement in human diseases and recent perspectives on epigenetic therapies in cancer, epigenetic targeting of rheumatoid arthritis synovial fibroblasts should be within future reach.
doi:10.1186/ar4596
PMCID: PMC4075141  PMID: 25165988
2.  Epigenetic contributions in the development of rheumatoid arthritis 
Rheumatoid arthritis (RA) is an autoimmune disease, characterized by chronic inflammation of the joints with severe pain and swelling, joint damage and disability, which leads to joint destruction and loss of function. Despite extensive research efforts, the underlying cause for RA is still unknown and current therapies are more or less effective in controlling symptoms but still fail to cure the disease. In recent years, epigenetic modifications were found to strongly contribute to the development of RA by affecting diverse aspects of the disease and modifying gene expression levels and behavior of several cell types, first and foremost joint resident synovial fibroblasts (SF). RASF are the most common cell type at the site of invasion. Owing to their aggressive, intrinsically activated phenotype, RASF are active contributors in joint damage. RASF are characterized by their ability to secrete cytokines, chemokines and joint-damaging enzymes. Furthermore, these cells are resistant to apoptosis, leading to hyperplasia of the synovium. In addition, RASF have invasive and migratory properties that could lead to spreading of the disease to unaffected joints. Epigenetic modifications, including DNA methylation and post-translational histone modifications, such as histone (de)acetylation, histone methylation and histone sumoylation were identified as regulatory mechanisms in controlling aggressive cell activation in vitro and in disease outcome in animal models in vivo. In the last 5 years, the field of epigenetics in RA has impressively increased. In this review we consider the role of diverse epigenetic modifications in the development of RA, with a special focus on epigenetic modifications in RASF.
doi:10.1186/ar4074
PMCID: PMC3674613  PMID: 23164162
3.  Resistin in idiopathic inflammatory myopathies 
Arthritis Research & Therapy  2012;14(3):R111.
Introduction
The purpose of this study was to evaluate and compare the serum levels and local expression of resistin in patients with idiopathic inflammatory myopathies to controls, and to determine the relationship between resistin levels, inflammation and disease activity.
Methods
Serum resistin levels were determined in 42 patients with inflammatory myopathies and 27 healthy controls. The association among resistin levels, inflammation, global disease activity and muscle strength was examined. The expression of resistin in muscle tissues from patients with inflammatory myopathies and healthy controls was evaluated. Gene expression and protein release from resistin-stimulated muscle and mononuclear cells were assessed.
Results
In patients with inflammatory myopathies, the serum levels of resistin were significantly higher than those observed in controls (8.53 ± 6.84 vs. 4.54 ± 1.08 ng/ml, P < 0.0001) and correlated with C-reactive protein (CRP) levels (r = 0.328, P = 0.044) and myositis disease activity assessment visual analogue scales (MYOACT) (r = 0.382, P = 0.026). Stronger association was observed between the levels of serum resistin and CRP levels (r = 0.717, P = 0.037) as well as MYOACT (r = 0.798, P = 0.007), and there was a trend towards correlation between serum resistin and myoglobin levels (r = 0.650, P = 0.067) in anti-Jo-1 positive patients. Furthermore, in patients with dermatomyositis, serum resistin levels significantly correlated with MYOACT (r = 0.667, P = 0.001), creatine kinase (r = 0.739, P = 0.001) and myoglobin levels (r = 0.791, P = 0.0003) and showed a trend towards correlation with CRP levels (r = 0.447, P = 0.067). Resistin expression in muscle tissue was significantly higher in patients with inflammatory myopathies compared to controls, and resistin induced the expression of interleukins (IL)-1β and IL-6 and monocyte chemoattractant protein (MCP)-1 in mononuclear cells but not in myocytes.
Conclusions
The results of this study indicate that higher levels of serum resistin are associated with inflammation, higher global disease activity index and muscle injury in patients with myositis-specific anti-Jo-1 antibody and patients with dermatomyositis. Furthermore, up-regulation of resistin in muscle tissue and resistin-induced synthesis of pro-inflammatory cytokines in mononuclear cells suggest a potential role for resistin in the pathogenesis of inflammatory myopathies.
doi:10.1186/ar3836
PMCID: PMC3446487  PMID: 22577940
4.  7th meeting of the global arthritis research network 
Last October, the 7th meeting of the Global Arthritis Research Network was held in Zurich, Switzerland. European and American experts who have made major recent contributions to molecular biology got together to provide insights into novel technologies and approaches useful for biomedical research, especially for research on arthritis and related conditions.
doi:10.1186/ar3340
PMCID: PMC3239332  PMID: 21892971
12.  Cell culture and passaging alters gene expression pattern and proliferation rate in rheumatoid arthritis synovial fibroblasts 
Introduction
Rheumatoid arthritis synovial fibroblasts (RASF) are key players in synovial pathophysiology and are therefore examined extensively in various experimental approaches. We evaluated, whether passaging during culture and freezing has effects on gene expression and cell proliferation.
Methods
RASF were passaged for up to 8 passages. RNA was isolated after each passage and cDNA arrays were performed to evaluate the RNA expression pattern during passaging. In addition, doubling time of the cells was also measured.
Results
From passages 2-4, mRNA expression did not change significantly. Gene expression in RASF started to change in passages 5-6 with 7-10% differentially expressed genes. After passages 7-8, more than 10% of the genes were differentially expressed. The doubling rate was constant for up to 5 passages and decreased after passages 6-8. After freezing, gene expression of the second passage is comparable to gene expression prior to freezing.
Conclusions
The results of this study show, that experiments, which examine gene expression of RASF and shall reflect or imitate an in vivo situation, should be limited to early culture passages to avoid cell culture effects. It is not necessary to stop culturing SF after a few passages, but to keep the problems of cell culture in mind to avoid false positive results. Especially, when large-scale screening methods on mRNA level are used. Of note, freezing does not affect gene expression substantially.
doi:10.1186/ar3010
PMCID: PMC2911867  PMID: 20462438
13.  Hypoxia. Hypoxia in the pathogenesis of systemic sclerosis 
Autoimmunity, microangiopathy and tissue fibrosis are hallmarks of systemic sclerosis (SSc). Vascular alterations and reduced capillary density decrease blood flow and impair tissue oxygenation in SSc. Oxygen supply is further reduced by accumulation of extracellular matrix (ECM), which increases diffusion distances from blood vessels to cells. Therefore, severe hypoxia is a characteristic feature of SSc and might contribute directly to the progression of the disease. Hypoxia stimulates the production of ECM proteins by SSc fibroblasts in a transforming growth factor-β-dependent manner. The induction of ECM proteins by hypoxia is mediated via hypoxia-inducible factor-1α-dependent and -independent pathways. Hypoxia may also aggravate vascular disease in SSc by perturbing vascular endothelial growth factor (VEGF) receptor signalling. Hypoxia is a potent inducer of VEGF and may cause chronic VEGF over-expression in SSc. Uncontrolled over-expression of VEGF has been shown to have deleterious effects on angiogenesis because it leads to the formation of chaotic vessels with decreased blood flow. Altogether, hypoxia might play a central role in pathogenesis of SSc by augmenting vascular disease and tissue fibrosis.
doi:10.1186/ar2598
PMCID: PMC2688169  PMID: 19473554
14.  Increased levels of circulating microparticles in primary Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis and relation with disease activity 
Arthritis Research & Therapy  2009;11(5):R156.
Introduction
Cell stimulation leads to the shedding of phosphatidylserine (PS)-rich microparticles (MPs). Because autoimmune diseases (AIDs) are characterized by cell activation, we investigated level of circulating MPs as a possible biomarker in primary Sjögren's syndrome (pSS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).
Methods
We measured plasma levels of total, platelet and leukocyte MPs by prothrombinase capture assay and flow cytometry in 43 patients with pSS, 20 with SLE and 24 with RA and in 44 healthy controls (HCs). Secretory phospholipase A2 (sPLA2) activity was assessed by fluorometry. Soluble CD40 ligand (sCD40L) and soluble P-selectin (sCD62P), reflecting platelet activation, were measured by ELISA.
Results
Patients with pSS showed increased plasma level of total MPs (mean ± SEM 8.49 ± 1.14 nM PS equivalent (Eq), P < 0.0001), as did patients with RA (7.23 ± 1.05 n PS Eq, P = 0.004) and SLE (7.3 ± 1.25 nM PS Eq, P = 0.0004), as compared with HCs (4.13 ± 0.2 nM PS Eq). Patients with AIDs all showed increased level of platelet MPs (P < 0.0001), but only those with pSS showed increased level of leukocyte MPs (P < 0.0001). Results by capture assay and flow cytometry were correlated. In patients with high disease activity according to extra-glandular complications (pSS), DAS28 (RA) or SLEDAI (SLE) compared with low-activity patients, the MP level was only slightly increased in comparison with those having a low disease activity. Platelet MP level was inversely correlated with anti-DNA antibody level in SLE (r = -0.65; P = 0.003) and serum β2 microglobulin level in pSS (r = -0.37; P < 0.03). The levels of total and platelet MPs were inversely correlated with sPLA2 activity (r = -0.37, P = 0.0007; r = -0.36, P = 0.002, respectively). sCD40L and sCD62P concentrations were significantly higher in pSS than in HC (P ≤ 0.006).
Conclusions
Plasma MP level is elevated in pSS, as well as in SLE and RA, and could be used as a biomarker reflecting systemic cell activation. Level of leukocyte-derived MPs is increased in pSS only. The MP level is low in case of more severe AID, probably because of high secretory phospholipase A2 (sPLA2) activity, which leads to consumption of MPs. Increase of platelet-derived MPs, sCD40L and sCD62P, highlights platelet activation in pSS.
doi:10.1186/ar2833
PMCID: PMC2787287  PMID: 19832990
15.  Epigenetic modifications in rheumatoid arthritis 
Over the last decades, genetic factors for rheumatoid diseases like the HLA haplotypes have been studied extensively. However, during the past years of research, it has become more and more evident that the influence of epigenetic processes on the development of rheumatic diseases is probably as strong as the genetic background of a patient. Epigenetic processes are heritable changes in gene expression without alteration of the nucleotide sequence. Such modifications include chromatin methylation and post-translational modification of histones or other chromatin-associated proteins. The latter comprise the addition of methyl, acetyl, and phosphoryl groups or even larger moieties such as binding of ubiquitin or small ubiquitin-like modifier. The combinatory nature of these processes forms a complex network of epigenetic modifications that regulate gene expression through activation or silencing of genes. This review provides insight into the role of epigenetic alterations in the pathogenesis of rheumatoid arthritis and points out how a better understanding of such mechanisms may lead to novel therapeutic strategies.
doi:10.1186/ar2500
PMCID: PMC2592785  PMID: 18947370
16.  DREAM is reduced in synovial fibroblasts of patients with chronic arthritic pain: is it a suitable target for peripheral pain management? 
Introduction
The endogenous pain-relieving system depends in part on the regulation of nociceptive signals through binding of opioids to the corresponding opioid receptor. Interfering with the trans-repression effect of downstream regulatory element antagonist modulator (DREAM) on the transcription of the opioid dynorphin-encoding prodynorphin (pdyn) gene might enhance pain relief in the periphery.
Methods
Expression levels were measured in osteoarthritis (OA) synovial fibroblast-like cells (SFLCs) (n = 8) and in peripheral blood mononuclear cells (PBMCs) from OA patients (n = 53) and healthy controls (n = 26) by real-time polymerase chain reaction. Lysed OA SFLCs were analyzed by immunoprecipitation. Translation of DREAM mRNA was inhibited by small interfering RNAs (siRNAs). Expressions of DREAM, pdyn, and c-fos mRNAs were measured at 24, 48, and 72 hours after transfection.
Results
The expression of DREAM mRNA was shown in both healthy and OA SFLCs as well as PBMCs. Inhibiting transcription using siRNAs led to a marked reduction in DREAM expression after 24, 48, and 72 hours. However, no significant changes in c-fos and pdyn expression occurred. In addition, DREAM mRNA expression was significantly reduced in OA patients with chronic pain (pain intensity as measured by a visual analog scale scale of greater than 40), but no pdyn expression was detectable.
Conclusion
To our knowledge, this is the first report showing the expression of DREAM in SFLCs and PBMCs on the mRNA level. However, DREAM protein was not detectable. Since repression of pdyn transcription persists after inhibiting DREAM translation, DREAM appears to play no functional role in the kappa opioid receptor system in OA SFLCs. Therefore, our data suggest that DREAM appears not to qualify as a target in peripheral pain management.
doi:10.1186/ar2431
PMCID: PMC2483451  PMID: 18507845
17.  Cells of the synovium in rheumatoid arthritis. Synovial fibroblasts 
For some time synovial fibroblasts have been regarded simply as innocent synovial cells, mainly responsible for synovial homeostasis. During the past decade, however, a body of evidence has accumulated illustrating that rheumatoid arthritis synovial fibroblasts (RASFs) are active drivers of joint destruction in rheumatoid arthritis. Details regarding the intracellular signalling cascades that result in long-term activation and synthesis of proinflammatory molecules and matrix-degrading enzymes by RASFs have been analyzed. Molecular, cellular and animal studies have identified various interactions with other synovial and inflammatory cells. This expanded knowledge of the distinct role played by RASFs in the pathophysiology of rheumatoid arthritis has moved these fascinating cells to the fore, and work to identify targeted therapies to inhibit their joint destructive potential is underway.
doi:10.1186/ar2337
PMCID: PMC2246247  PMID: 18177509
18.  Proteinases in the joint: clinical relevance of proteinases in joint destruction 
Proteinases are involved in essential steps in cartilage and bone homeostasis. Consequently, efforts have been made to establish their potential role in the pathology of rheumatic conditions such as rheumatoid arthritis, osteoarthritis and spondyloarthritis. Matrix metalloproteinases (MMPs) are sensitive markers of disease severity and response to treatment, and therefore they have potential in the assessment of rheumatic diseases. Despite disappointing early results with synthetic inhibitors of MMPs, there is still much scope for developing effective and safe MMPs inhibitors, and consequently to deliver new options to inhibit joint destruction.
doi:10.1186/ar2304
PMCID: PMC2212555  PMID: 18001502
21.  Safety concerns on the development of novel therapeutic drugs 
Along with recent innovative approaches resulting in the development of new therapies such as small molecular inhibitors, therapeutic antibodies, recombinant proteins and gene therapy, there is increasing need for improved understanding of the basic molecular mechanisms that are exploited by such treatments. Helpful tools in the analysis of drug effects include high-throughput screening techniques such as microarrays, which are used in transcriptomics and pharmacogenomics. Although we are far from using these extensive and costly tests in our daily clinical routine, their application in basic research nevertheless takes us closer to individualized therapeutic strategies, in which the optimal therapeutic regimen is identified for each individual patient.
doi:10.1186/ar2032
PMCID: PMC1779448  PMID: 16968526
22.  Somatic mutations in mitochondria: the chicken or the egg? 
Arthritis Research & Therapy  2005;7(5):179-180.
Somatic mutations of mitochondrial DNA have been detected in various pathologies such as cancer, neurodegenerative diseases, cardiac disorders and aging in general. Now it has been found that patients with rheumatoid arthritis also have a higher incidence of mitochondrial mutations in synoviocytes and synovial tissue compared with patients with osteoarthritis. Furthermore, it has been shown that these mutations possibly result in changed peptides that are presented by major histocompatibility complex II and thus might be recognized as non-self by the immune system. Further studies will show whether these mutations are actually able to trigger autoimmune inflammation in rheumatoid arthritis or whether they must be considered epiphenomena of cellular damage in chronic inflammation.
doi:10.1186/ar1809
PMCID: PMC1257449  PMID: 16207343
23.  Higher susceptibility to Fas ligand induced apoptosis and altered modulation of cell death by tumor necrosis factor-α in periarticular tenocytes from patients with knee joint osteoarthritis 
Arthritis Research & Therapy  2003;5(5):R253-R261.
The aim of the present study was to investigate the expression of Fas in periarticular tenocytes of patients with osteoarthritis (OA) and to study their susceptibility to Fas ligand-mediated apoptosis. Tendon samples were obtained from the quadriceps femoris muscle of patients with knee OA and used for histological evaluation, for immunohistochemical detection of Fas, and to establish tenocyte cultures. The expression of Fas mRNA was determined by quantitative PCR. Levels of soluble Fas and soluble tumour necrosis factor (TNF) receptor I were measured using ELISA. Apoptosis was induced with recombinant human Fas ligand and measured by a histone fragmentation assay and flow cytometry. The effects of TNF-α were studied by stimulation with TNF-α alone or 24 hours before the induction of apoptosis. Tendon samples from non-OA patients were used as controls. Histological evaluation revealed degenerative changes in the tendons of all OA patients but not in the controls. Fas was detected by immunohistochemistry in all specimens, but quantitative PCR revealed significantly higher levels of Fas mRNA in OA tenocytes. In contrast, lower levels of soluble Fas were found in OA tenocytes by ELISA. OA tenocytes were significantly more susceptible to Fas ligand induced apoptosis than were control cells. TNF-α reduced the Fas ligand induced apoptosis in OA tenocytes but had no effects on control tenocytes. These data suggest that knee OA is associated with higher susceptibility of periarticular tenocytes to Fas ligand induced apoptosis because of higher expression of Fas but lower levels of apoptosis-inhibiting soluble Fas. These changes may contribute to decreased cellularity in degenerative tendons and promote their rupturing. The antiapoptotic effects of TNF-α in OA tenocytes most likely reflect regenerative attempts and must be taken into account when anti-TNF strategies are considered for OA.
PMCID: PMC193726  PMID: 12932288
apoptosis; osteoarthritis; Fas ligand; tenocytes; tumour necrosis factor-α
24.  Osteoclast-independent bone resorption by fibroblast-like cells 
Arthritis Research & Therapy  2003;5(3):R163-R173.
To date, mesenchymal cells have only been associated with bone resorption indirectly, and it has been hypothesized that the degradation of bone is associated exclusively with specific functions of osteoclasts. Here we show, in aseptic prosthesis loosening, that aggressive fibroblasts at the bone surface actively contribute to bone resorption and that this is independent of osteoclasts. In two separate models (a severe combined immunodeficient mouse coimplantation model and a dentin pit formation assay), these cells produce signs of bone resorption that are similar to those in early osteoclastic resorption. In an animal model of aseptic prosthesis loosening (i.e. intracranially self-stimulated rats), it is shown that these fibroblasts acquire their ability to degrade bone early on in their differentiation. Upon stimulation, such fibroblasts readily release acidic components that lower the pH of their pericellular milieu. Through the use of specific inhibitors, pericellular acidification is shown to involve the action of vacuolar type ATPases. Although fibroblasts, as mesenchymal derived cells, are thought to be incapable of resorbing bone, the present study provides the first evidence to challenge this widely held belief. It is demonstrated that fibroblast-like cells, under pathological conditions, may not only enhance but also actively contribute to bone resorption. These cells should therefore be considered novel therapeutic targets in the treatment of bone destructive disorders.
doi:10.1186/ar752
PMCID: PMC165048  PMID: 12723988
aseptic prosthesis loosening; bone resorption; dentin; fibroblasts; severe combined immunodeficient mouse

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