12 efficiency determined by flow cytometry was in the range of 25-70%.Visualised by immunofluorescence, RASF transfected with SENP1-GFP revealed lowered levels of acetylated histone H4 when compared both to nontransfected as well as GFP transfected cells (n=3). Using flow cytometry analysis we could further confirm that the levels of H4 acetylation were significantly decreased by 40% when compared to nontransfected cells (n=3) (p
elated with the expression of LINE-1 ORF1p in the respective synovial tissues (r = 0.83, p< 0.05, by sequencing 6 RA and 1 OA clones in a total of 7 patients). Most important, the promoter methylation array showed that genes coding for maspin, talin and interferon regulatory factor 7 (IRF7) were hypomethylated in RA-SF, in comparison to normal SF (55-58% RA-SF and 100% normal SF). In contrast, 7 out of 83 gene promoters were methylated in both RA-SF and normal SF, whereas hypomethylated genes in both cell types included p16, p21, Ras, MLH1, E-cadherin, MGMT, TIMP-3 and COX-2. Conclusion: These results clearly show that global hypomethylation occurs in RA synovial tissues and especially in RA-SF. The specific hypomethylation of maspin, a novel serine protease inhibitor, talin, a cytoskeletal protein, and IRF7, a regulator of interferon genes, may contribute to the activated phenotype of RA-SF and their aggressive behaviour. 06 FIBRIN PROMOTES INVASIVENESS OF RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS BY THE INDUCTION OF MATRIX METALLOPROTEINASES 1 AND 3 Olga Sanchez-Pernaute 1 , Emmanuel Karouzakis 1 , Astrid Juengel 1 , Peter Kuenzler 1 , Renate E. Gay 1 , Christoph Kolling 2 , Gabriel Herrero-Beaumont 3 , Steffen Gay 1 , Michel Neidhart 1 . 1 Center of Experimental <strong>Rheuma</strong>tology, University Hospital, Zurich, Switzerland; 2 Department of Orthopedic Surgery, Schulthess Klinik, Zurich, Switzerland; 3 <strong>Rheuma</strong>tology Section, Fundacion Jimenez Di¬az, Madrid, Spain Purpose: Fibrin has been identified as a major autoantigen in rheumatoid arthritis (RA) as a result of its citrullination inside joints. We have previously hypothesized that fibrin could account not only for autoimmunity but also for the destructive behaviour of RA synovial cells. Our aim in the present work was to study the expression of the matrix metalloproteinases (MMP)1 and 3 in synovial fibroblasts upon stimulation with fibrin. Methods: Synovial fibroblasts from patients with RA (RASF) or osteoarthritis (OASF) were cultured and stimulated with fibrin freshly clotted by adding 0.75 U/ml thrombin to 1 mg/ml purified fibrinogen. The gene expression of MMP1, MMP3 and the alpha 5 integrin chain were determined by real-time PCR. By immunoblotting, we studied the appearance of pro- and active MMP1 and MMP3, as well as levels of the mitogen activated kinase p38 (phosphorylated and non phosphorylated isoforms) in RASF exposed to fibrin at different time points. Co-localisation studies of fibrin and MMP1 and 3 were performed in synovial specimens from RA patients by immunohistochemistry. Results: In cultured RASF, a 12 h stimulation with fibrin resulted in a 23-fold increase of the gene expression of MMP1 (n = 4, p < 0.03) and a 27-fold increase of the gene expression of MMP3 (n = 4, p < 0.03). A slight induction was also found in OASF, although it was only significant for the MMP3 mRNA (2-fold up-regulation, n = 4, p < 0.03). The alpha 5 integrin chain, which acts as receptor for several extracellular matrix proteins, was found up-regulated in RASF (1.8-fold, p < 0.03) but not in OASF. An increase in MMP1 protein levels followed fibrin treatment in RASF, peaking at 18 h (4-fold and 11-fold for latent and active isoforms, respectively), while the release of MMP3 to supernatants was 2-fold increased at 18 h of treatment compared to untreated RASF. Additionally, phosphorylation of p38 was observed between 1 h to 4 h of exposure to fibrin. In rheumatoid synovial tissues, interstitial immune-reactivity to both MMP1 and 3 was associated to fibrin deposits and both proteases colocalised with fibrin at the invasive interfaces. Fibroblast-like cells in fibrin-rich areas depicted a strong immune-reactivity to MMP1 and MMP3. Conclusion: Our observations support a role for fibrin in the aggressive potential of RASF, suggesting that these cells are stimulated by fibrin to enhance their production of proteases that mediate the destruction of cartilage and bone in RA. The selective up-regulation of the alpha 5 integrin observed in RASF, points to this molecule as a candidate receptor for the transduction of fibrin-dependent invasive signals in RASF. 07 PRODUCTION OF MATRIX METALLOPROTEINASES IN RHEUMATOID ARTHRITIS IS REGULATED BY PHOSPHATIDYLINOSITOL 3-KINASE GAMMA Noreen Pundt 1 , Marvin A. Peters 1 , Inga Kühnel 1 , Christina Wunrau 1 , Katja Neugebauer 1 , Lars H. Meyer 1 , Georg Schett 2 , Silvia Hayer 3 , Anja Baier 4 , Tzvetanka Bondeva 5 , Steffen Gay 6 ,Thomas Ruekle 7 , Montserrat Camps 7 , Matthias K. Schwarz 7 , Christian Rommel 7 , Reinhard Wetzker 5 , Thomas Pap 1 . 1 University Hospital Muenster, Muenster, Germany; 2 University Hospital Erlangen, Erlangen, Germany; 3 University Vienna, Vienna, Austria; 4 University Hospital Magdeburg, Magdeburg, Germany; 5 University Hospital Jena, Jena, Germany; 6 University Hospital Zurich, Zurich, Austria; 7 Serono Pharm Res Inst., Geneva, Switzerland Purpose: We analyzed the expression of the gamma isoform of PI3K in synovial fibroblasts from patient with RA (RASF) and OA (OASF). Further we investigated the effects of PI3K� on EGF induced Akt-phosphorylation and MMP expression as well as on the invasive behaviour of RASF and synovial fibroblasts from hTNFtg mice. METHODS: RASF and OASF were analyzed for the expression of mRNA for the catalytic p110 and regulatory p101 subunit of PI3K� by PCR and western blot. The involvement of PI3K� in Akt-phoshorylation was studied in EGFstimulated cells (10-100 ng/ml, 5 min) using the pan-PI3K-inhibitor LY294992 (200-500ng/ml) and a PI3KÁ specific inhibitor AS-252424 (50-100 mM). The expression of MMP-1 and MMP-3 was studied by ELISA following stimulation of synovial fibroblasts with EGF (10 ng/ml, 24 h). The invasive behaviour of EGF-stimulated RASF treated with AS-252424 was investigated using transepithelial resistant assay (TEER). Synovial Fibroblasts from hTNFtg mice crossed into PI3K�-/- mice were used to confirm these data in an animal model of RA. RESULTS: PCR and Western blot revealed the expression of p110 catalytic subunit of PI3K� but not of the regulatory p101 subunit in RASF. OASF showed only negligible levels of p110. Stimulation of RASF and OASF resulted in the phosphorylation of Akt with LY294002 inhibiting completely the EGF-mediated activation.AS-252424 reduced the EGF-stimulated phosphorylation of Akt in RASF but had no effect in OASF. Treatment of RASF with both LY294002 and AS-252424 significantly reduced the EGF-mediated induction of MMP-1 (63% and 42%, respectively) and MMP-3 (20% and 40%, respectively). Similarly, fibroblast from hTNFtg mice expressed significantly more MMP-3 than fibroblasts from hTNFtg/PI3K�-/- mice. As determined by TEER assay, AS-252424 significantly inhibited the invasive behaviour of RASF. CONCLUSIONS: These data suggest a disease-specific expression of PI3K� in RASF that contribute to EGF-mediated phosphorylation of Akt, induction of MMP-1 and MMP-3 and subsequent decreased invasive phenotype. Therefore specific inhibitors of PI3K� may be a possibility to interfere with the activation of RASF and to reduce cartilage destruction in RA. 08 THE NOVEL CHEMOKINE RECEPTOR CXCR7 IS EXPRESSED IN RHEUMATOID ARTHRITIS SYNOVIUM Yvonne Y. Rengel Colina 1 , Renate E. Gay 1 , Christoph Kolling 2 , Beat A. Michel 1 , Steffen Gay 1 , Caroline Ospelt 1 1 Center of Experimental <strong>Rheuma</strong>tology, University Hospital Zurich and Zurich Center of Integrative Human Physiology (ZIHP), Zurich, Switzerland; 2 Schulthess Clinic, Zurich Switzerland, Zurich, Switzerland Purpose: In rheumatoid arthritis (RA), the chemokine stromal cell-derived factor (SDF-1), also known as chemokine ligand 12 (CXCL12), has been related to cell recruitment, angiogenesis, proliferation, and to the production of matrix metalloproteinases by synovial fibroblasts (SF). It was generally believed that SDF-1 mediates these diverse processes via a single cell surface receptor known as chemokine receptor 4 (CXCR4). Recently, an alternative receptor for CXCL12 has been discovered in tumor cell lines, the chemokine receptor CXCR7. We analyzed the expression and regulation of CXCR7 in tissues from RA patients and RA synovial fibroblasts (RASF). Methods: Immunohistochemistry and Western blot with rabbit anti-human CXCR7 antibodies were used to detect the expression of CXCR7 protein in RA and osteoarthritis (OA) synovial tissues. To assay the expression of CXCR7 in synovial fibroblasts and macrophages, we performed a double staining with rabbit anti-human CXCR7 antibodies and mouse anti-vimentin or mouse anti-CD68 antibodies. The role of IL-1� (1ng/ml), TNF-� (10ng/ml) and TGF-� (10ng/ml) in the regulation of the expression of CXCR7 were analyzed by Real-time PCR (6, 12, 24h of stimulation) and Western blot (24 and 48h of stimulation). The expression of CXCR7 at protein level was measured by densitometry and a ratio CXCR7/·-tubulin was calculated. Results: By immunohistochemistry (4 RA, 3 OA), we demonstrate that CXCR7 is expressed in RA and OA synovium. RA synovial tissues exhibit a moderate to high expression of CXCR7, with a preferred localization in the lining layer. In contrast, OA samples showed a much weaker expression. However, around blood vessel, RA and OA samples showed a similar intensity of CXCR7. Double staining with vimentin and CD68 revealed that CXCR7 is 45–2007 13
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