J. E. Dubuc

148 FIBULIN-3 FRAGMENTS (FIB3-1 AND FIB3-2) ARE POTENTIAL NEW BIOMARKERS FOR THE DIAGNOSIS OF OSTEOARTHRITIS

By comparison with healthy subjects, Fib3-1 and FiB3-2 serum levels are found elevated in patients with knee OA. Furthermore, the immunohistochemical findings indicate that fibrillated cartilage is a major source of fibulin-3 fragments. Specific immunoassays were developed using two rabbit antisera with high specificity for Fib3-1 and Fib3-2 respectively, ELISAs were validated in serum population collected from healthy subjects and patients with knee OA. Antisera did not recognize native fibulin-3 and did not cross-react with each fragment. Immunohistochemistry analysis was performed on articular cartilage from tibial plateaus 20-25 26-30 31-35 36-40 41-45 46-50 51-55 56-60 61-65 40 60 80 100 **

FRI0058 Expression of specific pathways in the inflamed synovial membrane of osteoarthritis patient: Identification of new potential key intermediates

Background Synovitis is a key factor in osteoarthritis (OA) pathophysiology. Objectives We investigated the gene expression profiles of synovial cells from two different areas and identified differentially regulated pathways, using an original methodology comparing normal/reactive (N/R) and inflammatory (I) synovial membranes zones. Methods Synovial cells were isolated from OA synovial specimens obtained from 12 patients undergoing knee replacement. The inflammatory status of the synovial membrane was characterized according to a macroscopic criteria. The synovial membrane was dissected and biopsies from N/R and I areas cultured separately. Total RNA was extracted using the RNeasy Mini Kit. Gene expression profiling between N/R and I areas was performed using Illumina’s multi-sample format Human HT-12 BeadChip. Differential analysis was performed with the BRB array tools software. Class Comparison test between N/R and I areas was based on paired t-test where N/R and I were paired for each patient. The biological relevance of up- and down-regulated genes was analyses with Ingenuity Pathways Analysis (Ingenuity® Systems). Results From among 47000 probes, 17500 were filtered out. Probes with a p-value below than 0.005 were chosen and classified as up- or down-regulated ones. By this way, 896 differentially expressed genes between N/R and I zones were identified. Among these, 576 genes were upregulated (I/NR >1.5) and 320 downregulated (I/NR <0.75). With Ingenuity Pathways Analysis, a significant number of the top ranking differentially expressed genes were identified as inflammatory, Wnt and angiogenic pathways. Interleukin (IL)-6 and -8, chemokines (CXCL1, CXCL2, CXCL5, CXCL6, CXCL16) and arachidonate 5-lipoxygenase (ALOX5) were identified as the most upregulated in I zones in the inflammatory pathway. Interestingly, the alarmin S100A9 was found strongly upregulated in this pathway. Wnt5A and LRP (Low density lipoprotein receptor-related protein) 5 were upregulated whereas FZD (Frizzled homolog) 2 and DKK (dickkopf homolog) 3 were downregulated in the Wnt signaling pathway. Finally, stanniocalcin (STC)-1, an intermediate in angiogenesis was identified as the most upregulated gene in I zones compared to N/R zones. This difference of expression was confirmed at the protein level. Conclusions Using a unique culture system, this study is the first to identify different expression pattern between two areas of synovial membrane from the same OA patient. These differences concern several key pathways involved in OA pathogenesis, i.e. inflammation, Wnt and angiogenesis. This analysis also provided interesting information regarding new potent intermediates as S100A9 and STC-1. They could be potential targets for chondroitin sulfate, one of the most used molecules in the management of OA. New experiments are being perfomed at the moment to elucidate the potential effect of this molecule on these specific differentially expressed genes in the same culture system. Disclosure of Interest None Declared

AB0062 Investigation of potential new targets for the diagnosis and/or the treatment of osteoarthritis

Background Synovial inflammation plays a key role in the pathophysiology process of osteoarthritis (OA). We have previously compared the gene expression pattern of synovial cells isolated from inflammatory (I) or normal/reactive (N/R) areas of a synovial membrane harvested from the same OA patient. We identified a large number of mediators belonging to key pathways involved in OA pathogenesis. Objectives To validate different potential new targets for the diagnosis and/or the treatment OA. Methods Synovial cells (SC) were isolated from synovial biopsies from OA patients undergoing knee replacement. SC from N/R and I areas were cultured separately for a period of 7 days. Microarray gene expression profiling between N/R and I areas was performed. The biological relevance of regulated genes was analyzed with Ingenuity Pathways Analysis. Western blot and immunohistochemistry confirmed the most differentially expressed genes in the key pathways. Results 896 genes differentially expressed in N/R and I areas were identified. The key pathways were related to inflammation, cartilage metabolism, Wnt signaling and angiogenesis. In the inflammatory gene pattern, the triggering receptor expressed on myeloid cells-1 (TREM1) and the alarmin S100 calcium binding protein A9 (S100A9) were strongly upregulated. We validated the production of these proteins in OA synovial biopsies by Western blot. TREM1 and S100A9 were increased in I compared to N/R synovial cells culture. S100A9 was observed in the perivascular area and in sublining cells in I synovial biopsies, but not in N/R biopsies. An increased staining was also observed in the intima lining layer of I when compared to N/R biopsies. The most upregulated anabolism enzyme in I synovial biopsies was the hyaluronan synthase-1 (HAS1). Using immunohistochemistry, we observed in I areas an increase of the HAS1-positive cells mainly in the intima lining. We also studied the protein production of the wingless-type MMTV integration site family, member 5A (Wnt-5A), the most upregulated intermediate of Wnt signaling pathway. The protein level was increased in I compared to N/R areas. Finally, in the angiogenesis pathway, one the most up-regulated gene was the stanniocalcin 1 (STC1). A significant increase of STC1 production was observed in I areas compared to N/R areas by Western blot. This result was also supported by the immunohistochemical analysis. In I area, the staining for STC1 was more intense in perivascular and sublining cells. Conclusions Synovial membrane inflammation is a key target for OA treatments. In this work, we have identified proteins involved in the synovitis pathways like angiogenesis, cells infiltration and matrix remodeling. These proteins could be targeted by drugs and used as companion biomarkers for evaluating their efficacy. Although qualitative, our results could also yield to the identification of markers of the disease. This investigation has to be further pursued. Disclosure of Interest C. Lambert: None Declared, J.-E. Dubuc: None Declared, E. Montell Employee of: BIOIBÉRICA, S.A, Barcelona, Spain; Pre-Clinical R&D Manager, J. Vergès Employee of: BIOIBÉRICA, S.A, Barcelona, Spain; Medical Director, C. Munaut: None Declared, A. Noël: None Declared, Y. Henrotin Grant/research support from: This study was supported by a grant from BIOIBÉRICA, S.A, Barcelona, Spain

AB0061 Effects of chondroitin sulfate on the gene expression profile in il-1b stimulated synovial fibroblast cells cultures

Background Chondroitin sulfate (CS) is one the most used molecules in the management of OA. Its mechanism of action remains to be detailed. Objectives To perform a microarray analysis to identify a differential expression profile between control and IL-1β stimulated synovial fibroblast cells cultures and to investigate the effects of CS on this gene expression profile. Methods OA synovial specimens were obtained from 12 patients undergoing knee replacement. Synovial fibroblast cells (SFC) were enzymatically isolated and used after four passages (P4). SFC were pre-treated 1 hour with highly purified bovine CS (200 µg/ml, Bioibérica S.A., Barcelona, Spain) before treatment with IL-1β (1 ng/ml) for 24 hours. Gene expression profiling was performed using Illumina’s multi-sample format Human HT-12 BeadChip (Illumina Inc.). Differential analysis was performed with the BRB array tools software. Class comparison test between control (Ctl) and interleukin (IL)-1β conditions, Ctl and Ctl/CS and IL-1β and IL-1β/CS conditions was based on paired t-test where Ctl and IL-1β, Ctl and Ctl/CS and IL-1β and IL-1β/CS were paired for each patient. The biological relevance of regulated genes was analyzed with Ingenuity Pathways Analysis (Ingenuity® Systems). Probes with a p-value below 0.001 were chosen and classified as up- or down-regulated. Results 3308 genes were identified as differentially expressed genes between Ctl and IL-1β conditions. A differential profile of expression of major pathways involved in OA pathogenesis was observed. In the inflammatory network, the most upregulated cytokines were IL-8 and IL-6 (fold change: 156.25 and 58.8 respectively). We identified several chemokines, enzymes and metallothioneins (MTs). Complement factor B (CFB) and complement component 3 (C3) are two factors upregulated in the inflammatory complement cascade. We identified some genes implicated in the angiogenesis pathway. The most upregulated was Stanniocalcin 1 (STC1) (fold change: 9.09). The differential expression of intermediates in cartilage anabolism and catabolism revealed an imbalance in favour of catabolism. MMP-3 was largely upregulated (fold change: 62.5). Wnt 5A and low density lipoprotein receptor-related protein (LRP8) were significantly upregulated while frizzled homolog 2 (FZD2) and dickkopf homolog 3 (DKK3) were downregulated in the Wnt signaling. The class comparison test highlighted 660 differentially expressed genes between Ctl and Ctl/CS conditions and 241 genes between IL-1β and IL-1β/CS. Among them, our attention was focused on two genes upregulated in the presence of CS: lysyl oxidase-like 4 (LOXL4) and claudin 11 (CDLN11), two genes that negatively regulate cell invasion. Conclusions We here evidenced in synovial fibroblast cells the modulation of gene expression following IL-1β stimulation. We also demonstrated the modulatory effects of CS on gene expression and isolated several CS-modulated genes of interest such as LOXL4 and CDLN11, which could constitute new mechanisms of action of the molecule and contribute to explain the symptomatic efficacy of CS in the treatment of OA. Disclosure of Interest C. Lambert: None Declared, J.-E. Dubuc: None Declared, E. Montell Employee of: BIOIBÉRICA, S.A, Barcelona, Spain; Pre-Clinical R&D Manager, J. Vergés Employee of: BIOIBÉRICA, S.A, Barcelona, Spain; Medical Director, Y. Henrotin Grant/research support from: This study was supported by a grant from BIOIBÉRICA, S.A, Barcelona, Spain

AB0063 Effects of chondroitin sulfate on the gene expression profile in the inflamed synovial membrane

Background Chondroitin sulfate (CS) is one the most used molecules in the management of OA. Its mechanism of action remains to be detailed. Objectives To identify the differentially expressed genes between the inflammatory (I) and normal/reactive (N/R) synovial areas using a unique ex vivo culture model and to investigate the genetic modulatory effects of CS in this model. Methods Synovial cells (SC) were isolated from OA synovial specimens from 12 patients undergoing knee replacement. The synovial membrane was dissected and SC from N/R and I areas cultured separately for a period of 7 days with or without highly purified bovine CS (200 µg/ml, Bioibérica S.A., Barcelona, Spain). Gene expression profiling was performed using Illumina’s multi-sample format Human HT-12 BeadChip (Illumina Inc.). Differential analysis was performed with the BRB array tools software. Class Comparison test between N/R and I conditions, N/R and N/R-CS conditions and I and I-CS conditions was based on paired t-test where N/R and I, N/R and N/R-CS and I and I-CS were paired for each patient. The biological relevance of up- and down-regulated genes was analyses with Ingenuity Pathways Analysis (Ingenuity® Systems). Results From among 47000 probes, 18253 were filtered out. Probes with a p-value below than 0.005 were chosen and classified as up- or down-regulated ones. By this way, 465 differentially expressed genes between N/R and I areas were identified. Many inflammatory mediators appear differentially expressed. The interferon alpha-inductible protein 6 (IFI6) was the most up-regulated. We also identified the hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1), the cathepsin K (CTSK), the chemokine (C-X-C motif) ligand 1 (CXCL1) and the EBV-induced G-protein coupled receptor 2 (EBI2). The differential expression of intermediates involved in angiogenesis pathway was also revealed between N/R and I areas. Among them, R-spondin-3 (RSPO3), the secreted phopshoprotein 1 (SPP1) and aquaporin 9 (AQP9) were up-regulated whereas ADAMTS1 was down-regulated. Finally, in the Wnt signaling, RSPO3 was up-regulated unlike dickkopf homolog 3 (DKK3) which was in turn down-regulated. We next performed a class comparison test between N/R and N/R-CS in one hand and between I and I-CS the other hand. 489 genes were identified as differentially expressed genes between N/R and N/R-CS conditions while 219 genes were identified between I and I-CS conditions. In this latter, our attention was focused on the down-regulated genes. Among them, we identified a number implicated in angiogenesis and cell migration pathways. Thus, the endothelial cell-specific molecule-1 (ESM1), the Transmembrane-4-L-six-family-1 (TM4SF1), the 5’-Ectonucleotidase (NT5E) and the growth arrest-specific gene 6 (GAS6) were down-regulated by CS. Conclusions Our work demonstrates the differential gene expression profile between paired inflammatory and normal/reactive areas of synovial membrane as well as the modulatory effects of CS on gene expression in the inflammatory areas, especially regarding genes involved in both angiogenesis and cell migration. Disclosure of Interest C. Lambert: None Declared, J.-E. Dubuc: None Declared, E. Montell Employee of: BIOIBÉRICA, S.A, Barcelona, Spain; Pre-Clinical R&D Manager, J. Vergès Employee of: BIOIBÉRICA, S.A, Barcelona, Spain; Medical Director, Y. Henrotin Grant/research support from: This study was supported by a grant from BIOIBÉRICA, S.A, Barcelona, Spain