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