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

Purpose: Synovitis is a key factor in osteoarthritis (OA) pathophysiology, contributing to both patient symptoms and disease progression. In this study, using an original methodology comparing normal/reactive (N/R) and inflammatory (I) synovial membranes zones, we investigated the gene expression profiles of synovial cells from these areas and identified differentially regulated pathways. Methods: Synovial cells (SC) were isolated from OA synovial specimens obtained from 12 patients undergoing knee replacement. The inflammatory status of the synovial membrane was characterized by the surgeon according to macroscopic criteria including the synovial vascularization, the villi formation and the hypertrophic aspect of the tissue. At the surgery time, the synovial membrane was dissected and biopsies from N/R and I areas cultured separately for a period of 7 days. Total RNA was extracted using the RNeasy Mini Kit. RNA purity and quality were evaluated using the Experion RNA StdSens Analysis kit (Bio-rad Laboratories). Gene expression profiling between N/R and I areas 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 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). Western blot was performed to confirm certain intermediate expression. 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