Identification of synovium secretome factor(s) for the prevention of osteoarthritis

Abstract

Purpose: Previous studies have suggested that bioactive factors secreted from synoviocytes protect cartilage from interleukin-1 beta (IL-1β) - induced catabolism, but these bioactive secretome products have not been identified. We hypothesized that by using an unbiased bottom-up proteomics approach, chondroprotective factor(s) could be identified as potential novel therapeutics that could be used to alter the disease course of osteoarthritis (OA). Methods: Matched equine cartilage explants and synovial membrane were collected post-mortem from horses (n=4) with no history of lameness and normal joints at necropsy. Six groups were established: cartilage, synoviocytes, and cartilage+synoviocytes (co-culture) all +/- IL-1β. The catabolic effect of IL-1β was verified by measuring glycosaminoglycan (GAG) released from cartilage into media by the 1,9-dimethyl-methylene blue dye-binding microwell assay and cartilage toluidine blue histochemistry. Media from co-cultures +/- IL-1β were submitted for bottom-up proteomic analysis. Only co-culture media were used so that cell types were similar, and only IL-1β treatment was different between the groups for comparison. Data from MS/MS were searched against the Equus caballus database. Only high confidence peptides with a 1% false detection rate were considered for peptide ID. Final proteins ID contained protein groups that were filtered with at least 2 peptides per protein. A protein abundance ratio was calculated based on protein abundance in IL-1β treated co-cultures compared to controls. Results: As expected, IL-1β induced 2x GAG loss in cartilage cultures (data not shown). Matrix metachromasia loss was extensive in cartilage-only cultures treated with IL-1β, further validating culture conditions (Figs 1A, B). However, when cartilage was co-cultured with synoviocytes and treated with IL-1β, GAG was retained (Figs 1C, D). Using the abundance ratio, 226 proteins were identified as upregulated in IL-1β compared to control co-cultures. Protein distribution was 59% extracellular matrix (ECM), 12% structural, 14% catabolic, 9% chemokine, 3% growth factor (GF), and 3% ECM/structural (Fig 2). These proteins were then manually reviewed and searched in the literature for known or potential anti-catabolic effects for further analysis (Table 1). Presently, qPCR is being performed on RNA isolated from co-culture synoviocytes for the transcripts of these proteins which will be followed by gain and loss of function assays. Conclusions: We have identified proteins that are up-regulated in media of co-cultured synoviocyte and cartilage stimulated with IL-1β compared to control. Further evaluation will identify chondroprotective protein(s) to be explored as disease modifying OA therapeutics. Figure 1Synoviocytes protect cartilage from the catabolic effects of IL1-β. A) Cartilage with uniform matrix metachromasia, B) Cartilage + IL1-β with depletion of matrix metachromasia, C)Cartilage/synoviocyte co-culture with similar matrix metachromasia to cartilage in (A), D) Cartilage/synoviocyte co-culture + IL1-β stimulation with retained matrix metachromasia compared to cartilage in panel (B) demonstrating protection of cartilage from IL1-β when synoviocytes are present. Toluidine blue staining. Bar = 500 μm. Figure 2Protein distribution by function. Proteins (226) that were up regulated in IL1-β - treated co-cultures compared to control co-cultures. Proteins are categorized as 59% extracellular matrix (ECM), 12% structural, 14% catabolic, 9% chemokine, 3% growth factor (GF), and 3% ECM/structural.Table 1Proteins identified with potential chondroprotective properties.The 226 proteins that were upregulated in IL1-β - stimulated co-cultures were reviewed in the literature for known or potential roles in articular homeostasis and 12 were identified. These findings will be validated through PCR of synoviocytes, and then tested in gain/loss of function for chondroprotective mechanisms of action.Proteins identified with potential chondroprotective properties.gelsolininter-alpha-trypsin inhibitoralpha-2-macroglobulinlumicaninsulin-like growth factor-binding protein 2apolipoprotein B-100semaphorin-3Cvascular cell adhesion moleculethrombospondin-1insulin-like growth factor IImetalloproteinase inhibitor 3C-X-C motif chemokine 2 Open table in a new tab