Tissue growth factor β stimulates fibroblast-like synovial cells to produce extra domain A fibronectin in osteoarthritis

Introduction The pathophysiology of osteoarthritis (OA) involves wear and tear, and a state of low-grade inflammation. Wear and tear leads to tissue degradation and tissue repair responses, including tissue growth factor beta (TGFβ)-induced myofibroblast production of extracellular matrix (ECM). Fibronectins are an essential part of the ECM, and injection of fibronectin fragments into rabbit joints is a previously established animal model of OA. Alternatively-spliced fibronectin contains the ED-A domain (ED-A FN) and has been shown to activate Toll-like receptor 4. In this study, we tested the hypothesis that FN fragments containing the ED-A domain could be one mechanism transducing mechanical events into inflammatory signals in OA.

Methods Samples of synovial membrane and cartilage were obtained from patients with knee OA undergoing joint replacement surgery. Immunostaining for ED-A FN and the myofibroblast marker alpha smooth muscle actin (αSMA) was performed on synovial membranes and fibroblast-like synovial cells (FLS). FLS were stimulated with TGFβ, TNFα, lipopolysaccharide, IL-6, OA synovial fluid, or chondrocyte lysate, and analyzed for ED-A FN. Synovial cells isolated by enzymatic digestion and human monocyte-derived macrophages (MDM) were incubated with recombinant ED-A FN, plasmin, cellular FN, or cellular FN digested with plasmin; and culture supernatants were analyzed for MCP-1 and TNFα.

Results We hypothesized that ED-A FN is produced by OA FLS in response to factors found in the OA synovial joint. Indeed, the production of ED-A FN by OA FLS was increased by TGFβ, OA synovial fluid, and lysed chondrocytes in all experiments (n=3). ED-A FN co- localized with the myofibroblast marker αSMA in both the OA FLS (n=3) and in the OA synovial membranes (n=8). We further hypothesized that ED-A FN expression is associated with cellular density and expression of inflammatory molecules in OA. ED-A FN staining was associated with both number of lining layer cells (rho=0.85 and p=0.011) and sublining cells (rho=0.88 and p=0.007) in the OA synovium (n=8), and co-localized with both MCP-1 and TNFα (n=5). Recombinant ED-A FN increased the production of both MCP-1 and TNFα by MDM (n=3) and OA FLS (n=3). Finally, we demonstrated that the FN fragments containing the ED-A domain generated the same production of both MCP-1 and TNFα as recombinant ED-A FN.

Conclusion The disease process in OA shares features with the chronic wound healing response including myofibroblast differentiation and production of mediators that promote myofibroblast production of ED-A FN. We show that recombinant and plasmin-derived ED-A fragments stimulate FLS and MDM to produce pro-inflammatory mediators. Our findings support utilizing ED-A FN for drug delivery or therapeutic targeting of the formation of ED- A FN or the enzymatic fragmentation of FN to reduce pro-inflammatory responses in OA.