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

Abstract

2 Introduction: The pathophysiology of osteoarthritis (OA) involves wear and tear, and a state 3 of low-grade inflammation. Wear and tear leads to tissue degradation and tissue repair 4 responses, including tissue growth factor beta (TGFβ)-induced myofibroblast production of 5 extracellular matrix (ECM). Fibronectins are an essential part of the ECM, and injection of 6 fibronectin fragments into rabbit joints is a previously established animal model of OA.7 Alternatively-spliced fibronectin contains the ED-A domain (ED-A FN) and has been shown 8 to activate Toll-like receptor 4. In this study, we tested the hypothesis that FN fragments 9 containing the ED-A domain could be one mechanism transducing mechanical events into 10 inflammatory signals in OA. 11 12 Methods: Samples of synovial membrane and cartilage were obtained from patients with 13 knee OA undergoing joint replacement surgery. Immunostaining for ED-A FN and the 14 myofibroblast marker alpha smooth muscle actin (αSMA) was performed on synovial 15 membranes and fibroblast-like synovial cells (FLS). FLS were stimulated with TGFβ, TNFα, 16 lipopolysaccharide, IL-6, OA synovial fluid, or chondrocyte lysate, and analyzed for ED-A 17 FN. Synovial cells isolated by enzymatic digestion and human monocyte-derived 18 macrophages (MDM) were incubated with recombinant ED-A FN, plasmin, cellular FN, or 19 cellular FN digested with plasmin; and culture supernatants were analyzed for MCP-1 and 20 TNFα.21 22 Results: We hypothesized that ED-A FN is produced by OA FLS in response to factors found 23 in the OA synovial joint. Indeed, the production of ED-A FN by OA FLS was increased by 24 TGFβ, OA synovial fluid, and lysed chondrocytes in all experiments (n=3). ED-A FN co-25 localized with the myofibroblast marker αSMA in both the OA FLS (n=3) and in the OA ED-A FN in OA 3 1 synovial membranes (n=8). We further hypothesized that ED-A FN expression is associated 2 with cellular density and expression of inflammatory molecules in OA. ED-A FN staining 3 was associated with both number of lining layer cells (rho=0.85 and p=0.011) and sublining 4 cells (rho=0.88 and p=0.007) in the OA synovium (n=8), and co-localized with both MCP-1 5 and TNFα (n=5). Recombinant ED-A FN increased the production of both MCP-1 and TNFα 6 by MDM (n=3) and OA FLS (n=3). Finally, we demonstrated that the FN fragments 7 containing the ED-A domain generated the same production of both MCP-1 and TNFα as 8 recombinant ED-A FN. 9 10 Conclusion: The disease process in OA shares features with the chronic wound healing 11 response including myofibroblast differentiation and production of mediators that promote 12 myofibroblast production of ED-A FN. We show that recombinant and plasmin-derived ED-A 13 fragments stimulate FLS and MDM to produce pro-inflammatory mediators. Our findings 14 support utilizing ED-A FN for drug delivery or therapeutic targeting of the formation of ED-15 A FN or the enzymatic fragmentation of FN to reduce pro-inflammatory responses in OA. 16 17

Defining the extracellular matrix in non-cartilage soft tissues in osteoarthritis – a systematic review

“…Synovial tissue was studied in several joints, including 17 studies on the knee [68,[92][93][94][95][96][97][98][99][100][101][102][103][104][105][106][107], 5 studies on hip [108][109][110][111][112], 6 studies used a combination of knee and hip synovium [113][114][115][116][117][118], 2 studies on TMJ [119,120], and 10 studies did not report the joint site [121][122][123][124][125][126][127][128][129][130]. The ECM components that were most often studied in human synovium were collagens, fibronectins, and laminins.…”