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AIM:To investigate the effect of Tenascin C(TNC)on the expression of pro-inflammatory cytokines and matrixmetalloproteinases in human cardiac myofibroblasts(CMF).METHODS:CMF were isolated and cultured from patients undergoing coronary artery bypass grafting.Cultured cells were treated with either TNC(0.1μmol/L,24 h)or a recombinant protein corresponding to different domains of the TNC protein;fibrinogen-like globe(FBG)and fibronectin typeⅢ-like repeats(TNⅢ5-7)(both 1μmol/L,24 h).The expression of the proinflammatory cytokines;interleukin(IL)-6,IL-1β,TNFαand the matrix metalloproteinases;MMPs(MMP1,2,3,9,10,MT1-MMP)was assessed using real time RT-PCR and western blot analysis.RESULTS:TNC increased both IL-6 and MMP3(P<0.01)mR NA levels in cultured human CMF but had no significant effect on the other markers studied.The increase in IL-6 mR NA expression was mirrored by an increase in protein secretion as assessed by enzymelinked immunosorbant assay(P<0.01).Treating CMF with the recombinant protein FBG increased IL-6mR NA and protein(P<0.01)whereas the recombinant protein TNⅢ5-7 had no effect.Neither FBG nor TNⅢ5-7 had any significant effect on MMP3 expression.The expression of toll-like receptor 4(TLR4)in human CMF was confirmed by real time RT-PCR,western blot and immunohistochemistry.Pre-incubation of cells with TLR4neutralising antisera attenuated the effect of both TNC and FBG on IL-6 mR NA and protein expression.CONCLUSION:TNC up-regulates IL-6 expression in human CMF,an effect mediated through the FBG domain of TNC and via the TLR4 receptor.
AIM: To investigate the effect of Tenascin C (TNC) on the expression of pro-inflammatory cytokines and matrix metalloproteinases in human cardiac myofibroblasts (CMF). METHODS: CMF were isolated and cultured from patients undergoing coronary artery bypass grafting. Cultured cells were treated with either TNC (0.1 μmol / L, 24 h) or a recombinant protein corresponding to different domains of the TNC protein; fibrinogen-like globe (FBG) and fibronectin type Ⅲ- ). The expression of the proinflammatory cytokines; interleukin (IL) -6, IL-1β, TNFαand the matrix metalloproteinases; MMPs (MMP1,2,3,9,10, MT1-MMP) was assessed using real time RT-PCR and Western blot analysis .RESULTS: TNC increased both IL-6 and MMP3 (P <0.01) mR NA levels in cultured human CMF but had no significant effect on the other markers studied. increase in IL-6 mR NA expression was mirrored by an increase in protein secretion as assessed by enzyme-linked immunosorbant assay (P <0.01) .Treating CMF with the re The recombinant protein TNIII5-7 had no effect. Neither FBG nor TNIII5-7 had any significant effect on MMP3 expression. The expression of toll-like receptor 4 (TLR4 ) in human CMF was confirmed by real time RT-PCR, western blot and immunohistochemistry. Pre-incubation of cells with TLR4 neutralization antisera attenuated the effect of both TNC and FBG on IL-6 mR NA and protein expression. CONCLUSION: TNC up-regulates IL-6 expression in human CMF, an effect mediated through the FBG domain of TNC and via the TLR4 receptor.