Synergistic effects of cyclic strain and Th1-like cytokines on tenascin-C production by rheumatic aortic valve interstitial cells

Abstract

Tenascin-C (TN-C) is a key component of extracellular matrix (ECM) and its expression process is poorly understood during rheumatic heart valvular disease (RHVD). In this study, we found that interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and TN-C concentrations in patients with RHVD were significantly higher than in normal controls. More IFN-gamma receptors and TNF receptors were found being expressed on rheumatic aortic valves interstitial cells than on non-rheumatic ones and their expression was patients' sera dependent. Antibodies neutralizing IFN-gamma or TNF-alpha could attenuate patients' sera-induced TN-C transcription by isolated rheumatic aortic valves interstitial cells. By application with different protein kinase inhibitors, we found that combined with cyclic strain, TNF-alpha and IFN-gamma induced TN-C transcription through the RhoA/ROCK signalling pathway. At the same time, p38 mitogen-activated protein kinase was involved in TNF-alpha and IFN-gamma induced TN-C transcription. TNF-alpha also increased TN-C mRNA level by additional PKC and ERK 1/2 activation. Our finding revealed a new insight into ECM remodelling during RHVD pathogenesis and new mechanisms involved in the clinical anti-IFN-gamma and anti-TNF-alpha therapy.

Fig. 1

Mechanical ventricular assist device (VAD) and multiwelled equi-biaxial strain device. (a) Appearance of mechanical right ventricular assist device (RVAD). (b) Structure details of RVAD. Arrows indicate flow direction. (1) Upper adaptor. (2) Pulmonary artery frame with valves. (3) Valves. (4) Lower adaptor. (5) One-way valve. (6) Mimic right ventricle. (7) Diaphragm. (8) Air dynamic chamber. (9) Motivity entrance. (c) Multiwelled device to generate equi-biaxial strain on a fibronectin-coated silicone membrane onto which cells have been plated. The membrane is fitted between the frame and base plate of the six-well dish, forming the bottom of the wells (top view, upper of figure). The dish with cultured cells is mounted on the upper platform of the device (side view, bottom of figure). Frequency and amplitude of cyclic strain can be adjusted.

Fig. 2

TN-C distribution in sera from the normal subjects (lane 1), patients with the malformed valves (lane 2) and with RHVD (lane 3) with method of immunoprecipitation and Western blot analysis. L, largest variants; S, smallest variants.

Fig. 3

TN-C expression patterns in the normal, malformed and rheumatic valves. The normal (Fig. 2A and B), malformed (Fig. 2C and D) and rheumatic (Fig. 2E and F) valves were stained with polyclonal anti-TN-C antibodies as described in Materials and Methods. Magnification of 2A, 2C and 2E is 100-fold and that of 2B, 2D and 2F is 200-fold.

Fig. 4

IFN-γ receptor 1 and TNF receptor I expression pattern and effects of antibodies neutralizing IFN-γ or TNF-α on TN-C transcription. (a) IFN-γ R1 on malformed valve interstitial cells (M). (b) TNF RI on M. (c) IFN-γ R1 on rheumatic valve interstitial cells (R). (d) TNF RI on R. (e) IFN-γ R1 on R after starvation. (f) TNF RI on R after starvation. (g) Antibodies neutralizing IFN-γ or TNF-α blocked TN-C transcription. Antibody against IFN-γ or TNF-α were added and incubated for the indicated time course according to antibody: targeting molecule mole ratio equal to 2:1 respectively. TN-C mRNA level was tested by quantitative real-time RT-PCR.

Fig. 5

Cyclic strain increased TN-C mRNA level through the RhoA/ROCK pathway and its synergistic effects with cytokines. (a) TN-C mRNA was induced by cyclic strain within isolated malformed (M) or rheumatic (R) valve interstitial cells for the indicated time course. TN-C mRNA level was tested by quantitative real-time RT-PCR. (b) Induction of tenascin-C mRNA by cyclic strain was attenuated by ROCK antagonist. After inhibitors were added for 60 min, cells were stimulated by cyclic strain (10%, 0·3 Hz) (S) or left at rest (R) for 6 h. Data represent the average of five independent experiments; error bars indicate standard errors of the mean. $(P < 0·01), significant difference to the resting control (R, no inhibitor); *(P < 0·01), significant difference to the other four strained groups. (c) Cyclic strain and cytokines had synergistic effects on TN-C transcription. $(P < 0·01), significant difference to the stimulated control (S, no cytokines); *(P < 0·01), significant difference to resting control (R, no cytokines).

Fig. 6

RhoA/ROCK and p38 MAPK pathways were involved in IFN-γ-inducing TN-C transcription. (a) TN-C mRNA was induced by IFN-γ within isolated rheumatic (R) other than malformed (M) valve interstitial cells. The Figure is representative of the assays. (b) Induction of tenascin-C mRNA by IFN-γ was attenuated by ROCK and p38 MAPK antagonist. After inhibitors were added for 60 min, cells were stimulated by IFN-γ for 12 h (+), the others left untreated (−). Data represent the average of five independent experiments; error bars indicate standard errors of the mean. *(P < 0·01), significant difference to the other three IFN-γ-treated groups.

Fig. 7

RhoA/ROCK, p38 MAPK, ERK 1/2 and PKC pathways were involved in TNF-α-inducing TN-C transcription. (a) TN-C mRNA was induced by TNF-α within isolated rheumatic (R) other than malformed (M) valve interstitial cells. The figure is representative of the assays. (b) Induction of tenascin-C mRNA by TNF-α was attenuated by ROCK, p38 MAPK, ERK 1/2 and PKC antagonist. After inhibitors were added for 60 min, cells were stimulated by TNF-α for 12 h (+), the others left untreated (−). Data represent the average of five independent experiments; error bars indicate standard errors of the mean. *(P < 0·01), significant difference to the TNF-α-treated control group.

Fig. 8

Signalling pathways and synergistic effects induced by cyclic strain, IFN-γ and TNF-α on tenascin-C production by rheumatic aortic valve interstitial cells.