HGF depress the production of α-SMA and extracellular matrix induced by TGF-β1 in fibroblasts of the flexor tendon sheath

Abstract

Objective To examine the effectiveness of HGF in blocking TGF-β1 induced α-SMA and extracellular matrix production in fibroblasts of the flexor tendon sheath. Methods Seven adult male New Zealand white rabbits (3.75-4.00 kg) were used for this study. Both of their front feet were sterilised and the middle digit flexor digitorum profundus tendon equivalents were identified and isolated. These specimens were used to establish primary cell cultures. Sheath fibroblasts were obtained from rabbit flexor tendons. After the cells reached confluence, cells were detached with trypsin/ethylenediamine tetra-acetic acid. All experiments were performed using the cells at the third passage. At 70% confluence the medium was supplemented with 5 ng/ml of TGF-β1 along with increasing doses of HGF (10-40 ng/ml). After 72 hours incubation, the productions of α-SMA were assayed by Western-Blot. The productions of collagen Ⅰ and fibronectin in supernatants culture were examined using ELISA. Results Evaluation of protein expression revealed that TGF-β1 markedly induced α-SMA expression in cultured rabbit flexor tendon sheath fibroblasts. TGF-β1 treated fibroblasts expressed 1.8-fold more protein compared to non-treated fibroblasts (P ＜ 0.05). However, simultaneous incubation of HGF significantly abrogated TGF-β1 induced α-SMA expression in a dose-dependent manner (P＜ 0.05). Treatment with TGF-β1 significantly stimulated collagen Ⅰ and fibronectin production in flexor tendon sheath fibroblasts (P ＜ 0.01). Remarkably, the addition of HGF reduced productions of all components induced by TGF-β1 in a dose-dependent manner (P ＜ 0.05). Conclusion HGF antagonizes TGF-β1 induced α-SMA, collagen Ⅰ, and fibronectin production in flexor tendon sheath fibroblasts. The findings provide a cellular and molecular basis for HGF's acting as a therapeutic agent for adhesions in flexor tendons. Key words: Hepatocyte growth factor; Transforming growth factor-β; Flexor tendon; Fibroblast; α-smooth muscle actin