Effect of Pirfenidone on TGF-β1-Induced Myofibroblast Differentiation and Extracellular Matrix Homeostasis of Human Orbital Fibroblasts in Graves' Ophthalmopathy.

Shi-Bei Wu,Chieh-Chih Tsai,Hui-Chuan Kau,Tzu-Yu Hou

doi:10.3390/biom11101424

Abstract

Pirfenidone is a pyridinone derivative that has been shown to inhibit fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Its effect on orbital fibroblasts remains poorly understood. We investigated the in vitro effect of pirfenidone in transforming growth factor-β1 (TGF-β1)-induced myofibroblast transdifferentiation and extracellular matrix (ECM) homeostasis in primary cultured orbital fibroblasts from patients with Graves’ ophthalmopathy (GO). The expression of fibrotic proteins, including α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), fibronectin, and collagen type I, was determined by Western blots. The activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) responsible for the ECM homeostasis were examined. After pretreating the GO orbital fibroblasts with pirfenidone (250, 500, and 750 μg/mL, respectively) for one hour followed by TGF-β1 for another 24 h, the expression of α-SMA, CTGF, fibronectin, and collagen type I decreased in a dose-dependent manner. Pretreating the GO orbital fibroblasts with pirfenidone not only abolished TGF-β1-induced TIMP-1 expression but recovered the MMP-2/-9 activities. Notably, pirfenidone inhibited TGF-β1-induced phosphorylation of p38 and c-Jun N-terminal kinase (JNK), the critical mediators in the TGF-β1 pathways. These findings suggest that pirfenidone modulates TGF-β1-mediated myofibroblast differentiation and ECM homeostasis by attenuating downstream signaling of TGF-β1.

Highlights

Graves’ ophthalmopathy (GO) occurs in 25–50% of patients with Graves’ disease [1].The pathological processes include inflammation of orbital connective tissues in the early course, followed by soft tissue expansion and fibrosis in the orbit, wherein orbital fibroblasts proliferate and differentiate into myofibroblasts, adipogenesis increases, and glycosaminoglycans (GAGs) accumulate in the extracellular matrix (ECM) [2,3,4]
We have shown that transforming growth factor-β1 (TGF-β1) but not TGF-β2 could induce the expression of fibrotic proteins, including connective tissue growth factor (CTGF), fibronectin, and α-smooth muscle actin (α-SMA), in the GO orbital fibroblasts [8]
Our prior research showed that TGF-β1 induced differentiation of orbital fibroblasts into myofibroblasts, which dominated the pathogenic processes of tissue remodeling and into myofibroblasts, which dominated the pathogenic processes of tissue remodeling and fibrosis in GO [8,10]

Summary

Introduction

Graves’ ophthalmopathy (GO) occurs in 25–50% of patients with Graves’ disease [1].The pathological processes include inflammation of orbital connective tissues in the early course, followed by soft tissue expansion and fibrosis in the orbit, wherein orbital fibroblasts proliferate and differentiate into myofibroblasts, adipogenesis increases, and glycosaminoglycans (GAGs) accumulate in the extracellular matrix (ECM) [2,3,4]. TGF-β can induce tissue remodeling and fibrosis through my-

Methods

Results

Conclusion