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

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.

Reactivities of a Prostanoid EP2 Agonist, Omidenepag, Are Useful for Distinguishing between 3D Spheroids of Human Orbital Fibroblasts without or with Graves’ Orbitopathy

“…Since the orbital fibroblast should spread toward 3D spaces within the orbital cone during its differentiation, a 3D cell culture technology is more desirable than conventional 2D cell culture methods for this research purpose. Nevertheless, as far as we know, no related studies were conducted using a 3D cell culture technology because of their technical difficulties, although there were several studies using a 2D cell culture [24][25][26][27]. Previously, to understand the molecular pathology of GO, our group first succeeded in obtaining the 3D spheroids of n-HOF and GHOF using a 3D drop culture method, in which no scaffold was required [10].…”

Research progress on the pathogenesis of Graves’ ophthalmopathy: Based on immunity, noncoding RNA and exosomes

“…Wu et al. showed that TGF-β1 can induce the transdifferentiation of OFs into myofibroblasts through the MAPK signaling pathway, characterized by enhanced expression of fibrotic proteins such as α-smooth muscle actin, connective tissue growth factor, and fibronectin ( 26 ). In addition, their study showed that TGF-β1 induces the up-regulated of type I collagen with the accumulation of ECM proteins.…”

Pirfenidone Attenuates the EMT Process and the Secretion of VEGF in TGF-β2-Induced ARPE-19 Cells via Inhibiting the Activation of the NF-κB/Snail Signaling Pathway

“…Recently, a small synthetic pyridone compound named pirfenidone (PFD) was used as a therapeutic strategy to treat ophthalmologic diseases [ 10 ]. PFD (5-methyl-1-phenyl-2-[1H]-pyridone) was first used as an antifibrotic drug for idiopathic pulmonary fibrosis and was shown to have multiple benefits, including antioxidant, antifibrotic activity, and anti-inflammatory effects [ 11 , 12 ].…”