Suppression of TGF-β pathway by pirfenidone decreases extracellular matrix deposition in ocular fibroblasts in vitro

Abstract

In glaucoma surgery, fibrotic processes occur, leading to impairment of liquid outflow. Activated fibroblasts are responsible for postoperative scarring. The transforming growth factor-β (TGF-β) pathway plays a key role in fibroblast function, differentiation and proliferation. The aim of this study was the characterization of the fibrotic potential of two subtypes of primary human ocular fibroblasts and the attempt to inhibit fibrotic processes specifically, without impairing cell viability. For fibrosis inhibition we focused on the small molecule pirfenidone, which has been shown to prevent pulmonary fibrosis by the decrease of the expression of TGF-β1, TGF-β2 and TGF-β3 cytokines. For in vitro examinations, isolated human primary fibroblasts from Tenon capsule and human intraconal orbital fat tissues were used. These fibroblast subpopulations were analyzed in terms of the expression of matrix components responsible for postoperative scarring. We concentrated on the expression of collagen I, III, VI and fibronectin. Additionally, we analyzed the expression of α-smooth muscle actin, which serves as a marker for fibrosis and indicates transformation of fibroblasts into myofibroblasts. Gene expression was analyzed by rtPCR and synthesized proteins were examined by immunofluorescence and Western blot methods. Proliferation of fibroblasts under different culture conditions was assessed using BrdU assay. TGF-β1 induced a significant increase of cell proliferation in both cell types. Also the expression of some fibrotic markers was elevated. In contrast, pirfenidone decreased cell proliferation and matrix synthesis in both fibroblast subpopulations. Pirfenidone slightly attenuated TGF-β1 induced expression of fibronectin and α-smooth muscle actin in fibroblast cultures, without impairing cell viability. To summarize, manipulation of the TGF-β signaling pathway by pirfenidone represents a specific antifibrotic approach with no toxic side effects in two human orbital fibroblast subtypes. We presume that pirfenidone is a promising candidate for the treatment of fibrosis following glaucoma surgery.

Development of a biodegradable antifibrotic local drug delivery system for glaucoma microstents

“…The same applies for PFD, where in vitro studies demonstrated inhibition of cell proliferation, myofibroblast differentiation, collagen contractility, cytokine suppression, decreased collagen and fibronectin expression, and migratory ability of cardiac, renal, lung, hepatic, and ocular fibroblasts [31,32,62–66]. An antifibrotic effect in vivo has also been described in a trabeculectomy model in rabbits, where postoperative application of PFD was associated with less scarring and improved bleb survival [67] and following GDI implantation in a rabbit model [68].…”

Reconstruction of biological functions: novel implant concepts for cardiovascular, ophthal-mologic and otolaryngologic applications

“…The systems comprise in situ polymerizing drug carriers, and will be applied by minimal invasive injection systems [11]. In parallel, in-depth understanding of fibrotic processes and drug distribution mechanisms is gained through extensive in vitro and ex vivo experimentation [12][13][14]. Restoration of near and far vision in cataract patients, or as an elective procedure in presbyopes, shall be achieved through reconstruction of the enucleated capsular bag.…”

α-SMA Expression Increased Over Cell Passages and Decreased by Exogenous TGF-β1, In Vitro Studies on Myofibroblast Derived from Orbital Socket Contracture

“…Increased growth factor activity not only leads to an increase in wound healing rate, but also a higher risk for scar formation. Therefore, TGF-β is one of the most potential targets for scarring intervention [8,9,10,11].…”

“…TGF-β1 is a major profibrotic cytokine and increased expression plays an important role in the deposition of collagen and extracellular matrix, wound healing and scar formation in the socket contracture tissue [6]. TGF-β1 is a key mediator that induce α-SMA [11,12]. Alpha-smooth muscle actin (α-SMA) may serve as a reliable marker for myofibroblast cells [12], although others considered it as not a consistent marker for contractile fibroblasts and lots of collagen [13].…”

“…Fibroblasts appear on day 2-3 following injury, while myofibroblasts appear on day 12 when wound contractions are nearly 80% complete [11,12].…”