title: 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.
authors.affiliation: Department of Ophthalmology, Rostock University Medical Center, Rostock, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Stahnke
authors.affiliation: Department of Ophthalmology, Rostock University Medical Center, Rostock, Germany.
authors.forename: Bhavani S
authors.initials: BS
authors.lastname: Kowtharapu
authors.affiliation: Department of Ophthalmology, Rostock University Medical Center, Rostock, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Stachs
authors.affiliation: Institute for Biomedical Engineering, Rostock University Medical Center, Rostock, Germany.
authors.forename: Klaus-Peter
authors.initials: KP
authors.lastname: Schmitz
authors.affiliation: Department of Anatomy, Rostock University Medical Center, Rostock, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Wurm
authors.affiliation: Department of Anatomy, Rostock University Medical Center, Rostock, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Wree
authors.affiliation: Department of Ophthalmology, Rostock University Medical Center, Rostock, Germany.
authors.forename: Rudolf Friedrich
authors.initials: RF
authors.lastname: Guthoff
authors.affiliation: Institute for Biomedical Engineering, Rostock University Medical Center, Rostock, Germany.
authors.forename: Marina
authors.initials: M
authors.lastname: Hovakimyan
mesh_terms: D000199:Actins / Q000032:analysis / Q000235:genetics; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D005353:Fibronectins / Q000032:analysis / Q000235:genetics; D005355:Fibrosis; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D011728:Pyridones / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D000894:Anti-Inflammatory Agents, Non-Steroidal; D005353:Fibronectins; D011728:Pyridones; D016212:Transforming Growth Factor beta; C093844:pirfenidone