PURPOSE: In proliferative vitreoretinopathy retinal pigment epithelial (RPE) cells undergo epithelial-mesenchymal transformation (EMT). Vitreous and transforming growth factor-beta (TGFbeta) have been implicated in this EMT. The role of TGFbeta in the vitreous-mediated transformation of low-passage human RPE cells was investigated. METHODS: Cells were treated with vitreous or TGFbeta2. SB431542 was used to inhibit TGFbeta signaling. Morphology was investigated using phase-contrast or confocal microscopy. Motility was measured using a monolayer-wounding assay. Invasion was determined using basement membrane matrix-based assays. Gene expression was measured by quantitative PCR, immunohistochemistry, or immunoblotting. RESULTS: Changes in phosphorylation or cellular localization of Smad -2, -3, or -4 indicated a TGFbeta-like activity in vitreous. Cortical actin filaments in untreated cells were replaced by stress fibers after TGFbeta treatment, but peripheral actin aggregates were seen in vitreous-treated cells. SB431542 did not block the morphologic change induced by vitreous. Vitreous-treated cells exhibited increased motility and invasion, whereas TGFbeta-treated cells did not. However, SB431542 decreased vitreous-meditated changes in motility and invasion. The levels of mRNA for genes indicative of myofibroblast differentiation (alpha-SMA and CTGF) were increased by treatment with TGFbeta but suppressed by vitreous. TGFbeta or vitreous caused increased expression of Snail1. CONCLUSIONS: Vitreous or TGFbeta caused a fibroblast-like morphology and induced Snail1, a marker of EMT. TGFbeta activity in vitreous was necessary but not sufficient for the vitreous-induced motile, invasive phenotype. However, differences in the cytoskeletal organization and in the expression of CTGF and alpha-SMA suggested that TGFbeta-treatment caused differentiation along a myofibroblast pathway, whereas vitreous treatment suppressed myofibroblast formation.
PURPOSE: In proliferative vitreoretinopathy retinal pigment epithelial (RPE) cells undergo epithelial-mesenchymal transformation (EMT). Vitreous and transforming growth factor-beta (TGFbeta) have been implicated in this EMT. The role of TGFbeta in the vitreous-mediated transformation of low-passage human RPE cells was investigated. METHODS: Cells were treated with vitreous or TGFbeta2. SB431542 was used to inhibit TGFbeta signaling. Morphology was investigated using phase-contrast or confocal microscopy. Motility was measured using a monolayer-wounding assay. Invasion was determined using basement membrane matrix-based assays. Gene expression was measured by quantitative PCR, immunohistochemistry, or immunoblotting. RESULTS: Changes in phosphorylation or cellular localization of Smad -2, -3, or -4 indicated a TGFbeta-like activity in vitreous. Cortical actin filaments in untreated cells were replaced by stress fibers after TGFbeta treatment, but peripheral actin aggregates were seen in vitreous-treated cells. SB431542 did not block the morphologic change induced by vitreous. Vitreous-treated cells exhibited increased motility and invasion, whereas TGFbeta-treated cells did not. However, SB431542 decreased vitreous-meditated changes in motility and invasion. The levels of mRNA for genes indicative of myofibroblast differentiation (alpha-SMA and CTGF) were increased by treatment with TGFbeta but suppressed by vitreous. TGFbeta or vitreous caused increased expression of Snail1. CONCLUSIONS:Vitreous or TGFbeta caused a fibroblast-like morphology and induced Snail1, a marker of EMT. TGFbeta activity in vitreous was necessary but not sufficient for the vitreous-induced motile, invasive phenotype. However, differences in the cytoskeletal organization and in the expression of CTGF and alpha-SMA suggested that TGFbeta-treatment caused differentiation along a myofibroblast pathway, whereas vitreous treatment suppressed myofibroblast formation.
Authors: Robert Hoerster; Philipp S Muether; Sarah Vierkotten; Manuel M Hermann; Bernd Kirchhof; Sascha Fauser Journal: Graefes Arch Clin Exp Ophthalmol Date: 2013-05-17 Impact factor: 3.117