Differential Effects of TGFβ and Vitreous on the Transformation of Retinal Pigment Epithelial Cells

PURPOSE. In proliferative vitreoretinopathy retinal pigment epithelial (RPE) cells undergo epithelial-mesenchymal transformation (EMT). Vitreous and transforming growth factor-β (TGFβ) have been implicated in this EMT. The role of TGFβ in the vitreous-mediated transformation of low-passage human RPE cells was investigated. METHODS. Cells were treated with vitreous or TGFβ2. SB431542 was used to inhibit TGFβ 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 TGFβ-like activity in vitreous. Cortical actin filaments in untreated cells were replaced by stress fibers after TGFβ 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 TGFβ-treated cells did not. However, SB431542 decreased vitreous-meditated changes in motility and invasion. The levels of mRNA for genes indicative of myofibroblast differentiation (α-SMA and CTGF) were increased by treatment with TGFβ but suppressed by vitreous. TGFβ or vitreous caused increased expression of Snail1. CONCLUSIONS. Vitreous or TGFβ caused a fibroblast-like morphology and induced Snail1, a marker of EMT. TGFβ 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 α-SMA suggested that TGFβ-treatment caused differentiation along a myofibroblast pathway, whereas vitreous treatment suppressed myofibroblast formation.