Blockade of the TGF-? pathway by galunisertib inhibits the glial-mesenchymal transition in Muller glial cells

Aging increases the risks for developing fibrocontractile membranes on the retina, which causes significant macular distortion, as in the idiopathic epiretinal membrane (iERM). Retinal Muller glial cells are components of these membranes and may play a key role in the iERM pathogenesis. The transforming growth factor-beta (TGF-beta) induces Muller cell transdifferentiation into myofibroblast, reducing glial cell markers (glutamine synthetase, GS, and glial fibrillary acidic protein, GFAP) and increasing alpha-smooth muscle actin (alpha-SMA). Our aim was to investigate the effect of the TGF-beta inhibitor galunisertib (LY2157299) on the glial-mesenchymal transition and contraction of Muller cells.MIO-M1 human Muller cells were treated with TGF-beta 1 (10 ng/mL), galunisertib (5, 10 and 20 mu M) and TGF beta 1+galunisertib for 24h and 48h. Galunisertib cytotoxicity was analyzed by MTT and trypan blue, and TGF-beta 1 blockade by phospho-SMAD3 immunofluorescence. Caspase-3 (cell death indicator), GS, GFAP and alpha-SMA expression was examined by immunofluorescence, Western blotting, and qPCR analysis. Cell contractility was determined by collagen gel contraction assay with Muller cells incorporated.Galunisertib did not show cytotoxicity at the concentrations evaluated and maintained the Muller cells phenotype, ensuring the GS expression. Galunisertib inhibited the TGF-beta 1 pathway by decreasing phosphoSMAD3 immunoreactivity, attenuated the alpha-SMA expression, and prevented the contraction of Muller cells in collagen gel.Although more studies are needed, in vitro assays suggest that galunisertib may be a potential candidate to attenuate the formation of fibrocontractile membranes and prevent retinal detachment and consequent loss of vision.