da Silva Rafael André, Roda Vinicius Moraes de Paiva, Akamine Priscilla Sayami, da Silva Daniela Simões, Siqueira Paula Veloso, Matsuda Monique, Hamassaki Dânia Emi
Department of Cell & Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil.
Department of Cell & Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil; Laboratory of Investigation in Ophthalmology (LIM-33), Division of Ophthalmology, University of São Paulo Faculty of Medicine, São Paulo, SP, Brazil.
Exp Eye Res. 2023 Jan;226:109336. doi: 10.1016/j.exer.2022.109336. Epub 2022 Nov 29.
Aging increases the risks for developing fibrocontractile membranes on the retina, which causes significant macular distortion, as in the idiopathic epiretinal membrane (iERM). Retinal Müller glial cells are components of these membranes and may play a key role in the iERM pathogenesis. The transforming growth factor-β (TGF-β) induces Müller cell transdifferentiation into myofibroblast, reducing glial cell markers (glutamine synthetase, GS, and glial fibrillary acidic protein, GFAP) and increasing α-smooth muscle actin (α-SMA). Our aim was to investigate the effect of the TGF-β inhibitor galunisertib (LY2157299) on the glial-mesenchymal transition and contraction of Müller cells. MIO-M1 human Müller cells were treated with TGF-β1 (10 ng/mL), galunisertib (5, 10 and 20 μM) and TGF-β1+galunisertib for 24h and 48h. Galunisertib cytotoxicity was analyzed by MTT and trypan blue, and TGF-β1 blockade by phospho-SMAD3 immunofluorescence. Caspase-3 (cell death indicator), GS, GFAP and α-SMA expression was examined by immunofluorescence, Western blotting, and qPCR analysis. Cell contractility was determined by collagen gel contraction assay with Müller cells incorporated. Galunisertib did not show cytotoxicity at the concentrations evaluated and maintained the Müller cells phenotype, ensuring the GS expression. Galunisertib inhibited the TGF-β1 pathway by decreasing phospho-SMAD3 immunoreactivity, attenuated the α-SMA expression, and prevented the contraction of Müller 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.
衰老会增加视网膜上形成纤维收缩膜的风险，这会导致明显的黄斑变形，如特发性视网膜前膜（iERM）。视网膜穆勒神经胶质细胞是这些膜的组成部分，可能在iERM发病机制中起关键作用。转化生长因子-β（TGF-β）诱导穆勒细胞转分化为肌成纤维细胞，减少神经胶质细胞标志物（谷氨酰胺合成酶，GS，和胶质纤维酸性蛋白，GFAP）并增加α-平滑肌肌动蛋白（α-SMA）。我们的目的是研究TGF-β抑制剂加芦尼塞（LY2157299）对穆勒细胞神经胶质-间充质转化和收缩的影响。将MIO-M1人穆勒细胞用TGF-β1（10 ng/mL）、加芦尼塞（5、10和20 μM）以及TGF-β1+加芦尼塞处理24小时和48小时。通过MTT和台盼蓝分析加芦尼塞的细胞毒性，并通过磷酸化SMAD3免疫荧光分析TGF-β1阻断情况。通过免疫荧光、蛋白质印迹和qPCR分析检测半胱天冬酶-3（细胞死亡指标）、GS、GFAP和α-SMA的表达。通过掺入穆勒细胞的胶原凝胶收缩试验测定细胞收缩性。在所评估的浓度下，加芦尼塞未显示细胞毒性并维持了穆勒细胞表型，确保了GS表达。加芦尼塞通过降低磷酸化SMAD3免疫反应性抑制TGF-β1途径，减弱α-SMA表达，并防止穆勒细胞在胶原凝胶中收缩。尽管需要更多研究，但体外试验表明，加芦尼塞可能是减轻纤维收缩膜形成、预防视网膜脱离及随之而来的视力丧失的潜在候选药物。
