Rationale: The Notch and transforming growth factor-β (TGFβ) signaling pathways are two intracellular mechanisms that control fibrosis in general but whether they play a major role in retinal fibrosis is less clear. Here we study how these two signaling pathways regulate Müller cell-dominated retinal fibrosis in vitro and in vivo./nMethods: Human MIO-M1 Müller cells were treated with Notch ligands and TGFβ1, either alone or in combination. Western blots were performed to study changes in γ-secretase proteases, Notch downstream effectors, endogenous TGFβ1, phosphorylated Smad3 (p-Smad3) and extracellular matrix (ECM) proteins. We also studied the effects of RO4929097, a selective γ-secretase inhibitor, on expression of ECM proteins after ligand stimulation. Müller cell viability was studied by AlamarBlue and cytotoxicity by lactate cytotoxicity assays. Finally, we studied changes in Notch and TGFβ signaling and tested the effect of intravitreal injections of the Notch pathway inhibitor RO4929097 on retinal fibrosis resulted from Sodium iodate (NaIO₃)-induced retinal injury in mice. We also studied the safety of intravitreal injections of RO4929097 in normal mice./nResults: Treatment of Müller cells with Notch ligands upregulated γ-secretase proteases and Notch downstream effectors, with increased expression of endogenous TGFβ1, TGFβ receptors and p-Smad3. TGFβ1 upregulated the expression of proteins associated with both signaling pathways in a similar manner. Notch ligands and TGFβ1 had additive effects on overexpression of ECM proteins in Müller cells which were inhibited by RO4929097. Notch and TGFβ ligands stimulated Müller cell proliferation which was inhibited by RO4929097 without damaging the cells. NaIO₃-induced retinal injury activated both Notch and TGFβ signaling pathways in vivo. Intravitreal injection of RO4929097 prevented Müller cell gliosis and inhibited overexpression of ECM proteins in this murine model. We found no safety concerns for up to 17 days after an intravitreal injection of RO4929097./nConclusions: Inhibiting Notch signaling might be an effective way to prevent retinal fibrosis. This study is of clinical significance in developing a treatment for preventing fibrosis in proliferative vitreoretinopathy, proliferative diabetic retinopathy and wet age-related macular degeneration.

中文翻译：

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在体外和体内靶向 Notch 和 TGF-β 信号通路以预防视网膜纤维化。

基本原理： Notch 和转化生长因子-β (TGFβ) 信号通路是一般控制纤维化的两种细胞内机制，但它们是否在视网膜纤维化中起主要作用尚不清楚。在这里，我们研究了这两种信号通路如何在体外和体内调节 Müller 细胞主导的视网膜纤维化。/n方法：人类 MIO-M1 Müller 细胞用 Notch 配体和 TGFβ1 单独或组合处理。进行蛋白质印迹以研究 γ-分泌酶蛋白酶、Notch 下游效应物、内源性 TGFβ1、磷酸化 Smad3 (p-Smad3) 和细胞外基质 (ECM) 蛋白的变化。我们还研究了 RO4929097（一种选择性 γ-分泌酶抑制剂）对配体刺激后 ECM 蛋白表达的影响。Müller 细胞活力通过 AlamarBlue 进行研究，细胞毒性通过乳酸细胞毒性试验进行研究。最后，我们研究了 Notch 和 TGFβ 信号传导的变化，并测试了玻璃体内注射 Notch 通路抑制剂 RO4929097 对碘酸钠 (NaIO ₃)-诱导的小鼠视网膜损伤。我们还研究了正常小鼠玻璃体内注射 RO4929097 的安全性。/n结果：用 Notch 配体处理 Müller 细胞可上调 γ-分泌酶蛋白酶和 Notch 下游效应物，增加内源性 TGFβ1、TGFβ 受体和 p-Smad3 的表达。TGFβ1 以类似的方式上调与两种信号通路相关的蛋白质的表达。Notch 配体和 TGFβ1 对被 RO4929097 抑制的 Müller 细胞中 ECM 蛋白的过表达具有累加作用。Notch 和 TGFβ 配体刺激了被 RO4929097 抑制的 Müller 细胞增殖，而不会损害细胞。NaIO ₃诱导的视网膜损伤在体内激活了 Notch 和 TGFβ 信号通路. 玻璃体内注射 RO4929097 可防止 Müller 细胞神经胶质增生并抑制该小鼠模型中 ECM 蛋白的过度表达。我们在玻璃体内注射 RO4929097 后长达 17 天没有发现安全问题。/n结论：抑制 Notch 信号可能是预防视网膜纤维化的有效方法。该研究对于开发一种预防增殖性玻璃体视网膜病变、增殖性糖尿病视网膜病变和湿性年龄相关性黄斑变性纤维化的治疗方法具有临床意义。