Targeting the Notch and TGF-β signaling pathways to prevent retinal fibrosis in vitro and in vivo

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 Muller cell-dominated retinal fibrosis in vitro and in vivo. Methods: Human MIO-M1 Muller 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. Muller 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. Results: Treatment of Muller 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 Muller cells which were inhibited by RO4929097. Notch and TGFβ ligands stimulated Muller 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 Muller 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. Conclusions: 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.