Theranostics 2020; 10(18):7956-7973. doi:10.7150/thno.45192

Research Paper

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

Jiawen Fan1,2, Weiyong Shen1✉, So-Ra Lee1, Ashish Easow Mathai1, Rui Zhang1, Gezhi Xu2, Mark C. Gillies1✉

1. The University of Sydney, Save Sight Institute, Discipline of Ophthalmology, Sydney Medical School, Sydney, New South Wales, Australia.
2. Department of Ophthalmology and Vision Sciences and Key Laboratory of Myopia of State Health Ministry, Eye and ENT Hospital, Shanghai Medical College, Fudan University, Shanghai, People's Republic of China.

This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Citation:
Fan J, Shen W, Lee SR, Mathai AE, Zhang R, Xu G, Gillies MC. Targeting the Notch and TGF-β signaling pathways to prevent retinal fibrosis in vitro and in vivo. Theranostics 2020; 10(18):7956-7973. doi:10.7150/thno.45192. Available from http://www.thno.org/v10p7956.htm

File import instruction

Abstract

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.

Methods: 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.

Results: 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.

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.

Keywords: Notch, transforming growth factor β, signalling pathway, Müller cells, retina, fibrosis