Retinal pigment epithelial (RPE) cells maintain the health and functional integrity of both photoreceptors and the choroidal vasculature Loss of RPE differentiation has long been known to play a critical role in numerous retinal diseases, including inherited rod-cone degenerations, inherited macular degeneration, age-related macular degeneration, and proliferative vitreoretinopathy Recent studies in post-mortem eyes have found upregulation of critical epithelial-mesenchymal transition (EMT) drivers such as TGF-β, Wnt, and Hippo As RPE cells become less differentiated, they begin to exhibit the defining characteristics of mesenchymal cells, namely, the capacity to migrate and proliferate A number of preclinical studies, including animal and cell culture experiments, also have shown that RPE cells undergo EMT Taken together, these data suggest that RPE cells retain the reprogramming capacity to move along a continuum between polarized epithelial cells and mesenchymal cells We propose that movement along this continuum toward a mesenchymal phenotype be defined as RPE Dysfunction Potential mechanisms include impaired tight junctions, accumulation of misfolded proteins and dysregulation of several key pathways and molecules, such as TGF-β pathway, Wnt pathway, nicotinamide, microRNA 204/211 and extracellular vesicles This review synthesizes the evidence implicating EMT of RPE cells in post-mortem eyes, animal studies, primary RPE, iPSC-RPE and ARPE-19 cell lines

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Role of Epithelial-Mesenchymal Transition in Retinal Pigment Epithelium Dysfunction.

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

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

Under physiological conditions, retinal pigment epithelium (RPE) is a cellular monolayer composed of mitotically quiescent cells. Tight junctions and adherens junctions maintain the polarity of RPE cells, and are required for cellular functions. In proliferative vitreoretinopathy (PVR), upon retinal tear, RPE cells lose cell-cell contact, undergo epithelial-mesenchymal transition (EMT), and ultimately transform into myofibroblasts, leading to the formation of fibrocellular membranes on both surfaces of the detached retina and on the posterior hyaloids, which causes tractional retinal detachment. In PVR, RPE cells are crucial contributors, and multiple signaling pathways, including the SMAD-dependent pathway, Rho pathway, MAPK pathways, Jagged/Notch pathway, and the Wnt/β-catenin pathway are activated. These pathways mediate the EMT of RPE cells, which play a key role in the pathogenesis of PVR. This review summarizes the current body of knowledge on the polarized phenotype of RPE, the role of cell-cell contact, and the molecular mechanisms underlying the RPE EMT in PVR, emphasizing key insights into potential approaches to prevent PVR.

Polarity and epithelial-mesenchymal transition of retinal pigment epithelial cells in proliferative vitreoretinopathy.

SIX1 promotes epithelial-mesenchymal transition in colorectal cancer through ZEB1 activation

MicroRNA-34a inhibits epithelial-mesenchymal transition of lens epithelial cells by targeting Notch1.

Elevated Notch signaling has been verified in a large range of fibrotic diseases developed in the kidney, liver, and lung, inducing the development of the epithelial–mesenchymal transition (EMT). The aim of this study was to observe the involvement of Notch signaling in the EMT of retinal pigment epithelial (RPE) cells and the pathogenesis of proliferative vitreoretinopathy (PVR). In vitro cultivated human RPE cells (ARPE-19) were treated with 10 ng/mL transforming growth factor (TGF)-β1 for 24, 48, and 72 h. The expression levels of ZO-1, α-SMA, vimentin, Notch1 intracellular domain (NICD1), and Hes-1 were evaluated with quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence staining or Western blot. TGF-β1 induced EMT and the activation of Notch signaling in ARPE-19 cells. To examine the effect of Notch inhibition on TGF-β1-induced EMT and PVR formation, ARPE-19 cells were preincubated with γ-secretase inhibitor LY411575 before TGF-β1 treatment. Mouse PVR model was used for in vivo study. ARPE-19 cells were injected intravitreously with or without the LY411575 to examine the effect of Notch inhibition on PVR formation. LY411575 significantly attenuated EMT by inhibiting the Notch signaling activation in vitro. PVR was induced by intravitreal injections of ARPE-19 cells, while LY411575 inhibited mouse PVR formation in vivo. Notch signaling plays a critical role in TGF-β1-induced EMT in vitro and mice PVR model, which provides a novel insight into the pathogenesis of PVR. The specific inhibition of Notch signaling by γ-secretase inhibitor may provide a new approach for the prevention of PVR.

Gongqiang Yuan

Papers

Notch signaling modulates proliferative vitreoretinopathy via regulating retinal pigment epithelial-to-mesenchymal transition