Abstract
The epithelial to mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a critical event in the pathogenesis of proliferative vitreoretinopathy and neovascular age-related macular degeneration, which are the leading causes of severe vision loss. Endoplasmic reticulum (ER) stress has been implicated in the EMT of many cell types and various ocular diseases. However, the relationship between ER stress and EMT in RPE cells remains unknown. Therefore, in the study, we explored the impact of ER stress on EMT in RPE cells. Different concentrations of tunicamycin (TM) and thapsigargin (TG) were used to induce ER stress in human RPE cells. The expression of epithelial marker, mesenchymal markers and some of genes/proteins involved in TGF-β/Smad signaling were analized by qPCR, western blot or immunostaining at the condition with or without stimulation of TGF-β2 (10 ng/mL). Boyden chamber and scratch assay were used to evaluate the migration of RPE cells, while cell viability and apoptosis of RPE cells were measured by MTT and TUNEL assay, respectively. Treatment of RPE cells with TM and TG (24 h) reduced the expression of α -SMA and FN, and increased the expression of Occludin in a dose dependent manner at protein level, which was highly associated with the expression of GRP78. Treatment with TGF-β2 significantly increased the expression of α-SMA and FN, and decreased the expression of Occludin both in protein and mRNA levels, which was significantly inhibited by a 4h pre-treatment with TM. In addition, the expression of TGF-βRII and Smad2/3, and mRNAs of TGF-βRII and Smad3 were also decreased by the TM treatment. TM-induced ER stress inhibited RPE cell migration, and high concentrations of TM and TG reduced cell viability and induced apoptosis of RPE cells. Chemical induction of ER stress inhibited EMT and migration in RPE cells, possibly by inactivation of TGF-β signaling, suggesting that regulation of ER stress in RPE cells may be a new approach to prevent the development of intraocular fibrosis.
Highlights
Intraocular fibrotic diseases, such as proliferative vitreoretinopathy (PVR), late-stage neovascular age-related macular degeneration, and proliferative diabetic retinopathy (PDR), can occur in response to ocular trauma, ischemia, inflammation, and degenerative disease
Exposure of retinal pigment epithelial (RPE) cells to different concentrations of TM and TG reduced the expression of Alpha-smooth muscle actin (α-SMA) and FN, and increased the expression of occludin significantly (p < 0.05). These phenomena were strongly associated with the expression of Glucose regulated p rotein 78 kD (GRP78) (Fig. 1). These results suggested that Endoplasmic reticulum (ER) stress inhibited epithelial to mesenchymal transition (EMT) in RPE cells
To investigate whether ER stress can inhibit TGF-β2-induced EMT of RPE cells, we examined the expression of epithelial markers and mesenchymal markers in RPE cells at both the mRNA and protein levels
Summary
Intraocular fibrotic diseases, such as proliferative vitreoretinopathy (PVR), late-stage neovascular age-related macular degeneration (nAMD), and proliferative diabetic retinopathy (PDR), can occur in response to ocular trauma, ischemia, inflammation, and degenerative disease. The formation of subretinal, epiretinal, or intravitreal fibrotic membranes due to extensive wound healing responses has been identified as an important step in the pathogenesis of these fibrotic diseases This process orchestrates the interaction of several cellular components, including RPE cells, glial cells, fibroblasts, and inflammatory cells [1], with many inflammatory cytokines and growth factors, resulting in overproduction or remodeling of the extracellular matrix (ECM). In the development of PVR and at the end-stage of nAMD, with the stimulation of various growth factors and cytokines, the RPE cells, which are a monolayer of highly polarized hexagonal cells under physiological conditions, undergo EMT This enables them to lose their epithelial characteristics, transdifferentiate into fibroblast-like cells, and migrate. Conclusions: Chemical induction of ER stress inhibited EMT and migration in RPE cells, possibly by inactivation of TGF-β signaling, suggesting that regulation of ER stress in RPE cells may be a new approach to prevent the development of intraocular fibrosis
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