In-vitro effect of aflibercept and dexamethasone on the expression level of genes associated with epithelial-mesenchymal transition in retinal pigment epithelial cells

Role of Retinal Pigment Epithelial Cell β-Catenin Signaling in Experimental Proliferative Vitreoretinopathy

Immunological Properties of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.

Elimination of senescent cells inhibits epithelial-mesenchymal transition of retinal pigment epithelial cells

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.

The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is one of the significant pathogenic mechanisms for the formation of subretinal fibrosis in age-related macular degeneration (AMD). Multiple signaling pathways that promote EMT have been well described, yet the endogenous signaling pathways that inhibit EMT within RPE cells remain largely elusive. In this study, we confirmed the expression of CRX in human RPE cells and human embryonic stem cell-derived RPE (ESC-RPE) cells. By employing sub-culture to disrupt intercellular connections and thereby inhibit the Hippo signaling pathway, combined with TGF-β1 treatment in vitro to mimic the microenvironment for the formation of subretinal fibrosis, it was revealed that Hippo/YAP1 and TGF-β1 synergistically promoted the nuclear translocation of β-catenin, and the latter bound to TCF7 to inhibit the expression of CRX. Overexpression of CRX was capable of suppressing the occurrence of EMT in ESC-RPE cells. CRX exerted its inhibitory effect on EMT partly by upregulating the expression of PPP2R2B. In the laser-induced choroidal neovascularization mouse model, the nuclear translocation of CRX took place in RPE cells, and overexpression of CRX played an inhibitory role in the formation of subretinal fibrosis. This study has identified CRX as an endogenous signaling molecule that inhibits EMT in RPE cells and has provided a new research target and treatment strategy for the treatment of wet AMD and the inhibition of subretinal fibrosis formation.

Proliferative vitreoretinopathy (PVR) is a disease that causes severe blindness and is characterized by the formation of contractile fibrotic subretinal or epiretinal membranes. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a hallmark of PVR. This work aims to examine the role of a long noncoding RNA (lncRNA) named EMT-related lncRNA in RPE (ERLR, LINC01705-201 (ENST00000438158.1)) in PVR and to explore the underlying mechanisms. In this study, we found that ERLR is upregulated in RPE cells stimulated with transforming growth factor (TGF)-β1 as detected by lncRNA microarray and RT-PCR. Further studies characterized full-length ERLR and confirmed that it is mainly expressed in the cytoplasm. In vitro, silencing ERLR in RPE cells attenuated TGF-β1-induced EMT, whereas overexpressing ERLR directly triggered EMT in RPE cells. In vivo, inhibiting ERLR in RPE cells reduced the ability of cells to induce experimental PVR. Mechanistically, chromatin immunoprecipitation (ChIP) assays indicated that the transcription factor TCF4 directly binds to the promoter region of ERLR and promotes its transcription. ERLR mediates EMT by directly binding to MYH9 protein and increasing its stability. TCF4 and MYH9 also mediate TGF-β1-induced EMT in RPE cells. Furthermore, ERLR is also significantly increased in RPE cells incubated with vitreous PVR samples. In clinical samples of PVR membranes, ERLR was detected through fluorescent in situ hybridization (FISH) and colocalized with the RPE marker pancytokeratin (pan-CK). These results indicated that lncRNA ERLR is involved in TGF-β1-induced EMT of human RPE cells and that it is involved in PVR. This finding provides new insights into the mechanism and treatment of PVR.

Nintedanib inhibits normal human vitreous-induced epithelial-mesenchymal transition in human retinal pigment epithelial cells

The abnormal growth factors-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells was known as a vital pathogenesis of proliferative vitreoretinopathy (PVR). This study aims to explore how survivin inhibition affects EMT induced by epidermal growth factor (EGF) in RPE cells. Human primary RPE cells were identified in vitro. EMT in RPE cells was induced by EGF. Inhibition of survivin in RPE cells was accomplished through the use of a survivin inhibitor (YM155) and survivin siRNA. The viability, proliferation and migration of RPE cells was detected by methylthiazol tetrazolium assay, bromodeoxyuridine labeling assay, and wound healing assay, respectively. The EGF receptor /mitogen-activated protein kinase (EGFR/MAPK) proteins and EMT-related proteins were measured by western blot and immunofluorescence assay. EGF induced significant EMT in RPE cells, activated the phosphorylation of EGFR/MAPK signaling proteins, and caused changes to EMT-related proteins. YM155 suppressed RPE cells' viability, proliferation, and migration; induced the phosphorylation of EGFR, JNK, and P38MAPK; and down regulated EGFR and phosphorylated ERK. YM155 also increased expression of E-cadherin and ZO-1 proteins and reduced expression of N-cadherin, Vimentin, and α-SMA proteins. The EGF-induced increase of RPE cell proliferation and migration was constrained by survivin inhibition. Moreover, survivin inhibition in RPE cells suppressed the EGF-caused phosphorylation of EGFR/MAPK proteins and attenuated the EGF-induced reduction of E-cadherin and ZO-1 proteins and increase of N-cadherin, Vimentin, and α-SMA proteins. Survivin inhibition attenuates EGF-induced EMT of RPE cells by affecting the EGFR/MAPK signaling pathway. Survivin might be a promising target for preventing PVR.

Apigenin suppresses epithelial-mesenchymal transition in high glucose-induced retinal pigment epithelial cell by inhibiting CBP/p300-mediated histone acetylation

The proliferation of retinal pigment epithelium (RPE) cells following epithelial-mesenchymal transition (EMT) is critical in proliferative vitreoretinopathy (PVR), which results in retinal detachment and the loss of vision. The current study was conducted to examine the importance of transforming growth factor β-1 (TGF-β1)-activated kinase 1 (TAK1) inhibitor (LYTAK1) in regulating EMT and the proliferation of RPE cells. RPE cells were pre-treated with increasing concentrations of LYTAK1 prior to treatment with TGF-β1 for 24 h. The effect of LYTAK1 on RPE cell proliferation was examined using a Cell Counting kit-8 assay. The expression levels of TAK1, smooth muscle actin, fibronectin, p-Smad2, p-Smad3, nuclear factor (NF)-κB p65 and IκB kinase α were detected by western blotting. LYTAK1 suppressed the proliferation and migration of RPE cells. Additionally, LYTAK1 significantly prevented TGF-β1-induced EMT by decreasing the levels of fibronectin and α-smooth muscle actin. It was demonstrated that the effects of LYTAK1 were via the Smad signaling pathway. The present study also determined, that the underlying mechanism of the effects of LYTAK1 on EMT in RPE cells involves downregulation of the NF-κB signaling pathway. In conclusion, TAK1 transcription factor was shown to be important in TGF-β1-induced EMT in human RPE cells. Thus, the results of this study aid in elucidating the pathogenesis of human PVR. In addition, this study suggests that specific inhibition by LYTAK1 may provide a novel approach for the treatment and prevention of PVR.

Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is inevitable change of age-related macular degeneration (AMD). Smoking is a major risk factor for the development of EMT in several diseases, including lung cancer. Cigarette smoke-induced stress promotes the production of epidermal growth factor (EGF) in RPE cells. However, the underlying signaling pathways induced by aberrant EGF receptor (EGFR) expression in cigarette smoke-exposed RPE cells remain largely unknown. In the present study, the morphological transformation and production of EMT-associated cytokines were investigated to analyze the effect of smoking on the retina. Furthermore, EGF-treated or cigarette smoke-exposed RPE cells, as well as the downstream targets of EGFR, were investigated to identify the key molecules involved in EMT of cigarette smoke-stimulated RPE cells via immunoblotting. Exposure of RPE cells to cigarette smoke extract (CSE) induced secretion of VEGF and TGF-β1, and increased the expression of EMT markers. CSE-mediated focal adhesion kinase (FAK) activation resulted in the phosphorylation and activation of spleen associated tyrosine kinase (Syk)/Src proto-oncogene, non-receptor tyrosine kinase (Src), leading to migration and invasion of RPE cells. Knockdown of FAK or pharmacological inhibition of Syk/Src abrogated CSE-mediated VEGF and TGF-β1 production and blocked the phosphorylation of Smad2/3 in CSE-stimulated RPE cells. Erlotinib (an EGFR inhibitor) suppressed EGF and CSE-mediated switch from an epithelial to mesenchymal phenotype. Baicalein, an inhibitor of 12/15-lipooxygenase, also efficiently suppressed CSE-induced EMT processes by inhibiting EGFR-associated downstream signaling transduction. The results identified a novel signaling pathway mediated by EGFR in CSE-activated RPE cells, and suggest baicalein as a potential new therapeutic drug for CSE-associated retinopathy.

Exosomes have recently emerged as a pivotal mediator of many physiological and pathological processes. However, the role of exosomes in proliferative vitreoretinopathy (PVR) has not been reported. In this study, we aimed to investigate the role of exosomes in PVR. Transforming growth factor beta 2 (TGFß‐2) was used to induce epithelial‐mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, as an in vitro model of PVR. Exosomes from normal and EMTed RPE cells were extracted and identified. We incubated extracted exosomes with recipient RPE cells, and co‐cultured EMTed RPE cells and recipient RPE cells in the presence of the exosome inhibitor GW4869. Both experiments suggested that there are further EMT‐promoting effects of exosomes from EMTed RPE cells. MicroRNA sequencing was also performed to identify the miRNA profiles in exosomes from both groups. We identified 34 differentially expressed exosomal miRNAs (P <. 05). Importantly, miR‐543 was found in exosomes from EMTed RPE cells, and miR‐543‐enriched exosomes significantly induced the EMT of recipient RPE cells. Our study demonstrates that exosomal miRNA is differentially expressed in RPE cells during EMT and that these exosomal miRNAs may play pivotal roles in EMT induction. Our results highlight the importance of exosomes as cellular communicators within the microenvironment of PVR.

Subretinal fibrosis contributes to the loss of vision associated with age-related macular degeneration (AMD). Retinal pigment epithelial (RPE) cells play a key role in the pathogenesis of AMD including the fibrotic reaction. We examined the role of retinoic acid receptor-γ (RAR-γ) in the epithelial-mesenchymal transition (EMT) and other fibrosis-related processes in mouse RPE cells cultured in a type I collagen gel. Transforming growth factor-β2 (TGF-β2)-induced collagen gel contraction mediated by the RPE cells was inhibited by the RAR-γ agonist R667 in a concentration- and time-dependent manner. Expression of the mesenchymal markers α-smooth muscle actin and fibronectin, the release of interleukin-6, and the phosphorylation of paxillin, mitogen-activated protein kinases (ERK, p38, and JNK), Smad2, and AKT induced by TGF-β2 were also suppressed by the RAR-γ agonist. Furthermore, gelatin zymography and immunoblot analysis revealed that the TGF-β2-induced release of matrix metalloproteinase (MMP)-2, MMP-3, MMP-8, and MMP-9 from RPE cells was inhibited by R667, and the MMP inhibitor GM6001 attenuated TGF-β2-induced RPE cell contraction. Finally, immunohistofluorescence analysis with antibodies to glial fibrillary acidic protein showed that R667 inhibited the development of subretinal fibrosis in a mouse model in vivo. Our results thus suggest that RAR-γ agonists may prove effective for the treatment of subretinal fibrosis associated with AMD. RAR-γ agonist R667 suppressed collagen gel contraction mediated by RPE cells. Epithelial-mesenchymal transition (EMT) in RPE cells was inhibited by RAR-γ agonist R667. RAR-γ agonist R667 inhibited fibrosis-related processes in RPE cells. RAR-γ agonists may attenuate AMD-associated fibrosis.

Nano-graphene oxide (Nano-GO) is an extensively studied multifunctional carbon nanomaterial with attractive applications in biomedicine and biotechnology. However, few studies have been conducted to assess the epithelial-to-mesenchymal transition (EMT) in the retinal pigment epithelium (RPE). We aimed to determine whether Nano-GO induces EMT by regulating phospholipase D (PLD) signaling in human RPE (ARPE-19) cells. The physicochemical characterization of Nano-GO was performed using a Zetasizer, X-ray diffraction, Fourier-transform infrared spectroscopy, and transmission electron microscopy. RPE cell viability assays were performed, and the migratory effects of RPE cells were evaluated. RPE cell collagen gel contraction was also determined. Intracellular reactive oxygen species (ROS) levels were determined by fluorescence microscopy and flow cytometry. Immunofluorescence staining and western blot analysis were used to detect EMT-related protein expression. Phospholipase D (PLD) enzymatic activities were also measured. Nano-GO significantly enhanced the scratch-healing ability of RPE cells, indicating that the RPE cell migration ability was increased. Following Nano-GO treatment, the RPE cell penetration of the chamber was significantly promoted, suggesting that the migratory ability was strengthened. We also observed collagen gel contraction and the generation of intracellular ROS in RPE cells. The results showed that Nano-GO induced collagen gel contraction and intracellular ROS production in RPE cells. Moreover, immunofluorescence staining and western blot analysis revealed that Nano-GO significantly regulated key molecules of EMT, including epithelial-cadherin, neural-cadherin, α-smooth muscle actin, vimentin, and matrix metalloproteinases (MMP-2 and MMP-9). Interestingly, Nano-GO-induced RPE cell migration and intracellular ROS production were abrogated in PLD-knockdown RPE cells, indicating that PLD activation played a crucial role in the Nano-GO-induced RPE EMT process. We demonstrate for the first time that Nano-GO promotes RPE cell migration through PLD-mediated ROS production. We provide preliminary evidence to support the hypothesis that Nano-GO has adverse health effects related to RPE damage.

PurposeTo investigate the role of protein misfolding in retinal pigment epithelial (RPE) cell dysfunction, the effects of R345W-Fibulin-3 expression on RPE cell phenotype were studied.MethodsPrimary RPE cells were cultured to confluence on Transwells and infected with lentivirus constructs to express wild-type (WT)- or R345W-Fibulin-3. Barrier function was assessed by evaluating zonula occludens-1 (ZO-1) distribution and trans-epithelial electrical resistance (TER). Polarized secretion of vascular endothelial growth factor (VEGF), was measured by Enzyme-linked immunosorbent assay (ELISA). Differentiation status was assessed by qPCR of genes known to be preferentially expressed in terminally differentiated RPE cells, and conversion to an epithelial–mesenchymal transition (EMT) phenotype was assessed by a migration assay.ResultsCompared to RPE cells expressing WT-Fibulin-3, ZO-1 distribution was disrupted and TER values were significantly lower in RPE cells expressing R345W-Fibulin-3. In cells expressing mutant Fibulin-3, VEGF secretion was attenuated basally but not in the apical direction, whereas Fibulin-3 secretion was reduced in both the apical and basal directions. Retinal pigment epithelial signature genes were downregulated and multiple genes associated with EMT were upregulated in the mutant group. Migration assays revealed a faster recovery rate in ARPE-19 cells overexpressing R345W-Fibulin-3 compared to WT.ConclusionsThe results suggest that expression of R345W-Fibulin-3 promotes EMT in RPE cells.