Vaccinia-related kinase 1/snail family transcriptional repressor 1 regulates epithelial-mesenchymal transition and inflammation in proliferative vitreoretinopathy.

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.

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

Age-related macular degeneration (AMD), the leading cause of blindness among the elderly, is without treatment for early disease. Degenerative retinal pigment epithelial (RPE) cell heterogeneity is a well-recognized but understudied pathogenic factor. Due to the daily phagocytosis of photoreceptor outer segments, unique photo-oxidative stress, and high metabolism for maintaining vision, the RPE has robust macroautophagy/autophagy, and mitochondrial and antioxidant networks. However, the autophagy subtype, mitophagy, in the RPE and AMD is understudied. Here, we found decreased PINK1 (PTEN induced kinase 1) in perifoveal RPE of early AMD eyes. PINK1-deficient RPE have impaired mitophagy and mitochondrial function that triggers death-resistant epithelial-mesenchymal transition (EMT). This reprogramming is mediated by novel retrograde mitochondrial-nuclear signaling (RMNS) through superoxide, NFE2L2 (NFE2 like bZIP transcription factor 2), TXNRD1 (thioredoxin reductase 1), and phosphoinositide 3-kinase (PI3K)-AKT (AKT serine/threonine kinase) that induced canonical transcription factors ZEB1 (zinc finger E-box binding homeobox 1) and SNAI1 (Snail family transcriptional repressor 1) and an EMT transcriptome. NFE2L2 deficiency disrupted RMNS that paradoxically normalized morphology but decreased function and viability. Thus, RPE heterogeneity is defined by the interaction of two cytoprotective pathways that is triggered by mitophagy function. By neutralizing the consequences of impaired mitophagy, an antioxidant dendrimer tropic for the RPE and mitochondria, EMT (a recognized AMD alteration) was abrogated to offer potential therapy for early AMD, a stage without treatment. Abbreviations: ACTB: actin beta; AKT: AKT serine/threonine kinase; AMD: age-related macular degeneration; CCCP: cyanide m-chlorophenyl hydrazone; CDH1: cadherin 1; DAVID: Database for Annotation, Visualization and Integrated Discovery; DHE: dihydroethidium; D-NAC: N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; ECAR: extracellular acidification rate; EMT: epithelial-mesenchymal transition; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSEA: Gene Set Enrichment Analysis; HSPD1: heat shock protein family D (Hsp60) member 1; IVT: intravitreal; KD: knockdown; LMNA, lamin A/C; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MMP: mitochondrial membrane potential; NAC: N-acetyl-l-cysteine; NQO1: NAD(P)H quinone dehydrogenase 1; NFE2L2: NFE2 like bZIP transcription factor 2; O2 −: superoxide anion; OCR: oxygen consumption rate; PI3K: phosphoinositide 3-kinase; PINK1: PTEN induced kinase 1; RMNS: retrograde mitochondrial-nuclear signaling; ROS: reactive oxygen species; RPE: retinal pigment epithelium; SNAI1: snail family transcriptional repressor 1; TJP1: tight junction protein 1; TPP-D-NAC: triphenyl phosphinium and N-acetyl-l-cysteine conjugated to a poly(amido amine) dendrimer; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; Trig: trigonelline; TXNRD1: thioredoxin reductase 1; VIM: vimentin; WT: wild-type; ZEB1: zinc finger E-box binding homeobox 1

Proliferative vitreoretinopathy (PVR) progression is associated with TGF-β2-induced epithelial–mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells. In cancer cells, miR-4516 downregulates orthodenticle homeobox 1 (OTX1)-mediated cell invasion. Moreover, OTX1 is shown to be involved in invasion and EMT. The purpose of this study was to assess whether microRNA (miR-4516) suppresses EMT in RPE cells. EMT features were assessed using Western blotting, immunocytochemical staining, scratch-wound healing, modified Boyden chamber assay, and collagen gel contraction assay. For in vivo testing, a rabbit model was used, which involved induction of PVR by injection of transfected spontaneously arising RPE (ARPE) cells into the vitreous chamber. The putative target of miR-4516 was identified by luciferase reporter assay. Results showed that TGF-β2-induced transdifferentiation and migration of RPE cells was inhibited by miR-4516 delivery. Overexpression of miR-4516 led to upregulation of zonula occludens-1, downregulation of α-smooth muscle actin and vimentin, and cell contractility—all EMT features—in the TGF-β2-treated ARPE-19 cells. MiR-4516 regulated OTX1 expression negatively by binding to its 3’-UTR. TGF-β2-induced phosphorylated ERK was inhibited in miR-4516-overexpressing ARPE-19 cells. MiR-4516 suppressed experimental PVR in vitro and in vivo. In conclusion, the overexpression of miR-4516 suppresses TGF-β2-induced EMT in a PVR model, and its role in PVR depends on OTX1/ERK. Further research is needed to develop a feasible treatment method to prevent and treat PVR.

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.

Oxidative stress causes retinal pigment epithelium (RPE) cell dysfunction and is a major risk factor leading to the development of dry-type age-related macular degeneration. Taking pharmacological and genetic approaches, we address the mechanisms by which sublethal oxidative stress inhibits RPE cell phagocytosis. Sublethal oxidative stress dose-dependently inhibited RPE cell phagocytosis of photoreceptor outer segments (POS) and activated AMP-activated protein kinase (AMPK) as determined by increased Thr172 and Ser79 phosphorylation of AMPKalpha and its substrate acetyl-CoA carboxylase, respectively. Similar to oxidative stress, 5-aminoimidazole-4-carboxamide riboside (AICAR), a pharmacological activator of AMPK, inhibited RPE cell phagocytosis of POS in a dose-dependent manner. Inhibition of RPE cell phagocytosis by AICAR was fully reversed by blockade of AICAR translocation into cells by dipyridamole or inhibition of AICAR conversion to ZMP by adenosine kinase inhibitor 5-iodotubercidin. In agreement, AICAR-induced activation of AMPK was abolished by preincubation with dipyridamole or 5-iodotubercidin. Knock-out experiments further revealed that alpha2 but not alpha1 AMPK was involved in RPE cell phagocytosis and that activation of alpha2 AMPK contributed to the inhibition of RPE cell phagocytosis by oxidative stress. Inhibition of RPE cell phagocytosis by activation of alpha2 AMPK was associated with a dramatic increase in acetyl-CoA carboxylase phosphorylation. In comparison, AMPK had no role in oxidative stress-induced breakdown of RPE barrier function. Taken together, reduction in POS load under oxidative stress might direct RPE cells to a self-protected status. Thus, activating AMPK could have therapeutic potential in treating dry macular degeneration.

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.

Proliferative vitreoretinopathy (PVR) is the major cause of treatment failure in individuals who undergo surgery for retinal detachment. The epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells contributes to the pathogenesis of PVR. Oxidative stress is thought to play a role in the progression of retinal diseases including PVR. We have now examined the effects of oxidative stress on the EMT and related processes in the human RPE cell line. We found that H2 O2 induced the contraction of RPE cells in a three-dimensional collagen gel. Analysis of a cytokine array revealed that H2 O2 specifically increased the release of macrophage migration inhibitory factor (MIF) from RPE cells. Reverse transcription-polymerase chain reaction and immunoblot analyses showed that H2 O2 increased the expression of MIF in RPE cells. Immunoblot and immunofluorescence analyses revealed that H2 O2 upregulated the expression of α-SMA and vimentin and downregulated that of ZO-1 and N-cadherin. Consistent with these observations, the transepithelial electrical resistance of cell was reduced by exposure to H2 O2 . The effects of oxidative stress on EMT-related and junctional protein expression as well as on transepithelial electrical resistance were inhibited by antibodies to MIF, but they were not mimicked by treatment with recombinant MIF. Finally, analysis with a profiling array for mitogen-activated protein kinase signalling revealed that H2 O2 specifically induced the phosphorylation of p38 mitogen-activated protein kinase. Our results thus suggest that MIF may play a role in induction of the EMT and related processes by oxidative stress in RPE cells and that it might thereby contribute to the pathogenesis of PVR. Proliferative vitreoretinopathy is a major complication of rhegmatogenous retinal detachment, and both oxidative stress and induction of the EMT in RPE cells are thought to contribute to the pathogenesis of this condition. We have now examined the effects of oxidative stress on the EMT and related processes in the human RPE cell line ARPE19. Our results thus implicate MIF in induction of the EMT and related processes by oxidative stress in RPE cells and the regulated expression of EMT markers. They further suggest that MIF may play an important role in the pathogenesis of PVR.

Proliferative vitreoretinopathy (PVR) is a complex ocular disease that leads to detached retinas and irreversible vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells plays a critical role in PVR occurrence. However, the core targets driven by the EMT process that lead to the pathogenesis of PVR remain unclear. In our study, the relationship between embryonic stem cell-expressed Ras (ERas) and EMT in RPE cells was investigated. The subretinal and epiretinal membrane specimens of human PVR were examined for ERas and hallmarks of autophagy and EMT using Western blotting and immunofluorescence. EMT was induced by transforming growth factor (TGF)-β1 or epidermal growth factor (EGF) in ARPE-19 cells. Autophagy was inhibited by U0126 or bafilomycin A1 in ARPE-19 cells. ERas was decreased and the classical autophagy biomarker microtubule associated protein 1 light chain 3 alpha (LC3) was upregulated in the subretinal and epiretinal membranes of PVR patients in vivo. Moreover, ERas was downregulated and autophagy was activated in RPE ARPE-19 cells in response to transforming growth factor (TGF)-β1 and epidermal growth factor (EGF) induction. Finally, overexpression of ERas in RPE cells inhibited autophagy via impaired formation of autophagosomes and lysosomes. Our study revealed the role of ERas in the pathogenesis of PVR through EMT and provided a novel therapeutic target for PVR prevention and treatment.

This study aimed to explore the impact of HSPA13 on epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells and proliferative vitreoretinopathy (PVR) development, along with its associated molecular mechanisms. HSPA13 expression was evaluated in epiretinal membranes (ERMs) from patients with PVR using immunohistochemistry. The effects of HSPA13 knockdown on TGFβ1-induced EMT in hESC-RPE cells were studied through quantitative PCR (qPCR), Western blot, and wound healing assays. Intracellular Ca2+ levels were measured using Fluo-8/AM incubation. A rat PVR model was induced by the intravitreal injection of RPE cells combined with platelet-rich plasma (PRP). RNA-seq was applied to study the molecular mechanism of HSPA13 knockdown-mediated EMT inhibition. HSPA13 was found in human ERMs and its expression increased with TGFβ1 treatment in hESC-RPE cells. Knockdown of HSPA13 inhibited TGFβ1-induced EMT and migration. In the PVR rat model, HSPA13 was expressed in the ERMs and its knockdown in RPE cells reduced the development of PVR. Consistent with these observations, RNA-seq showed a global suppression of TGFβ1-induced EMT and migration by shHSPA13 in RPE cells. Mechanistically, TGFβ1 treatment increased intracellular Ca2+ levels, leading to an upregulation of HSPA13 expression. Downregulation of HSPA13 hindered the phosphorylation of PI3K/Akt in TGFβ1-induced RPE cells. Our study revealed the involvement of HSPA13 in PVR development, as well as in TGFβ1-induced EMT of RPE through the PI3K/Akt signaling pathway. Targeting HSPA13-related pathways involved in regulating EMT in RPE cells could serve as a novel therapeutic approach for patients with PVR.

Epithelial-mesenchymal transition (EMT) is a crucial pathological process that contributes to proliferative vitreoretinopathy (PVR), and research indicates that factors present in the vitreous that target cells play pivotal roles in regulating EMT. Experimental studies have confirmed that rabbit vitreous (RV) promotes EMT in human retinal pigment epithelial (RPE) cells. The long noncoding RNA (lncRNA) MALAT1 has been implicated in EMT in various diseases. Thus, this study aimed to investigate the involvement of lncRNA MALAT1 in vitreous-induced EMT in RPE cells. MALAT1 was knocked down in ARPE-19 cells by short hairpin RNA (shRNA) transfection. Reverse transcription PCR (RT‒PCR) was used to evaluate MALAT1 expression, and Western blotting analysis was used to measure the expression of EMT-related proteins. Wound-healing, Transwell, and cell contraction assays were conducted to assess cell migration, invasion, and contraction, respectively. Additionally, cell proliferation was assessed using the CCK-8 assay, and cytoskeletal changes were examined by immunofluorescence. MALAT1 expression was significantly increased in ARPE-19 cells cultured with RV. Silencing MALAT1 effectively suppressed EMT and downregulated the associated factors snail1 and E-cadherin. Furthermore, silencing MALAT1 inhibited the RV-induced migration, invasion, proliferation, and contraction of ARPE-19 cells. Silencing MALAT1 also decreased RV-induced AKT and P53 phosphorylation. In conclusion, lncRNA MALAT1 participates in regulating vitreous-induced EMT in human RPE cells; these results provide new insight into the pathogenesis of PVR and offer a potential direction for the development of antiproliferative drugs.

Particulate matter 2.5 (PM2.5) exposure can promote epithelial-mesenchymal transition (EMT) in human retinal pigment epithelial (RPE) cells. The flowers of Tagetes erecta Linn, commonly known as marigold, are rich in diverse flavonoids and carotenoids and play a significant role in preventing cellular damage induced by oxidative stress, but the role of their extracts in RPE cells has not been reported. This study aimed to evaluate the influence of an ethanol extract of T. erecta Linn flower (TE) on PM2.5-induced EMT processes in RPE ARPE-19 cells. To investigate the protective effect of TE against ARPE-19 cell damage following PM2.5 treatment, cells were exposed to TE for 1 h before exposure to PM2.5 for 24 h. We investigated whether the efficacy of TE on suppressing PM2.5-induced EMT was related to antioxidant activity and the effect on the expression changes of factors involved in EMT regulation. Additionally, we further explored the role of intracellular signaling pathways associated with EMT inhibition. TE significantly blocked PM2.5-induced cytotoxicity while effectively preventing mitochondrial dysfunction, increased reactive oxygen species (ROS) generation, and mitochondrial membrane potential disruption. TE inhibited PM2.5-induced EMT and inflammatory response by suppressing the ROS-mediated transforming growth factor-β/suppressor of mothers against decapentaplegic/mitogen-activated protein kinases signaling pathway. Our results suggest that marigold extract is a highly effective in protection against PM2.5-induced eye damage.

To explore the effect of glucosamine (GlcN) on transforming growth factor (TGF)-β signalling and several processes involved in proliferative vitreoretinopathy (PVR). We evaluated the surface levels of TGF-β receptor and its binding of TGF-β in ARPE-19 cells. Release of cytokines and collagen, and expression of signalling intermediates were quantified. Migration was qualitatively and quantitatively examined. The morphology of cells undergoing PVR in vitro and in a mouse PVR model was observed. Glucosamine reduced the surface levels of TGF-β receptor and the ability of ARPE-19 cells to bind TGF-β. In ARPE-19 cells, TGF-β1 plus epidermal growth factor (EGF) or TGF-β2 increased the expression of alpha-smooth muscle actin (α-SMA) and decreased the expression of zona occludens protein (ZO-1). Transforming growth factor-(β2) also caused the release of platelet-derived growth factor (PDGF), connective tissue growth factor (CTGF) and type 1 collagen and increased the phosphorylation of SMAD2 and SMAD3. Platelet-derived growth factor and CTGF stimulated cell migration, and TGF-β2 stimulated wound closure, contraction of collagen and changes in cell morphology. Treatment with GlcN counteracted all of these effects, and its administration in the mouse model reduced the morphologic appearance of PVR. Glucosamine could inhibit the TGF-β signalling pathway in retinal pigment epithelium cells and several of the downstream events associated with epithelial-mesenchymal transition and PVR.

BackgroundThe proliferation of retinal pigment epithelium (RPE) cells resulting from an epithelial-mesenchymal transition (EMT) plays a key role in proliferative vitreoretinopathy (PVR), which leads to complex retinal detachment and the loss of vision. Genes of Snail family encode the zinc finger transcription factors that have been reported to be essential in EMT during embryonic development and cancer metastasis. However, the function of Snail in RPE cells undergoing EMT is largely unknown.Principal FindingsTransforming growth factor beta(TGF-β)-1 resulted in EMT in human RPE cells (ARPE-19), which was characterized by the expected decrease in E-cadherin and Zona occludin-1(ZO-1) expression, and the increase in fibronectin and α-smooth muscle actin (α-SMA) expression, as well as the associated increase of Snail expression at both mRNA and protein levels. Furthermore, TGF-β1 treatment caused a significant change in ARPE-19 cells morphology, with transition from a typical epithelial morphology to mesenchymal spindle-shaped. More interestingly, Snail silencing significantly attenuated TGF-β1-induced EMT in ARPE-19 cells by decreasing the mesenchymal markers fibronectin and a-SMA and increasing the epithelial marker E-cadherin and ZO-1. Snail knockdown could effectively suppress ARPE-19 cell migration. Finally, Snail was activated in epiretinal membranes from PVR patients. Taken together, Snail plays very important roles in TGF-β-1-induced EMT in human RPE cells and may contribute to the development of PVR.SignificanceSnail transcription factor plays a critical role in TGF-β1-induced EMT in human RPE cells, which provides deep insight into the pathogenesis of human PVR disease. The specific inhibition of Snail may provide a new approach to treat and prevent PVR.