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AIM: To investigate the proliferation regulatory effect of cone-rod homeobox (CRX) in retinal pigment epithelium (RPE) and retinoblastoma (RB) cells to explore the potential application and side effect (oncogenic potential) of CRX-based gene therapy in RPE-based retinopathies. METHODS: Adult human retinal pigment epithelial (ARPE)-19 and human retinal pigment epithelial (RPE)-1 cells and Y79 RB cell were used in the study. Genetic manipulation was performed by lentivirus-based technology. The cell proliferation was determined by a CellTiter-Glo Reagent. The mRNA and protein levels were determined by quantitative real-time polymerase chain reaction (qPCR) and Western blot assay. The transcriptional activity of the promoter was determined by luciferase reporter gene assay. The bindings between CRX and transcription factor 7 (TCF7) promoter as well as TCF7 and the promoters of TCF7 target genes were examined by chromatin immunoprecipitation (ChIP) assay. The transcription of the TCF7 was determined by a modified nuclear run-on assay. RESULTS: CRX overexpression and knockdown significantly increased (n=3, P

Age-related ocular diseases such as age-related macular degeneration, glaucoma, and diabetic retinopathy are major causes of irreversible vision impairment in the elderly. Conventional treatments focus on symptom relief and disease slowdown, often involving surgery, but fall short of providing a cure, leading to substantial vision loss. Regenerative medicine, particularly mesenchymal stem cells (MSCs), holds promise for ocular disease treatment. This study investigates the synergistic potential of combining placenta-derived MSCs (PD-MSCs) with Achyranthis radix extract (ARE) from Achyranthes japonica to enhance therapeutic outcomes. In a 24-h treatment, ARE significantly increased the proliferative capacity of PD-MSCs and delayed their senescence (* p < 0.05). ARE also enhanced antioxidant capabilities and increased the expression of regeneration-associated genes in an in vitro injured model using chemical damages on human retinal pigment epithelial cell line (ARPE-19) (* p < 0.05). These results suggest that ARE-primed PD-MSC have the capability to enhance the activation of genes associated with regeneration in the injured eye via increasing antioxidant properties. Taken together, these findings support the conclusion that ARE-primed PD-MSC may serve as an enhanced source for stem cell-based therapy in ocular diseases. [ABSTRACT FROM AUTHOR]

In this immunohistological study on the peripheral retina of 3-year-old beagle dogs, excised retina specimens were immunostained with antibodies against nestin, Oct4, Nanog, Sox2, CDX2, cytokeratin 18 (CK 18), RPE65, and YAP1, as well as hematoxylin and DAPI, two nuclear stains. Our findings revealed solitary cysts of various sizes in the inner retina. Intriguingly, a mass of small round cells with scant cytoplasms was observed in the cavity of small cysts, while many disorganized cells partially occupied the cavity of the large cysts. The small cysts were strongly positive for nestin, Oct4, Nanog, Sox2, CDX2, CK18, and YAP1. RPE65-positive cells were exclusively observed in the tissue surrounding the cysts. Since RPE65 is a specific marker of retinal pigment epithelial (RPE) cells, the surrounding cells of the peripheral cysts were presumably derived from RPE cells that migrated intraretinally. In the small cysts, intense positive staining for nestin, a marker of retinal stem cells, seemed to indicate that they were derived from retinal stem cells. The morphology and positive staining for markers of blastocyst and RPE cells indicated that the small cysts may have formed structures resembling the blastocyst, possibly caused by the interaction between retinal stem cells and migrated RPE cells. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy (DR) is a secondary complication of diabetes that can lead to visual impairment and blindness. The retinal pigment epithelium (RPE) is a monolayer of pigment cells that forms the blood–retinal barrier (BRB) via tight junction (TJ) proteins and plays a crucial role in the physiological function of the retina. Hyperglycemia induces RPE death and BRB breakdown, which accelerates the process of DR. Curcumin, an active extract of Curcuma longa, has anti-inflammatory, antioxidant, antiapoptotic, and neuroprotective properties. However, the effect of Curcumin on the BRB under high glucose conditions remains unknown. This study aimed to investigate the protective effects of Curcumin on RPE physiology in vitro and in vivo. Curcumin significantly alleviated cell viability inhibition under high glucose conditions. Moreover, high glucose reduced extracellular signal-regulated kinase and Akt pathways activation to diminish RPE cell growth but reversed by Curcumin treatment. Curcumin protected not only TJ integrity but also retinoid regeneration through TJ proteins and isomerase modulation in diabetic retina. Furthermore, Curcumin decreased the expression of angiogenic factor to inhibit retinal neovascularization. Finally, Curcumin treatment markedly reduced apoptosis during hyperglycemia. In conclusion, Curcumin can alleviate the progression of DR by promoting RPE survival, TJ integrity, retinoid isomerase activity, RPE senescence inhibition, and neovascularization. Therefore, Curcumin exhibits high potential for use as a therapeutic agent for early DR.

Background: Retinal pigment epithelial cells (RPECs) are a type of retinal cells that structurally and physiologically support photoreceptors. However, hyperglycemia has been shown to play a critical role in the progression of diabetic retinopathy (DR), which is one of the leading causes of vision impairment. In the diabetic eye, the high glucose environment damages RPECs via the induction of oxidative stress, leading to the release of excess reactive oxygen species (ROS) and triggering apoptosis. In this study, we aim to investigate the antioxidant mechanism of Vitamin C in reducing hyperglycemia-induced stress and whether this mechanism can preserve the function of RPECs. Methods and results: ARPE-19 cells were treated with high glucose in the presence or absence of Vitamin C. Cell viability was measured by MTT assay. Cleaved poly ADP-ribose polymerase (PARP) was used to identify apoptosis in the cells. ROS were detected by the DCFH-DA reaction. The accumulation of sorbitol in the aldose reductase (AR) polyol pathway was determined using the sorbitol detection assay. Primary mouse RPECs were isolated from adult mice and identified by Rpe65 expression. The mitochondrial damage was measured by mitochondrial membrane depolarization. Our results showed that high glucose conditions reduce cell viability in RPECs while Vitamin C can restore cell viability, compared to the vehicle treatment. We also demonstrated that Vitamin C reduces hyperglycemia-induced ROS production and prevents cell apoptosis in RPECs in an AR-independent pathway. Conclusions: These results suggest that Vitamin C is not only a nutritional necessity but also an adjuvant that can be combined with AR inhibitors for alleviating hyperglycemic stress in RPECs. [ABSTRACT FROM AUTHOR]

Diabetic retinopathy (DR) is a secondary complication of diabetes that can lead to visual impairment and blindness. The retinal pigment epithelium (RPE) is a monolayer of pigment cells that forms the blood–retinal barrier (BRB) via tight junction (TJ) proteins and plays a crucial role in the physiological function of the retina. Hyperglycemia induces RPE death and BRB breakdown, which accelerates the process of DR. Curcumin, an active extract of Curcuma longa , has anti-inflammatory, antioxidant, antiapoptotic, and neuroprotective properties. However, the effect of Curcumin on the BRB under high glucose conditions remains unknown. This study aimed to investigate the protective effects of Curcumin on RPE physiology in vitro and in vivo. Curcumin significantly alleviated cell viability inhibition under high glucose conditions. Moreover, high glucose reduced extracellular signal-regulated kinase and Akt pathways activation to diminish RPE cell growth but reversed by Curcumin treatment. Curcumin protected not only TJ integrity but also retinoid regeneration through TJ proteins and isomerase modulation in diabetic retina. Furthermore, Curcumin decreased the expression of angiogenic factor to inhibit retinal neovascularization. Finally, Curcumin treatment markedly reduced apoptosis during hyperglycemia. In conclusion, Curcumin can alleviate the progression of DR by promoting RPE survival, TJ integrity, retinoid isomerase activity, RPE senescence inhibition, and neovascularization. Therefore, Curcumin exhibits high potential for use as a therapeutic agent for early DR. [ABSTRACT FROM AUTHOR]

Retinal ischemia followed by reperfusion (IR) is a common cause of many ocular disorders, such as age‐related macular degeneration (AMD), which leads to blindness in the elderly population, and proper therapies remain unavailable. Retinal pigment epithelial (RPE) cell death is a hallmark of AMD. Hyperbaric oxygen (HBO) therapy can improve IR tissue survival by inducing ischemic preconditioning responses. We conducted an in vitro study to examine the effects of HBO preconditioning on oxygen–glucose deprivation (OGD)‐induced IR‐injured RPE cells. RPE cells were treated with HBO (100% O2 at 3 atmospheres absolute for 90 min) once a day for three consecutive days before retinal IR onset. Compared with normal cells, the IR‐injured RPE cells had lower cell viability, lower peroxisome proliferator activator receptor‐alpha (PPAR‐α) expression, more severe oxidation status, higher blood‐retinal barrier disruption and more elevated apoptosis and autophagy rates. HBO preconditioning increased PPAR‐α expression, improved cell viability, decreased oxidative stress, blood‐retinal barrier disruption and cellular apoptosis and autophagy. A specific PPAR‐α antagonist, GW6471, antagonized all the protective effects of HBO preconditioning in IR‐injured RPE cells. Combining these observations, HBO therapy can reverse OGD‐induced RPE cell injury by activating PPAR‐α signalling. [ABSTRACT FROM AUTHOR]

During recent years, evidence has emerged that immune privileged sites such as the CNS and the retina may be more integrated in the systemic response to infection than was previously believed. In line with this, it was recently shown that a systemic acute virus infection leads to infiltration of CD8 T cells in the brains of immunocompetent mice. In this study, we extend these findings to the neurological tissue of the eye, namely the retina. We show that an acute systemic virus infection in mice leads to a transient CD8 T cell infiltration in the retina that is not directed by virus infection inside the retina. CD8 T cells were found throughout the retinal tissue, and had a high expression of CXCR6 and CXCR3, as also reported for tissue residing CD8 T cells in the lung and liver. We also show that the pigment epithelium lining the retina expresses CXCL16 (the ligand for CXCR6) similar to epithelial cells of the lung. Thus, our results suggest that the retina undergoes immune surveillance during a systemic infection, and that this surveillance appears to be directed by mechanisms similar to those described for non-privileged tissues. [ABSTRACT FROM AUTHOR]

In this immunohistological study on the peripheral retina of 3-year-old beagle dogs, excised retina specimens were immunostained with antibodies against nestin, Oct4, Nanog, Sox2, CDX2, cytokeratin 18 (CK 18), RPE65, and YAP1, as well as hematoxylin and DAPI, two nuclear stains. Our findings revealed solitary cysts of various sizes in the inner retina. Intriguingly, a mass of small round cells with scant cytoplasms was observed in the cavity of small cysts, while many disorganized cells partially occupied the cavity of the large cysts. The small cysts were strongly positive for nestin, Oct4, Nanog, Sox2, CDX2, CK18, and YAP1. RPE65-positive cells were exclusively observed in the tissue surrounding the cysts. Since RPE65 is a specific marker of retinal pigment epithelial (RPE) cells, the surrounding cells of the peripheral cysts were presumably derived from RPE cells that migrated intraretinally. In the small cysts, intense positive staining for nestin, a marker of retinal stem cells, seemed to indicate that they were derived from retinal stem cells. The morphology and positive staining for markers of blastocyst and RPE cells indicated that the small cysts may have formed structures resembling the blastocyst, possibly caused by the interaction between retinal stem cells and migrated RPE cells.

Age-related macular degeneration (AMD) causes vision loss in the elderly population. Dry AMD leads to the formation of Drusen, while wet AMD is characterized by cell proliferation and choroidal angiogenesis. The retinal pigment epithelium (RPE) plays a key role in AMD pathogenesis. In particular, helioreceptor renewal depends on outer segment phagocytosis of RPE cells, while RPE autophagy can protect cells from oxidative stress damage. However, when the oxidative stress burden is too high and homeostasis is disturbed, the phagocytosis and autophagy functions of RPE become damaged, leading to AMD development and progression. Hence, characterizing the roles of RPE cell phagocytosis and autophagy in the pathogenesis of AMD can inform the development of potential therapeutic targets to prevent irreversible RPE and photoreceptor cell death, thus protecting against AMD. [ABSTRACT FROM AUTHOR]

During recent years, evidence has emerged that immune privileged sites such as the CNS and the retina may be more integrated in the systemic response to infection than was previously believed. In line with this, it was recently shown that a systemic acute virus infection leads to infiltration of CD8 T cells in the brains of immunocompetent mice. In this study, we extend these findings to the neurological tissue of the eye, namely the retina. We show that an acute systemic virus infection in mice leads to a transient CD8 T cell infiltration in the retina that is not directed by virus infection inside the retina. CD8 T cells were found throughout the retinal tissue, and had a high expression of CXCR6 and CXCR3, as also reported for tissue residing CD8 T cells in the lung and liver. We also show that the pigment epithelium lining the retina expresses CXCL16 (the ligand for CXCR6) similar to epithelial cells of the lung. Thus, our results suggest that the retina undergoes immune surveillance during a systemic infection, and that this surveillance appears to be directed by mechanisms similar to those described for non-privileged tissues.

Abstract Background We previously reported higher plasma levels of complement fragments C3a and C5a in neovascular Age-related Macular Degeneration (nAMD) patients with macular fibrosis. This study aimed to understand whether complement activation contributes to the development of macular fibrosis and the underlying mechanisms involved. Methods Complement activation was blocked using a C5 neutralizing antibody (BB5.1) in C57BL/6J mice after induction of subretinal fibrosis using the two-stage laser protocol. Fibrotic lesions were examined 10 days after the 2nd laser through fundus examination and immunohistochemistry. The expression of C5aR in fibrotic lesions and retinal pigment epithelial (RPE) cultures were examined by confocal microscopy. Primary murine RPE cells were treated with C3a or C5a (10–100 ng/mL) or TGF-β2 (10 ng/mL). Epithelial-to-mesenchymal transition (EMT) was assessed through various readouts. The expression of E-cadherin, vimentin, fibronectin, α-SMA, Slug, ERK/AKT and pSMAD2/3 were determined by Western blot and immunocytochemistry. Collagen contraction and wound-healing assays were used as functional readouts of EMT. The production of IL-6, TGF-β1, TGF-β2 and VEGF by RPE cells were determined by ELISA. PMX53 was used to block C5aR in RPE cultures and in vivo in mice with subretinal fibrosis. Results Extensive C5b-9 deposition was detected at the site of subretinal fibrosis. BB5.1 treatment completely abrogated complement activation and significantly reduced subretinal fibrosis. C5aR was detected in RPE and infiltrating MHC-II+ cells in subretinal fibrosis. In vitro, RPE cells constitutively express C5/C5a and C5aR, and their expression was increased by TGF-β2 treatment. C5a but not C3a increased fibronectin, α-SMA, vimentin and Slug expression, and decreased E-cadherin expression in RPE cells. C5a treatment also increased the contractility and migration of RPE cells and enhanced the production of VEGF and TGF-β1/2. C5a treatment induced pSmad2/3 and pERK1/2 expression in RPE cells and this was blocked by PMX53. PMX53 treatment significantly reduced sodium fluorescein leakage in the subretinal fibrosis model, while collagen-I+ lesions only mildly reduced. Conclusions Complement activation is critically involved in the development of subretinal fibrosis, partially through C5a–C5aR-mediated EMT in RPE cells. Targeting complement activation rather than C5a may be a novel approach for the management of macular fibrosis.

Esculetin is a coumarin-derived compound with antioxidant and anti-inflammatory properties. The current study aims to evaluate the therapeutic implications of esculetin on retinal dysfunction and uncover the underlying mechanisms. Tert-butyl hydroperoxide (t-BHP) at a concentration of 300 μM was used to induce oxidative stress in human retinal pigment epithelial cell line (ARPE-19) cells. Esculetin at concentrations below 250 μM did not cause cytotoxicity to ARPE-19 cells. Cell viability analysis confirmed that t-BHP induced oxidative injury of ARPE-19 cells. However, ARPE-19 cells were protected from t-BHP-induced oxidative injury by esculetin in a concentration-dependent manner. As a result of the TUNEL assay to confirm apoptosis, esculetin treatment reduced the number of TUNEL-positive cells. Esculetin down-regulated the expression levels of Bax, Caspase-3, and PARP and up-regulated the expression level of Bcl2. Collectively, this study demonstrates that esculetin exerts potent antioxidant properties in ARPE-19 cells, inhibiting t-BHP-induced apoptosis under the regulation of apoptotic factors. [ABSTRACT FROM AUTHOR]

AIM: To investigate the synthesis method of curcumin nanoparticles grafted with deoxycholic acid and the effect of curcumin nanoparticles on human retinal pigment epithelial(hRPE)cells. METHODS: The synthesis and performance analysis of Cur/Chit-DC. The relationship between FITC/Chit-DC and hRPE cells was observed under an inverted fluorescence microscope after treating hRPE cells with FITC(FITC/Chit-DC)and Cur/Chit-DC(FITC/Cur/Chit-DC)for 24h, keeping them in dark for 1, 3 and 5d respectively.RESULTS: By mixing Cur and Chit-DC, the nanoparticles containing chitosan derivatives were light yellow. The drug release from the nanoparticles reached equilibrium after 96h, and the cumulative drug release amount was 31.6%. After FITC/Chit-DC was treated with hRPE cells for 1d, most of Chit-DC nanoparticles were still located near the cell membrane. After 3d, the nanoparticles gradually converged towards the nucleus and most of them were located around the nucleus. After 5d, it was observed that Chit-DC nanoparticles had entered the nucleus and were partially degraded under the action of intracellular lysosomes. The relationship between Cur/Chit-DC and cellular action is roughly the same as the relationship between Chit-DC and cellular action. CONCLUSION: Cur can be continuously released from Cur/Chit-DC nanoparticle, which has long-lasting sustained-release function.

Inflammation-induced choroidal neovascularization followed by the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPEs) is a cause of neovascular age-related macular degeneration (nAMD). RPE-derived myofibroblasts overproduce extracellular matrix, leading to subretinal fibrosis. We already have demonstrated that benzylphenylurea (BPU) derivatives inhibit the function of cancer-associated fibroblasts. Here, we investigated the anti-myofibroblast effects of BPU derivatives and examined such BPU activity on subretinal fibrosis. A BPU derivative, BPU17, exhibits the most potent anti-myofibroblast activity among dozens of BPU derivatives and inhibits subretinal fibrosis in a mouse model of retinal degeneration. Investigations with primary cultured RPEs reveal that BPU17 suppresses cell motility and collagen synthesis in RPE-derived myofibroblasts. These effects depend on repressing the serum response factor (SRF)/CArG-box-dependent transcription. BPU17 inhibits the expression of SRF cofactor, cysteine and glycine-rich protein 2 (CRP2), which activates the SRF function. Proteomics analysis reveals that BPU17 binds to prohibitin 1 (PHB1) and inhibits the PHB1-PHB2 interaction, resulting in mild defects in mitochondrial function. This impairment causes a decrease in the expression of CRP2 and suppresses collagen synthesis. Our findings suggest that BPU17 is a promising agent against nAMD and the close relationship between PHB function and EMT. [Display omitted] • BPU17 inhibits EMT of RPEs and exhibits antifibrotic activity in vivo. • This effect is due to the inhibition of SRF/CArG-box-dependent transcription. • BPU17 causes mild mitochondrial defects by inhibiting the PHB1-PHB2 interaction. • PHB1 is closely associated with RPE-derived myofibroblast function. [ABSTRACT FROM AUTHOR]

AIM: To investigate the relationship between autophagy and apoptosis in photoinduced injuries in retinal pigment epithelium (RPE) cells and how Lycium barbarum polysaccharide (LBP) contributes to the increased of RPE cells to photoinduced autophagy. METHODS: In vitro cultures of human RPE strains (ARPE-19) were prepared and randomly divided into the blank control, model, low-dose LBP, middle-dose LBP, high-dose LBP, and 3-methyladenine (3MA) groups. The viability of the RPE cells and apoptosis levels in each group were tested through cell counting kit-8 (CCK8) method with a flow cytometer (Annexin V/PI double staining technique). The expression levels of LC3II, LC3I, and P62 proteins were detected with the immunofluorescence method. The expression levels of beclin1, LC3, P62, PI3K, P-mTOR, mTOR, P-Akt, and Akt proteins were tested through Western blot. RESULTS: LBP considerably strengthens cell viability and inhibits the apoptosis of RPE cells after photoinduction. The PI3K/Akt/mTOR signal pathway is activated because of the upregulation of the phosphorylation levels of Akt and mTOR proteins, and thus autophagy is inhibited. CONCLUSION: LBP can inhibit the excessive autophagy in RPE cells by activating the PI3K/Akt/mTOR signaling pathways and thereby protect RPE cells from photoinduced injuries.

Exposure to artificial blue light, one of the most energetic forms of visible light, can increase oxidative stress in retinal cells, potentially enhancing the risk of macular degeneration. Retinal pigment epithelial (RPE) cells play a crucial role in this process; the loss of RPE cells is the primary pathway through which retinal degeneration occurs. In RPE cells, Kelch-like ECH-associated protein 1 (KEAP1) is located in both the nucleus and cytosol, where it binds to nuclear factor erythroid 2-related factor 2 (NRF2) and p62 (sequestosome-1), respectively. Blue light exposure activates the NRF2-heme oxygenase 1 (HMOX1) axis through both canonical and noncanonical p62 pathways thereby reducing oxidative damage, and initiates autophagy, which helps remove damaged proteins. These protective responses may support the survival of RPE cells. However, extended exposure to blue light drastically decreases the viability of RPE cells. This exposure diminishes the ability of KEAP1 to bind to p62 and reduces the level of KEAP1. Inhibition of autophagy does not prevent KEAP1 degradation, the NRF2-HMOX1 axis, or blue-light-induced cytotoxicity. However, proteasome inhibitor along with a transient increase in the amount of KEAP1 in RPE cells, partially restores the p62-KEAP1 complex and reduces blue-light-induced cytotoxicity. In vivo studies confirmed the downregulation of KEAP1 in damaged RPE cells. Mice subjected to periodic blue light exposure exhibited significant atrophy in the outer retina, particularly in the peripheral areas. Additionally, there was a significant decrease in c-wave electroretinography and pupillary light reflex, indicating functional impairments in both visual and nonvisual physiological processes. These data underscore the essential role of KEAP1 in managing oxidative defense and autophagy pathways triggered by blue light exposure in RPE cells., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aryl hydrocarbon receptors (AHRs), a class of ligand-dependent nuclear receptors that regulate cellular responses by inducing the expression of various target genes in response to external signals, are implicated in maintaining retinal tissue homeostasis. Previous studies have shown that the regulation of AHR-induced gene expression requires transcriptional co-regulators. However, it is not yet clear how chromatin remodelers, histone methyltransferases and coactivators interact during AHR-mediated gene expression in human retinal cells. In this study, we reveal that the histone methyltransferase MLL1 and the coactivator FLII are involved in AHR-mediated gene expression in retinal pigment epithelial cells. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) significantly increased the expression of CYP1A1, CYP1B1 and AHRR in ARPE-19 cells, whereas FLII or MLL1 depletion significantly reduced the expression of these genes induced by TCDD. Mechanistically, FLII binds to AHR in a ligand-dependent manner in ARPE-19 cells. In particular, the binding of FLII to MLL1 occurs through the GelB domain of FLII. In addition, MLL1 binds to AHR in a ligand-independent manner. FLII is involved in the recruitment of the BRG1 chromatin remodeler and MLL1 histone methyltransferase to the AHR-regulated CYP1A1 gene region in ARPE-19 cells and consequently, plays an important role in RNA polymerase II binding and transcriptional activity by modulating chromatin accessibility. Our results identify the functions and mechanisms of action of FLII and MLL1 in AHR-induced gene expression in human retinal pigment epithelial cells.

Background: We previously reported higher plasma levels of complement fragments C3a and C5a in neovascular Age-related Macular Degeneration (nAMD) patients with macular fibrosis. This study aimed to understand whether complement activation contributes to the development of macular fibrosis and the underlying mechanisms involved. Methods: Complement activation was blocked using a C5 neutralizing antibody (BB5.1) in C57BL/6J mice after induction of subretinal fibrosis using the two-stage laser protocol. Fibrotic lesions were examined 10 days after the 2nd laser through fundus examination and immunohistochemistry. The expression of C5aR in fibrotic lesions and retinal pigment epithelial (RPE) cultures were examined by confocal microscopy. Primary murine RPE cells were treated with C3a or C5a (10–100 ng/mL) or TGF-β2 (10 ng/mL). Epithelial-to-mesenchymal transition (EMT) was assessed through various readouts. The expression of E-cadherin, vimentin, fibronectin, α-SMA, Slug, ERK/AKT and pSMAD2/3 were determined by Western blot and immunocytochemistry. Collagen contraction and wound-healing assays were used as functional readouts of EMT. The production of IL-6, TGF-β1, TGF-β2 and VEGF by RPE cells were determined by ELISA. PMX53 was used to block C5aR in RPE cultures and in vivo in mice with subretinal fibrosis. Results: Extensive C5b-9 deposition was detected at the site of subretinal fibrosis. BB5.1 treatment completely abrogated complement activation and significantly reduced subretinal fibrosis. C5aR was detected in RPE and infiltrating MHC-II+ cells in subretinal fibrosis. In vitro, RPE cells constitutively express C5/C5a and C5aR, and their expression was increased by TGF-β2 treatment. C5a but not C3a increased fibronectin, α-SMA, vimentin and Slug expression, and decreased E-cadherin expression in RPE cells. C5a treatment also increased the contractility and migration of RPE cells and enhanced the production of VEGF and TGF-β1/2. C5a treatment induced pSmad2/3 and pERK1/2 expression in RPE cells and this was blocked by PMX53. PMX53 treatment significantly reduced sodium fluorescein leakage in the subretinal fibrosis model, while collagen-I+ lesions only mildly reduced. Conclusions: Complement activation is critically involved in the development of subretinal fibrosis, partially through C5a–C5aR-mediated EMT in RPE cells. Targeting complement activation rather than C5a may be a novel approach for the management of macular fibrosis. [ABSTRACT FROM AUTHOR]

AIM: To observe the role and mechanism of autophagy in retinal pigment epithelial cell (RPE) damaged by high glucose, so as to offer a new idea for the treatment of diabetic retinopathy (DR). METHODS: ARPE-19, a human RPE cell line cultured in vitro was divided into the normal control (NC), autophagy inhibitor 3-methyladenine (3-MA), high-glucose (HG), and HG+3-MA groups. Cell viability was detected by CCK-8 assay and the apoptosis rate was measured by flow cytometry. The protein expressions of apoptosis markers, including Bax, Bcl-2, and Caspase-3, as well as autophagy marker including microtubule-related protein 1 light chain 3 (LC3), p62, and mechanistic target of rapamycin (mTOR) were detected by Western blotting. Autophagic flux was detected by transfection with Ad-mCherry-GFP-LC3B. RESULTS: Under high glucose conditions, the viability of ARPE-19 was decreased, and the apoptosis rate increased, the protein expressions of Bax, Caspase-3, and LC3-II/LC3-I were all increased and the expressions of Bcl-2, p62 and p-mTOR decreased, and autophagic flux was increased compared with that of the controls. Treatment with 3-MA reversed all these changes caused by high glucose. CONCLUSION: The current study demonstrates the mechanisms of cell damage of ARPE-19 through high glucose/mTOR/autophagy/apoptosis pathway, and new strategies for DR may be developed based on autophagy regulation to manage cell death of RPE cells.

[Display omitted] • Small molecule SIRT1 activators protected RPE cells from NLRP3 inflammasome activation and cell death. • In vivo treatment with SIRT1 activators inhibited retinal inflammation, and reduced structural and functional deteriorations. • It is suggested that SIRT1 activators may be a novel therapeutic strategy for the treatment of retinal degeneration. The nicotinamide adenosine dinucleotide-dependent deacetylase Sirtuin 1 (SIRT1) has been identified as a protective factor that inhibits the activation of nucleotide-binding and oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NLRP3) inflammasome. However, whether pharmacological SIRT1 activators can protect retinal pigment epithelial (RPE) cells against oxidative and inflammatory injuries related to age-related macular degeneration remains to be explored. Two small molecule specific SIRT1 activators (SRT2104 and CAY10602) were tested, with resveratrol being used as a positive control. Mouse models with sodium iodate-induced retinal degeneration were constructed. ARPE-19 cells in culture were used for in vitro experiments. The effects of SIRT1 activators on H 2 O 2 -induced ARPE-19 cell injury were determined by reactive oxygen species quantification, western blotting, flow cytometry and immunofluorescence staining. In vivo , the severity of retinal damage was assessed using flash electroretinography and histopathological analysis. In vitro , SRT2104, CAY10602 and resveratrol significantly attenuated H 2 O 2 -induced cell death, nucleolar stress response, and reactive oxygen species accumulation. In H 2 O 2 -stimulated cells, SIRT1 activators reduced the level of NLRP3, inhibited the activation of caspase-1, and decreased the production of interleukin (IL)-1β and IL-18. The inhibitory effects of SIRT1 activators on caspase-1 activation and IL-1β production were blunted by SIRT1 gene silencing. In vivo, treatment with SRT2104 or CAY10602 in mice with sodium iodate-induced retinal degeneration markedly improved the retinal functions and reduced the loss of RPE cells. Our study suggests that small molecule SIRT1 activators are effective for protection of RPE cells against oxidative stress-induced NLRP3 inflammasome activation, highlighting potential applications in the treatment of macular degeneration associated RPE dysfunctions. [ABSTRACT FROM AUTHOR]

AIM: To investigate YM155's effect on retinal pigment epithelium (RPE) cells' viability and the potential regulatory mechanisms. METHODS: Human immortalized RPE cell lines (ARPE-19 cell line) were processed with YM155 and epidermal growth factor (EGF). ARPE-19 cell viability was detected by methyl thiazolyl tetrazolium assay, and apoptosis was tested by flow cytometry assay. ARPE-19 cell proliferation was assessed with bromodeoxyuridine tagged incorporation assay, and migration ability was evaluated via a wound-healing assay. Epidermal growth factor receptor (EGFR)/MAPK pathway proteins were tested via immunoblotting. EGFR localization was examined by immunofluorescence assay. RESULTS: YM155 suppressed ARPE-19 cells' viability in a time and concentration-dependent manner. A high dose of YM155 caused a small amount of ARPE-19 cell death. YM155 significantly diminished the ARPE-19 cells' proliferative and migrative capacity. YM155 down-regulated total EGFR and phosphorylated external signal-regulated protein kinase (ERK), and it up-regulated the phosphorylation of P38MAPK and c-Jun N-terminal kinase (JNK). YM155 induced endocytosis of EGFR in ARPE-19 cell. YM155 also attenuated EGF-induced ARPE-19 cells' proliferative and migrative capacity. Moreover, YM155 significantly decreased the expression of phosphorylated EGFR and ERK after treated by EGF. CONCLUSION: YM155 inhibits RPE cell survival, the cell proliferative and migrative capacity, and it effectuates a small amount of cell death through the EGFR/MAPK signaling pathway. YM155 might, therefore, be an agent to prevent and treat abnormal RPE cell survival in proliferative vitreoretinopathy.

Age-related macular degeneration (AMD) causes vision loss in the elderly population. Dry AMD leads to the formation of Drusen, while wet AMD is characterized by cell proliferation and choroidal angiogenesis. The retinal pigment epithelium (RPE) plays a key role in AMD pathogenesis. In particular, helioreceptor renewal depends on outer segment phagocytosis of RPE cells, while RPE autophagy can protect cells from oxidative stress damage. However, when the oxidative stress burden is too high and homeostasis is disturbed, the phagocytosis and autophagy functions of RPE become damaged, leading to AMD development and progression. Hence, characterizing the roles of RPE cell phagocytosis and autophagy in the pathogenesis of AMD can inform the development of potential therapeutic targets to prevent irreversible RPE and photoreceptor cell death, thus protecting against AMD.

The impact of plastic pollution on living organisms have gained significant research attention. However, the effects of nanoplastics (NPs) on retina remain unclear. This study aimed to investigate the effect of long-term polystyrene nanoparticles (PS-NPs) exposure on mouse retina. Eight weeks old C57BL/6 J mice were exposed to PS-NPs at the diameter of 100 nm and concentration of 10 mg/L in drinking water for 3 months. PS-NPs were able to penetrate the blood-retina barrier, accumulated at retinal tissue, caused increased oxidative stress level and reduced scotopic electroretinal responses without remarkable structural damage. PS-NPs exposure caused cytotoxicity and reactive oxygen species accumulation in cultured photoreceptor cell. PS-NPs exposure increased oxidative stress level in retinal pigment epithelial (RPE) cells, leading to changes of gene and protein expression indicative of compromised phagocytic activity and cell junction formation. Long-term PS-NPs exposure also aggravated light-induced photoreceptor cell degeneration and retinal inflammation. The transcriptomic profile of PS-NPs-exposed, light-challenged retinal tissue shared similar features with those of age-related macular degeneration (AMD) patients in the activation of complement-mediated phagocytic and proinflammatory responses. Collectively, these findings demonstrated the oxidative stress- and inflammation-mediated detrimental effect of PS-NPs on retinal function, suggested that long-term PS-NPs exposure could be an environmental risk factor contributing to retinal degeneration. [Display omitted] • Long-term PS-NPs exposure causes retinal ROS accumulation and decreases electroretinal responses in mice. • PS-NPs exposure affects gene and protein expression of photoreceptor cells and retinal pigment epithelial cells. • Long-term PS-NPs exposure exacerbates the retinal degenerative effect of light challenge. • Chronic MPs/NPs exposure could be a detrimental environmental factor contributing to retinal degeneration. [ABSTRACT FROM AUTHOR]

Purpose The damage of hyperglycemia to the retinal pigment epithelial (RPE) cells is a critical event in diabetic retinopathy (DR). Procyanidin (PC), a kind of polyphenol compounds, has shown to be effective in preventing and treating diabetes as well as its complications, in which autophagy disorder is involved in the pathological mechanism. However, it remains unclear whether PC can play a protective role in DR by regulating the autophagy of RPE. Here, the effect of PC on RPE under high glucose conditions and the role of autophagy were investigated. Materials and Methods: The cell viability of ARPE‐19, a human RPE cell line, was detected by cell counting kit‐8 (CCK‐8) and the apoptosis rate was measured by flow cytometry. The protein expressions of apoptosis markers, including Bax, Bcl‐2, and Caspase‐3, as well as autophagy markers including LC3, p62, p53, and mTOR were detected by Western blotting. Autophagic flux in ARPE‐19 cells was detected by transfection with Ad‐mCherry‐GFP‐LC3B. Results: Under high glucose conditions, the viability of ARPE‐19 was decreased and the apoptosis rate increased, the protein expressions of Bax, Caspase‐3, LC3‐II/LC3‐I, and p‐p53 were all increased and the expressions of Bcl‐2, p62, and p‐mTOR decreased, and autophagic flux was increased compared with that of the controls. Treatment with PC weakened all these changes caused by high glucose. When rapamycin (RPM), an autophagy agonist was added, the cell viability of ARPE‐19 by PC treatment was decreased while the apoptosis was increased. Conclusions: Our findings indicate that through the p53/mTOR autophagy pathway, PC may protect RPE cells from high glucose‐induced injury. [ABSTRACT FROM AUTHOR]

AIM: To investigate the effects of different wavelength of blue light on human retinal pigment epithelial(RPE)cells. METHODS: ARPE-19 cells cultured in vitro were randomly divided into four groups, which were control group, 447nm blue light group, 456nm blue light group and 468nm blue light group. The cells in control group were cultured under normal conditions whereas the cells in blue light group were irradiated with different wavelengths of OLED blue light with the illumination intensity of 200Lux for 72h. Live/Dead staining assay, CCK-8 assay and real-time PCR were performed to compare the effects of different wavelengths of blue light on the morphology, cell viability, proliferation capacity, mRNA expression level of visual cycle biomarkers and inflammatory biomarkers of ARPE-19 cells, respectively.RESULTS: After blue light irradiation, the abnormal morphology and the decrease of cell confluence of ARPE-19 cells were observed. Furthermore, with the decrease in the wavelength of blue light, the inhibition effect of blue light on RPE proliferation was enhanced, and the mRNA expression of the proliferation marker Ki-67 and visual cycle biomarkers LRAT, CRALBP, RDH and IRBP decreased. In addition, the mRNA expression levels of inflammatory factors MCP-1 and IL-6 in RPE cells were up-regulated with the decrease in the wavelength of blue light.CONCLUSION: Our data demonstrated that blue light in different wavelengths exerted detrimental effects on RPE cells. The shorter the wavelength of blue light was, the more severe damage it caused on the RPE cells.

Esculetin is a coumarin-derived compound with antioxidant and anti-inflammatory properties. The current study aims to evaluate the therapeutic implications of esculetin on retinal dysfunction and uncover the underlying mechanisms. Tert-butyl hydroperoxide (t-BHP) at a concentration of 300 μM was used to induce oxidative stress in human retinal pigment epithelial cell line (ARPE-19) cells. Esculetin at concentrations below 250 μM did not cause cytotoxicity to ARPE-19 cells. Cell viability analysis confirmed that t-BHP induced oxidative injury of ARPE-19 cells. However, ARPE-19 cells were protected from t-BHP-induced oxidative injury by esculetin in a concentration-dependent manner. As a result of the TUNEL assay to confirm apoptosis, esculetin treatment reduced the number of TUNEL-positive cells. Esculetin down-regulated the expression levels of Bax, Caspase-3, and PARP and up-regulated the expression level of Bcl2. Collectively, this study demonstrates that esculetin exerts potent antioxidant properties in ARPE-19 cells, inhibiting t-BHP-induced apoptosis under the regulation of apoptotic factors.

Diabetic retinopathy (DR) is a common diabetic complication known to cause vision impairment and blindness. Previous studies have demonstrated that proanthocyanidins (PACs), polyphenols that are naturally found in several plants and fruits, have powerful antioxidant and anti‐inflammatory effects on various cells. However, the effects and underlying mechanism of PACs against DR pathogenesis remain unknown. Here, we investigated the proliferation, apoptosis, and mechanisms of ARPE‐19 cells in response to oxidative stress and inflammation under high‐glucose conditions with or without PACs treatment. The Cell‐Counting Kit‐8 assay and western blot analysis showed that treatment with 10 μl PACs significantly increased cell proliferation and the expression level of Bcl‐2 in ARPE‐19 cells under high‐glucose conditions. Moreover, PACs attenuated the high glucose‐induced apoptosis, and the increased expression levels of caspase‐3 and Bax. Under high‐glucose conditions, the generation of reactive oxygen species (ROS) and levels of malondialdehyde increased, whereas the superoxide dismutase content decreased. Moreover, the expression level of the NLRP3 inflammasome, and the release of interleukin 1β (IL‐1β) and IL‐18 increased. PACs reversed all of these high glucose‐induced effects on ARPE‐19 cells. Additionally, exposure to nigericin sodium salt, an agonist of the NLRP3 inflammasome, upregulated expression of the NLRP3 inflammasome accompanied by the release of IL‐1β and IL‐18. Again, treatment with PACs markedly downregulated these effects. Collectively, these results demonstrate that PACs can prevent retinal pigment epithelial cells from high glucose‐induced injury via inhibiting the generation of ROS and activation of the NLRP3 inflammasome, suggesting PACs as a potential candidate for the management of DR. [ABSTRACT FROM AUTHOR]

Pro-inflammatory cytokine production by the retinal pigment epithelium (RPE) is a key etiology in retinal degenerative diseases, yet the underlying mechanisms are not well understood. TMEM97 is a scarcely studied transmembrane protein recently implicated in retinal degeneration. BAH domain coiled coil 1 (BAHCC1) is a newly discovered histone code reader involved in oncogenesis. A role for TMEM97 and BAHCC1 in RPE inflammation was not known. Here we found that they constitute a novel axis regulating pro-inflammatory cytokine expression in RPE cells. Transcriptomic analysis using a TMEM97−/− ARPE19 human cell line and the validation via TMEM97 loss- and gain-of-function revealed a profound role of TMEM97 in promoting the expression of pro-inflammatory cytokines, notably IL1β and CCL2, and unexpectedly BAHCC1 as well. Moreover, co-immunoprecipitation indicated an association between the TMEM97 and BAHCC1 proteins. While TMEM97 ablation decreased and its overexpression increased NFκB (p50, p52, p65), the master transcription factor for pro-inflammatory cytokines, silencing BAHCC1 down-regulated NFκB and downstream pro-inflammatory cytokines. Furthermore, in an RPE-damage retinal degeneration mouse model, immunofluorescence illustrated down-regulation of IL1β and CCL2 total proteins and suppression of glial activation in the retina of Tmem97−/− mice compared to Tmem97+/+ mice. Thus, TMEM97 is a novel determinant of pro-inflammatory cytokine expression acting via a previously unknown TMEM97- > BAHCC1- > NFκB cascade. Retinal pigment epithelium (RPE) inflammation can lead to blindness. We identify here a previously uncharacterized cascade that underlies RPE cell production of pro-inflammatory cytokines. Specifically, transmembrane protein TMEM97 positively regulates the recently discovered histone code reader BAHCC1, which in turn enhances pro-inflammatory cytokine expression via the transcription factor NFκB. [Display omitted] • TMEM97 promotes the expression of pro-inflammatory cytokines, most notably IL1β and CCL2. • TMEM97 positively regulates BAHCC1 expression and the two proteins co-immunoprecipitate. • The TMEM97/BAHCC1 axis positively regulates NFκB expression. • Retinal IL1β, CCL2, and glial activation are suppressed in oxidant-treated Tmem97−/− mice. [ABSTRACT FROM AUTHOR]

Aim: To investigate the association of the pair box 6 gene (PAX6) and hsa-miR-328-3p with optical density of macular pigment. Materials and Methods: We evaluated 112 individuals (34 with moderate myopia, eight with high-degree myopia, and 70 healthy individuals). The optical density of macular pigment was measured using single-wavelength reflectometry. DNA and RNA were extracted from whole blood samples. Expression of hsa-miR-328-3p and genotyping of single-nucleotide polymorphism of PAX6 (rs662702) were performed using Applied Biosystems 7900HT real-time polymerase chain reaction system. Optical density of retinal pigment epithelial cells was evaluated using Fundus plus camera. Results: In the group with myopia, with increasing ∆Ct hsa-miR-328-3p, the median optical density of the retinal pigment epithelium decreased statistically significantly (p<0.032). No statistically significant association was found between SNP rs662702 genotype variant of the PAX6 gene and the optical density of the retinal pigment epithelium. Conclusion: The increased expression of hsa-miR-328-3p in the blood indicates a decrease in the optical density of the retinal pigment epithelium in those with myopia. [ABSTRACT FROM AUTHOR]

Excessive blue light light-emitting diode (LED) exposure and consequent oxidative stress causes damage to retinal pigment epithelial (RPE) cells. Chitosan oligosaccharides (COSs) are the hydrolyzed products of chitin, which is abundant in the exoskeleton of crustaceans and cell walls of fungi. We investigated the protective effect of COSs on blue light LED-induced RPE cell damage. These cells were treated with various concentrations of COSs and then exposed to blue light LED. Our results confirmed that RPE cell apoptosis increased significantly with longer light exposure. Treatment with COSs significantly reduced apoptosis in a dose-dependent manner. These molecules also suppressed the production of reactive oxygen species and the expression of inflammation- and apoptosis-related proteins. Moreover, COSs stabilized the mitochondrial membrane potential of cells and down-regulated the NF-κB pathway. In this study, the protective effects of COSs on blue light LED-induced RPE cell damage, as well as the associated mechanisms, were demonstrated.

Objectives:To assess the effects of anti-vascular endothelial growth factor (VEGF) drugs on retinal pigment epithelium cell culture.Materials and Methods:Aflibercept (0.5 mg/mL), bevacizumab (0.3125 mg/mL), and ranibizumab (0.125 mg/mL) were applied to retinal pigment epithelium cell cultures isolated from the enucleated eyes of New Zealand white rabbits. Viability, apoptosis, proliferation, and senescence of the cells were evaluated in control and drug-treated cultures at the end of 72 hours.Results:Cells treated with aflibercept showed increased viability and decreased apoptosis compared to the control culture and both the bevacizumab- and ranibizumab-treated groups (p0.05).Conclusion:Anti-VEGF drugs did not affect senescence or proliferation of retinal pigment epithelium cells. Aflibercept was found to decrease apoptosis and increase cell viability, while ranibizumab and bevacizumab increased apoptosis and reduced cell viability in retinal pigment epithelium culture.

AIM: To observe the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells (hUCMSCs) on the expression of vascular endothelial growth factor-A (VEGF-A) in blue light injured human retinal pigment epithelial (RPE) cells and laser-induced choroidal neovascularization (CNV) in rats. METHODS: Exosomes were isolated from hUCMSCs and characterized by transmission electron microscope and Western blot. MSCs-derived exosomes were cultured with RPE cells exposed to blue light. The mRNA and protein expression of VEGF-A were determined by real time-polymerase chain reaction (PCR) and Western blot, respectively. Immunofluorescence assay was used for the detection of the expression level of VEGF-A. We injected different doses of MSCs-derived exosomes intravitreally to observe and compare their effects in a mouse model of laser-induced retinal injury. The histological structure of CNV in rats was inspected by hematoxylin-eosin (HE) staining and fundus fluorescein angiography. The expression of VEGF-A was detected by immunohistochemistry. RESULTS: Exosomes exhibited the typical characteristic morphology (cup-shaped) and size (diameter between 50 and 150 nm). The exosomes marker, CD63, and hUCMSCs marker, CD90, showed a robust presence. In vitro, MSCs-derived exosomes downregulated the mRNA(Exo-L: t=6.485, 7.959, 9.286; Exo-M: t=7.517, 10.170, 13.413; Exo-H: t=10.317, 12.234, 14.592, P

AIM:To investigate the effect of epidermal growth factor(EGF)on proliferation and migration of retinal pigment epithelial(RPE)cells. METHODS: Human RPE cell lines(ARPE-19 cell)were treated with different doses of EGF. The methyl thiazolyl tetrazolium(MTT)assay was used to detect cell viability. The 5-bromodeoxyuridine(BrdU)incorporation assay was used to detect cell proliferation and the “scratch-wound assay” was used to detect cell migration ability. The epidermal growth factor receptor(EGFR)and protein kinase B(AKT)proteins were detected by Western blot. RESULTS:The MTT assay results showed that treatment with 50 and 100ng/mL EGF for 12h increased ARPE-19 cell viability. The BrdU incorporation assay and the “scratch-wound assay” showed that treatment with 100ng/mL EGF for 24h increased ARPE-19 cell proliferation and migration. The Western blot results showed that treatment with 10-100ng/mL EGF for 12h or 100ng/mL EGF for 15-180min increased phosphorylation levels of EGFR and decreased total levels of EGFR. Similarly, treatment with 10-100ng/mL EGF for 12h or 100ng/mL EGF for 15-180min increased phosphorylation levels of AKT, but not affected total levels of AKT. CONCLUSION: EGF affects ARPE-19 cell viability, proliferation and migration through inducing phosphorylation of the EGFR/AKT signaling pathway. The EGFR/AKT signaling pathway might play an important role in abnormal proliferation and migration of RPE cells in proliferative vitreoretinopathy.

Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disease of the macula that leads to bilateral loss of central vision and is caused by mutations in the TIMP3 gene. However, the mechanisms by which TIMP3 mutations cause SFD are poorly understood. Here, we generated human induced pluripotent stem cell‐derived retinal pigmented epithelial (hiPSC‐RPE) cells from three SFD patients carrying TIMP3 p.(Ser204Cys) and three non‐affected controls to study disease‐related structural and functional differences in the RPE. SFD‐hiPSC‐RPE exhibited characteristic RPE structure and physiology but showed significantly reduced transepithelial electrical resistance associated with enriched expression of cytoskeletal remodelling proteins. SFD‐hiPSC‐RPE exhibited basolateral accumulation of TIMP3 monomers, despite no change in TIMP3 gene expression. TIMP3 dimers were observed in both SFD and control hiPSC‐RPE, suggesting that mutant TIMP3 dimerisation does not drive SFD pathology. Furthermore, mutant TIMP3 retained matrix metalloproteinase activity. Proteomic profiling showed increased expression of ECM proteins, endothelial cell interactions and angiogenesis‐related pathways in SFD‐hiPSC‐RPE. By contrast, there were no changes in VEGF secretion. However, SFD‐hiPSC‐RPE secreted higher levels of monocyte chemoattractant protein 1, PDGF and angiogenin. Our findings provide a proof‐of‐concept that SFD patient‐derived hiPSC‐RPE mimic mature RPE cells and support the hypothesis that excess accumulation of mutant TIMP3, rather than an absence or deficiency of functional TIMP3, drives ECM and angiogenesis‐related changes in SFD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Aims: Long non-coding RNA (lncRNA) VIM Antisense RNA 1 (VIM-AS1) has been reported to be correlated with type 2 diabetes (T2D) susceptibility, while the roles of this lncRNA in T2D and its complications remain unclear. This study aimed to explore the role of VIM-AS1 in diabetic retinopathy (DR). Methods: Gene expression levels in both human specimens and in vitro cultivated cells were determined by qPCR and western blot. Overexpression experiments were performed to analyze gene interactions. Cell apoptosis after transfections was detected by cell apoptosis assay. Results: We found that VIM-AS1 was significantly downregulated in T2D patients in comparison with that in healthy controls. Specifically, the expression levels of VIM-AS1 were lowest among T2D patients complicated with DR. Bioinformatics analysis showed that VIM-AS1 can interact with microRNA 29 (miR-29), which is a critical player in high glucose-induced apoptosis of human retinal pigment epithelial cells (RPEs). Dual-luciferase assay also revealed the direct interaction between them. High glucose treatment led to upregulated miR-29 and downregulated VIM-AS1. However, overexpression of VIM-AS1 and miR-29 did not affect the expression of each other. Cell apoptosis analysis showed that overexpression of VIM-AS1 reduced the enhancing effects of miR-29 overexpression on RPEs cell proliferation. Conclusions: Therefore, VIM-AS1 may sponge miR-29 to participate in DR. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus is a γ-herpes virus that infects primary B cells and can transform infected cells into immortalized lymphoblastoid cell lines (LCL). The role of EBV in malignancies such as Burkitt's lymphoma and nasopharyngeal carcinoma is well understood, however, its role in EBV-infected retinal cells remains poorly understood. Therefore, we investigated the effect of EBV on the growth of retinal cells. Previously, we established and reported a cell line model to address the relationship between EBV infection and retinal cell proliferation that used adult retinal pigment epithelium (ARPE-19) and EBV infection. To determine the effect of EBV on ARPE-19 cells, cell death was measured by propidium iodine/annexin V staining and reactive oxygen species (ROS) were measured by FACS, and protein expression was evaluated using western blot analysis. Also, downregulation of LMP1 and NADPH oxidase 4 (NOX4) expression was accomplished using siRNA technology. We found that ROS were dramatically increased in EBV-infected ARPE19 cells (APRE19/EBV) relative to the parental cell line. Additionally, the expression level of NOX4, a main source of ROS, was upregulated by EBV infection. Interestingly, downregulation of LMP1, one of the EBV viral onco-proteins, completely decreased EBV-induced ROS accumulation and the upregulation of NOX4. Treatment with APX-115A, a pan-NOX inhibitor, induced apoptotic cell death of only the EBV-infected ARPE19 cells but not the parental cell line. Pretreatment with z-VAD, a pan-caspase inhibitor, inhibited NOX inhibitor-induced cell death in ARPE19/EBV cells. Furthermore, APX-115A-induced cell death mediated the activation of JNK and ERK. Finally, we confirmed the expression level of NOX4, and APX-115A induced cell death of EBV-infected human primary retina epithelial cells and the activation of JNK and ERK. Taken together, these our results suggest that APX-115A could be a therapeutic agent for treating EBV-infected retinal cells or diseases by inhibiting LMP1-NOX4-ROS signaling. [ABSTRACT FROM AUTHOR]

Background: This study analysed the effectiveness of plasma rich in growth factors (PRGF) in reducing the oxidative stress induced by blue light exposition on retinal pigment epithelial (RPE) cells. Methods: Blood from six healthy donors was collected to obtain the PRGF. Retinal pigment epithelium (ARPE‐19) cells were exposed to blue light. Then, cells were incubated with PRGF or with control for 24 and 48 hours maintaining exposure to blue light. The cytoprotective effect of PRGF on ARPE cells was evaluated by measuring the cell viability, the reactive oxygen species (ROS) production and the expression of different proteins such as heme oxygenase 1 (HO‐1), catalase (CAT), superoxide dismutase (SOD‐1), apoptosis‐inducing factor (AIF), pigment epithelium‐derived factor (PEDF) and vascular endothelial growth factor (VEGF). Results: The cell viability increased significantly at 24 and 48 hours after PRGF treatment compared to the control group. ROS synthesis was significantly reduced in PRGF‐treated cells with respect to control. Furthermore, the levels of HO‐1, SOD‐1 and AIF were significantly reduced after PRGF treatment at both times of treatment. However, CAT levels were only significantly reduced after PRGF treatment at 48 hours. The high expression of VEGF by RPE cells exposed to blue light was only counterbalanced in the PRGF group by increasing the expression of PEDF in comparison to the control group. Conclusion: The present results show that PRGF treatment reduces the cytotoxic effects induced in RPE cells exposed to an oxidative stress environment. Furthermore, PRGF treatment preserves the mitochondrial activity and cell viability of RPE cells subjected to an oxidative stress. [ABSTRACT FROM AUTHOR]

Background: Geographic atrophy (GA) is a late‐stage symptom of an age‐related macular degeneration (AMD), characterized by the loss of retinal pigment epithelial (RPE) cells and photoreceptor functions. Despite being a major cause of blindness in individuals of 65 years of age and older, some forms of AMD, including GA, still lack targeted treatment. Our previous study demonstrated that mini‐αA peptide, which contains the functional site of αA‐crystallin, protected RPE cells from NaIO3‐induced apoptosis. Methods: To further investigate the underlying mechanism, we applied next‐generation sequencing analysis to identify miR‐1246 as a putative mediator of mini‐αA protective function. To investigate the role of miR‐1246 in RPE cell apoptosis, a stable miR‐1246‐low‐expression cell line was established by using miR‐1246 inhibitor. A 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay was used to investigate the proliferation of RPE cells, mRNA and miR‐1246 expression were detected by the quantitative reverse transcriptase‐polymerase chain reaction. Results: We have further identified caspase‐3 and caspase‐14 as molecular targets of miR‐1246 involved in regulation of apoptosis in NaIO3‐incubated cells. Interestingly, disruption of miR‐1246 expression enhanced anti‐apoptotic effect of mini‐αA on RPE cells during oxidative stress. Conclusions: Our results provide a mechanistic basis for evaluation of miR‐1246 as a new candidate target for the clinical treatment of AMD. [ABSTRACT FROM AUTHOR]

Purpose: Proliferative vitreoretinopathy (PVR) occurs in approximately 5–10% of patients after retinal detachment surgery. Neferine is a bis-benzylisoquinoline alkaloid found in the green seed embryos (Nelumbo nucifera) of the lotus flower and has various properties, such as being antithrombotic, antioxidant, neuroprotective, anticancerous, and anti-inflammatory. Although the effects of neferine on the proliferation and migration of cancer cells have been partially shown, their possible role and the mechanism of action on PVR remain unclear. Materials and methods: To mimic a PVR model in vitro, retinal pigment epithelial (RPE) cells were exposed to epidermal growth factor (EGF) and treated with various concentrations of neferine. Cell viability was determined by MTT test. Cell-cycle phase distribution and cell migration were examined by image-based cytometry and wound healing test, respectively. Messenger RNA (mRNA) and protein expression were determined by RT-qPCR and Western blotting, respectively. Results: Stimulation of the cells with EGF significantly increased the rate of proliferation, whilst treatment with low concentrations of neferine-reduced proliferation to a level equal to that seen in untreated cells. Neferine significantly downregulated EGF-increased cell viability, and survivin mRNA expression was depressed to the basal level. In addition, neferine treatment contributed to cell proliferation loss by upregulating p21 and p27 expression leading to cycle arrest at the G1 phase. The treatment significantly inhibited cell migration by upregulating the expression of epithelial markers, such as E-cadherin and occludin, and decreased MMP2, MMP9, α-SMA, and vimentin. Neferine treatment markedly reduced phosphotidyl inositol 3-kinase (PI3K), AKT, p-p38 mitogen-activated protein kinase (MAPK), and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) protein expression. Conclusion: It can be considered that neferine may be a potential candidate molecule in the treatment of PVR by inhibiting cell proliferation and the migration of EGF-induced RPE cells through the modulation of various transcriptional activities. [ABSTRACT FROM AUTHOR]

Glucocorticoids require the glucocorticoid receptor (GR), a type of ligand-dependent nuclear receptor to transmit their downstream effects. Upon glucocorticoid binding, GR associates with glucocorticoid response elements (GREs) and recruits other transcriptional coregulators to activate or repress target gene transcription. Many SET-domain family proteins have been demonstrated to contribute to GR-mediated transcriptional activity. However, whether histone H3K4-specific methyltransferase plays a cell-type-specific role in GR transcriptional regulation remains poorly understood. In this report, we examined MLL2 (KMT2D), a histone-lysine methyltransferase that catalyzes histone H3 lysine 4 methylation (H3K4me). Furthermore, we demonstrated that MLL2 specifically regulates the transcription of some GR target genes (e.g., ENACα and FLJ20371) in ARPE-19 cells, but has no effect in A549 cells. Mechanistically, co-immunoprecipitation assays revealed that MLL2 is associated with GR in a ligand-independent manner in APRE-19 cells. Moreover, chromatin immunoprecipitation analyses demonstrated that MLL2 could co-occupy glucocorticoid response elements (GREs) of GR target genes along with GR following Dex stimulation. Finally, the FAIRE-qPCR results illustrated that MLL2 is pivotal in establishing chromatin structure accessibility at the GREs of ARPE-19 specific genes in the presence of Dex. Taken together, our study determined that MLL2 regulates GR-mediated transcription in a cell-type-specific manner, and we provide a molecular mechanism to explain the specific role of MLL2 in regulating GR target gene expression in ARPE-19 cells. • MLL2 specifically regulates the transcription of some GR target genes in ARPE-19 cells. • MLL2 is recruited to glucocorticoid response elements (GREs) of ENACα genes along with GR. • MLL2 is pivotal in establishing chromatin structure accessibility at the GREs of ARPE-19 specific genes. [ABSTRACT FROM AUTHOR]

Aims: Oxidative damage plays a vital role in the pathogenesis of age-related macular degeneration (AMD). Exendin-4 (EX4), a glucagon-like peptide-1 receptor agonist, possesses several pharmacological functions, such as anti-inflammatory and antioxidative properties. However, the effects and mechanism of EX4 on oxidative stress in retinal pigment epithelial (RPE) cells induced by hydrogen peroxide (H2O2) remain unclear. The present study aimed to investigate the protective mechanism of EX4 on human RPE cells subjected to oxidative stress. Methods: Human RPE ARPE-19 cells were treated with H2O2 to induce oxidative damage. Cell viability was determined by Cell Counting Kit-8 and lactate dehydrogenase assay. Levels of intracellular reactive oxygen species (ROS), malonyldialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH) were measured using commercial kits. The expression of nuclear factor erythroid 2-related factor-2 (NRF2), heme oxygenase-1 (HO-1), and NAD(P)H:quinone oxidoreductase-1 (NQO-1) was measured using reverse transcription quantitative polymerase chain reaction assay and western blot, respectively. Results: H2O2 significantly induced oxidative stress to reduce viability of RPE cells and increased intracellular ROS generation. EX4 significantly ameliorated H2O2-induced oxidative damage by reducing intracellular ROS generation, decreasing MDA concentration, and increasing antioxidant enzymes activities (SOD and GSH). In addition, EX4 markedly increased expression of NRF2, HO-1, and NQO-1 and significantly improved protein expression of NRF2 and HO-1 in H2O2-treated ARPE-19 cells, caused by increased nuclear NRF2 protein expression. NRF2 knockdown by targeted siRNA alleviated EX4-mediated HO-1 expression and significantly nullified EX4-mediated RPE cell protection against H2O2. Conclusions: EX4 attenuated oxidative damage induced by H2O2 in ARPE-19 cells through the activation of the NRF2 signaling pathway. The findings suggested that EX4 may be a potential therapeutic agent for the treatment of AMD. [ABSTRACT FROM AUTHOR]

• atRAL activates autophagy in RPE cells. • atRAL-induced p62 accumulation is closely involved in the regulation of autophagy and apoptosis in RPE cells. • atRAL-induced autophagy aggravates intracellular oxidative stress and promote apoptosis in RPE cells. • atRAL-activated ER stress mediates autophagy via oxidative stress in RPE cells. Failure of all- trans -retinal (atRAL) clearance contributes to retina degeneration. However, whether autophagy can be activated by excess atRAL accumulation in retinal pigment epithelial (RPE) cells is not known. This study showed that atRAL provoked mitochondria-associated reactive oxygen species (ROS) production, activated the nuclear factor (erythroid-derived 2)-like 2 and apoptosis in a human RPE cell line, ARPE-19 cells. Moreover, we found that autophagic flux was functionally activated after atRAL treatment. The antioxidant N-acetylcysteine attenuated the expression of autophagy markers, suggesting that ROS triggered atRAL-activated autophagy. In addition, autophagic cell death was observed in atRAL-treated RPE cells, while inhibition of autophagy with 3-methyladenine or LC3 , Beclin1, p62 silencing ameliorated atRAL-induced cytotoxicity. Suppression of autophagy quenched mitochondrial ROS and inhibited HO-1 and γ-GCSh expression, indicating that atRAL-activated autophagy enhances intracellular oxidative stress, thereby promoting RPE cell apoptosis. Furthermore, we found that inhibiting endoplasmic reticulum (ER) stress suppressed atRAL-induced mitochondrial ROS generation, subsequently attenuated autophagy and apoptosis in RPE cells. Taken together, these results suggest that atRAL-induced oxidative stress and ER stress modulate autophagy, which may contribute to RPE degeneration. There may be positive feedback regulatory mechanisms between atRAL-induced oxidative stress and autophagy or ER stress. [ABSTRACT FROM AUTHOR]

AIM: To investigate the cross-talk between oxidative stress and the epidermal growth factor receptor (EGFR)/AKT signaling pathway in retinal pigment epithelial (RPE) cells. METHODS: Human RPE cell lines (ARPE-19 cell) were treated with different doses of epidermal growth factor (EGF) and hydrogen peroxide (H2O2). Cell viability was determined by a methyl thiazolyl tetrazolium assay. Cell proliferation was examined by a bromodeoxyuridine (BrdU) incorporation assay. EGFR/AKT signaling was detected by Western blot. EGFR localization was also detected by immunofluorescence. In addition, EGFR/AKT signaling was intervened upon by EGFR inhibitor (erlotinib), PI3K inhibitor (A66) and AKT inhibitor (MK-2206), respectively. H2O2-induced oxidative stress was blocked by antioxidant N-acetylcysteine (NAC). RESULTS: EGF treatment increased ARPE-19 cell viability and proliferation through inducing phosphorylation of EGFR and AKT. H2O2 inhibited ARPE-19 cell viability and proliferation and also suppressed EGF-stimulated increase of RPE cell viability and proliferation by affecting the EGFR/AKT signaling pathway. EGFR inhibitor erlotinib blocked EGF-induced phosphorylation of EGFR and AKT, while A66 and MK-2206 only blocked EGF-induced phosphorylation of AKT. EGF-induced phosphorylation and endocytosis of EGFR were also affected by H2O2 treatment. In addition, antioxidant NAC attenuated H2O2-induced inhibition of ARPE-19 cell viability through alleviating reduction of EGFR, and phosphorylated and total AKT proteins. CONCLUSION: Oxidative stress affects RPE cell viability and proliferation through interfering with the EGFR/AKT signaling pathway. The EGFR/AKT signaling pathway may be an important target in oxidative stress-induced RPE cell dysfunction.

AIM: To determine whether gypenosides have protective effects in experimental autoimmune optic neuritis (EAON). METHODS: Mice were randomly divided into seven groups: control group, model group, three different density gypenosides monotherapy, methylprednisolone monotherapy, combination of gypenosides and methylprednisolone group. The control group was subcutaneously injected with oil emulsion adjuvant and all other groups were subcutaneously immunized with an emulsified mixture of myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide to induce EAON. Mice in the gypenosides groups were administered injections daily with three concentrations (15 mg/kg, 30 mg/kg, 45 mg/kg) of gypenosides respectively. Mice in the methylprednisolone group and the combination treatment group were injected daily with methylprednisolone (20 mg/kg) or methylprednisolone (20 mg/kg) + gypenosides (30 mg/kg), respectively. After MOG immunization, visual evoked potential (VEP), optical coherence tomography (OCT), and histopathologic examination were performed at 14, 20, 30, and 40d post-inoculation (p.i.). All results were expressed as mean±SEM. The data were evaluated by one-way ANOVA followed by Tukey or Games-Howell test. RESULTS: Compared with the control group, p2 latency was prolonged in the model group (P=0.041). Combination treatment can alleviated the change in VEP at 20d p.i. (P=0.012). Average peripapillary retinal nerve fiber layer (RNFL) thickness was reduced in the model group (P= 0.000, 30d; P=0.000, 40d) and gypenosides treatment remarkably diminished the degree of RNFL degeneration at 30d and 40d p.i (P=0.000, 30d; P=0.000, 40d). The pathomorphological results showed a decrease in demye-lination (P=0.020) and inflammatory reactions in the combination group compared with the model group (20d p.i.). Gypenosides treatment also alleviated the degree of axonal loss (40d p.i.) (P=0.003). CONCLUSION: Treatment with gypenosides exerts protective effects on retinal nerve fibers and axons in EAON. When combined with gypenosides, methylprednisolone reduces demyelination in the acute stage of EAON.