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

Epithelial mesenchymal transition (EMT) is a critical process implicated in the pathogenesis of retinal fibrosis and the exacerbation of diabetic retinopathy (DR) within retinal pigment epithelium (RPE) cells. Apigenin (AP), a potential dietary supplement for managing diabetes and its associated complications, has demonstrated inhibitory effects on EMT in various diseases. However, the specific impact and underlying mechanisms of AP on EMT in RPE cells remain poorly understood. In this study, we have successfully validated the inhibitory effects of AP on high glucose-induced EMT in ARPE-19 cells and diabetic db/db mice. Notably, our findings have identified CBP/p300 as a potential therapeutic target for EMT in RPE cells and have further substantiated that AP effectively downregulates the expression of EMT-related genes by attenuating the activity of CBP/p300, consequently reducing histone acetylation alterations within the promoter region of these genes. Taken together, our results provide novel evidence supporting the inhibitory effect of AP on EMT in RPE cells, and highlight the potential of specifically targeting CBP/p300 as a strategy for inhibiting retinal fibrosis in the context of DR.

Cellular components of the idiopathic epiretinal membrane.

Idiopathic epiretinal membrane (iERM) is a fibrocellular proliferation on the inner surface of the retina, which leads to decreased visual acuity and even central visual loss. As iERM is associated to advanced age and posterior vitreous detachment, a higher prevalence is expected with increasing life expectancy and aging of the global population. Although various cell types of retinal and extra-retinal origin have been described in iERMs (Müller glial cells, astrocytes, hyalocytes, retinal pigment epithelium cells, myofibroblasts, and fibroblasts), myofibroblasts have a central role in collagen production and contractile activity. Thus, myofibroblast differentiation is considered a key event for the iERM formation and progression, and fibroblasts, Müller glial cells, hyalocytes, and retinal pigment epithelium have been identified as myofibroblast precursors. On the other side, the different cell types synthesize growth factors, cytokines, and extracellular matrix, which have a crucial role in ERM pathogenesis. In the present review, the major cellular components and their functions are summarized, and their possible roles in the iERM formation are discussed. By exploring in detail the cellular and molecular aspects of iERM, we seek to contribute for better understanding of this fibrotic disease and the origin of myofibroblasts, which may eventually drive to more targeted therapeutic approaches.

TNF-α released from retinal Müller cells aggravates retinal pigment epithelium cell apoptosis by upregulating mitophagy during diabetic retinopathy.

Retinal pigment epithelium (RPE) cell damage, including mitophagy-associated cell apoptosis, accelerates the pathogenesis of diabetic retinopathy (DR), a common complication of diabetes that causes blindness. Müller cells interact with RPE cells via pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α). Herein, we investigated the role of the RPE cell epidermal growth factor receptor (EGFR)/p38 mitogen-activated protein kinase (p38)/nuclear factor kappa B (NF-κB) pathway in Müller cell-derived TNF-α-induced mitophagy-associated apoptosis during DR. Our results showed that TNF-α released from Müller cells activated the EGFR/p38/NF-κB/p62 pathway to increase mitophagy and apoptosis in RPE cells under high glucose (HG) conditions. Additionally, blockade of the TNF-α/EGFR axis alleviates blood-retina barrier breakdown in diabetic mice. Our data further illustrate the effects of the Müller cell inflammatory response on RPE cell survival, implying potential molecular targets for DR treatment.

Disease-causing mutations associated with bestrophinopathies promote apoptosis in retinal pigment epithelium cells.

PURPOSE: Best vitelliform macular dystrophy (BVMD) and autosomal recessive bestrophinopathy (ARB) are two kinds of bestrophinopathies which are caused by BEST1 mutations and characterized by accumulation of lipofuscin-like materials on the retinal pigment epithelium cell-photoreceptor interface. In the past two decades, research about the pathogenesis of bestrophinopathies was mainly focused on the anion channel and intracellular Ca2+ signaling, but seldom concentrated on the function of retinal pigment epithelium (RPE) cells. In this study, we explored the possible effect of the three BEST1 mutations p.V143F, p.S142G, and p.A146T on the apoptosis in human fetal RPE cells. METHODS: Wild-type plasmid and mutant plasmids BEST1-pcDNA3.1 p.V143F, p.S142G, and p.A146T were transfected to human fetal RPE cells. The molecules caspase-3, phospho-Bcl-2, BAX, PARP, and AIF associated with apoptosis were determined by quantitative PCR and Western blot. Apoptotic rate and active Caspase-3 staining were analyzed by flow cytometry. RESULTS: Caspase-3 and PARP expression were significantly increased in BEST1-pcDNA3.1 p.S142G and p.A146T group. Flow cytometry showed that the apoptosis rates were significantly increased in the BEST1-pcDNA3.1 p.V143F, p.S142G, and p.A146T group compared with the wild-type group. CONCLUSIONS: For the first time, we found that the three mutations promoted RPE cell apoptosis. Furthermore, the results indicated that BEST1 mutations p.S142G and p.A146T may contribute apoptosis of RPE cells by targeting Caspase 3. Our observations suggested that the apoptosis of RPE cells may be one of the mechanisms in bestrophinopathies, which may provide a new potential therapeutic target for the treatment of this disease.

Transplantation of rat embryonic stem cell-derived retinal cells restores visual function in the Royal College of Surgeons rats.

AIM OF STUDY: To evaluate the feasibility of transplantation of embryonic stem cell (ESC)-derived retinal cells in the treatment of retinal degeneration. MATERIALS AND METHODS: Rat ESCs were isolated and induced into retinal progenitor cells (RPCs) in vitro, which were subsequently induced into retinal pigment epithelium cells (RPEs) and photoreceptors (PRCs). All cells were identified by Western blot detection of their specific markers. RPEs and PRCs were, respectively, injected into the retina of Royal College of Surgeons (RCSs) rats. Control group was injected with PBS. Post-transplantation visual function was determined by electroretinography (ERG). The histology of the whole eye was compared by H&E staining. RESULTS: RPEs and PRCs were successfully derived from rat ESCs through the two-step differentiation as indicated by the presence of ESC- (Oct-3/4, Nanog, TRA-1-60 and TRA-1-81), RPC- (Rx, Mitf, Pax6 and Chx10), RPE- (RPE65 and keratin) and PRC-specific markers (blue opsin, red/green opsin, recoverin and rhodopsin) in Western blot. The amplitude of ERG a- and b-wave in RPE- and PRC-transplanted groups at week 2 and 10 after transplantation was markedly higher compared with PBS controls. Retinal injury and vascular stress response was not detected in any of the RCS rats after transplantation. CONCLUSION: The developed stepwise protocol can derive retinal cells from ESCs. Transplantation of these retinal cells can restore visual function of RCS rats. Our study provides evidence for potential clinical application of ESC-based cell therapy for retinal degeneration.

Intermittent high glucose-induced oxidative stress modulates retinal pigmented epithelial cell autophagy and promotes cell survival via increased HMGB1.

BACKGROUND: In this study, we evaluated the effects of intermittent high glucose on oxidative stress production in retinal pigmented epithelial (RPE) cells and explored whether the mechanisms of autophagy and apoptosis in oxidative stress are associated with high-mobility group box 1 (HMGB1) protein. METHODS: Cultured human RPE cell line ARPE-19 cells were exposed to intermittent high glucose-induced oxidative stress. Reactive oxygen species (ROS) was determined by 2', 7'-dichlorofluorescin diacetate (DCFH-DA); and malonyldialdehyde (MDA), superoxide dismutase (SOD) by commercial kits. Transmission electron microscopy was used to observe the generation of autophagosome. And MTT assay was used to examine the effect of autophagy on cell viability. For the inhibition experiments, cells were pre-incubated with lysosomal inhibitors NH4Cl or N-acetyl cysteine (NAC).Western blot was used to measure the expression patterns of autophagic markers, including LC3 and p62. The expression of HMGB1 was detected by immunohistochemistry.Cells were pre-incubated with HMGB1 inhibitor ethyl pyruvate (EP) ,then detected the expression pattern of autophagic markers and level of cellular ROS. RESULTS: We found that intermittent high glucose significantly increased oxidative stress levels (as indicated by ROS, MDA, SOD), increased in the generation of autophagosome, decreased the level of p62, induced conversion of LC3 I to LC3 II. We further demonstrated that the NH4Cl/NAC inhibited intermittent high glucose-induced autophage by altered level of LC3 and p62. Intermittent high glucose-induced autophagy is independent of HMGB1 signaling, inhibition of HMGB1 release by EP decreased expression pattern of autophagic markers and level of cellular viability. CONCLUSIONS: Under intermittent high glucose condition, autophagy may be required for preventing oxidative stress-induced injury in RPE. HMGB1 plays important roles in signaling for both autophagy and oxidative stress.

Inhibition of EGFR attenuates EGF-induced activation of retinal pigment epithelium cell via EGFR/AKT signaling pathway.

AIM: To explore the effect of epidermal growth factor receptor (EGFR) inhibition by erlotinib and EGFR siRNA on epidermal growth factor (EGF)-induced activation of retinal pigment epithelium (RPE) cells. METHODS: Human RPE cell line (ARPE-19 cells) was activated by 100 ng/mL EGF. Erlotinib and EGFR siRNA were used to intervene EGF treatment. Cellular viability, proliferation, and migration were detected by methyl thiazolyl tetrazolium (MTT) assay, bromodeoxyuridine (BrdU) staining assay and wound healing assay, respectively. EGFR/protein kinase B (AKT) pathway proteins and N-cadherin, α-smooth muscle actin (α-SMA), and vimentin were tested by Western blot assay. EGFR was also determined by immunofluorescence staining. RESULTS: EGF treatment for 24h induced a significant increase of ARPE-19 cells' viability, proliferation and migration, phosphorylation of EGFR/AKT proteins, and decreased total EGFR expression. Erlotinib suppressed ARPE-19 cells' viability, proliferation and migration through down regulating total EGFR and AKT protein expressions. Erlotinib also inhibited EGF-induced an increase of proliferative and migrative ability in ARPE-19 cells and clearly suppressed EGF-induced EGFR/AKT proteins phosphorylation and decreased expression of N-cadherin, α-SMA, and vimentin proteins. Similarly, EGFR inhibition by EGFR siRNA significantly affected EGF-induced an increase of cell proliferation, viability, and migration, phosphorylation of EGFR/AKT proteins, and up-regulation of N-cadherin, α-SMA, and vimentin proteins. CONCLUSION: Erlotinib and EGFR-knockdown suppress EGF-induced cell viability, proliferation, and migration via EGFR/AKT pathway in RPE cells. EGFR inhibition may be a possible therapeutic approach for proliferative vitreoretinopathy (PVR).

Diabetes and high-glucose could upregulate the expression of receptor for activated C kinase 1 in retina.

BACKGROUND: Diabetic retinopathy (DR) is a major ocular complication of diabetes mellitus, leading to visual impairment. Retinal pigment epithelium (RPE) injury is a key component of the outer blood retinal barrier, and its damage is an important indicator of DR. Receptor for activated C kinase 1 (RACK1) activates protein kinase C-ε (PKC-ε) to promote the generation of reactive oxygen species (ROS) in RPE cells, leading to apoptosis. Therefore, we hypothesize that the activation of RACK1 under hypoxic/high-glucose conditions may promote RPE cell apoptosis by modulating PKC-ε/ROS, thereby disrupting the barrier effect of the outer blood retinal barrier and contributing to the progression of DR. AIM: To investigate the role and associated underlying mechanisms of RACK1 in the development of early DR. METHODS: In this study, Sprague-Dawley rats and adult RPE cell line-19 (ARPE-19) cells were used as in vivo and in vitro models, respectively, to explore the role of RACK1 in mediating PKC-ε in early DR. Furthermore, the impact of RACK1 on apoptosis and barrier function of RPE cells was also investigated in the former model. RESULTS: Streptozotocin-induced diabetic rats showed increased apoptosis and up-regulated expression of RACK1 and PKC-ε proteins in RPE cells following a prolonged modeling. Similarly, ARPE-19 cells exposed to high glucose and hypoxia displayed elevated mRNA and protein levels of RACK1 and PKC-ε, accompanied by an increases in ROS production, apoptosis rate, and monolayer permeability. However, silencing RACK1 significantly downregulated the expression of PKC-ε and ROS, reduced cell apoptosis and permeability, and protected barrier function. CONCLUSION: RACK1 plays a significant role in the development of early DR and might serve as a potential therapeutic target for DR by regulating RPE apoptosis and barrier function.

Inhibiting autophagy by miR-19a-3p/PTEN regulation protected retinal pigment epithelial cells from hyperglycemic damage.

OBJECTIVE: The catabolic process of autophagy is arousing the attention of researchers studying diabetic retinopathy (DR), but the role and molecular mechanism of autophagy in DR are still unclear. METHODS: An in vivo diabetic rat model and in vitro hyperglycemic-exposed retinal pigment epithelium (RPE) cell cultures were established to mimic early DR. Transmission electron microscopy and mRFP-GFP-LC3 adenovirus transfection were applied for autophagic flux analysis. MicroRNA (miR)-19a-3p, members of the phosphate and tensin homolog (PTEN)/Akt/mammalian target of rapamycin (mTOR) pathway, and the autophagy-related proteins light chain (LC)3II/I and p62 were detected. Annexin V, transwell, Cell Counting Kit-8, fluorescein isothiocyanate-dextran monolayer permeability assay, and transepithelial electrical resistance were performed to evaluate the effects of regulating autophagy on RPE cells under the DR condition. RESULTS: Autophagy was aberrantly activated in DR as evidenced by autophagosome accumulation. Further mechanistic experiments revealed that DR induced PTEN expression, thus inhibiting Akt/mTOR phosphorylation and stimulating aberrant autophagy and apoptosis. Notably, these events could be reversed by miR-19a-3p directly targeting PTEN. Downregulation of autophagy by miR-19a-3p overexpression, PTEN knockdown, or 3-methyladenine (3-MA) treatment inhibited autophagosome formation and thus effectively ameliorated hyperglycemia-induced RPE cell apoptosis, increased migration, inhibited viability, and enhanced monolayer permeability under the DR condition. CONCLUSIONS: Our findings suggest that upregulation of miR-19a-3p inhibits aberrant autophagy by directly targeting PTEN, thus protecting RPE cells against DR damage. miR-19a-3p may represent a novel therapeutic target for inducing protective autophagy in early DR.

Role of Epithelial-to-Mesenchymal Transition of Retinal Pigment Epithelial Cells in Glaucoma Cupping.

Optic nerve head (ONH) cupping is a clinical feature of glaucoma associated with extracellular matrix (ECM) remodelling and lamina cribrosa (LC) fibrosis. Peripapillary atrophy (PPA) occurs commonly in glaucoma, and is characterised by the loss of retinal pigment epithelium (RPE) adjacent to the ONH. Under pro-fibrotic conditions, epithelial cells throughout the body can differentiate into fibroblast-like cells through epithelial-to-mesenchymal transition (EMT) and contribute to ECM fibrosis. This is investigated here in the context of glaucoma and PPA. Human-donor ONH sections were assessed for the presence of the RPE cell-specific marker RPE65 using immunofluorescence. We examined the EMT response of ARPE-19 cells to the following glaucoma-related stimuli: cyclic mechanical stretch, mechanical stiffness, transforming growth factor beta (TGFß), and tumour necrosis factor alpha (TNFα). The gene expression was measured using the PCR of the epithelial tight junction marker zona occludens 1 (ZO-1) and the mesenchymal markers alpha smooth muscle actin (αSMA) and vimentin. A scratch assay was used to assess the ARPE-19 migration. Significant RPE-65 staining was demonstrated in the glaucomatous ONH. The cyclic stretching and substrate stiffness of the ARPE-19 cells caused a significant decrease in ZO-1 (p = 0.04), and an increase in αSMA (p = 0.04). The scratch assays demonstrated increased migration of ARPE19 in the presence of TNFα (p = 0.02). Furthermore, ARPE-19 cells undergo an EMT-like transition (gain of αSMA, loss of ZO-1 and increased migration) in response to glaucomatous stimuli. This suggests that during PPA, RPE cells have the potential to migrate into the ONH and differentiate into fibroblast-like cells, contributing to glaucomatous ONH cupping.

Updates on RPE cell damage in diabetic retinopathy (Review).

Diabetic retinopathy (DR) is a microvascular complication of diabetes. The retinal pigment epithelium (RPE) forms the outer layer of the blood­retinal barrier and serves a role in maintaining retinal function. RPE cell injury has been revealed in diabetic animal models, and high glucose (HG) levels may cause damage to RPE cells by increasing the levels of oxidative stress, promoting pro­inflammatory gene expression, disrupting cell proliferation, inducing the endothelial­mesenchymal transition, weakening tight conjunctions and elevating cell death mechanisms, such as apoptosis, ferroptosis and pyroptosis. Non­coding RNAs including microRNAs, long non­coding RNAs and circular RNAs participate in RPE cell damage caused by HG levels, which may provide targeted therapeutic strategies for the treatment of DR. Plant extracts such as citrusin and hesperidin, and a number of hypoglycemic drugs, such as sodium­glucose co­transporter 2 inhibitors, metformin and glucagon­like peptide­1 receptor agonists, exhibit potential RPE protective effects; however, the detailed mechanisms behind these effects remain to be fully elucidated. An in­depth understanding of the contribution of the RPE to DR may provide novel perspectives and therapeutic targets for DR.

Hydrogen peroxide-induced oxidative damage and protective role of peroxiredoxin 6 protein via EGFR/ERK signaling pathway in RPE cells.

Introduction: Damage to retinal pigment epithelium (RPE) cells caused by oxidative stress is closely related to the pathogenesis of several blinding retinal diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, and other inherited retinal degenerative conditions. However, the mechanisms of this process are poorly understood. Hence, the goal of this study was to investigate hydrogen peroxide (H2O2)-induced oxidative damage and protective role of peroxiredoxin 6 (PRDX6) protein via EGFR/ERK signaling pathway in RPE cells. Methods: Cells from a human RPE cell line (ARPE-19 cells) were treated with H2O2, and then cell viability was assessed using the methyl thiazolyl tetrazolium assay. Cell death and reactive oxygen species (ROS) were detected by flow cytometry. The levels of PRDX6, epidermal growth factor receptor (EGFR), P38 mitogen-activated protein kinase (P38MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) were detected by Western blot assay. PRDX6 and EGFR were also detected via immunofluorescence staining. Results: Our results show that H2O2 inhibited cell viability, induced cell death, and increased ROS levels in ARPE-19 cells. It was also found that H2O2 decreased the levels of PRDX6, EGFR, and phosphorylated ERK but increased the levels of phosphorylated P38MAPK and JNK. PRDX6 overexpression was found to attenuate H2O2-induced inhibition of cell viability and increased cell death and ROS production in ARPE-19 cells. PRDX6 overexpression also increased the expression of EGFR and alleviated the H2O2-induced decrease in EGFR and phosphorylated ERK. Moreover, inhibition of epidermal growth factor-induced EGFR and ERK signaling in oxidative stress was partially blocked by PRDX6 overexpression. Discussion: Our findings indicate that PRDX6 overexpression protects RPE cells from oxidative stress damage caused by decreasing ROS production and partially blocking the inhibition of the EGFR/ERK signaling pathway induced by oxidative stress. Therefore, PRDX6 shows promise as a therapeutic target for the prevention of RPE cell damage caused by oxidative stress associated with retinal diseases.

Characterization of Gelatin/Gellan Gum/Glycol Chitosan Ternary Hydrogel for Retinal Pigment Epithelial Tissue Reconstruction Materials.

The cellular transplantation approach to treat damaged or diseased retina is limited because of poor survival, distribution, and integration of cells after implantation to the sub-retinal space. To overcome this, it is important to develop a cell delivery system. In this study, a ternary hydrogel of gelatin (Ge)/gellan gum (GG)/glycol chitosan (CS) is suggested as a cell carrier for retinal tissue engineering (TE). Physicochemical properties such as porosity, swelling, sol fraction, and weight loss were measured. The mechanical study was performed with compressive strength and viscosity to confirm applicability in retinal TE. An in vitro experiment was carried out by encapsulating ARPE-19 in the designed hydrogel to measure viability and expression of retinal pigment epithelium-specific proteins and genes. The results showed that the ternary hydrogel system improves the mechanical properties and stability of the composite. Cell growth, survival, adhesion, and migration were enhanced as the CS was incorporated into the matrix. In particular, real-time polymerase chain reaction analysis showed a markedly improved specific gene expression rate in the Ge/GG/CS. Therefore, it is expected that the ternary system suggested in this study can be used as a promising material for retinal TE.

Reversed Senescence of Retinal Pigment Epithelial Cell by Coculture With Embryonic Stem Cell via the TGFß and PI3K Pathways.

Retinal pigment epithelium (RPE) cellular senescence is an important etiology of age-related macular degeneration (AMD). Aging interventions based on the application of stem cells to delay cellular senescence have shown good prospects in the treatment of age-related diseases. This study aimed to investigate the potential of the embryonic stem cells (ESCs) to reverse the senescence of RPE cells and to elucidate its regulatory mechanism. The hydrogen peroxide (H2O2)-mediated premature and natural passage-mediated replicative senescent RPE cells were directly cocultured with ESCs. The results showed that the proliferative capacity of premature and replicative senescent RPE cells was increased, while the positive rate of senescence-associated galactosidase (SA-ß-GAL) staining and levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were decreased. The positive regulatory factors of cellular senescence (p53, p21WAF1/CIP1, p16INK4a) were downregulated, while the negative regulatory factors of cellular senescence (Cyclin A2, Cyclin B1, Cyclin D1) were upregulated. Furthermore, replicative senescent RPE cells entered the S and G2/M phases from the G0/G1 phase. TGFß (TGFB1, SMAD3, ID1, ID3) and PI3K (PIK3CG, PDK1, PLK1) pathway-related genes were upregulated in premature and replicative senescent RPE cells after ESCs application, respectively. We further treated ESCs-cocultured premature and replicative senescent RPE cells with SB531542 and LY294002 to inhibit the TGFß and PI3K pathways, respectively, and found that p53, p21WAF1/CIP1 and p16INK4a were upregulated, while Cyclin A2, Cyclin B1, Cyclin D1, TGFß, and PI3K pathway-related genes were downregulated, accompanied by decreased proliferation and cell cycle transition and increased positive rates of SA-ß-GAL staining and levels of ROS and MMP. In conclusion, we demonstrated that ESCs can effectively reverse the senescence of premature and replicative senescent RPE cells by a direct coculture way, which may be achieved by upregulating the TGFß and PI3K pathways, respectively, providing a basis for establishing a new therapeutic option for AMD.

Comparison of Artificial Intelligence based approaches to cell function prediction.

Predicting Retinal Pigment Epithelium (RPE) cell functions in stem cell implants using non-invasive bright field microscopy imaging is a critical task for clinical deployment of stem cell therapies. Such cell function predictions can be carried out using Artificial Intelligence (AI) based models. In this paper we used Traditional Machine Learning (TML) and Deep Learning (DL) based AI models for cell function prediction tasks. TML models depend on feature engineering and DL models perform feature engineering automatically but have higher modeling complexity. This work aims at exploring the tradeoffs between three approaches using TML and DL based models for RPE cell function prediction from microscopy images and at understanding the accuracy relationship between pixel-, cell feature-, and implant label-level accuracies of models. Among the three compared approaches to cell function prediction, the direct approach to cell function prediction from images is slightly more accurate in comparison to indirect approaches using intermediate segmentation and/or feature engineering steps. We also evaluated accuracy variations with respect to model selections (five TML models and two DL models) and model configurations (with and without transfer learning). Finally, we quantified the relationships between segmentation accuracy and the number of samples used for training a model, segmentation accuracy and cell feature error, and cell feature error and accuracy of implant labels. We concluded that for the RPE cell data set, there is a monotonic relationship between the number of training samples and image segmentation accuracy, and between segmentation accuracy and cell feature error, but there is no such a relationship between segmentation accuracy and accuracy of RPE implant labels.

Protective effects of lipoic acid-niacin dimers against blue light-induced oxidative damage to retinal pigment epithelium cells.

AIM: To evaluate the protective effects of lipoic acid-niacin (N2L) dimers against blue light (BL)-induced oxidative damage to human retinal pigment epithelium (hRPE) cells in vitro. METHODS: hRPE cells were divided into a control group (CG), a BL group, an N2L plus BL irradiation group, an α-lipoic acid (ALA) plus BL group, an ALA-only group, and an N2L-only group. hRPE cellular viability was detected by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide assays, and apoptosis was evaluated by annexin-V-PE/7-AAD staining followed by flow cytometry. Ultrastructural changes in subcellular organelles were observed by transmission electron microscopy. Reactive oxygen species formation was assayed by flow cytometry. The expression levels of the apoptosis-related proteins BCL-2 associated X protein (BAX), B-cell leukmia/lymphoma 2 (BCL-2), and caspase-3 were quantified by Western blot analysis. RESULTS: BL exposure with a light density of 4±0.5 mW/cm2 exceeding 6h caused hRPE toxicity, whereas treatment with a high dose of N2L (100 mol/L) or ALA (150 mol/L) maintained cell viability at control levels. BL exposure caused vacuole-like degeneration, mitochondrial swelling, and reduced microvillus formation; however, a high dose of N2L or ALA maintained the ultrastructure of hRPE cells and their organelles. High doses of N2L and ALA also protected hRPE cells from BL-induced apoptosis, which was confirmed by Western blot analysis: BCL-2 expression significantly increased, while BAX and caspase-3 expression slightly decreased compared to the CG. CONCLUSION: High-dose N2L treatment (>100 mol/L) can reduce oxidative damage in degenerating hRPE cells exposed to BL with an efficacy similar to ALA.

JAM-C maintains VEGR2 expression to promote retinal pigment epithelium cell survival under oxidative stress.

Junctional adhesion molecule-C (JAM-C) has been shown to play critical roles during development and in immune responses. However, its role in adult eyes under oxidative stress remains poorly understood. Here, we report that JAM-C is abundantly expressed in adult mouse retinae and choroids in vivo and in cultured retinal pigment epithelium (RPE) and photoreceptor cells in vitro. Importantly, both JAM-C expression and its membrane localisation are downregulated by H2O2-induced oxidative stress. Under H2O2-induced oxidative stress, JAM-C is critically required for the survival of human RPE cells. Indeed, loss of JAM-C by siRNA knockdown decreased RPE cell survival. Mechanistically, we show that JAM-C is required to maintain VEGFR2 expression in RPE cells, and VEGFR2 plays an important role in keeping the RPE cells viable since overexpression of VEGFR2 partially restored impaired RPE survival caused by JAM-C knockdown and increased RPE survival. We further show that JAM-C regulates VEGFR2 expression and, in turn, modulates p38 phosphorylation. Together, our data demonstrate that JAM-C plays an important role in maintaining VEGR2 expression to promote RPE cell survival under oxidative stress. Given the vital importance of RPE in the eye, approaches that can modulate JAM-C expression may have therapeutic values in treating diseases with impaired RPE survival.

Variability of miRNA expression during the differentiation of human embryonic stem cells into retinal pigment epithelial cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells can be induced to differentiate into retinal pigment epithelium (RPE). MiRNAs have been characterized and found playing important roles in the differentiation process of ESCs, but their length and sequence heterogeneity (isomiRs), and their non-canonical forms of miRNAs are underestimated or ignored. In this report, we found some non-canonical miRNAs (dominant isomiRs) in all differentiation stages, and 27 statistically significant editing sites were identified in 24 different miRNAs. Moreover, we found marked major-to-minor arm-switching events in 14 pre-miRNAs during the hESC to RPE cell differentiation phases. Our study for the first time reports exploring the variability of miRNA expression during the differentiation of hESCs into RPE cells and the results show that miRNA variability is a ubiquitous phenomenon in the ESC differentiation.

Docosahexaenoic acid aggravates photooxidative damage in retinal pigment epithelial cells via lipid peroxidation.

Docosahexaenoic acid (DHA, 22:6n-3), a long-chain polyunsaturated fatty acid (PUFA) with important functions in normal human retinal activity and vision development, is recommended to promote brain and eye development. However, recent research has revealed that increased DHA level in the retina due to linoleic acid-rich diet heightens the vulnerability of the retina to photooxidative stress. Thus, many scholars have analyzed the potential risks of DHA intake on retinal damage. This study evaluated the potential adverse effects of DHA intake on individuals usually exposed to high-light intensity conditions using a visible light-induced retinal pigment epithelium (RPE) cell damage model in vitro. Results showed that DHA promoted the proliferation of RPE cells without any cytotoxicity under dark conditions. However, DHA supplement elicited deleterious effects on RPE cells under high-intensity light conditions. That is, DHA supplement inhibited cellular proliferation, destroyed cell membrane integrity, enhanced cellular senescence, increased vascular endothelial growth factor (VEGF) release, and decreased phagocytic function. Moreover, DHA supplement increased the intracellular and extracellular levels of reactive oxygen species and the extracellular level of lipid peroxidation products under high-intensity light conditions. These results demonstrate that DHA increases the vulnerability of the retina to light damage through lipid peroxidation.

Adhesive protein-free synthetic hydrogels for retinal pigment epithelium cell culture with low ROS level.

Engineering of human retinal pigment epithelium (RPE) cell monolayer with low level of reactive oxygen species (ROS) is important for regenerative RPE-based therapies. However, it is still challenging to culture RPE monolayer with low ROS level on soft substrates in vitro. To address this, we developed cytocompatible hydrogels to culture human RPE cell monolayer for future use in regenerative RPE-based therapies. The cell adhesion, proliferation, monolayer formation, morphology, survival, and ROS level of human ARPE-19 cells cultured on the surfaces of negatively charged poly (2-acrylamido-2-methyl propane sulfonic sodium) (PNaAMPS) and neutral poly(N,N-dimethylacrylamide) (PDMAAm) hydrogels with different stiffness were investigated. The importance of hydrogel stiffness on the cell function was firstly highlighted on the base of determined optimal Young's modulus for cultivation of RPE cell monolayer with relatively low ROS level. The construction of RPE cell monolayer with low ROS level on the PNaAMPS hydrogel may hold great potential as promising candidates for transplantation of RPE cell monolayer-hydrogel construct into the subretinal space to repair retinal functions.