Primary Human Retinal Pigment Epithelial Cells Research Articles

Protective Effects of Fucoidan on Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells and Progression of Proliferative Vitreoretinopathy

Background/Aims: Proliferative vitreoretinopathy (PVR) is a severe blinding complication of rhegmatogenous retinal detachment. Epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is thought to play a pivotal role in the pathogenesis of PVR. Fucoidan, a marine extract, reportedly has many benefits effects in a variety of tissues and organs such as anti-inflammation, anti-oxidative stress, and anti-carcinogenesis. In this study, we investigated the potential role of fucoidan on EMT in RPE cells and its effect on the development of PVR. Methods: MTS, Transwell, and collagen gel contraction assays were employed to measure the viability, migration, and contraction of RPE cells, respectively. mRNA and protein expression were evaluated via real-time quantitative PCR and western blot analysis, respectively. In vivo, a pigmented rabbit model of PVR was established to examine the anti-PVR effect of fucoidan. Results: Fucoidan reversed the transforming growth factor (TGF)-β1-induced EMT of RPE cells, including the increased expression of α-smooth muscle actin (α-SMA) and fibronectin and down-regulation of E-cadherin in human primary RPE cells. Moreover, the upregulation of phosphorylated Smad2/3 induced by TGF-β1 was suppressed by fucoidan. Fucoidan also inhibited the migration and contraction of RPE cells induced by TGF-β1. In vivo, fucoidan inhibited the progression of experimental PVR in rabbit eyes. Histological findings showed that fucoidan suppressed the formation of α-SMA-positive epiretinal membranes. Conclusion: Our findings regarding the protective effects of fucoidan on the EMT of RPE cells and experimental PVR suggest the potential clinical application of fucoidan as an anti-PVR agent.

The Antibiotic-free pFAR4 Vector Paired with the Sleeping Beauty Transposon System Mediates Efficient Transgene Delivery in Human Cells

The anti-angiogenic and neurogenic pigment epithelium-derived factor (PEDF) demonstrated a potency to control choroidal neovascularization in age-related macular degeneration (AMD) patients. The goal of the present study was the development of an efficient and safe technique to integrate, ex vivo, the PEDF gene into retinal pigment epithelial (RPE) cells for later transplantation to the subretinal space of AMD patients to allow continuous PEDF secretion in the vicinity of the affected macula. Because successful gene therapy approaches require efficient gene delivery and stable gene expression, we used the antibiotic-free pFAR4 mini-plasmid vector to deliver the hyperactive Sleeping Beauty transposon system, which mediates transgene integration into the genome of host cells. In an initial study, lipofection-mediated co-transfection of HeLa cells with the SB100X transposase gene and a reporter marker delivered by pFAR4 showed a 2-fold higher level of genetically modified cells than when using the pT2 vectors. Similarly, with the pFAR4 constructs, electroporation-mediated transfection of primary human RPE cells led to 2.4-fold higher secretion of recombinant PEDF protein, which was still maintained 8 months after transfection. Thus, our results show that the pFAR4 plasmid is a superior vector for the delivery and integration of transgenes into eukaryotic cells.

Polarized Human Retinal Pigment Epithelium Exhibits Distinct Surface Proteome on Apical and Basal Plasma Membranes.

Surface proteins localized on the apical and basal plasma membranes are required for a cell to sense its environment and relay changes in ionic, cytokine, chemokine, and hormone levels to the inside of the cell. In a polarized cell, surface proteins are differentially localized on the apical or the basolateral sides of the cell. The retinal pigment epithelium (RPE) is an example of a polarized cell that performs a variety of functions that are dependent on its polarized state including trafficking of ions, fluid, and metabolites across the RPE monolayer. These functions are absolutely crucial for maintaining the health and integrity of adjacent photoreceptors, the photosensitive cells of the retina. Here we present a series of approaches to identify and validate the polarization state of cultured primary human RPE cells using immunostaining for RPE apical/basolateral markers, polarized cytokine secretion, electrophysiology, fluid transport, phagocytosis, and identification of plasma membrane proteins through cell surface capturing technology. These approaches are currently being used to validate the polarized state and the epithelial phenotype of human induced pluripotent stem (iPS) cell derived RPE cells. This work provides the basis for developing an autologous cell therapy for age-related macular degeneration using patient specific iPS cell derived RPE.

Activation of KGFR-Akt-mTOR-Nrf2 signaling protects human retinal pigment epithelium cells from Ultra-violet

Ultra-violet (UV) radiation causes oxidative injuries to human retinal pigment epithelium (RPE) cells. We tested the potential effect of keratinocyte growth factor (KGF) against the process. KGF receptor (KGFR) is expressed in ARPE-19 cells and primary human RPE cells. Pre-treatment with KGF inhibited UV-induced reactive oxygen species (ROS) production and RPE cell death. KGF activated nuclear-factor-E2-related factor 2 (Nrf2) signaling in RPE cells, causing Nrf2 Ser-40 phosphorylation, stabilization and nuclear translocation as well as expression of Nrf2-dependent genes (HO1, NOQ1 and GCLC). Nrf2 knockdown (by targeted shRNAs) or S40T mutation almost reversed KGF-induced RPE cell protection against UV. Further studies demonstrated that KGF activated KGFR-Akt-mTORC1 signaling to mediate downstream Nrf2 activation. KGFR shRNA or Akt-mTORC1 inhibition not only blocked KGF-induced Nrf2 Ser-40 phosphorylation and activation, but also nullified KGF-mediated RPE cell protection against UV. We conclude that KGF-KGFR activates Akt-mTORC1 downstream Nrf2 signaling to protect RPE cells from UV radiation.

The formation of a functional retinal pigment epithelium occurs on porous polytetrafluoroethylene substrates independently of the surface chemistry

Subretinal transplantation of functioning retinal pigment epithelial (RPE) cells may have the potential to preserve or restore vision in patients affected by blinding diseases such as age-related macular degeneration (AMD). One of the critical steps in achieving this is the ability to grow a functioning retinal pigment epithelium, which may need a substrate on which to grow and to aid transplantation. Tailoring the physical and chemical properties of the substrate should help the engineered tissue to function in the long term. The purpose of the study was to determine whether a functioning monolayer of RPE cells could be produced on expanded polytetrafluoroethylene substrates modified by either an ammonia plasma treatment or an n-Heptylamine coating, and whether the difference in surface chemistries altered the extracellular matrix the cells produced. Primary human RPE cells were able to form a functional, cobblestone monolayer on both substrates, but the formation of an extracellular matrix to exhibit a network structure took months, whereas on non-porous substrates with the same surface chemistry, a similar appearance was observed after a few weeks. This study suggests that the surface chemistry of these materials may not be the most critical factor in the development of growth of a functional monolayer of RPE cells as long as the cells can attach and proliferate on the surface. This has important implications in the design of strategies to optimise the clinical outcomes of subretinal transplant procedures.Graphical

Survival Improvement in Human Retinal Pigment Epithelial Cells via Fas Receptor Targeting by miR-374a.

Oxidative conditions of the eye could contribute to retinal cells loss through activating the Fas-L/Fas pathway. This phenomenon is one of the leading causes of some ocular diseases like age-related macular degeneration (AMD). By targeting proteins at their mRNA level, microRNAs (miRNAs) can regulate gene expression and cell function. The aim of the present study is to investigate Fas targeting by miR-374a and find whether it can inhibit Fas-mediated apoptosis in primary human retinal pigment epithelial (RPE) cells under oxidative stress. So, the primary human RPE cells were transfected with pre-miR-374a pLEX construct using polymeric carrier and were exposed to H2 O2 (200 μM) as an oxidant agent for induction of Fas expression. Fas expression at mRNA and protein level was evaluated by quantitative real-time PCR and Western blot analysis, respectively. These results revealed that miR-374a could prevent Fas upregulation under oxidative conditions. Moreover, Luciferase activity assay confirmed that Fas could be a direct target of miR-374a. The cell viability studies demonstrated that caspase-3 activity was negligible in miR-374a treated cells compared to the controls. Our data suggest miR-374a is a negative regulator of Fas death receptor which is able to enhance the cell survival and protect RPE cells against oxidative conditions. J. Cell. Biochem. 118: 4854-4861, 2017. © 2017 Wiley Periodicals, Inc.

Hypoxia and inflammation in the release of VEGF and interleukins from human retinal pigment epithelial cells.

Retinal diseases are closely associated with both decreased oxygenation and increased inflammation. It is not known if hypoxia-induced vascular endothelial growth factor (VEGF) expression in the retina itself evokes inflammation, or whether inflammation is a prerequisite for the development of neovascularization. Human ARPE-19 cell line and primary human retinal pigment epithelium (RPE) cells were used. ARPE-19 cells were kept either under normoxic (24h or 48h) or hypoxic conditions (1% O2, 24h). Part of the cells were re-oxygenated (24h). Some ARPE-19 cells were additionally pre-treated with bacterial lipopolysaccharide (LPS). The levels of IL-6, IL-8, IL-1β, and IL-18 were determined from medium samples by an enzyme-linked immunosorbent assay (ELISA) method. Primary human RPE cells were exposed to hypoxia for 24h, and the subsequent release of IL-6 and IL-8 was measured with ELISA. VEGF secretion from ARPE-19 cells was determined up to 24h. Hypoxia induced significant (P<0.01) increases in the levels of both IL-6 and IL-8 in ARPE-19 cells, and LPS pre-treatment further enhanced these responses. Hypoxia exposure did not affect the IL-1β or IL-18 release irrespective of LPS pre-treatment. If primary RPE cells were incubated for 4h in hypoxic conditions, IL-6 and IL-8 concentrations were increased by 7 and 8-fold respectively. Hypoxia increased the VEGF secretion from ARPE-19 cells in a similar manner with or without pre-treatment with LPS. Hypoxia causes an inflammatory reaction in RPE cells that is potentiated by pre-treatment with the Toll-like receptor-activating agent, LPS. The secretion of VEGF from these cells is regulated directly by hypoxia and is not mediated by inflammation.

Progranulin increases phagocytosis by retinal pigment epithelial cells in culture.

Retinal pigment epithelium (RPE) cells take part in retinal preservation, such as phagocytizing the shed photoreceptor outer segments (POS), every day. The incomplete phagocytic function accelerates RPE degeneration and formation of the toxic by-product lipofuscin. Excessive lipofuscin accumulation is characteristic of various blinding diseases in the human eye. Progranulin is a cysteine-rich protein that has multiple biological activities, and it has a high presence in the retina. Progranulin has been recognized to be involved in macrophage phagocytosis in the brain. The purpose of this study is to determine whether progranulin influences phagocytosis by RPE cells. All experiments were performed on primary human RPE (hRPE) cells in culture. pHrodo was used to label the isolated porcine POS, and quantification of pHrodo fluorescence was used to determine the degree of phagocytosis. Western blotting and immunohistochemistry of key proteins involved in phagocytosis were used to clarify the mechanism of progranulin. Progranulin increased RPE phagocytosis in hydrogen peroxide-treated and nontreated RPE cells. The phosphorylated form of Mer tyrosine kinase, which is important for POS internalization, was significantly increased in the progranulin-exposed cells. This increase was attenuated by SU11274, an inhibitor of hepatic growth factor receptor. Under the oxidative stress condition, exposure to progranulin led to an approximately twofold increase in integrin alpha-v, which is associated with the first step in recognition of POS by RPE cells. These results suggest that progranulin could be an effective stimulator for RPE phagocytosis and could repair RPE function. © 2017 Wiley Periodicals, Inc.

Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy.

p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative stress and functions, in part, by modulating ATG10 expression. NFκB p65 activity may be a critical upstream initiator of p62 expression in RPE cells under oxidative stress.

Salvianolic Acid B (Sal B) Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Cell Death by Activating Glutaredoxin 1 (Grx1).

Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H2O2-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA) significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H2O2-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage.

Clearance of autophagy-associated dying retinal pigment epithelial cells - a possible source for inflammation in age-related macular degeneration.

Retinal pigment epithelial (RPE) cells can undergo different forms of cell death, including autophagy-associated cell death during age-related macular degeneration (AMD). Failure of macrophages or dendritic cells (DCs) to engulf the different dying cells in the retina may result in the accumulation of debris and progression of AMD. ARPE-19 and primary human RPE cells undergo autophagy-associated cell death upon serum depletion and oxidative stress induced by hydrogen peroxide (H2O2). Autophagy was revealed by elevated light-chain-3 II (LC3-II) expression and electron microscopy, while autophagic flux was confirmed by blocking the autophago-lysosomal fusion using chloroquine (CQ) in these cells. The autophagy-associated dying RPE cells were engulfed by human macrophages, DCs and living RPE cells in an increasing and time-dependent manner. Inhibition of autophagy by 3-methyladenine (3-MA) decreased the engulfment of the autophagy-associated dying cells by macrophages, whereas sorting out the GFP-LC3-positive/autophagic cell population or treatment by the glucocorticoid triamcinolone (TC) enhanced it. Increased amounts of IL-6 and IL-8 were released when autophagy-associated dying RPEs were engulfed by macrophages. Our data suggest that cells undergoing autophagy-associated cell death engage in clearance mechanisms guided by professional and non-professional phagocytes, which is accompanied by inflammation as part of an in vitro modeling of AMD pathogenesis.

Inflammasome priming increases retinal pigment epithelial cell susceptibility to lipofuscin phototoxicity by changing the cell death mechanism from apoptosis to pyroptosis

Progressive death of retinal pigment epithelium (RPE) cells is a hallmark of age-related macular degeneration (AMD), the leading cause of blindness in all developed countries. Photooxidative damage and activation of the NLRP3 inflammasome have been suggested as contributing factors to this process. We investigated the effects of inflammasome activation on oxidative damage-induced RPE cell death. In primary human RPE cells and ARPE-19 cells, lipofuscin accumulated following incubation with oxidatively modified photoreceptor outer segments. Oxidative stress was induced by blue light irradiation (dominant wavelength: 448nm, irradiance: 0.8mW/cm2, duration: 3 to 6h) of lipofuscin-loaded cells and resulted in cell death by apoptosis. Prior inflammasome priming by IL-1α or complement activation product C5a altered the cell death mechanism to pyroptosis and resulted in a significant increase of the phototoxic effect. Following IL-1α priming, viability 24h after irradiation was reduced in primary RPE cells and ARPE-19 cells from 65.3% and 56.7% to 22.6% (p=0.003) and 5.1% (p=0.0002), respectively. Inflammasome-mediated IL-1β release occurred only in association with pyroptotic cell lysis. Inflammasome priming by conditioned media of pyroptotic cells likewise increased cell death. Suppression of inflammasome activation by inhibition of caspase-1 or cathepsins B and L significantly reduced cell death in primed cells. In summary, inflammasome priming by IL-1α, C5a, or conditioned media of pyroptotic cells increases RPE cell susceptibility to photooxidative damage-mediated cell death and changes the mechanism of induced cell death from apoptosis to pyroptosis. This process may contribute to RPE degeneration in AMD and provide new targets for intervention.

Chloroquine and Hydroxychloroquine Are Novel Inhibitors of Human Organic Anion Transporting Polypeptide 1A2

Chloroquine (CQ) and hydroxychloroquine (HCQ) are widely used to treat malaria and inflammatory diseases, long-term usage of which often causes severe side effects, especially retinopathy. Solute carrier transporters (SLCs) are important proteins responsible for the cellular uptake of endogenous and exogenous substances. Inhibitors competing with transporter substrates for SLCs often results in unfavorable toxicities and unsatisfactory therapeutic outcomes. We investigated the inhibitory effect of CQ and HCQ on substrate uptake mediated through a range of important SLC transporters in overexpressing human embryonic kidney (HEK293) cells. Our data revealed that both CQ and HCQ potently inhibit the uptake activity of organic anion transporting polypeptide 1A2 (OATP1A2). We recently reported OATP1A2 to be expressed in human retinal pigment epithelium (RPE), where it mediates cellular uptake of all-trans-retinol (atROL), a key step in the classical visual cycle. In this study, we demonstrate that CQ and HCQ could markedly impair atROL uptake in OATP1A2-expressing HEK293 cells and more importantly, in primary human RPE cells. Our study shows that CQ and HCQ are novel inhibitors of OATP1A2 and significantly impair OATP1A2-mediated substrate uptake, particularly transport of atROL into the RPE. This effect may compromise the function of the classic visual cycle leading to vision impairment and contribute to the retinopathy observed clinically in patients using CQ or HCQ.

The novel triterpenoid RTA 408 protects human retinal pigment epithelial cells against H2O2-induced cell injury via NF-E2-related factor 2 (Nrf2) activation

Oxidative stress-induced retinal pigment epithelial (RPE) cell damage is an important factor in the pathogenesis of age-related macular degeneration (AMD). Previous studies have shown that RTA 408, a synthetic triterpenoid compound, potently activates Nrf2. This study aimed to investigate the protective effects of RTA 408 in cultured RPE cells during oxidative stress and to determine the effects of RTA 408 on Nrf2 and its downstream target genes. Primary human RPE cells were pretreated with RTA 408 and then incubated in 200μM H2O2 for 6h. Cell viability was measured with the WST-8 assay. Apoptosis was quantitatively measured by annexin V/propidium iodide (PI) double staining and Hoechst 33342 fluorescent staining. Reduced (GSH) and oxidized glutathione (GSSG) were measured using colorimetric assays. Nrf2 activation and its downstream effects on phase II enzymes were examined by Western blot. Treatment of RPE cells with nanomolar ranges (10 and 100nM) of RTA 408 markedly attenuated H2O2-induced viability loss and apoptosis. RTA 408 pretreatment significantly protected cells from oxidative stress-induced GSH loss, GSSG formation and decreased ROS production. RTA 408 activated Nrf2 and increased the expression of its downstream genes, such as HO-1, NQO1, SOD2, catalase, Grx1, and Trx1. Consequently, the enzyme activities of NQO1, Grx1, and Trx1 were fully protected by RTA 408 pretreatment under oxidative stress. Moreover, knockdown of Nrf2 by siRNA significantly reduced the cytoprotective effects of RTA 408. In conclusion, our data suggest that RTA 408 protect primary human RPE cells from oxidative stress-induced damage by activating Nrf2 and its downstream genes.

Complement Component C5a Primes Retinal Pigment Epithelial Cells for Inflammasome Activation by Lipofuscin-mediated Photooxidative Damage

Complement activation, oxidative damage, and activation of the NLRP3 inflammasome have been implicated in retinal pigment epithelium (RPE) pathology in age-related macular degeneration (AMD). Following priming of RPE cells, the NLRP3 inflammasome can be activated by various stimuli such as lipofuscin-mediated photooxidative damage to lysosomal membranes. We investigated whether products of complement activation are capable of providing the priming signal for inflammasome activation in RPE cells. We found that incubation of primary human RPE cells and ARPE-19 cells with complement-competent human serum resulted in up-regulation of C5a receptor, but not C3a receptor. Furthermore, human serum induced expression of pro-IL-1β and enabled IL-1β secretion in response to lipofuscin phototoxicity, thus indicating inflammasome priming. Complement heat-inactivation, C5 depletion, and C5a receptor inhibition suppressed the priming effect of human serum whereas recombinant C5a likewise induced priming. Conditioned medium of inflammasome-activated RPE cells provided an additional priming effect that was mediated by the IL-1 receptor. These results identify complement activation product C5a as a priming signal for RPE cells that allows for subsequent inflammasome activation by stimuli such as lipofuscin-mediated photooxidative damage. This molecular pathway provides a functional link between key factors of AMD pathogenesis including lipofuscin accumulation, photooxidative damage, complement activation, and RPE degeneration and may provide novel therapeutic targets in this disease.

Reduced Metabolic Capacity in Aged Primary Retinal Pigment Epithelium (RPE) is Correlated with Increased Susceptibility to Oxidative Stress.

One of the affected tissues in age-related macular degeneration (AMD) is the retinal pigment epithelium (RPE), a tissue that consists of terminally differentiated cells and that accumulates damage over time. In all tissues, mitochondria (mt), which play an essential role in both cell health (energy) and death (initiator of apoptosis), undergo an aging process through the accumulation of mtDNA damage, changes in mitochondrial dynamics, a reduction in biogenesis, and mitophagy, leading to an overall reduction in mitochondrial energy production and other non-energy-related functions. Here we have compared energy metabolism in primary human RPE cells isolated from aborted fetus or aged donor eyes and grown as stable monolayers. H2O2 treatment resulted in the generation of reactive oxygen species and superoxide, an effect that was significantly augmented by age. Mitochondrial metabolism, as analyzed by Seahorse respirometry, revealed reduced mitochondrial oxygen consumption (ATP production) at baseline and a complete loss of reserve capacity in aged cells. Likewise, glycolysis was blunted in aged cells. Taken together, these studies showed that RPE cells derived from aged donor eyes are more susceptible to oxidative stress, and exhibit a loss in mitochondrial respiratory reserve capacity and a reduction in glycolysis. These data suggest that while old cells may have sufficient energy at rest, they cannot mount a stress response requiring additional ATP and reducing agents. In summary, these data support the hypothesis that mitochondria or energy metabolism is a valid target for therapy in AMD.

Illumination from light-emitting diodes (LEDs) disrupts pathological cytokines expression and activates relevant signal pathways in primary human retinal pigment epithelial cells

Age-related macular degeneration (AMD) is the leading cause of blindness in the aged people. The latest systemic review of epidemiological investigations revealed that excessive light exposure increases the risk of AMD. With the drastically increasing use of high-energy light-emitting diodes (LEDs) light in our domestic environment nowadays, it is supposed to pose a potential oxidative threat to ocular health. Retinal pigment epithelium (RPE) is the major ocular source of pathological cytokines, which regulate local inflammation and angiogenesis. We hypothesized that high-energy LED light might disrupt the pathological cytokine expression of retinal pigment epithelium (RPE), contributing to the pathogenesis of AMD. Primary human RPE cells were isolated from eyecups of normal eye donors and seeded into plate wells for growing to confluence. Two widely used multichromatic white light-emitting diodes (LEDs) with correlated color temperatures (CCTs) of 2954 and 7378 K were used in this experiment. The confluent primary RPE cells were under white LEDs light exposure until 24 h. VEGF-A, IL-6, IL-8 and MCP-1 proteins and mRNAs were measured using an ELISA kit and RT-PCR, respectively. Activation of mitogen-activated protein kinases (MAPKs), Akt, Janus kinase (JAK)2 and Nuclear factor (NF)-κB signal pathways after LEDs illumination were evaluated by western blotting analysis. The level of reactive oxygen species (ROS) using chloromethyl- 2′,7′-dichlorodihydrofluorescein diacetate. Inhibitors of relevant signal pathways and anti-oxidants were added to the primary RPE cells before LEDs illumination to evaluate their biological functions. We found that 7378 K light, but not 2954 K upregulated the VEGF-A, IL-6, IL-8 and downregulated MCP-1 proteins and mRNAs levels in a time-dependent manner. In parallel, initial activation of MAPKs and NF-κB signal pathways were also observed after 7378 K light exposure. Mechanistically, antioxidants for eliminating reactive oxygen species (ROS) and targeted inhibitors of MAPKs and NF-κB significantly blocked 7378 K light-induced changes of specific cytokines, respectively. Our findings suggest that 7378 K light, not 2954 K induced upregulation of VEGF-A, IL-6, IL-8 and downregulation of MCP-1 via ROS accumulation, activating MAPKs and NF-κB signal pathways.

Transposon-Based, Targeted Ex Vivo Gene Therapy to Treat Age-Related Macular Degeneration (TargetAMD).

Transposon-Based, Targeted Ex Vivo Gene Therapy to Treat Age-Related Macular Degeneration (TargetAMD).

Glutaredoxin 1 (Grx1) Protects Human Retinal Pigment Epithelial Cells From Oxidative Damage by Preventing AKT Glutathionylation.

Glutaredoxin 1 (Grx1) belongs to the oxidoreductase family and is a component of the endogenous antioxidant defense system. However, its physiological function remains largely unknown. In this study, we investigated whether and how Grx1 overexpression protects the retinal pigment epithelial (RPE) cells against H2O2-induced apoptosis. Human retinal pigment epithelial (ARPE-19) cells were transfected with either a Grx1-containing plasmid or an empty vector. Primary human RPE cells were transfected with Grx1 small interfering RNA (siRNA) or scrambled siRNA. Cell viability was measured with the WST8 assay. Apoptosis was quantitatively measured by annexin V/propidium iodide (PI) double staining. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Protein kinase B (AKT) activation was examined by Western blot. Protein kinase B glutathionylation was detected by immunoprecipitation followed by immunoblotting with anti-PSSG antibody. Glutaredoxin 1 overexpression protected ARPE-19 cells from H2O2-induced cell viability loss. Conversely, Grx1 gene knockdown sensitized primary human RPE cells to H2O2. Assessment of apoptosis indicated that cells transfected with the Grx1-containing plasmid were more resistant to H2O2 with fewer cells undergoing apoptosis as compared to empty vector-transfected cells. Hydrogen peroxide-induced PSSG accumulation was also attenuated by Grx1 enrichment. Furthermore, Grx1 overexpression prevented H2O2-induced AKT glutathionylation, resulting in a sustained phospho-AKT elevation in RPE cells. Glutaredoxin 1 can protect RPE cells against oxidative stress-induced apoptosis. The mechanism of this protection is associated with its ability to stimulate the phosphorylation of AKT by preventing AKT glutathionylation. Considering Grx1's protective abilities in RPE cells, Grx1 could be a potential pharmacological target for retinal degenerative diseases.

Light induces NLRP3 inflammasome activation in retinal pigment epithelial cells via lipofuscin-mediated photooxidative damage.

Photooxidative damage and chronic innate immune activation have been implicated in retinal pigment epithelium (RPE) dysfunction, a process that underlies blinding diseases such as age-related macular degeneration (AMD). To identify a potential molecular link between these mechanisms, we investigated whether lipofuscin-mediated phototoxicity activates the NLRP3 inflammasome in RPE cells in vitro. We found that blue light irradiation (dominant wavelength 448 nm, irradiance 0.8 mW/cm2, duration 6 h) of lipofuscin-loaded primary human RPE cells and ARPE-19 cells induced photooxidative damage, lysosomal membrane permeabilization (79.5 % of cells vs. 3.8 % in nonirradiated controls), and cytosolic leakage of lysosomal enzymes. This resulted in activation of the inflammasome with activation of caspase-1 and secretion of interleukin-1β (14.6 vs. 0.9 pg/ml in nonirradiated controls) and interleukin-18 (87.7 vs. 0.2 pg/ml in nonirradiated controls). Interleukin secretion was dependent on the activity of NLRP3, caspase-1, and lysosomal proteases cathepsin B and L. These results demonstrate that accumulation of lipofuscin-like material in vitro renders RPE cells susceptible to phototoxic destabilization of lysosomes, resulting in NLRP3 inflammasome activation and secretion of inflammatory cytokines. This new mechanism of inflammasome activation links photooxidative damage and innate immune activation in RPE pathology and may provide novel targets for therapeutic intervention in retinal diseases such as AMD.Key message• Visible light irradiation of lipofuscin-loaded RPE cells activates inflammasome.• Inflammasome activation results from lysosomal permeabilization and enzyme leakage.• Inflammasome activation induces secretion of inflammatory cytokines by RPE cells.• Photooxidative damage by visible light as new mechanism of inflammasome activation.• Novel link between hallmark pathogenetic features of retinal degenerative diseases.