Protective effects of cynaroside on oxidative stress in retinal pigment epithelial cells

Cynaroside is a flavonoid compound proved to possess antioxidant activity, but its protective effect on age‐related macular degeneration still remains unclear. In this study, the protective effects of cynaroside on oxidative stress and apoptosis in retinal pigment epithelial (RPE) cells induced by hydrogen peroxide (H₂O₂) were investigated. Results showed that cynaroside effectively attenuated the decrease of cell activity induced by H₂O₂. The total reactive oxygen species can be remitted by decreasing malondialdehyde level, as well as increasing glutathione level, and superoxide dismutase and catalase activities. In addition, Western blot analysis indicated that cynaroside protected ARPE‐19 cells from apoptosis through downregulation of caspase‐3 protein activation which was controlled by the upstream proteins Bcl‐2 and Bax. It was finally proved that cynaroside could enhance the antioxidant and antiapoptotic ability in ARPE‐19 cells by promoting the expression of p‐Akt.     

Human retinal pigment epithelial cells

''Human retinal pigment epithelial cells'' is the first set of guidelines on human retinal pigment epithelial cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies technical requirements, test methods, inspection rules, instructions for usage, labelling requirements, packaging requirements, storage requirements and transportation requirements and waste disposal requirements for human retinal pigment epithelial cells, which is applicable to quality control during the process of manufacturing and testing of human retinal pigment epithelial cells. It was originally released by the Chinese Society for Cell Biology on 9 January 2021. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols and accelerate the international standardization of human retinal pigment epithelial cells for applications.     

Potassium currents in cultured rabbit retinal pigment epithelial cells

Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26±12 pF (sd, n=92). The resting membrane potential averaged ?31±15 mV (n=37), but it was as high as ?60 mV in some cells. When K⁺ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K⁺ selective. The most frequently observed outward K⁺ current was a voltage- and time-dependent outward current (I _K) which resembled the delayed rectifier K⁺ current described in other cells. I _K was blocked by tetraethylammonium ions (TEA) and barium (Ba²⁺) and reduced by 4-aminopyridine (4-AP). In a few cells (3–4%), depolarization to ?50 mV or more negative potentials evoked an outwardly rectifying K⁺ current (I _Kt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K⁺ current (I _KI) was also present in 41% of cells. I _KI was blocked by extracellular Ba²⁺ or Cs⁺ and exhibited time-dependent decay, due to Na⁺ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K⁺ currents. The K⁺ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space.     

Hydroxytyrosol protects retinal pigment epithelial cells from acrolein-induced oxidative stress and mitochondrial dysfunction

Hydroxytyrosol (HTS) is a natural polyphenol abundant in olive oil. Increasing evidence indicates HTS has beneficial effect on human health for preventing various diseases. In the present study, we investigated the protective effects of HTS on acrolein-induced toxicity in human retinal pigment epithelial cell line, ARPE-19, a cellular model of smoking- and age-related macular degeneration. Acrolein, a major component of the gas phase cigarette smoke and also a product of lipid peroxidation in vivo , at 75 μmol/L for 24 h caused significant loss of cell viability, oxidative damage (increase in oxidant generation and oxidative damage to proteins and DNA, decrease in antioxidants and antioxidant enzymes, and also inactivation of the Keap1/Nrf2 pathway), and mitochondrial dysfunction (decrease in membrane potential, activities of mitochondrial complexes, viable mitochondria, oxygen consumption, and factors for mitochondrial biogenesis, and increase in calcium). Pre-treatment with HTS dose dependently and also time dependently protected the ARPE-19 cells from acrolein-induced oxidative damage and mitochondrial dysfunction. A short-term pre-treatment with HTS (48 h) required > 75 μmol/L for showing protection while a long-term pre-treatment (7 days) showed protective effect from 5 μmol/L on. The protective effect of HTS in this model was as potent as that of established mitochondria-targeting antioxidant nutrients. These results suggest that HTS is also a mitochondrial-targeting antioxidant nutrient and that dietary administration of HTS may be an effective measure in reducing and or preventing cigarette smoke-induced or age-related retinal pigment epithelial degeneration, such as age-associated macular degeneration.     

Lysosome-associated membrane protein-2 deficiency increases the risk of reactive oxygen species-induced ferroptosis in retinal pigment epithelial cells

Lysosome-associated membrane protein-2 (LAMP2), is a highly glycosylated lysosomal membrane protein involved in chaperone mediated autophagy. Mutations of LAMP2 cause the classic triad of myopathy, cardiomyopathy and encephalopathy of Danon disease (DD). Additionally, retinopathy has also been observed in young DD patients, leading to vision loss. Emerging evidence show LAMP2-deficiency to be involved in oxidative stress (ROS) but the mechanism remains obscure. In the present study, we found that tert-butyl hydroperoxide or antimycin A induced more cell death in LAMP2 knockdown (LAMP2-KD) than in control ARPE-19 cells. Mechanistically, LAMP2-KD reduced the concentration of cytosolic cysteine, resulting in low glutathione (GSH), inferior antioxidant capability and mitochondrial lipid peroxidation. ROS induced RPE cell death through ferroptosis. Inhibition of glutathione peroxidase 4 (GPx4) increased lethality in LAMP2-KD cells compared to controls. Cysteine and glutamine supplementation restored GSH and prevented ROS-induced cell death of LAMP2-KD RPE cells.     

Lycopene inhibits PDGF-BB-induced retinal pigment epithelial cell migration by suppression of PI3K/Akt and MAPK pathways

Retinal pigment epithelial (RPE) cells play a dominant role in the development of proliferative vitreoretinopathy (PVR), which is the leading cause of failure in retinal reattachment surgery. Several studies have shown that platelet-derived growth factor (PDGF) exhibits chemotaxis and proliferation effects on RPE cells in PVR. In this study, the inhibitory effect of lycopene on PDGF-BB-induced ARPE19 cell migration is examined. In electric cell-substrate impedance sensing (ECIS) and Transwell migration assays, significant suppression of PDGF-BB-induced ARPE19 cell migration by lycopene is observed. Cell viability assays show no cytotoxicity of lycopene on RPE cells. Lycopene shows no effect on ARPE19 cell adhesion and is found to inhibit PDGF-BB-induced tyrosine phosphorylation and the underlying signaling pathways of PI3K, Akt, ERK and p38 activation. However, PDGF-BB and lycopene show no effects on JNK activation. Taken together, our results demonstrate that lycopene inhibits PDGF-BB-induced ARPE19 cell migration through inhibition of PI3K/Akt, ERK and p38 activation.     

Bioengineered Bruch's-like extracellular matrix promotes retinal pigment epithelial differentiation

In the eye, the retinal pigment epithelium (RPE) adheres to a complex protein matrix known as Bruch's membrane (BrM). The aim of this study was to provide enriched conditions for RPE cell culture through the production of a BrM-like matrix. Our hypothesis was that a human RPE cell line would deposit an extracellular matrix (ECM) resembling BrM. The composition and structure of ECM deposited by ARPE19 cells (ARPE19-ECM) was characterized. To produce ARPE19-ECM, ARPE19 cells were cultured in the presence dextran sulphate. ARPE19-ECM was decellularized using deoxycholate and characterized by immunostaining and western blot analysis. Primary human RPE and induced pluripotent stem cells were seeded onto ARPE19-ECM or geltrex coated surfaces and examined by microscopy or RT-PCR. Culture of ARPE19 cells with dextran sulphate promoted nuclear localization of SOX2, formation of tight junctions and deposition of ECM. ARPE19 cells deposited ECM proteins found in the inner layers of BrM, including fibronectin, vitronectin, collagens IV and V as well as laminin-alpha-5, but not those found in the middle elastic layer (elastin) or the outer layers (collagen VI). ARPE19-ECM promoted pigmentation in human RPE and pluripotent stem cell cultures. Expression of RPE65 was significantly increased on ARPE19-ECM compared with geltrex in differentiating pluripotent stem cell cultures. ARPE19 cells deposit ECM with a composition and structure similar to BrM in the retina. Molecular cues present in ARPE19-ECM promote the acquisition and maintenance of the RPE phenotype. Together, these results demonstrate a simple method for generating a BrM-like surface for enriched RPE cell cultures.     

Neutral sphingomyelinase inhibition decreases ER stress-mediated apoptosis and inducible nitric oxide synthase in retinal pigment epithelial cells

Endoplasmic reticulum (ER) stress and excessive nitric oxide production via the induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of ocular diseases characterized by retinal degeneration. Previous studies have revealed the sphingomyelinase/ceramide pathway in the regulation of NOS2 induction. Thus, the objective of this study was to determine the activity of the sphingomyelinase/ceramide pathway, assess nitric oxide production, and examine apoptosis in human retinal pigment epithelial (RPE) cells undergoing ER stress. Sphingomyelinase (SMase) activity; nuclear factor κB (NF-κB) activation; NOS2, nitrite/nitrate, and nitrotyrosine levels; and apoptosis were determined in cultured human RPE cell lines subjected to ER stress via exposure to tunicamycin. Induction of ER stress was confirmed by increased intracellular levels of ER stress markers including phosphorylated PKR-like ER kinase, C/EBP-homologous protein, and 78-kDa glucose-regulated protein. ER stress increased nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, and nitrotyrosine formation and caused apoptosis in RPE cell lines. Inhibition of neutral SMase (N-SMase) activity via GW 4869 treatment caused a significant reduction in nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, nitrotyrosine formation, and apoptosis in ER-stressed RPE cells. In conclusion, N-SMase inhibition reduced nitrative stress and apoptosis in RPE cells undergoing ER stress. Obtained data suggest that NOS2 can be regulated by N-SMase in RPE cells experiencing ER stress.     

Glucose deprivation induces mitochondrial dysfunction and oxidative stress in PC12 cell line

Glucose metabolism plays a pivotal role in many physiological and pathological conditions. To investigate the effect of hypoglycemia (obtained by glucose deprivation) on PC12 cell line, we analyzed the cell viability, mitochondrial function (assessed by MTT reduction, cellular ATP level, mitochondrial transmembrane potential), and the level of reactive oxygen species (ROS) after glucose deprivation (GD). Upon exposure to GD, ROS level increased and MTT reduction decreased immediately, intracellular ATP level increased in the first 3 hours, followed by progressive decrease till the end of GD treatment, and the mitochondrial transmembrane potential (ΔΨ_m) dropped after 6 hours. Both necrosis and apoptosis occurred apparently after 24 hours which was determined by nuclei staining with propidium iodide(PI) and Hoechst 33342. These data suggested that cytotoxity of GD is mainly due to ROS accumulation and ATP depletion in PC12 cells.     

Long noncoding RNA BDNF‐AS inversely regulated BDNF and modulated high‐glucose induced apoptosis in human retinal pigment epithelial cells

In this study, we characterized the functional role of long noncoding RNA (lncRNA), brain derived neurotrophic factor anti‐sense (BDNF‐AS) in regulating D‐glucose‐induced (DGI) apoptosis in human retinal pigment epithelial (RPE) cells. Human RPE cell line, ARPE‐19 cells were cultured in vitro and treated with various concentrations of D‐glucose for 24 h. A TUNEL assay was applied with immunohistochemical and quantitative approaches to assess the apoptotic effect of D‐glucose. Under the condition of 50 mM D‐glucose, qPCR was used to assess gene expression of BDNF and BDNF‐AS in ARPE‐19 cells. Using siRNA transfection, BDNF‐AS was endogenously knocked down in ARPE‐19 cells. The effects of BDNF‐AS downregulation on DGI apoptosis and BDNF expression were assessed by TUNEL assay, qPCR, and Western blot, respectively. Furthermore, in BDNF‐AS‐downregulated ARPE‐19 cells, secondary siRNA transfection was conducted to knock down endogenous BDNF expression. Its effect on BDNF‐AS‐associated apoptotic regulation was further evaluated. High concentrations of D‐glucose induced significant apoptosis in ARPE‐19 cells in vitro. With treatment of 50 mM D‐glucose, BDNF was markedly downregulated whereas BDNF‐AS upregulated in ARPE‐19 cells. SiRNA‐mediated BDNF‐AS downregulation ameliorated DGI apoptosis and upregulated BDNF in ARPE‐19 cells. In addition, inhibiting BDNF reversed the protective effect of BDNF‐AS downregulation on DGI apoptosis. Our results suggest that BDNF‐AS, through inverse regulation of BDNF, might play a critical role in the process of DGI apoptosis in diabetic retinopathy.     

Erythropoietin protects retinal pigment epithelial cells against the increase of permeability induced by diabetic conditions: essential role of JAK2/ PI3K signaling

The outer blood-retinal barrier is formed by retinal pigment epithelial (RPE) cells and its disruption significantly contributes to the development of diabetic macular edema (DME). The aim of the study was to explore whether erythropoietin (Epo) has beneficial effects on the barrier function of human RPE cells and the main downstream pathways involved. ARPE-19 cells were cultured in standard conditions and under conditions leading to the disruption of the monolayer [25 mmol/L d-glucose plus IL-1β (10 ng/mL)]. Epo (200 mU/mL/day) was added during the last 2 days of the experiment. The experiments were repeated in the presence of an Epo neutralizing antibody and specific inhibitors of JAK2 and PI3K (AG490 and LY294002, respectively). Permeability was evaluated by fluorescein isothiocyanate dextran (70 kDa) movements. Distribution of tight junction proteins was examined by immunofluorescence. Changes in cytosolic Ca²⁺ induced by Epo were also measured. Epo treatment was able to prevent but not to restore the increase of permeability induced by high glucose plus IL-1β. The protective effect of Epo on RPE barrier function was completely blocked by AG490 and almost completely abolished by LY294002. In addition, Epo was able to increase cytosolic Ca²⁺ with dependence on extracellular calcium influx and this effect was blocked by either JAK2 or PI3K inhibition. We conclude that RPE disruption induced by high glucose plus IL-1β is prevented by Epo through the downstream signaling of JAK2 and PI3K/AKT pathways.     

Lipoamide protects retinal pigment epithelial cells from oxidative stress and mitochondrial dysfunction

alpha-Lipoic acid (LA) has been widely studied as an agent for preventing and treating various diseases associated with oxidative disruption of mitochondrial functions. To investigate a related mitochondrial antioxidant, we compared the effects of lipoamide (LM), the neutral amide of LA, with LA for measures of oxidative damage and mitochondrial dysfunction in a human retinal pigment epithelial (RPE) cell line. Acrolein, a major component of cigarette smoke and a product of lipid peroxidation, was used to induce oxidative mitochondrial damage in RPE cells. Overall, using comparable concentrations, LM was more effective than LA at preventing acrolein-induced mitochondrial dysfunction and oxidative stress. Relative to LA, LM improved ATP levels, membrane potentials, and activities of mitochondrial complexes I, II, and V and dehydrogenases that had been decreased by acrolein exposure. LM reduced acrolein-induced oxidant generation, calcium levels, protein oxidation, and DNA damage to a greater degree than LA. And, total antioxidant capacity, glutathione content, glutathione S-transferase, and superoxide dismutase activities and expression of nuclear factor-E2-related factor 2 were increased by LM relative to LA. These results suggest that LM is a more potent mitochondrial-protective agent and antioxidant than LA in protecting RPE from oxidative damage.     

Oxidative stress induces Z-DNA-binding protein 1–dependent activation of microglia via mtDNA released from retinal pigment epithelial cells

Oxidative stress, inflammation, and aberrant activation of microglia in the retina are commonly observed in ocular pathologies. In glaucoma or age-related macular degeneration, the chronic activation of microglia affects retinal ganglion cells and photoreceptors, respectively, contributing to gradual vision loss. However, the molecular mechanisms that cause activation of microglia in the retina are not fully understood. Here we show that exposure of retinal pigment epithelial (RPE) cells to chronic low-level oxidative stress induces mitochondrial DNA (mtDNA)-specific damage, and the subsequent translocation of damaged mtDNA to the cytoplasm results in the binding and activation of intracellular DNA receptor Z-DNA-binding protein 1 (ZBP1). Activation of the mtDNA/ZBP1 pathway triggers the expression of proinflammatory markers in RPE cells. In addition, we show that the enhanced release of extracellular vesicles (EVs) containing fragments of mtDNA derived from the apical site of RPE cells induces a proinflammatory phenotype of microglia via activation of ZBP1 signaling. Collectively, our report establishes oxidatively damaged mtDNA as an important signaling molecule with ZBP1 as its intracellular receptor in the development of an inflammatory response in the retina. We propose that this novel mtDNA-mediated autocrine and paracrine mechanism for triggering and maintaining inflammation in the retina may play an important role in ocular pathologies. Therefore, the molecular mechanisms identified in this report are potentially suitable therapeutic targets to ameliorate development of ocular pathologies.     

Oxidative stress in retinal pigment epithelium cells increases exosome secretion and promotes angiogenesis in endothelial cells

The retinal pigment epithelium (RPE), a monolayer located between the photoreceptors and the choroid, is constantly damaged by oxidative stress, particularly because of reactive oxygen species (ROS). As the RPE, because of its physiological functions, is essential for the survival of the retina, any sustained damage may consequently lead to loss of vision. Exosomes are small membranous vesicles released into the extracellular medium by numerous cell types, including RPE cells. Their cargo includes genetic material and proteins, making these vesicles essential for cell‐to‐cell communication. Exosomes may fuse with neighbouring cells influencing their fate. It has been observed that RPE cells release higher amounts of exosomes when they are under oxidative stress. Exosomes derived from cultured RPE cells were isolated by ultracentrifugation and quantified by flow cytometry. VEGF receptors (VEGFR) were analysed by both flow cytometry and Western blot. RT‐PCR and qPCR were conducted to assess mRNA content of VEGFRs in exosomes. Neovascularization assays were performed after applying RPE exosomes into endothelial cell cultures. Our results showed that stressed RPE cells released a higher amount of exosomes than controls, with a higher expression of VEGFR in the membrane, and enclosed an extra cargo of VEGFR mRNA. Angiogenesis assays confirmed that endothelial cells increased their tube formation capacity when exposed to stressed RPE exosomes.     

Involvement of STIM1 and Orai1 in EGF-mediated cell growth in retinal pigment epithelial cells

Background

In non-excitable cells, one major route for calcium entry is through store-operated calcium (SOC) channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca²⁺ store. STIM1 and Orai1 are major regulators of SOC channels. In this study, we explored the functions of STIM1 and Orai1 in epidermal growth factor (EGF)-induced cell proliferation and migration in retinal pigment epithelial cells (ARPE-19 cell line).

Results

EGF triggers cell proliferation and migration in ARPE-19 cells. Cell proliferation and migration involve STIM1 and Orai1, as well as phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2, and Akt. Pharmacological inhibitors of SOC channels and siRNA of Orai1 and STIM1 suppress cell proliferation and migration. Pre-treatment of mitogen-activated protein kinase kinase (MEK) inhibitors and a phosphatidylinositol 3 kinases (PI3K) inhibitor attenuated cell proliferation and migration. However, inhibition of the SOC channels failed to prevent EGF-mediated ERK 1/2 and Akt phosphorylation.

Conclusions

Our results showed that STIM1, Orai1, ERK 1/2, and Akt are key determinants of EGF-mediated cell growth in ARPE-19 cells. EGF is a potent growth molecule that has been linked to the development of PVR, and therefore, STIM1, Orai1, as well as the MEK/ERK 1/2 and PI3K/Akt pathways, might be potential therapeutic targets for drugs aimed at treating such disorders.