Retinal Pigment Epithelium Cell Damage Research Articles

Eupatilin prevents H2O2-induced oxidative stress and apoptosis in human retinal pigment epithelial cells.

Eupatilin, a pharmacologically active flavone derived from the Artemisia plant species, is known to possess anti-oxidant activity. However, the effects of eupatilin on oxidative stress-induced retinal damage in retinal pigment epithelium (RPE) cells and the potential mechanisms involved have not been explored. Therefore, the aim of this study was to investigate the effects of eupatilin on oxidative stress-induced retinal damage in RPE cells. Our results showed that eupatilin significantly attenuated H2O2-induced cell injury and ROS production in ARPE-19 cells. In addition, eupatilin pretreatment greatly upregulated Bcl-2 expression, downregulated Bax expression, as well as suppressed caspase-3 activity in ARPE-19 cells exposed to H2O2. Furthermore, eupatilin pretreatment markedly enhanced phosphorylation levels of PI3K and Akt in ARPE-19 cells exposed to H2O2. In conclusion, our data showed that eupatilin protected against H2O2-induced oxidative stress and apoptosis through the activation of PI3K/Akt signaling pathway in ARPE-19 cells. Thus, eupatilin may be useful for the prevention or treatment of proliferative vitreoretinopathy (PVR).

The Protective Effect of Brown-, Gray-, and Blue-Tinted Lenses against Blue LED Light-Induced Cell Death in A2E-Laden Human Retinal Pigment Epithelial Cells

A2E-laden ARPE-19 cells were exposed to a blue light to induce cytotoxicity, in order to investigate the protective effects of various tinted ophthalmic lenses against photo-induced cytotoxicity in human retinal pigment epithelial (RPE) cells laden with A2E, known to be among the etiologies of age-related macular degeneration (AMD). Different-colored tinted lenses with varying levels of tint and different filtering characteristics, such as polarized, blue-cut, and photochromatic lenses, were placed over the cells, and the protective efficacies thereof were evaluated by lactate dehydrogenase assay. When tinted lenses were placed over ARPE-19 cells, there were different reductions in cytotoxicity according to the colors and tint levels. The level of protection afforded by brown-tinted lenses was 6.9, 36.1, and 49% with a tint level of 15, 50, and 80%, respectively. For gray-tinted lenses, the protective effect was 16.3, 35, and 43.4% for the corresponding degree of tint, respectively. In the case of blue-tinted lenses, a protective effect of 20% was observed with 80% tinted lenses, but 15 and 50% tinted lenses provided no significant protection. In addition, photochromic lenses showed a protective effect but blue-cut lenses and polarized lenses provided no significant protection. Tinted lenses significantly reduced cytotoxicity in RPE cells irradiated with blue light. The protection was more efficient in lenses with a brown or gray tint than in blue-tinted lenses. Tinted glasses may provide significant protection against potential blue-light-induced photochemical and photo-oxidative damage in RPE cells.

Lycopene inhibits ICAM-1 expression and NF-κB activation by Nrf2-regulated cell redox state in human retinal pigment epithelial cells

AimsAge-related macular degeneration (AMD) is one of the most common diseases leading to blindness in elderly people. The progression of AMD may be prevented through anti-inflammation and antioxidation in retinal pigment epithelium (RPE) cells. Lycopene, a carotenoid, has been shown to possess both antioxidative and anti-inflammatory properties. This research was conducted to detail the mechanisms of these effects of lycopene-treated RPE cells. Main methodsWe exposed ARPE-19 cells to TNFα after pretreatment with lycopene, and measured monocyte adhesion, ICAM-1 expression, NF-κB nuclear translocation, and transcriptional activity. Cell viability was assayed with Alamar Blue. The cell redox state was tested by glutathione (GSH) and reactive oxygen species (ROS) levels. The importance of the Nrf2 pathway was tested in nuclear translocation, promoter reporter assay, and siRNA. Key findingsLycopene could reduce TNF-α-induced monocyte adhesion and H2O2– induced cell damage in RPE cells. Furthermore, lycopene inhibits ICAM-1 expression and abolishes NF-κB activation for up to 12h in TNFα-treated RPE cells. Lycopene upregulates Nrf2 levels in nuclear extracts and increases the transactivity of antioxidant response elements. The use of Nrf2 siRNA blocks the inhibitory effect of lycopene in TNF-α-induced ICAM-1 expression and NF-κB activation. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the de novo synthesis of GSH. We found that lycopene increases intracellular GSH levels and GCL expression. Following lycopene treatment, TNF-α-induced ROS production was abolished. SignificanceThe Nrf2-regulated antioxidant property plays a pivotal role in the anti-inflammatory mechanism underlying the inhibition of NF-κB activation in lycopene-treated ARPE-19 cells.

Protective Effects of Resveratrol against UVA‐induced Damage in ARPE19 Cells

Exposure to solar ultraviolet (UV) radiation is believed to induce the production of reactive oxygen species (ROS) that may cause oxidative stress to retinal pigment epithelial (RPE) cells; oxidative injury to the retinal pigment epithelium has been implicated to play a contributory role in age‐related macular degeneration (AMD). Prior studies have suggested that resveratrol, an abundant and active component of red grapes, can protect several cell types from oxidative stress. This study is to evaluate if resveratrol can protect RPE cells from UVA induced damage, and be useful in the prevention of early AMD. Cell viability of adult RPE cells pretreated with different concentrations of resveratrol was determined using cell viability assay after UVA radiation. Intracellular H2O2 levels were measured by flow cytometry, and UVA induced signaling pathways were detected using Western blotting. Results show that resveratrol inhibited UVA‐induced RPE cell death. The generation of intracellular H2O2 induced by UVA irradiation in RPE cells was suppressed by resveratrol in a concentration‐dependent manner. In addition, resveratrol inhibited the activation of UVA‐induced extracellular signal‐regulated kinase, c‐jun‐NH2 terminal kinase and p38 kinase in RPE cells. There was also a reduction in UVA‐induced cyclooxygenase‐2 (COX‐2) expression in RPE cells treated with resveratrol. This study suggests that resveratrol is effective in preventing UVA‐induced damage in RPE cells and may be suitable for further developments as a chemoprotective factor for the primary prevention of early AMD.

PRMT1 and PRMT4 Regulate Oxidative Stress-Induced Retinal Pigment Epithelial Cell Damage in SIRT1-Dependent and SIRT1-Independent Manners.

Oxidative stress-induced retinal pigment epithelial (RPE) cell damage is involved in the progression of diabetic retinopathy. Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) has emerged as an important histone modification involved in diverse diseases. Sirtuin (SIRT1) is a protein deacetylase implicated in the onset of metabolic diseases. Therefore, we examined the roles of type I PRMTs and their relationship with SIRT1 in human RPE cells under H2O2-induced oxidative stress. H2O2 treatment increased PRMT1 and PRMT4 expression but decreased SIRT1 expression. Similar to H2O2 treatment, PRMT1 or PRMT4 overexpression increased RPE cell damage. Moreover, the H2O2-induced RPE cell damage was attenuated by PRMT1 or PRMT4 knockdown and SIRT1 overexpression. In this study, we revealed that SIRT1 expression was regulated by PRMT1 but not by PRMT4. Finally, we found that PRMT1 and PRMT4 expression is increased in the RPE layer of streptozotocin-treated rats. Taken together, we demonstrated that oxidative stress induces apoptosis both via PRMT1 in a SIRT1-dependent manner and via PRMT4 in a SIRT1-independent manner. The inhibition of the expression of type I PRMTs, especially PRMT1 and PRMT4, and increased SIRT1 could be therapeutic approaches for diabetic retinopathy.

Effect of leptin on the oxidative damage in human retinal pigment epithelial cells

Objective To investigate the effects of leptin on the oxidative damage in human retinal pigment epithelial (RPE) cells.Methods Human RPE cells (ARPE-19) were cultured in vitro,and randomly divided into control group and insulin resistance group.RPE cells were treated with 0,10,100 ng/mL leptin for 24,48,72 hours respectively.Then the levels of reactive oxygen species (ROS) expression in RPE cells were detected by 2',7'-dichlorofluorescin-diacetate (DCFH-DA),and the levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) expression in RPE cells were observed by immunocytochemistry (ICC),and the levels of human 8-oxoguanine DNA glycosylase 1 (hOGG1) expression in lysate were measured by Western blot.Results After 24,48,72 hours,the level of ROS (Control group:F=37.136,37.178,49.634; P＜0.05.Insulin resistance group:F=9.822,28.881,71.150;P＜0.05),8-OHdG (Control group:F =88.643,390.920,1039.276; P ＜ 0.05.Insulin resistance group:F =273.311,299.155,82.237;P＜0.05) and hOGGl (Control group:F=470.062,1073.113,295.456;P＜0.05.Insulin resistance group:F =240.032,592.389,527.760 ; P＜0.05) expression increased significantly with the increase of leptin concentration in control group and insulin resistance group.Under the same leptin concentration,the level of 8-OHdG has a trend that it was higher in the insulin resistance group than the control group.After 24 hours,the difference of hOGGl expression between control group and insulin resistance group was not significant (F=23.392,P＞0.05).After 72 hours,the level of hOGGl expression was significantly higher in the insulin resistance group than the control group (F=129.394,P＜0.05).The level of hOGGl expression was significantly higher at 48 hours than that at 24 hours and 72 hours (P＜ 0.05).Conclusion Leptin could induce the oxidative damage of RPE cells in normal and insulin resistance status.With the increase of leptin concentration and time extended,the degree of oxidative damage and its repair were both increased.The degree of oxidative repair increased with the increase of leptin concentration,but decreased with time extended. Key words: Retinal pigment epithelium/injuries; Leptin/poisoning; Reactive oxygen species/poisoning

Expression of Endoplasmic Reticulum Stress Markers GRP78 and CHOP Induced by Oxidative Stress in Blue Light-Mediated Damage of A2E-Containing Retinal Pigment Epithelium Cells

Aims: Age-related lipofuscin N-retinylidene-N-retinylethanolamine (A2E) accumulated in human retinal pigment epithelium (RPE) cells confers susceptibility to blue light-mediated damage, which represents one pathogenesis of age-related macular degeneration. This study investigated the expression of 2 best-characterized endoplasmic reticulum (ER) stress markers, glucose-related protein 78 (GRP78) and C/EBP homologous protein (CHOP), as well as their regulation by oxidative stress after blue light-mediated damage of A2E-containing RPE cells. Methods: ARPE-19 cells were incubated with A2E (10, 25, 50 μ<smlcap>M</smlcap>) for 2 h and exposed to blue light for 20 min. A2E distributions in RPE cells were assessed via laser scanning confocal microscope and liquid chromatography-mass spectrometry. Cell viability was measured by a Cell Titer 96 Aqueous One Solution cell proliferation assay. The quantity of intracellular reactive oxygen species (ROS) was detected by dihydroethidium fluorescence using flow cytometry. Expressions of GRP78 and CHOP were measured at both mRNA and protein levels. To examine the role of oxidative stress in regulating GRP78 and CHOP expression, RPE cells were pretreated with the antioxidant N-acetylcysteine (NAC) for 2 h. RNA interference of GRP78 performed by short hairpin RNA was used to evaluate the effect of GRP78 in blue light-mediated damage of RPE cells. Results: After blue light exposure, A2E-treated RPE cells showed a gradual decrease in cell viability and a particular increase in ROS levels. Meanwhile, the expressions of GRP78 and CHOP in A2E-treated RPE cells were significantly increased at different time points after illumination. Pretreatment with NAC attenuated the expression of 2 ER stress markers, especially CHOP in A2E and blue light-treated RPE cells. Silencing of GRP78 by RNA interference upregulated CHOP and caspase-12 expression as well as aggravated the blue light-mediated damage of A2E-laden RPE cells. Conclusion: RPE cells exhibited ROS accumulation and subsequent elevation of GRP78 and CHOP expression after A2E and blue light-induced damage. The ROS scavenger NAC diminished ER stress protein expression, suggesting a connection between ER and oxidative stress in blue light-mediated damage of A2E-containing RPE cells. Besides, GRP78 may play a protective role in it.

Inhibition of autophagy induces retinal pigment epithelial cell damage by the lipofuscin fluorophore A2E

In this study, we show augmented autophagy in the retinal pigment epithelial cell line ARPE-19 when cultured in the presence of the lipofuscin pigment A2E. A2E alone does not induce RPE cell death, but cell death was induced in the presence of A2E with the autophagy inhibitor 3-methyladenine (3MA), with a concomitant increase in the generation of mitochondrial reactive oxygen species. On the other hand, the ATP production capacity of mitochondria was decreased in the presence of A2E, and pharmacological inhibition of autophagy had no additional effects. The altered mRNA expression level of mitochondrial function markers was confirmed by real-time polymerase chain reaction, which showed that the antioxidant enzymes SOD1 and SOD2 were not reduced in the presence of A2E alone, but significantly suppressed with the addition of 3MA. Furthermore, transmission electron micrography revealed autophagic vacuole formation in the presence of A2E, and inhibition of autophagy resulted in the accumulation of abnormal mitochondria with loss of cristae. Spheroid culture of human RPE cells demonstrated debris accumulation in the presence of A2E, and this accumulation was accelerated in the presence of 3MA. These results indicate that autophagy in RPE cells is a vital cytoprotective process that prevents the accumulation of damaged cellular molecules.

Alpha-melanocyte stimulating hormone protects retinal pigment epithelium cells from oxidative stress through activation of melanocortin 1 receptor–Akt–mTOR signaling

Patients with age related macular degeneration (AMD) will develop vision loss in the center of the visual field. Reactive oxygen species (ROS)-mediated retinal pigment epithelium (RPE) cell apoptosis is an important contributor of AMD. In this study, we explored the pro-survival effect of α-melanocyte stimulating hormone (α-MSH) on oxidative stressed RPE cells. We found that α-MSH receptor melanocortin 1 receptor (MC1R) was functionally expressed in primary and transformed RPE cells. RPE cells were response to α-MSH stimulation. α-MSH activated Akt/mammalian target of rapamycin (mTOR) and Erk1/2 signalings in RPE cells, which were inhibited by MC1R siRNA knockdown. α-MSH protected RPE cells from hydrogen peroxide (H2O2)-induced apoptosis, an effect that was almost abolished when MC1R was depleted by siRNA. α-MSH-mediated S6K1 activation and pro-survival effect against H2O2 was inhibited by Akt inhibitors (perifosine, MK-2206 and LY294002). Further, mTOR inhibition by rapamycin, or by mTOR siRNA knockdown, diminished α-MSH’s pro-survival effect in RPE cells. Thus, Akt and its downstream mTOR signaling mediates α-MSH-induced survival in RPE cells. In summary, we have identified a new α-MSH–MC1R physiologic pathway that reduces H2O2-induced RPE cell damage, and might minimize the risk of developing AMD.

The association of blue light-induced human retinal pigment epithelium cell damage with intercellular free Ca2+ change in vitro

Background Investigating the association of blue light-induced damage of retinal pigmenepithelial (RPE) cellwith intracellulaCa2+ conteniimportanfounderstanding the mechanism of retinal disorders.Objective Thistudy wato establish blue light-induced damage model of human RPE celland explore the relationship between the damage of RPE cell and intracellulaCa2+ content.MethodHuman RPE cellwere cultured and passaged.Cell vitality waassayed by trypan blue staining.Fourth-generation cellwere used in these experiments.The cellwere exposed to blue lighwith an intensity of (2000±500)lx fo3,6,9 o12 hours,and the rate of apoptosiwaassayed by TUNEL to assesthe optimal irradiation time focellcultured.The cellwere then randomized into the withouirradiation group,irradiation only group,nifedipine group,ligh+ nifedipine group,(-) BayK8644 group and ligh+ (-) BayK8644 group.The laseconfocal microscope waused to determine the fluorescence intensity of intracellulafree Ca2+ aan excitation wavelength of 488 nm and an emission wavelength of 505 nm.The cell imagewere analyzed using computesoftware.The differenceof fluorescence intensity among the differengroupwere compared by one-way analysiof variance.ResultTrypan blue staining showed thathe viability of RPE cellwamore than 90％ afteculturing and passaging.No apoptoticell waseen aftelighexposure fo3 hours.However,differennumberof apoptoticellappeared aftelighexposure fo6,9 and 12 hours.The fluorescence intensity of intracellulafree Ca2+ in the nifedipine group wasignificantly lowethan thaof the withouirradiation group othe ligh+ nifedipine group(both aP=0.000).Lasescanning confocal microscopy showed thathe fluorescence intensitieof intracellulafree Ca2+ in the irradiation only group,(-) BayK8644 group,ligh+ (-)BayK8644 group were highethan thaof the withouirradiation group,with statistical significancebetween them(all P=0.000).No significandifferencewere found in the fluorescence intensity of intracellulafree Ca2+ between the ligh+ nifedipine group and withouirradiation group awell abetween the (-)BayK8644 group and the ligh+(-) BayK8644 group(P =0.339,P =0.410).ConclusionThe optical conditionfoblue light-induced RPE cell damage were exposure of blue-ligha(2000± 500) lx fo6 hourand culturing the cellfo24 hours.Blue lighexposure can induce damage of human RPE cellprobably by triggering the increase of intracellulafree Ca2+ concentration. Key words: Light/damage; Retinal pigmented epithelium; Calcium ion/intercellular, free, antagonist,agonist ; Apoptosis

The role of SIRT1/AKT/ERK pathway in ultraviolet B induced damage on human retinal pigment epithelial cells

Ultraviolet (UV)-induced damage plays a major role in ocular diseases, such as cataracts and retinal degeneration. UVB may also cause retinal phototoxicity and photic retinopathy. In this study, we explored the effects of UVB on the cell cycle and the role of silent mating type information regulation 2 homolog 1 (SIRT1) in the UVB-induced damage. UVB dose-dependently suppressed the growth of retinal pigment epithelial (RPE) cells by activating the phosphatidylinositol 3-kinase (PI3K) pathway and triggering cell cycle arrest at the S phase. SIRT1, an NAD-dependent histone deacetylase, is involved in multiple biological processes, such as the stress response and the regulation of the cell cycle. However, its role in the effects of UVB on RPE cells is unclear. We showed that UVB down-regulates SIRT1 expression in a dose-dependent manner. Resveratrol, an SIRT1 activator, prevented the UVB-induced damage by inhibiting AKT and ERK phosphorylation. A specific PI3K inhibitor attenuated the UVB-induced ERK1/2 and p53 phosphorylation. Finally, UVB activated the PI3K/AKT/ERK pathway by reducing the expression of SIRT1 in ARPE-19 cells. Our study, therefore, illustrated the molecular mechanisms of UVB-induced phototoxicity and damage of RPE cells. SIRT1 and resveratrol may be significant regulators, protecting against UVB-induced injury.

Protective effect of pancreatic β MIN-6 cell on retinal pigment epithelial cell from high glucose-induced damage

Background Diabetic retinopathy (DR) associated with this disease closely approximates the oxidative damage inflicted on retinal pigment epithelial (RPE) cells by high glucose,in recent years,people devote themselves to the protection of RPE cells extensively. Objective To investigate the protective effect of pancreatic β MIN-6 cells on retinal pigment epithelial(RPE) cells from high glucose-induced damage. Methods RPE cells were incubated with normal medium for 4 d and divided into 3 groups:normal glucose group,high glucose group and MIN-6 cells group.RPE cell were exposed with 5 mmol/L normal glucose in normal glucose group,exposed with 30 mmol/L high glucose in high glucose group,and exposed with 5 × 104 MIN-6 cells and 30 mmol/L high glucose in MIN-6 cells group.After 24 h,cell viability of RPE cells was determined by MTT cell viability assay. Results More than 95％ cells showed the brown staining by ABC method.After incubation for another 24 hours,the A550 value was 0.44±0.02,0.30±0.01 and 0.41±0.01 in the normal glucose group,high glucose group and MIN-6 cells group respectively with a significant difference among these three groups (F =19.94,P＜ 0.01 ).The A55o value was significantly higher in the normal glucose group and the MIN-6 cells group compared with the high glaucose group (t =6.85,5.62,P＜0.01 ).The survival rate of RPE cells in the normal glucose group was(97.5±3.3 )％,and that in the high glucose group was ( 68.2 ± 4.5 ) ％,showing significant difference between them ( t =11.30,P＜0.01 ).Conclusions High glucose-induced damage of RPE cells is abrogated,and MIN-6 cells can protect RPE cells from high glucose-induced damage. Key words: High glucose; MIN-6 cell; Retinal pigment epithelial cell

Blueberry anthocyanins: protection against ageing and light-induced damage in retinal pigment epithelial cells

Retinal pigment epithelium (RPE) cells are vital for retinal health. However, they are susceptible to injury with ageing and exposure to excessive light, including UV (100-380 nm) and visible (380-760 nm) radiation. To evaluate the protective effect of blueberry anthocyanins on RPE cells, in vitro cell models of replicative senescent and light-induced damage were established in the present study. After purification and fractionation, blueberry anthocyanin extracts (BAE) were yielded with total anthocyanin contents of 31·0 (SD 0·5) % and were used in this study. Replicative senescence of RPE cells was induced by repeatedly passaging cells from the fourth passage to the tenth. From the fifth passage, cultured RPE cells began to enter a replicative senescence, exhibiting reduced cell proliferation along with an increase in the number of β-galactosidase-positive cells. RPE cells maintained high cell viability (P < 0·01) and a low (P < 0·01) percentage of β-galactosidase-positive cells when treated with 0·1 μg/ml BAE. In contrast, after exposure to 2500 (SD 500) lx light (420-800 nm) for 12 h, RPE cells in the positive control (light exposure, no BAE treatment) exhibited premature senescence, low (P < 0·01) cell viability and increased (P < 0·01) vascular endothelial growth factor (VEGF) release compared with negative control cells, which were not subjected to light irradiation and BAE exposure. Correspondingly, BAE is beneficial to RPE cells by protecting these cells against light-induced damage through the suppression of ageing and apoptosis as well as the down-regulation of the over-expressed VEGF to normal level. These results demonstrate that BAE is efficacious against senescence and light-induced damage of RPE cells.

Visible light induce oxidative damage to human retinal pigment epithelial cells

Objective To observe the effect of visible light (white light, red light, blue light) on the expression of reactive oxygen species (ROS), 8-OHdG and hOGG1 in cultured human retinal pigment epithelial (RPE) cells. Methods Cultured human RPE-19 cells (4th-6th generations) were divided into white light, red light, blue light and control group. The illumination was 600 Lux. The cells of experimental groups were exposed to white light or red light for 6, 12, 24 and 48 hours, and exposed to blue light for 1, 3, 6 and 12 hours, while cells of the control group were cultured in foil packaged dishes to avoid light. The levels of ROS expression were detected by 2′, 7′-dichlorofluorescin-diacetate (DCFH-DA), the levels of 8-OHdG protein expression were observed by immunocytochemistry (ICC), and the levels of hOGG1 were measured by western blot. Results Compared to the control group, the ROS expression in RPE cells were increased in white and red light group after 12, 24 and 48 hours and in blue light group after 1, 3, 6 and 12 hours (Fwhite light=11. 611, Fred light =6.706, Fblue light =23. 259; P＜0.05 ). Additionally, the ROS expression had a tendency to increase gradually along with exposure time. Compared to the control group, the 8-OHdG expression in RPE cells were increased significantly in both white and red light group after 12, 24 and 48 hours and in blue light group after 1, 3, 6 and 12 hours (Fwhite light =16. 032,Fred light =6. 378, Fblue light =19. 484; P＜0.05). Additionally, the 8-OHdG expression in white and red light group were increased gradually with exposure time but decreased when exposure time was up to 48 hours, while that in blue light group was increased firstly though it started to decrease when exposure time was up to 6hours. Compared to the control group, the hOGG1 expression in RPE cells were increased in white and red light group after 12, 24 and 48 hours and in blue light group after 6 and 12 hours (Fwhite light =15. 121,Fred light=21. 041,Fblue light12. 479; P＜0.05). ConclusionsExposure to white, red or blue light could induce ROS production and DNA oxidative damage in RPE cells in a time-dependent way. Exposure to visible light could switch on self protection of RPE cells against DNA oxidative damage by up-regulating of the hOGG1 expression. Key words: Pigment epithelium of eye/ cytology; Reactive oxygen species; Deoxyguanine nucleotides; Cell culture techniques

LEDGF1-326 Decreases P23H and Wild Type Rhodopsin Aggregates and P23H Rhodopsin Mediated Cell Damage in Human Retinal Pigment Epithelial Cells

BackgroundP23H rhodopsin, a mutant rhodopsin, is known to aggregate and cause retinal degeneration. However, its effects on retinal pigment epithelial (RPE) cells are unknown. The purpose of this study was to determine the effect of P23H rhodopsin in RPE cells and further assess whether LEDGF1-326, a protein devoid of heat shock elements of LEDGF, a cell survival factor, reduces P23H rhodopsin aggregates and any associated cellular damage.MethodsARPE-19 cells were transiently transfected/cotransfected with pLEDGF1-326 and/or pWT-Rho (wild type)/pP23H-Rho. Rhodopsin mediated cellular damage and rescue by LEDGF1-326 was assessed using cell viability, cell proliferation, and confocal microscopy assays. Rhodopsin monomers, oligomers, and their reduction in the presence of LEDGF1-326 were quantified by western blot analysis. P23H rhodopsin mRNA levels in the presence and absence of LEDGF1-326 was determined by real time quantitative PCR.Principal FindingsP23H rhodopsin reduced RPE cell viability and cell proliferation in a dose dependent manner, and disrupted the nuclear material. LEDGF1-326 did not alter P23H rhodopsin mRNA levels, reduced its oligomers, and significantly increased RPE cell viability as well as proliferation, while reducing nuclear damage. WT rhodopsin formed oligomers, although to a smaller extent than P23H rhodopsin. Further, LEDGF1-326 decreased WT rhodopsin aggregates.ConclusionsP23H rhodopsin as well as WT rhodopsin form aggregates in RPE cells and LEDGF1-326 decreases these aggregates. Further, LEDGF1-326 reduces the RPE cell damage caused by P23H rhodopsin. LEDGF1-326 might be useful in treating cellular damage associated with protein aggregation diseases such as retinitis pigmentosa.

Impact of HMG‐CoA reductase inhibition on oxidant‐induced injury in human retinal pigment epithelium cells

In addition to cholesterol-lowering effect, HMG-CoA reductase inhibition by statins has been shown to have protective effect in many cells type. The loss of vision in retinal degeneration disease associates with oxidative stress and apoptosis in retinal pigment epithelium (RPE) cell. This study was designed to examine the effect of statins on oxidant-induced damage in human RPE cells. Cultured human ARPE-19 (ARPE) cells were challenged with hydrogen peroxide (H(2) O(2) ) plus tumor necrosis factor alpha (TNFα) in the presence or absence of statins or various stress signaling inhibitors, including anti-oxidants N-acetyl cysteine (NAC), the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium (DPI), and the p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580. Apoptosis was evaluated by TUNEL analysis and cell viability was determined by MTT assay. Reactive oxygen species (ROS) were detected by 2',7'-dichlorodihydrofluorescein diacetate (H(2) DCFH-DA). Expression of p-p38 MAPK protein was measured by Western blot analysis. Our findings indicate that statins treatment significantly suppressed oxidant-induced ROS accumulation and RPE apoptosis. Statins increased cell viability in a dose-dependent manner. In addition, statins treatment prevented the activation of NADPH oxidase and p38 MAPK signaling induced by oxidative stress. These results suggest that statins protects ARPE cells from oxidative stress via an NADPH oxidase and/or p38 MAPK-dependent mechanisms, which may contribute to statins-induced beneficial effects on RPE function.

Xanthophylls protect against induced oxidation in cultured human retinal pigment epithelial cells

Lutein and zeaxanthin are dietary carotenoids which accumulate in human retina, including the retinal pigment epithelial (RPE) cells. Oxidative damage of RPE cells is involved in the pathogenesis age related macular degeneration (AMD). The purpose of this study was to evaluate the potential protective effect of lutein and zeaxanthin against hydrogen peroxide induced oxidative stress in cultured human RPE cells. Human D407 RPE cells were pretreated with lutein and zeaxanthin (10μM) for 24h, and exposed for 1h to 500μM hydrogen peroxide. Cell viability, reactive oxygen species (ROS), the lipid peroxidation and the activity of antioxidant enzymes were determined in basal and oxidative stress conditions. Lutein and zeaxanthin pretreatment protected RPE cells against hydrogen peroxide induced cytoxicity, and inhibited the intracellular ROS generation and lipid peroxidation. Xanthophylls induced a significant increase of superoxide dismutase and glutathione peroxidase activity and of the level of reduced glutathione, but did not significantly modify the catalase activity. These results suggest that lutein and zeaxanthin supplementation can contribute to the prevention of age-related macular degeneration, not only by increasing the macular pigment density, but also by protecting retinal pigment epithelium against oxidative stress.

The new paradigm: retinal pigment epithelium cells generated from embryonic or induced pluripotent stem cells

Compared with neural crest-derived melanocytes, retinal pigment epithelium (RPE) cells in the back of the eye are pigment cells of a different kind. They are a part of the brain, form an epithelial monolayer, respond to distinct extracellular signals, and provide functions that far exceed those of a light-absorbing screen. For instance, they control nutrient and metabolite flow to and from the retina, replenish 11-cis-retinal by re-isomerizing all-trans-retinal generated during photoconversion, phagocytose daily a portion of the photoreceptors' outer segments, and secrete cytokines that locally control the innate and adaptive immune systems. Not surprisingly, RPE cell damage is a major cause of human blindness worldwide, with age-related macular degeneration a prevalent example. RPE replacement therapies using RPE cells generated from embryonic or induced pluripotent stem cells provide a novel approach to a rational treatment of such forms of blindness. In fact, RPE-like cells can be obtained relatively easily when stem cells are subjected to a two-step induction protocol, a first step that leads to a neuroectodermal fate and a second to RPE differentiation. Here, we discuss the characteristics of such cells, propose criteria they should fulfill in order to be considered authentic RPE cells, and point out the challenges one faces when using such cells in attempts to restore vision.

Effect of Tetramethylpyrazine on RPE degeneration, choroidal blood flow and oxidative stress of RPE cells.

To study the effects of Tetramethylpyrazine (TMP) on retinal pigment epithelium (RPE) degeneration, choroidal blood flow and oxidative stress of RPE cells. The 35mg/kg NaIO(3)-induced RPE degeneration rat eyes was given 25µg 1% TMP eye drops 3 times a day for 7 days before NaIO(3) injection, and then 2 to 4 weeks after NaIO(3) injection. RPE function was measured with c-wave of electroretinogram (ERG). Colored microsphere technique was used for in vivo experiments to determine the choroidal blood flow in ocular hypertensive (40mmHg) rabbit eyes. Methylthiazoltetrazolium (MTT) assay was used to study in vitro effect of TMP on various oxidants induced injury in the hRPE (ARPE-19 (ATCC, Manassas, VA, USA)). Two weeks after NaIO(3) injection, the amplitude of ERG c-wave fell markedly in NaIO(3) group to 36% of control group(P<0.01). No apparent difference was observed in TMP+NaIO(3) group. Four weeks later, the NaIO(3) group fell to 46% of control group (P<0.01), while the TMP+NaIO(3) group fell to only 77% of control group (P<0.01). There was a 67% reversal of the ERG c-wave by TMP as compared to NaIO(3) group(P<0.01). The choroidal blood flow was significantly increased at all time points (at 30, 60 and 120 minutes after TMP instillation) as compared with corresponding controls. TMP had no effect on hypoxia-(1% O(2)), t-BHP- and H(2)O(2)-induced damage in RPE cells. 10(g/mL TMP could reverse 1 and 3mM NaN(3)-induced loss of viability of RPE by 18.5% (P<0.01) and 23% (P<0.01), respectively. 30µg/mL TMP could reverse 30 and 100mM NaIO(3) induced loss of viability of RPE by 18.1% (P<0.05) and 16.8% (P<0.01), respectively. TMP can significantly protect RPE from NaIO(3) induced degeneration in vivo and oxidative stress in vitro and can increase choroidal blood flow markedly in vivo.

Molecular regulation of cigarette smoke induced-oxidative stress in human retinal pigment epithelial cells: implications for age-related macular degeneration

Cigarette smoke is the most important environmental risk factor for developing age-related macular degeneration (AMD). Damage to the retinal pigment epithelium (RPE) caused by cigarette smoke may underlie the etiology of AMD. This study investigated the molecular and cellular effects of cigarette smoke exposure on human RPE cells. ARPE-19 or primary human RPE cells were exposed to cigarette smoke extract (CSE) or hydroquinone (HQ), a component of cigarette smoke. The effect of this exposure on key aspects of RPE vitality including viability, cell size, mitochondrial membrane potential (DeltaPsi(m)), superoxide production, 4-hydroxy-2-nonenal (4-HNE), vascular endothelial growth factor (VEGF), and heme oxygenase-1 (HO-1) expression was determined. Exposure of RPE cells to CSE or HQ caused oxidative damage and apoptosis, characterized by a reduction in cell size and nuclear condensation. Evidence of oxidative damage also included increased lipid peroxidation (4-HNE) and mitochondrial superoxide production, as well as a decrease in intracellular glutathione (GSH). Exogenous administration of antioxidants (GSH and N-acetyl-cysteine) prevented oxidative damage to the RPE cells caused by CSE. Cigarette smoke also induced expression of VEGF, HO-1, and the transcription factor nuclear factor erythroid-derived 2, like 2 (NRF2). However, NRF2 was only modestly involved in CSE-induced HO-1 expression, as shown by the NRF2 small interfering RNA studies. These new findings demonstrate that cigarette smoke is a potent inducer of oxidative damage and cell death in human RPE cells. These data support the hypothesis that cigarette smoke contributes to AMD pathogenesis by causing oxidative damage and cell death to RPE cells.