Human Pigment Epithelial Cells Research Articles

The effect of blue light on Ca2+-protein kinase C signaling pathway in human retinal pigment epithelial cells in vitro

Objective To investigate the effect of blue light on Ca2+-protein kinase C (PKC)signaling pathway in human retinal pigment epithelial (RPE) cells in vitro.Methods Primary human RPE cells were cultured in vitro and characterized.The experiments were carried out using the 4th generation of human RPE cells.The PKC protein level was measured by Western blot to determine the most appropriate concentration of phorbol ester (PMA) and calcium phosphate binding protein (calphostin C) on PKC expression.Non-radioactive isotope method was used to determine the effect of blue light on PKC expression of cultured cells.Blue-light damage model of human RPE cells was established by 6 hour irradiation of medical blue-light lamp [20 W,450-500 nm wavelength,(2000± 500) Lux],and 24 hours prolongation of post-exposure culture.The human RPE cells were randomly divided into 5 groups.Group A did not receive light irradiation,group B only received blue light irradiation,group C was blue light irradiation and 0.1 mmol/L nifedipine treatment,group D was blue light irradiation and 100.0 nmol/L calphostin C treatment,group E was blue light irradiation and 100.0 nmol/L PMA treatment.Intracellular Ca2-concentration was measured by acetoxymethyl ester (Fluo 3-AM) labelling and confocal microscope imaging.Results The PKC protein expression in 100.0 nmol/L or 200.0 nmol/L PMA-treated groups was higher than 0.1,1.0,10.0,and 50.0 nmol/L PMA-treated groups,the difference was statistically significant (F=217.537,P＜0.05),but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L PMA-treated groups (P =0.072).The PKC protein expression in 100.0 nmol/L or 200.0 nmol/L calphostin C-treated groups was lower than 5.0,25.0,50.0,and 75.0 nmol/L calphostin C-treated groups,the difference was statistically significant (F=164.543,P＜0.05),but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L calphostin C treated groups (P=0.385).PKC level in blue light group was higher than non-light group,the difference was statistically significant (t =-9.869,P＜0.05).The Ca2+ fluorescence intensity values in group B,C,D and E was higher than group A,the difference was statistically significant (F=26 764.92,P＜0.05).The Ca2+ fluorescence intensity values in group E was higher than group B,C and D (P＜0.05),and that in group B was higher than group C and D (P＜0.05).Conclusions The PKC activity and intracellular Ca2+ concentration in human RPE cells increase after blue light irradiation.Both calcium channel inhibitor nifedipine and PKC inhibitor calphostin C can reduce intracellular Ca2+ concentration in human RPE cells.PMA can induce intracellular Ca2+ concentration in human RPE cells after blue light irradiation. Key words: Eye injuries/physiopathology; Retinal pigment epithelium; Apoptosis; Photic stimulation

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

Antiproliferative effect of curcumin on human retinal pigment epithelial cell

Background Curcumin derives from the rhizome of curcuma longa.It has proven to have an antiproliferative effect in previous studies on vast majority of endothelial and epithelial cells,however,the study of its inhibiting effect on the proliferation of retinal pigment epithelial (RPE) cells and underlying mechanism is rare.Objective Aim of this study was to investigate the potential inhibitory effect of curcumin on the proliferation of cuhured human RPE cells in vitro and its possible mechanism.Methods Human RPE cells harvested by trpsinEDTA were suspended in DMEM/F12 medium with serial dilutions of curcumin (5,10,15,20 mg/L),and the human RPE cells cultured by DMEM/F12 without curcumin were used as control.The proliferation value of human RPE cells (A value) was measured by water-soluble tetrazole-1 (WST-1) assay,the optimized dose of antiproliferation of curcumin was determined and applied for further experimental process.Apoptosis and cell cycle of human RPE cells were detected by flow cytometric analysis at 48 hours and 72 hours after curcumin treatment.The ultrastructure profile of the cells were examined by transmission electron microscopy (TEM).Western blot analysis was performed to measure the relative expressing level of the pro-apoptotic factors p53,p21 WAF1/CIP1 and proliferating cell nuclear antigen (PCNA) in the cells,respectively.Factorial design of two factor analysis of variance of SPSS 17.0 software was used to compare the difference of A values of the cells among the various groups and time points,and independent-sample t test was used to compare the differences of apoptosis rate and cell ratio in different cycles between curcumin group and control group.Results WST-1 assay showed that the A value was gradually reduced with the increase of curcumin dose (F tion =96.55,P =0.00),and gradually increased with the lapse of time (Ftime =4634.28,P =0.00).The early apoptotic rate of the cells was (13.37±1.26) ％ in the curcumin group 48 hours after treated by 15 mg/L curcumin,and that of the control group was (7.03 ±0.37) ％,with a significant difference between them (t =8.33,P=0.00).In 72 hours after treated by 15 mg/L curcumin,the early and middle-late apoptotic rates of the cells were (15.97±0.16) ％ and (0.26±0.03) ％,which were significantly higher than those of the control group (7.29±0.37) ％ and (0.14±0.02) ％ (t=37.80,P=0.00;t=7.44,P=0.00).The cell ratio of G0/G1 phase in the curcumin group was (57.17±1.17)％ 48 hours after treated by 15 mg/L curcumin,and that in the control group was (67.73± 1.10)％,showing a significant difference (t =11.40,P =0.00).M itochondrial swelling and vacuolar degeneration were seen in the cells after treated by 15 mg/L curcumin.The relative expression levels of p53 and p21WAF1/CIP1 protein in the cells were higher in the curcumin group than those of the control group at 24,48 and 72 hours (all at P＜0.05),but the expression levels of PCNA protein were lower in the curcumin group than those of the control group in various time points (all at P ＜ 0.05).Conclusions Curcumin can effectively inhibit the proliferation of human pigment epithelial cells in a dose-and time-dependent manner.P53 pathway may participate in anti-proliferating process. Key words: Curcumin; Retinal pigment epithelial cell; Apoptosis

The anti-aging effect of lycium barbarum polysaccharide on human retinal pigment epithelial cell

Background The pathogenesiof age-related maculadegeneration (AMD) iassociated with the senility of human retinal pigmenepithelium (RPE) cells.Seeking drug to arresRPE cell senility iof significance fothe prevention and treatmenof AMD.Research showed thathe lycium barbarum polysaccharide (LBP) can delay senility,buitinfluence on RPE cell aging iunclear.Objective Thistudy wato discusthe protective effecand mechanism of LBP on RPE cell aging.MethodPorcine retinal neural epithelial layewaisolated,and photoreceptooutesegmen(POS) waextracted by density gradiencentrifugation and marked by FITC.The POwathen co-cultured with RPE cellin the medium containing 0.01,0.10 and 1.00 g/L LBP fo24 hours.The areof fluorescence,representing the amounof POphagocytosed by RPE cells,wameasured undethe fluorescenmicroscope to evaluate the influence of LBP on the phagocytifunction of RPE cells.The POS-induced RPE lipofuscin-uptake cell model waestablished by co-culturing human RPE cellwith porcine POfo3 weeks.The RPE-POco-culture cell model waincubated in medium containing 0.01,0.10 o1.00 g/L LBP,and the autofluorescence caused by lipofuscin up-taken into RPE cellwadetected with flow cytometry.cell counting kiwaused to assescell proliferation and viability (value) 24,48 and 72 hourafteculturing.ResultPorcine POpresented athin rodundethe lighmicroscope and appeared abilayedisc-like structureundethe transmission electron microscope,and itFITC-labeled yellow-green autofluorescence waobserved undethe fluorescenmicroscope.No POwaup-taken into the RPE cellin the normal control group,buthe areof POphagocytosed by RPE cellwagradually enlarged with increasing doseof LBP,showing significandifference among the group(F =21.425,P =0.006).Compared with the POcontrol group,the phagocytosed areincreased avariouconcentrationof LBP+POgroup(P＜0.01).Flow cytometry showed thathe autofluorescence value in the POcontrol group wamore highethan thaof the normal control group.Athe LBP dose increased,the autofluorescence value in the RPE celldeclined gradually and iwaneathe normal value in the 1.00 g/L LBP+ POgroup.The rate of proliferation of the lipofuscin RPE cellvaried with the increase of doseof LBP with the maximal value in the normal RPE group and minimal value in the lipofuscin RPE group,and the rate of proliferation of the lipofuscin RPE cellascended with increasing doseof LBP until neathe normal value in the 1.00 g/L LBP + lipofuscin RPE cellgroup (P＞0.05).ConclusionLBP enhance the anti-aging effecof human RPE cellby strengthening the phagocytiability to POand the ability to remove lipofuscin and by heightening the proliferation of human RPE cells. Key words: Retinal pigment epithelial cell; Lycium barbarum polysaccharide; Senility; Photoreceptor

Exploring phospho‐signaling pathways in patients with agerelated macular degeneration (AMD), human pigment epithelial cell line ARPE‐19 and porcine ocular tissue

The pig has been used extensively in experimental studies of human eye diseases. Pig retina shares many morphological features with the human eye. Retinal pigment epithelial cells constitute an important component of the blood‐retinal barrier and play a pivotal role in the development of AMD. Understanding the underlying molecular mechanisms is a prerequisite for developing therapeutic strategies. We investigated cytokine‐induced changes of the JAK/STAT and NF‐kappa B signaling pathways.MethodsPorcine ocular cells, human ARPE‐19 cell line, eyes with AMD were used to study the nuclear translocation of STAT and NF‐kB proteins by gel‐shift assay, immunohistochemistry, confocal laser microscopy and flow cytometry.ResultsHistological examination of AMD patients revealed a strong formation of choroidal neovascular membranes in the macular region. Strong activation of STAT3 was observed in these membranes. Strong STAT1 activation was demonstrated upon exposure to interferon‐γ in ARPE‐19 and pig ocular cells in vitro. Human recombinant interferon‐α upregulated STAT1, STAT2 and STAT3, while IL‐6 and IL‐4 activated STAT3 and STAT6, respectively. Confocal microscopy identified the nuclear translocation of the STAT proteins.ConclusionOur data demonstrate the upregulation of the members of the JAK‐STAT signaling pathway in AMD patients, porcine ocular cells and ARPE‐19 cell line.

Effects of amniotic membrane on proliferation and differentiation of human retinal pigment epithelial cell

Background Human retinal pigment epithelial (RPE) cell transplantation treating retinal degenerative diseases is a researching topic,and the source of human RPE cells is a key problem.Many biological carriers can be used for the preparation of RPE cell layer.However,some advantages,such as cytotoxicity,lack of stability and immunologic reaction etc.are still existed.To study an ideal biological carrier is very important.Objective This experimental was to determine the effects of amniotic membrane on the proliferation and differentiation of human RPE cells and the possibility as a scaffold for RPE cell transplantation.Methods ARPE19 cell line cells were cultured and passaged in DMEM/F12 medium with 10％ fetal bovine serum,and 8-12generation of cells were used.The cells were divided into two groups.One group of cells were incubated on the denuded amniotic membrane,and the other group of cells were cultured in the medium (control group).MTT was performed to detect the A492 value of RPE cells for the evaluation of cell proliferation ability 24,48,72,96 hours after culture.Cell morphology was compared by histopathological examination 3 weeks after culture.The mRNA expression of pigment epithelium-derived factor (PEDF),N-cadherin,β-catenin and cell connection related proteins in the cells of both groups were assayed using reverse transcription polymerase chain reaction (RT-PCR).Ultrastructure of the cells was observed under the transmission and scan electronic microscope 3 weeks after culture.Results The number of ARPE-19 cells cultured on denuded amniotic membrane was decreased significantly in comparison with the normal culture plate(F=41.760,P =0.000).Histopatholy also showed that the cell density on amniotic membrane was lower than of normal cells on plate surface.Moreover,the expression level of claudin 1 mRNA,N-cadherin mRNA and PEDF mRNA were significantly up-regulated in denuded amniotic membrane group in comparison with control group (t=15.828,P=0.000 ;t=6.839,P=0.002 ;t=14.667,P=0.000),but the expression of Connexin 43 mRNA was down-regulated in denuded amniotic membrane group compared with control group(t=3.358,P=0.024).Ultrastructural examination revealed that ARPE-19 cells cultured on amniotic membrane exhibited a polygonal epithelial phenotype with cilium on the apical side,however,the cells cultured on normal culture plate displayed fusiform shape and uneven thickness.Conclusions Amniotic membrane plays a promoting effect on the differentiation of ARPE-19 cells and a inhibitory effect on the proliferation of ARPE-19 cells,suggesting that amniotic membrane might be an useful scaffold for the preparation of functionally mature RPE cells for clinical transplantation. Key words: Amniotic membrane; Retinal pigment epithelium cell; Proliferation; Differentiation

Effects of PACAP on Intracellular Signaling Pathways in Human Retinal Pigment Epithelial Cells Exposed to Oxidative Stress

The integrity of retinal pigment epithelial cells is critical for photoreceptor survival and vision. Pituitary adenylate cyclase activating polypeptide (PACAP) exerts retinoprotective effects against several types of injuries in vivo, including optic nerve transection, retinal ischemia, excitotoxic injuries, UVA-induced lesion, and diabetic retinopathy. In a recent study, we have proven that PACAP is also protective in oxidative stress-induced injury in human pigment epithelial cells (ARPE-19 cells). The aim of the present study was to investigate the possible mechanisms of this protection. ARPE cells were exposed to a 24-h hydrogen peroxide treatment. Expressions of kinases and apoptotic markers were studied by complex array kits and Western blot. Oxidative stress induced the activation of several apoptotic markers, including Bad, Bax, HIF-1α, several heat shock proteins, TNF-related apoptosis-inducing ligand, and Fas-associated protein with death domain, while PACAP treatment decreased them. The changes in the expression of MAP kinases showed that PACAP activated the protective ERK1/2 and downstream CREB, and decreased the activation of the pro-apoptotic p38MAPK and c-Jun N-terminal kinase, an effect opposite to that observed with only oxidative stress. Furthermore, PACAP increased the activation of the protective Akt pathway. In addition, the effects of oxidative stress on several other signaling molecules were counteracted by PACAP treatment (Chk2, Yes, Lyn, paxillin, p53, PLC, STAT4, RSK). These play a role in cell death, cell cycle, inflammation, adhesion, differentiation and proliferation. In summary, PACAP, acting at several levels, influences the balance between pro- and anti-apoptotic factors in favor of anti-apoptosis, thereby providing protection in oxidative stress-induced injury of human retinal pigment epithelial cells.

Mechanism of the aging phenomenon with passage of human retinal pigment epithelial cell

Background Retinal pigment epithelial (RPE) cell senescence damage the metabolism of photoreceptor,leading to retinal dysfunction and loss of vision.To understand RPE cell senescence mechanism will contribute to the study of age-related macular degeneration ( AMD). Objective The present study was to prepare the ageing RPE cell model with passage and explore its potential mechanisms. Methods This study was approved by the Ethic Committee of Qingdao University Medical College,and the informed consent was obtained from each gravida.Six human eyeballs were obtained from artificial labor fetusl with the gestational age 16-28 weeks.RPE cells were isolated,cultured and passaged in vitro to establish the cell replicative aging model.The third to twelfth cells were collected to be used to this experiment.Human keratin was used to identify the cells by immunochemistry,and MTT method was utilized to assess the proliferation and viability of different generations of cells as the A490 value.The cellular cycles and transmembrane potential (△ψm) of mitochondrion (△ψm) with passage were detected and compared using Flow Cytometry. Results Cultured and passaged cells showed the hexagon in shape with the melanin in 1-2 generations of cells and presented with the brown staining in cytoplasm for human keratin.The melanin was absent in the third generation cells.Vibrant growth statues were seen from the 3-6 generations cells and thereafter the proliferation ability reduced.The cells of G0/G1 phase were gradually elevated with the passage from 3 - 12 generations with a percentage of 68.40％ in the third generation of cells to 87.33％ in the twelfth generation of cells,showing a significant difference among various generations ( F =180.43,P =0.00),and that of the sixth,ninth and twelfth generation of cells was significant higher than the third,sixth and ninth generation respectively (t =4.002,P＜0.05 ; t=12.885,P＜0.01 ;t=16.387,P＜0.01 ).MTT assay showed that of RPE cells were significantly declined with the passage ( F =38.77,P =0.00),and the A490 value of the ninth,twelfth generations of cells was considerably lower in comparison with sixth and ninth generation respectively ( t =5.991,11.983,P＜0.01 ).From 3 through 12 generations of cells,the staining intensity of rhodamine 123 was gradually decreased ( F =121.68,P =0.00 ),and the staining intensity in the sixth,ninth and twelfth generation of cells was significant lower than that the third,sixth and ninth generation respectively(t=6.918,7.620,11.207,P＜0.01 ). Conclusions A replicative aging model can be successfully created by the passage in vitro using human fetal RPE cells.The reduce of transmembrane potential and damage of mitochondria might be one of mechanisms of senescence of RPE cells. Key words: Retina/pigment epithelium; Cell ageing; Cell cycle; Mitochondria; Transmembrane potential

Effect of Acid-sensing ion channel 1a in cultural human retinal pigment epithelial cells during oxidative stress

Objective To investigated whether Acid-sensing ion channel 1 a (ASIC1a) is expressed in the retinal pigment epithelium (RPE) cells and whether it is involved in the function of these cells.Methods Real time revere transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to analyze the ASIC1a expression in ARPE-19 cells during oxidative stress induced by hydrogen peroxide (H2O2 ).Furthermore,blockage or overexpression of ASIC1a in RPE cells were obtained using inhibitors (amiloride and PCTxl ) or by the transfection of cDNA encoding hASIC1a.Cell viability was determined using the methyl thiazol tetrazolium (MTT) assay.Results The RT-PCR and Western blot results showed that both the mRNA and the protein of ASIC1a are expressed in RPE.Blockade of ASICs by amiloride in normal RPE cells resulted in cell death,indicating that ASICs play an important physiological role in RPE cells.Furthermore,overexpression of ASIC1a in RPE increased cell survival after oxidative stress induced by H2O2.Conclusion ASIC1a is functionally expressed in RPE cells and may play an important role in the physiological function of RPE cells in protecting them from oxidative stress. Key words: Acid-sensing ion channel 1a; Retinal pigment epithelium; Oxidative stress

The effects of rapamycin on the proliferation and apoptosis of human retinal pigment epithelium cells in vitro

Background Rapamycin(RAPA)is a specific inhibitor of the mammalian target of rapamycin (mTOR).Researches showed that RAPA inhibits the proliferation of lens epithelium cells(LECs)and tumor cells and induces apoptosis of tumor cells.To investigate whether rapamycin has the inhibitory effect on retinal pigment epithelium(RPE)cells is very important for the prevention and management of proliferative vitreoretinopathy (PVR).Objective This study was to investigate the effects of RAPA on the proliferation and apoptosis of human RPE cells in vitro.Methods Human RPE cells(D407 strain)were cultured and passaged and then divided into regular culture group(blank control group),DMSO control group(0.1‰ DMSO +regular culture),and different concentrations RAPA-treatment groups(5,10,20,40,80,160,320 nmol/L).The proliferation(A490)of human RPE cells was detected using MTT,and the inhibitory rates of RAPA on the proliferation of RPE cells were calculated and compared among different groups at 12,24 and 48 hours.The apoptosis rates of the cells were analyzed among various groups by Hoechst staining after 12,24,48 hours.Results The inhibitory rates of RAPA on RPE cells were significantly different among various groups(F=484.451,P＜0.01)and evidently elevated in 20-320 nmol/L RAPA groups compared with DMSO control group(P ＜ 0.01).The inhibition of RAPA on the cells was considerably enhanced as the lapse of time(F=232.262,P＜0.01)with more dominant effects in 24 and 48 hours compared to 12 hours after addition of RAPA(P＜0.05-0.01).Compared with blank control group and DMSO control group,the apoptotic rates of the cells were evidently increased in 12,24,48 hours in 10 nmol/L RAPA group(all P＜0.05),and higher cellular apoptotic rates were found in 20-320 nmol/L RAPA groups(all P＜0.01).The alteration of cellular apoptotic rate showed a gradually incremental trend as the acting time of RAPA(F =625.584,P＜0.01).Karyorrhexis and mass-like density staining and chromatin substance were seen in RPE cells under the fluorescence microscope in ≥ 10 nmoL/L RAPA groups.Conclusions RAPA suppresses the proliferation and induces the apoptosis of human RPE cells in concentration-and time-dependent manner in vitro. Key words: Rapamycin ; Retinal pigment epithelial cells; Proliferative vitreoretinopathy ; Apoptosis

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

Effects of different wave-length lights on proliferation and secretion of growth factors in human retinal pigment epithelium cells

Background The study of myopia development is always the hotspot worldwide. Recently,scientist found that some growth factor secreted by retinal nerve epithelium cells and retinal pigment epithelium(RPE)cells are associated with the development of myopia. Whenever, the absorption of RPE cells to different wave-length lights is different. ObjectiveThis study was to investigate the effects of different wave-lengths lights on the proliferation of human RPE cells, and explore the influence of different wave-lengths lights on RPE cells secreting hepatocyte growth factor(HGF) ,basic fibroblast growth factor(bFGF) and transforming growth factor-β(TGF-β).Methods The fourth to fifth passages of human embryonic RPE cells were exposed to blue light( λ =480 nm),red light( λ =775 nm) and white light. The cells of control group were harvested in normal condition. The proliferation and growth of RPE cells were assayed by MTT,and the ultrastructure of cells was examined under the transmission electron microscopy at 48 hours after light exposure of RPE cells. Enzyme linked immunosorbent assay(ELISA) was adopted to determine the concentrations of HGF,bFGF and TGF-β in the culture medium in 12,24,48,72 hours. The expression of HGF mRNA in RPE cells was detected by RT-PCR. This study was approved by Ethic committee of Fudan University. Results The A490 values of the cells exposed to blue light,red light,white light and white light were 0. 0218±0. 0014 ;0. 0353±0. 0025 ;0. 0371 ±0. 0024 and 0. 0445 +0. 0046 respectively with the significant difference among 4 groups ( F =12. 579, P＜0.05 ), and A490 value in blue light group, red light group were significantly lower than that of the control group ( t =2.043 ; t =2.024, P＜0.05 ). ELISA showed that the concentrations of HGF and TGF-β in culture medium were evidently elevated as the prolongation of light exposure in various light exposure groups in 72 hours(HGF) and 48 hours(TGF-β) compared with 12 hours with a predominating rise in the control group. The statistically significant differences were found in the concentrations of HGF and TGF-β between control group and blue light group or red light group in the( all P＜0. 05 ). The bFGF level was decreased with the time increase of various light exposure with the significant differences in 72 hours compared with 12 hours( P＜0.05 ). RT-PCR revealed the considerable difference about expression of HGF mRNA in RPE cells among these four groups( P＜0. 05 ), and the lest expression in HGF mRNA was in the blue light group compared with control group( t =3. 972,P＜0.05 ). Thinning of the chromatin, decreasing of organelle and loss of cellular membrane were seen in the blue light group, but no obvious change of ultrastructure of human embryo RPE cells was found in the ret and white light groups. Conclusions The irradiation of different wave-length light can effect the growth and proliferation and secretion of HGF,bFGF and TGFβ in human RPE cells in vitro,implying myopia formation is associated to exposure of different wave-length light. Key words: Myopia; Retinal pigment epithelial cell; Hepatocyte growth factor; Basic fibroblast growth factor; Transforming growth factor-β ; Light exposure

Simvastatin upregulates the antioxidant defense protein heme oxygenase-1 in human retinal pigment epithelial cells

Simvastatin upregulates the antioxidant defense protein heme oxygenase-1 in human retinal pigment epithelial cells

NKCC1 cotransporters: keeping an ‘ion’ them

NKCC1 cotransporters: keeping an ‘ion’ them

Water cotransport in pigmented epithelial cells

Water cotransport in pigmented epithelial cells

Comparative Effects of Six Intraocular Vital Dyes on Retinal Pigment Epithelial Cells

To evaluate and compare the effects of the following dyes on human pigmented epithelial cells: indocyanine green (ICG), infracyanine green (IfCG), trypan blue (TB), bromophenol blue (BrB), patent blue (PB), and Brilliant Blue G (BBG). ARPE-19 cells cultured in vitro were exposed to these dyes, and acute and chronic toxicity were evaluated. Cell viability was measured by colorimetry (MTT assay), morphology was observed by phase-contrast microscopy, membrane permeability (CMP) was evaluated by flow cytometry with propidium iodide (PI), and mitochondrial membrane potential (ΔΨm) was measured with 3,3'-dihexyloxacarbocyanine (DiOC(6)(3)). Each of the studied dyes exhibited toxicity after acute exposure at surgical doses. The presence of light often reduced cell viability, especially when measured 3 hours after incubation in the case of ICG, TB, BrB, and BBG. Morphologic changes were induced by ICG, IfCG, and BBG. Both CMP and ΔΨm were altered after exposure to surgical doses of ICG, TB, PB, and a fourfold surgical dose of BrB. Chronic exposure to residual amounts of some dyes was associated with reduced proliferation and even cell death. It appears to be prudent to use the lowest possible dose of each dye, to minimize the risk of toxic effects. This precaution may be particularly important in the case of BrB, which should not be used in excess of 0.5%. In addition, abundant irrigation of the vitreous cavity after surgery to completely remove traces of dye may be of crucial importance, particularly in the case of ICG, in minimizing chronic toxicity.

Integrin α5 expression induced by epidermal growth factor affect proliferation and migration of human retinal pigment epithelium cells

Objective To observe the effect of epidermal growth factor (EGF) on integrin α5 expression and its influence on human retinal pigment epithelium (RPE) cells. Methods Human RPE cells were treated in vitro with 0.1, 1.0, 10.0, 20.0 and 100.0 ng/ml of EGF, the mRNA and protein of integrin α5 was measured by reverse transcription polymerase chain reaction(RT-PCR)and flow cytometry. Human RPE cells were cultured under 4 conditions including DMEM/F12, DMEM/F12 + 10 ng/ml EGF, DMEM/F12 + 10 ng/ml EGF+ rabbit anti-human integrin α5 antibody (1: 100), DMEM/F12 + 10 ng/ml EGF+ rabbit anti-human vimentin antibody (1: 100), and their proliferation and migration were measured by methyl-thiazole tetrazolium(MTT)and Boyden chamber. Results The integrin α5 mRNA level of human RPE cells was not changed after 12 hours of EGF stimulation (F=0.618, P = 0. 687), however it was induced in a dose-dependent manner after 24 and 48 hours of EGF stimulation (F=465. 303, 212. 340; P= 0. 000, 0. 000). The protein level of integrinα5 was higher in 10 ng/ml EGF stimulation compared with the control group and 0. 1 ng/ml group (P<0. 01). MTT and Boyden chamber showed that the integrin α5 expression increased the proliferation and migration of human RPE cells. Conclusion EGF can induce integrin α5 expression, thus increase the proliferation and migration of human RPE cells. Key words: Epidermal growth factor; Pigment epithelium of eye; Integrin alphα5; Cell proliferation; Cell movement

The relationship between MERTK gene and protein kinase C in the phagocytic process of human retinal pigment epithelial cells

To investigate relationship of the expression of MERTK gene and the activity of protein kinase C (PKC) in the phagocytic process of human retinal pigment epithelial (hRPE) cells. Cultured hRPE cells were incubated with rod outer segments (ROS) suspension (containing ROS 1x10(10)/L) at 37 degrees C, then cells were rinsed at different times to terminate the phagocytosis. The kinetic of phagocytosis was measured by double-fluorescent labeling. The activity of PKC and the expression level of MERTK gene were measured by counting gamma-32P radio-activity with liquid scintillation and RT-PCR respectively. Change of MERTK gene expression was measured after hRPE was treated cells with stimulator or inhibitor of PKC. Statistical analysis was performed by SPSS 13.0 software. The phagocytic assay showed that the quality of bound and ingested ROS by hRPE cells increased. The quality of ingested ROS by hRPE cells at 24 hours was (2.85+/-0.11)x10(6), which reached maximum contrast with control (0.00+/-0.00)x10(6) (t=47.64, P<0.05). The activity of PKC (both in cytoplasm and on membrane) decreased during all the incubation periods compared with control [cytoplasm: (329.63+/-14.26) nmolxg(-1)xmin(-1)and on membrane: (467.67+/-68.87) nmolxg(-1)xmin(-1)], and reached the minimum at 24 h[cytoplasm: (151.13+/-17.67) nmolxg(-1)xmin(-1) and on membrane: (152.45+/-64.83) nmolxg(-1)xmin(-1); cytoplasm t=89.66 and membrane t=10.31, P<0.05]. The level of MERTK mRNA increased in pulse-chase and long-time incubation test. The gray level for 90 min was 1.8853+/-0.0077, contrasted with control 0.7246+/-0.0062, F=16,060.2167 and P<0.05. The gray level for 24 h was 0.5946+/-0.0082, contrasted with control 0.3343+/-0.0064, F=919.8421 and P<0.05. When up-regulating the activity of PKC in hRPE cells, the level of MERTK mRNA was decreased in the proceeding incubating with ROS contrasted with control (pulse-chase group F=17,142.2331, long time group F=1886.4614; P<0.05). After down-regulating the activity of PKC in hRPE cells, the level of MERTK mRNA waved between 4.4670+/-0.0092 and 5.7034+/-0.0095 in the first 30 min of incubating with ROS, which lower than control 0.9117+/-0.0021 (F=199,012.9138, P<0.05). The lower activity of PKC and the higher expression MERTK gene are very important for sustaining phagocytic process of ROS by hRPE cells. MERTK gene and PKC both as up-stream regulators are negative-correlated in the phagocytic process of hRPE.

Interleukin-1β stimulates the secretion of vascular endothelial growth factor and basic fibroblast growth factor in human retinal pigment epithelial cells

Objective To investigate the effect of interleukin (IL-1β) on vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) secretion in cultured human retinal pigment epithelial (RPE) cells. Methods Enzyme linked immunosorbent assay (ELISA) was used to detect the production of VEGF and bFGF by RPECs. The cells were cultured in the presence of IL-1β at different concentrations,which were 0,1,10, and 20 ng/ml, respectively. VEGF and bFGF levels were measured at various time points. Semiquantitative reverse transcription -polymerase chain reaction (RT-PCR) was conducted todetect the expression of VEGF and bFGF mRNA in RPE cells with and without IL-1β stimulation. The proliferation of cells in all groups was measured by the MTT assay and expressed as light absorption values. Results IL-1β significantly stimulated the secretion of VEGF at the concentration of 1 ng/ml and 10 ng/ml (P 0.05). Conclusion Pro-inflammatory cytokine IL-1β increased the secretion of VEGF and bFGF by human RPE cells to some extent. Key words: Pigment epithelium of eye/physiology; Interleukin-lbeta/administration & dosage; Vascular endothelial growth factors; Fibroblast growth factor 2

Targeting retinal and choroid neovascularization using the small molecule inhibitor carboxyamidotriazole

Neovascular ocular diseases as exemplified by proliferative diabetic retinopathy (PDR), exudative age-related macular degeneration (AMD), and retinopathy of prematurity (ROP) are severe diseases affecting all age groups in the US. We asked whether a small molecule, carboxyamidotriazole (CAI) known for its anti-angiogenic and anti-tumor effects and its ability to be administered orally in humans, could have anti-angiogenic effects in ocular in vitro and in vivo angiogenesis models. The anti-proliferative effects of CAI were examined by BrdU incorporation using human retinal and dermal endothelial cells and human pigment epithelial cells. The effect of CAI was determined using the Matrigel tube formation assay. The mouse model of choroidal neovascularization (CNV) initiated by laser rupture of Bruch's membrane was used to quantify in vivo effects of aqueous beta-hydroxypropyl cyclodextrin (bHPCD) formulations of CAI on neovascularization. The pharmacokinetics (PK) of CAI after intravitreal administration of bHPCD-CAI was studied in rabbit. The intravitreal toxicology of bHPCD-CAI was also examined in rat ocular tissue. We observed that CAI treatment of human endothelial cells decreased cell proliferation in a dose-dependent manner. In the in vivo tests bHPCD-CAI treatment reduced choroidal neovascular lesion volume, also in a dose-dependent manner. The intravitreal PK of bHPCD-CAI demonstrated that highly efficacious concentrations of CAI are reached in the vitreous compartment. No ocular toxicology was observed with intravitreous injection of CAI. These studies support the potential of developing intravitreal CAI in an bHPCD ocular formulation for treatment of proliferative retinopathies in humans.