AIM: To observe the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells(h UCMSCs) on the expression of vascular endothelial growth factor-A(VEGF-A) in blue light injured human retinal pig...AIM: To observe the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells(h UCMSCs) on the expression of vascular endothelial growth factor-A(VEGF-A) in blue light injured human retinal pigment epithelial(RPE) cells and laser-induced choroidal neovascularization(CNV) in rats.METHODS: Exosomes were isolated from h UCMSCs and characterized by transmission electron microscope and Western blot. MSCs-derived exosomes were cultured with RPE cells exposed to blue light. The m RNA and protein expression of VEGF-A were determined by real time-polymerase chain reaction(PCR) and Western blot, respectively. Immunofluorescence assay was used for the detection of the expression level of VEGF-A. We injected different doses of MSCs-derived exosomes intravitreally to observe and compare their effects in a mouse model of laserinduced retinal injury. The histological structure of CNV in rats was inspected by hematoxylin-eosin(HE) staining and fundus fluorescein angiography. The expression of VEGF-A was detected by immunohistochemistry.RESULTS: Exosomes exhibited the typical characteristic morphology(cup-shaped) and size(diameter between 50 and 150 nm). The exosomes marker, CD63, and h UCMSCs marker, CD90, showed a robust presence. In vitro, MSCsderived exosomes downregulated the m RNA(Exo-L: t=6.485, 7.959, 9.286; Exo-M: t=7.517, 10.170, 13.413; Exo-H: t=10.317, 12.234, 14.592, P<0.05) and protein(Exo-L: t=2.945, 4.477, 6.657; Exo-M: t=4.713, 6.421, 8.836; Exo-H:t=6.539, 12.194, 12.783; P<0.05) expression of VEGF-A in RPE cells after blue light stimulation. In vivo, we found that the MSCs-derived exosomes reduced damage, distinctly downregulated VEGF-A(Exo-H: t=0.957, 1.382; P<0.05), and gradually improved the histological structures of CNV for a better visual function(Exo-L: 0.346, Exo-M: 3.382, Exo-H: 8.571; P<0.05). CONCLUSION: MSCs-derived exosomes ameliorate blue light stimulation in RPE cells and laser-induced retinal injury via downregulation of VEGF-A.展开更多
AIM:To investigate the cross-talk between oxidative stress and the epidermal growth factor receptor(EGFR)/AKT signaling pathway in retinal pigment epithelial( RPE) cells.METHODS:Human RPE cell lines(ARPE-19 cell) were...AIM:To investigate the cross-talk between oxidative stress and the epidermal growth factor receptor(EGFR)/AKT signaling pathway in retinal pigment epithelial( RPE) cells.METHODS:Human RPE cell lines(ARPE-19 cell) were treated with different doses of epidermal growth factor(EGF) and hydrogen peroxide(H2O2).Cell viability was determined by a methyl thiazolyl tetrazolium assay.Cell proliferation was examined by a bromodeoxyuridine(Brd U) incorporation assay.EGFR/AKT signaling was detected by Western blot.EGFR localization was also detected by immunofluorescence.In addition,EGFR/AKT signaling was intervened upon by EGFR inhibitor(erlotinib),PI3 K inhibitor(A66) and AKT inhibitor(MK-2206),respectively.H2O2-induced oxidative stress was blocked by antioxidant N-acetylcysteine(NAC).RESULTS:EGF treatment increased ARPE-19 cell viabili ty and proliferation through inducing phosphorylation of EGFR and AKT.H2O2 inhibited ARPE-19 cell viability and proliferation and also suppressed EGF-stimulated increase of RPE cell viability and proliferation by affecting the EGFR/AKT signaling pathway.EGFR inhibitor erlotinib blocked EGF-induced phosphorylation of EGFR and AKT,while A66 and MK-2206 only blocked EGF-induced phosphorylation of AKT.EGF-induced phosphorylation andendocytosis of EGFR were also affected by H2O2 treatment.In addition,antioxidant NAC attenuated H2O2-induced inhibition of ARPE-19 cell viability through all eviating reduction of EGFR,and phosphorylated and total AKT proteins.CONCLUSION:Oxidative stress affects RPE cell viability and proliferation through interfering with the EGFR/AKT signaling pathway.The EGFR/AKT signaling pathway may be an important target in oxidative stress-induced RPE cell dysfunction.展开更多
AIM: To investigate the role of oxidative stress in regulating the functional expression of P-glycoprotein(P-gp) in mitochondria of D407 cells.METHODS: D407 cells were exposed to different ranges of concentrations of ...AIM: To investigate the role of oxidative stress in regulating the functional expression of P-glycoprotein(P-gp) in mitochondria of D407 cells.METHODS: D407 cells were exposed to different ranges of concentrations of H_2O_2. The mitochondrial location of P-gp in the cells subjected to oxidative stress was detected by confocal analysis. Expression of P-gp in isolated mitochondria was assessed by Western blot. The pump activity of P-gp was evaluated by performing the efflux study on isolated mitochondria with Rhodamine 123(Rho-123) alone and in the presence of P-gp inhibitor(Tariquidar) using flow cytometry analysis. The cells were pretreated with 10 mmol/L N-acetylcysteine(NAC) for 30 min before exposing to H_2O_2, and analyzed the mitochondrial extracts by Western blot and flow cytometry.RESULTS: P-gp was co-localized in the mitochondria by confocal laser scanning microscopy, and it was also detected in the mitochondria of D407 cells using Western blot. Exposure to increasing concentrations of H_2O_2 led to gradually increased expression and location of P-gp in the mitochondria of cells. Rho-123 efflux assay showed higher uptake of Rho-123 on isolated mitochondria in the presence of Tariquidar both in normal and oxidative stress state. H_2O_2 up-regulated P-gp in D407 cells, which could be reversed by NAC treatment. CONCLUSION: H_2O_2 could up-regulate the functional expression of P-gp in mitochondria of D407 cells, while antioxidants might suppress oxidative-stress-induced over-expression of functional P-gp. It is indicative that limiting the mitochondrial P-gp transport in retinal pigment epithelium cells would be to improve the effect of mitochondria-targeted antioxidant therapy in age-related macular degeneration-like retinopathy.展开更多
AIM:To investigate the effect of anti-vascular epithelial growth factor(VEGF)agents on the expression of fibrosisrelated inflammatory mediators under normoxic and hypoxic conditions,and to further clarify the mechanis...AIM:To investigate the effect of anti-vascular epithelial growth factor(VEGF)agents on the expression of fibrosisrelated inflammatory mediators under normoxic and hypoxic conditions,and to further clarify the mechanism underlying fibrosis after anti-VEGF therapy. METHODS:Human retinal pigment epithelial(RPE)cells were incubated under normoxic and hypoxic conditions.For hypoxia treatment,CoCl_2 at 200μmol/L was added to the media. ARPE-19 cells were treated as following:1)control group:no treatment; 2)bevacizumab group:bevacizumab at 0.25 mg/mL was added to the media; 3)hypoxia group:CoCl_2 at 200 μmol/L was added to the media; 4)hypoxia+bevacizumab group:CoCl_2 at 200 μmol/L and bevacizumab at 0.25 mg/mL were added to the media.The expression of interleukin(IL)-1β,IL-6,IL-8 and tumor necrosis factor(TNF)-α were evaluated using real-time polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA)at 6,12,24 and 48 h. RESULTS:Both m RNA and protein levels of IL-1β,IL-6 and IL-8 were statistically significantly higher in the bevacizumab group than in the control group at each time point,and TNF-α gene and protein expression was only significantly higher only at 24 and 48h(P<0.05). Under hypoxic conditions,bevacizumab significantly increased the expression of IL-1β,IL-6,IL-8 and TNF-α at 6,12,24 and 48h(P<0.05). IL-1β,IL-8 and TNF-α peaked at 24 h and IL-6 peaked at 12 h after the bevacizumab treatment under both normoxic and hypoxic conditions. CONCLUSION:Treatment of ARPE-19 cells with bevacizumab can significantly increase the expression of fibrosis-related inflammatory mediators under bothnormoxic and hypoxic conditions. Inflammatory factors might be involved in the process of fibrosis after antiVEGF therapy,and the up-regulation of inflammatory factors induced by anti-VEGF drugs might promote the fibrosis process.展开更多
AIM: To explore whether resveratrol(Res) can inhibit human retinal pigment epithelial cell(ARPE-19 cell)proliferation and migration, and to research the molecular mechanisms.·METHODS: ARPE-19 cells were pretreate...AIM: To explore whether resveratrol(Res) can inhibit human retinal pigment epithelial cell(ARPE-19 cell)proliferation and migration, and to research the molecular mechanisms.·METHODS: ARPE-19 cells were pretreated with various concentrations at 0, 50, 100, 150, 200 and 300 μmol/L of Res, and with 0 μmol/L Res as the control for 24, 48 and72 h. The cell proliferation, apoptosis and migration were measured with cell counting kit-8(CCK-8), flow cytometry, and wound-healing and Transwell assays,respectively. The expression of proliferating cell nuclear antigen(PCNA), P21 and P27, as well as matrix metalloproteinase-9(MMP-9) and p38 mitogen-activated protein kinases(p38MAPK) was identified by Western blot.·RESULTS: Cell proliferation was effectively inhibited by Res(P <0.05). When pretreated with Res, cells arrested in S-phase increased remarkably(P <0.05), but the apoptosis ratios showed no significant difference between the treatment and control groups(P >0.05). Cell migration was suppressed by Res both in wound-healing assay and Transwell migration assay(P <0.05). Decreases of PCNA, MMP-9 and p38 MAPK, as well as increases of P21 and P27 were detected by Western blot(P <0.05).·CONCLUSION: Res can inhibit APRE-19 cell proliferation and migration in a concentration-dependent manner with up-regulation of the expression of P21 and P27, and down-regulation of PCNA, MMP-9 and p38 MAPK.展开更多
AIM: To evaluate whether protein tyrosine phosphatase1B(PTP1B) contributed to initiate human retinal pigment epithelium cells(ARPE)-19 migration and investigate the signaling pathways involved in this process.·ME...AIM: To evaluate whether protein tyrosine phosphatase1B(PTP1B) contributed to initiate human retinal pigment epithelium cells(ARPE)-19 migration and investigate the signaling pathways involved in this process.·METHODS: ARPE-19 cells were cultured and treated with the si RNA-PTP1 B. Expression of PTP1 B was confirmed by quantitative reverse transcriptase-polymerase chain reaction(q RT-PCR). AG1478 [a selective inhibitor of epidermal growth factor receptor(EGFR)] and PD98059(a specific inhibitor of the activation of mitogen-activated protein kinase) were used to help to determine the PTP1 B signaling mechanism.Western blot analysis verified expression of EGFR and extracellular signal-regulated kinase(ERK) in ARPE-19 cells. The effect of si RNA-PTP1 B on cell differentiation was confirmed by immunostaining for α-smooth muscle actin(α-SMA) and q RT-PCR. Cell migration ability was analyzed by transwell chamber assay.·RESULTS: The m RNA levels of PTP1 B were reduced by si RNA-PTP1 B as determined by q RT-PCR assay.Si RNA-PTP1 B activated EGFR and ERK phosphorylation.α-SMA staining and q RT-PCR assay demonstrated that si RNA-PTP1 B induced retinal pigment epithelium(RPE)cells to differentiate toward better contractility and motility. Transwell chamber assay proved that PTP1 B inhibition improved migration activity of RPE cells.Treatment with AG1478 and PD98059 abolished si RNA-PTP1B-induced activation of EGFR and ERK, α-SMA expression and cell migration.·CONCLUSION: PTP1 B inhibition promoted myofibroblast differentiation and migration of ARPE-19 cells, and EGFR/ERK signaling pathway played important role in migration process.展开更多
AIM: To investigate the effects of high glucose levels and anti-vascular endothelial growth factor(VEGF) agents(bevacizumab,ranibizumab and aflibercept) on retinal pigment epithelium(RPE) cells.METHODS: ARPE-19 cells ...AIM: To investigate the effects of high glucose levels and anti-vascular endothelial growth factor(VEGF) agents(bevacizumab,ranibizumab and aflibercept) on retinal pigment epithelium(RPE) cells.METHODS: ARPE-19 cells were cultured at different glucose levels(5.5 mmol/L,25 mmol/L,and 75 mmol/L).Cell viability was evaluated by MTT assay at 3d after treatment with D-glucose.Cell migration ability was measured by wound healing assay at 3d.A cell death detection kit was used to assess apoptosis at 3 and 14 d.Cell proliferation was assessed by EdU assay at 3d.The culture medium was treated with anti-VEGF agents at clinically relevant concentrations.The experiment was then repeated at a different glucose level.RESULTS: The viability and migration of ARPE-19 cells were significantly decreased in the presence of 75 mmol/L as compared to 5.5 mmol/L glucose.The percentage of TUNEL-positive cells was significantly increased and the proliferative potential was decreased with 75 mmol/L compared to 5.5 mmol/L glucose.There were no significant differences in the results between 25 mmol/L and 5.5 mmol/L glucose.In the presence of 75 mmol/L glucose,the groups treated with anti-VEGF showed decreased cell viability and proliferation and increased apoptosis.However,there were no significant differences between the anti-VEGF groups.CONCLUSION: High glucose level decreases the viability,wound healing ability,and proliferation of RPE cells,while increasing apoptosis.Furthermore,anti-VEGF agents interfered with the physiological functions of RPE cells under high-glucose conditions,accompanied by decreases in cell viability and proliferation.展开更多
AIM:To study the effects of cytokeratin 17(CK17) on sodium iodate(NalO_3) induced rat retinal pigment epithelium(RPE) degeneration,laser induced rat choroidal neovascularization(CNV),and oxidative stress of human reti...AIM:To study the effects of cytokeratin 17(CK17) on sodium iodate(NalO_3) induced rat retinal pigment epithelium(RPE) degeneration,laser induced rat choroidal neovascularization(CNV),and oxidative stress of human retinal pigment epithelium cells(ARPE-19) and human umbilical vein endothelial cell(HUVEC).· METHODS:Thirty 8-week-old male Brown Norway rats were randomly divided into 3 groups,10 rats in control group treated with solvent alone;10 rats in NalO_3 group treated with solvent and 35 mg/kg NalO_3 injection through hypoglossal vein and 10 rats in CK17+NalO_3group treated with 1%CK17 eye drop 3 times a day for1 wk before and 4wk after NalO_3 injection.RPE function was measured with c-wave of electroretinogram(ERG).Another 20 rats were randomly divided into 2 groups.Of them 10 rats in CK17 group were anesthetized to receive Nd:YAG laser and given 1%CK17 eye drop before same as above;10 rats in control were received Nd:YAG and treated with solvent.The development of choroidal neovascularization(CNV) was determined by fundus fluorescein angiography(FFA) performed on 4wk after laser.Methylthiazoly tetrazolium(MTT) assay was used to study effect of CK17 on various oxidants induced injury in ARPE-19 and HUVEC in vitro.· RESULTS:Four weeks after NalO_3 injection,the cwave amplitude of ERG was 0.393±0.02 V in the control group,0.184 ±0.018 V in NalO_3 group and 0.3±0.01 V in CK17+NalO_3 group.There was a significant reversal of the c-wave by CK17 as compared to NalO_3 group(P<0.01).Four weeks after laser,the size of the CNV lesion was2.57±0.27 mm^2 in control group and 1.64±0.08 mm_2 in CK17 group.The lesion size significantly diminished in CK17 group(P<0.01).The in vitro results showed CK17 also reversed the various oxidants induced injuries in ARPE-19 at the dose of 100 μg/mL and enhanced the injury in HUVECs at different concentrations.CONCLUSION:CK17 can significantly protect RPE from NalO_3 induced degeneration in vivo and in vitro and also could reverse the various oxidants induced injuries in vitro.It inhibits the development of CNV in rat model,interfered with vascular endothelial cell proliferation in vitro.展开更多
Age-related macular degeneration(AMD) is the leading cause of vision loss in the elderly throughout the world. Treatment of AMD utilizing retinal pigment epithelium(RPE) transplantation represents a promising therapy....Age-related macular degeneration(AMD) is the leading cause of vision loss in the elderly throughout the world. Treatment of AMD utilizing retinal pigment epithelium(RPE) transplantation represents a promising therapy. However, simplex RPE transplantation can only replace the diseased RPE cells, but has no abilities to stop the development of AMD. It has been indicated that oxidization triggers the development of AMD by inducing the dysfunction and degeneration of RPE cells, which results in the upregulation of local monocyte chemotactic protein-1(MCP-1) expression. MCP-1 induces macrophage recruiment which triggers local inflammation. As a result, the expression of vascular endothelial growth factor(VEGF) is upregulated by MCP-1mediated inflammation and results in the formation of choroidal neovascularization(CNV). We accordingly propose a targeted therapy of AMD by subretinal transplanting the compound of RPE cell, MCP-1 antibody, and VEGF antibody and using a magnetic system to guide RPE cell compounds conjugated with superparamagnetic iron oxide nanoparticles(SPIONs). Furthermore, SPION-labelled RPE cells can be tracked and detected in vivo by non-invasive magnetic resonance imaging(MRI). This novel RPE cell transplantation methodology seems very promising to provide a new therapeutic approach for the treatment of AMD.展开更多
AIM:To investigate the effect of Htr A1 on the proliferation,migration and apoptosis of human retinal pigment epithelium(RPE) cells in the light injured model,as well as the expression of the apoptosis related molecul...AIM:To investigate the effect of Htr A1 on the proliferation,migration and apoptosis of human retinal pigment epithelium(RPE) cells in the light injured model,as well as the expression of the apoptosis related molecules.METHODS:The human RPE cell line ARPE-19 was exposed to blue light to establish the light injured model.The cells were transfected with Htr A1 si RNA to knockdown Htr A1 expression.Subsequent expression of Htr A1 was determined by real-time polymerase chain reaction(RTPCR) and Western blot,respectively.Changes in cell proliferation,migration and apoptosis were assessed by cell counting kit-8(CCK-8),Transwell assay and flow cytometry respectively,as well as changes in the m RNA and protein levels of Bax,Caspase-3 and Bcl-2 expression.RESULTS:Htr A1 was highly expressed in ARPE-19 cells after blue light irradiation.Knockdown of Htr A1 expression inhibited the proliferation,migration and apoptosis of the blue-light-irradiated ARPE-19 cells(P<0.05).Bax and Caspase-3 expression were significantly reduced both at m RNA and protein levels(P<0.05) after si RNA treatment.Bcl-2 expression significantly increased in blue-lightirradiated ARPE-19 cells after si RNA interference(P<0.05).CONCLUSION:Silence of Htr A1 may inhibit the proliferation,migration and apoptosis of ARPE-19 cells in light injured model.Moreover,Htr A1 suppression in blue-lightirradiated ARPE-19 cells may ameliorate cell apoptosis through down-regulation of Bax and Caspase-3,and upregulation of Bcl-2 expression.展开更多
AIM: To determine the effect of different concentrations of the acetylcholinesterase(ACh E)inhibitors tacrine and donepezil on retinal protection in AChE+/- mice(ACh E knockout mice) of various ages.·METHODS: Cul...AIM: To determine the effect of different concentrations of the acetylcholinesterase(ACh E)inhibitors tacrine and donepezil on retinal protection in AChE+/- mice(ACh E knockout mice) of various ages.·METHODS: Cultured ARPE-19 cells were treated with hydrogen peroxide(H2O2) at concentrations of 0, 250,500, 1000 and 2000 μmol/L and protein levels were measured using Western blot. Intraperitoneal injections of tacrine and donepezil( 0. 1 mg / m L, 0. 2 mg / m L and0.4 mg/m L) were respectively given to AChE+/- mice aged2 mo and 4mo and wild-type S129 mice for 7d; phosphate buffered saline(PBS) was administered to the control group. The mice were sacrificed after 30 d by in vitro cardiac perfusion and retinal samples were taken. ACh Edeficient mice were identified by polymerase chain reaction(PCR) analysis using specific genotyping protocols obtained from the Jackson Laboratory website.H&E staining, immunofluorescence and Western blot were performed to observe ACh E protein expression changes in the retinal pigment epithelial(RPE) cell layer.·RESULTS: Different concentrations of H2O2 induced ACh E expression during RPE cell apoptosis. AChE+/- mice retina were thinner than those in wild-type mice(P <0.05); the retinal structure was still intact at 2mo but became thinner with increasing age(P <0.05); furthermore,AChE+/- mice developed more slowly than wild-type mice(P <0.05). Increased concentrations of tacrine and donepezil did not significantly improve the protection of the retina function and morphology(P >0.05).·CONCLUSION: In vivo, tacrine and donepezil can inhibit the expression of ACh E; the decrease of ACh E expression in the retina is beneficial for the development of the retina.展开更多
AIM: To investigate the roles of PKC-α/ezrin signals in phagocytosis crisis of retinal pigment epithelium(RPE) cells in light damage model. METHODS: Light induced mice RPE injury model was established by continuously...AIM: To investigate the roles of PKC-α/ezrin signals in phagocytosis crisis of retinal pigment epithelium(RPE) cells in light damage model. METHODS: Light induced mice RPE injury model was established by continuously irradiating cool white light at different exposure time(0, 4, 8h light intensity: 4.18×10-6 J/cm2). In vitro, human ARPE-19 cells treated with the doses and intensity(1.57×10-6 J/cm2) of laser irradiation. Histology analysis was evaluated by hematoxylin and eosin(HE) staining. In vivo RPE phagocytosis was quantified by measuring the accumulation of photoreceptor outer segments in the sub-retinal space. In vitro RPE phagocytosis was assessed by calculating the relative fluorescence intensity of FITC-labeled microspheres in ARPE-19 cells. To further investigate the molecular mechanism, the activation of PKC-α/ezrin signal was evaluated by Western blot in vivo and in vitro.RESULTS: HE staining revealed that the thickness of outer nuclear layer decreased significantly after 4 and 8h light exposure. By immunostaining with rhodopsin, a significant greater accumulation of photoreceptor outer segment was noticed after light injury. In vitro, light injuredRPE cells had less phagocytic activity in a dose dependent manner than that of the normal control(P<0.01). Western blot suggested the activation of PKC-α/ezrin signaling was down-regulated in a dose-dependent manner after light exposure. CONCLUSION: Our data suggest that light induced phagocytic crisis of RPE cells may result from the downregulation of PKC-α/ezrin signaling.展开更多
AIM:To identify the effects of interleukin(IL)-13 on retinal pigment epithelial(RPE) cells and the IL-13 level in aqueous humor of age-related macular degeneration(AMD) patients.METHODS:IL-13 levels in aqueous humor s...AIM:To identify the effects of interleukin(IL)-13 on retinal pigment epithelial(RPE) cells and the IL-13 level in aqueous humor of age-related macular degeneration(AMD) patients.METHODS:IL-13 levels in aqueous humor specimens from AMD patients were detected with enzyme-linked immunosorbent assay(ELISA).ARPE-19 cells were treated with 10 ng/m L IL-13 for 12,24,and 48 h.The cell proliferaton was evaluated by the MTS method.The m RNA and protein levels of α-SMA and ZO-1 were evaluated with quantitative real-time polymerase chain reaction(q RT-PCR) and Western blot respectively.The expression of tumor necrosis factor-α(TNF-α),transforming growth factor-β(TGF-β) and vascular endothelial growth factor(VEGF) were assessed by ELISA.RESU LTS:IL-13 levels in the aqueous humor of patients with AMD were significantly higher than those in the control(167.33±17.64 vs 27.12±5.65 pg/m L;P<0.01).In vit ro,IL-13 of high concentrations(10,15,and 20 ng/m L) inhibited ARPE-19 cell proliferation.α-SMA m RNA in ARPE-19 cell were increased(1.017±0.112 vs 1.476±0.168;P<0.001) and ZO-1 decreased(1.051±0.136 vs 0.702±0.069;P<0.001) after treated with 10 ng/m L IL-13 for 48 h.The protein expression of α-SMA and ZO-1 also showed the same tendency(α-SMA:P=0.038;ZO-1:P=0.008).IL-13 significantly reduced the level of TNF-α(44.70±1.67 vs 31.79±3.53 pg/m L;P=0.005) at 48 h,but the le vel of TGF-β2 was significantly increased from 34.44±2.92 to 57.61±6.31 pg/m L at 24h(P=0.004) and from 61.26±1.11 to 86.91±3.59 pg/m L at 48h(P<0.001).While expressions of VEGF didn't change after IL-13 treatment.CONCLUSION:IL-13 in vitr o inhibit ARPE-19 cell proliferation and expression in the aqueous may be associated with AMD.展开更多
基金Supported by the National Natural Science Foundation of China(No.81700846)Tianjin Science and Technology Project of China(No.14JCYBJC27400)Science and technology Project of Tianjin Municipal Health Bureau(No.2015KZ073)
文摘AIM: To observe the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells(h UCMSCs) on the expression of vascular endothelial growth factor-A(VEGF-A) in blue light injured human retinal pigment epithelial(RPE) cells and laser-induced choroidal neovascularization(CNV) in rats.METHODS: Exosomes were isolated from h UCMSCs and characterized by transmission electron microscope and Western blot. MSCs-derived exosomes were cultured with RPE cells exposed to blue light. The m RNA and protein expression of VEGF-A were determined by real time-polymerase chain reaction(PCR) and Western blot, respectively. Immunofluorescence assay was used for the detection of the expression level of VEGF-A. We injected different doses of MSCs-derived exosomes intravitreally to observe and compare their effects in a mouse model of laserinduced retinal injury. The histological structure of CNV in rats was inspected by hematoxylin-eosin(HE) staining and fundus fluorescein angiography. The expression of VEGF-A was detected by immunohistochemistry.RESULTS: Exosomes exhibited the typical characteristic morphology(cup-shaped) and size(diameter between 50 and 150 nm). The exosomes marker, CD63, and h UCMSCs marker, CD90, showed a robust presence. In vitro, MSCsderived exosomes downregulated the m RNA(Exo-L: t=6.485, 7.959, 9.286; Exo-M: t=7.517, 10.170, 13.413; Exo-H: t=10.317, 12.234, 14.592, P<0.05) and protein(Exo-L: t=2.945, 4.477, 6.657; Exo-M: t=4.713, 6.421, 8.836; Exo-H:t=6.539, 12.194, 12.783; P<0.05) expression of VEGF-A in RPE cells after blue light stimulation. In vivo, we found that the MSCs-derived exosomes reduced damage, distinctly downregulated VEGF-A(Exo-H: t=0.957, 1.382; P<0.05), and gradually improved the histological structures of CNV for a better visual function(Exo-L: 0.346, Exo-M: 3.382, Exo-H: 8.571; P<0.05). CONCLUSION: MSCs-derived exosomes ameliorate blue light stimulation in RPE cells and laser-induced retinal injury via downregulation of VEGF-A.
基金Supported by the National Natural Science Foundation of China(N o.81570875No.31170685)+4 种基金the China Postdoctoral Science Foundation Funded Project(No.2015M582044)the Health Systems Young Personnel Training Projects Foundation of Fujian Province,China(No.2013-ZQN-JC-37)the Science and Technology Program Foundation of Xiamen City in China(No.3502720144044)the Scientific Research Foundation of the State Human Resource Ministrythe Scientific Research Staring Foundation for the Returned Overseas Chinese Scholars,Ministry of Education of China
文摘AIM:To investigate the cross-talk between oxidative stress and the epidermal growth factor receptor(EGFR)/AKT signaling pathway in retinal pigment epithelial( RPE) cells.METHODS:Human RPE cell lines(ARPE-19 cell) were treated with different doses of epidermal growth factor(EGF) and hydrogen peroxide(H2O2).Cell viability was determined by a methyl thiazolyl tetrazolium assay.Cell proliferation was examined by a bromodeoxyuridine(Brd U) incorporation assay.EGFR/AKT signaling was detected by Western blot.EGFR localization was also detected by immunofluorescence.In addition,EGFR/AKT signaling was intervened upon by EGFR inhibitor(erlotinib),PI3 K inhibitor(A66) and AKT inhibitor(MK-2206),respectively.H2O2-induced oxidative stress was blocked by antioxidant N-acetylcysteine(NAC).RESULTS:EGF treatment increased ARPE-19 cell viabili ty and proliferation through inducing phosphorylation of EGFR and AKT.H2O2 inhibited ARPE-19 cell viability and proliferation and also suppressed EGF-stimulated increase of RPE cell viability and proliferation by affecting the EGFR/AKT signaling pathway.EGFR inhibitor erlotinib blocked EGF-induced phosphorylation of EGFR and AKT,while A66 and MK-2206 only blocked EGF-induced phosphorylation of AKT.EGF-induced phosphorylation andendocytosis of EGFR were also affected by H2O2 treatment.In addition,antioxidant NAC attenuated H2O2-induced inhibition of ARPE-19 cell viability through all eviating reduction of EGFR,and phosphorylated and total AKT proteins.CONCLUSION:Oxidative stress affects RPE cell viability and proliferation through interfering with the EGFR/AKT signaling pathway.The EGFR/AKT signaling pathway may be an important target in oxidative stress-induced RPE cell dysfunction.
基金Supported by National Natural Science Foundation of China(No.81400424)Guangdong Medical Science Foundation(No.A2015151)
文摘AIM: To investigate the role of oxidative stress in regulating the functional expression of P-glycoprotein(P-gp) in mitochondria of D407 cells.METHODS: D407 cells were exposed to different ranges of concentrations of H_2O_2. The mitochondrial location of P-gp in the cells subjected to oxidative stress was detected by confocal analysis. Expression of P-gp in isolated mitochondria was assessed by Western blot. The pump activity of P-gp was evaluated by performing the efflux study on isolated mitochondria with Rhodamine 123(Rho-123) alone and in the presence of P-gp inhibitor(Tariquidar) using flow cytometry analysis. The cells were pretreated with 10 mmol/L N-acetylcysteine(NAC) for 30 min before exposing to H_2O_2, and analyzed the mitochondrial extracts by Western blot and flow cytometry.RESULTS: P-gp was co-localized in the mitochondria by confocal laser scanning microscopy, and it was also detected in the mitochondria of D407 cells using Western blot. Exposure to increasing concentrations of H_2O_2 led to gradually increased expression and location of P-gp in the mitochondria of cells. Rho-123 efflux assay showed higher uptake of Rho-123 on isolated mitochondria in the presence of Tariquidar both in normal and oxidative stress state. H_2O_2 up-regulated P-gp in D407 cells, which could be reversed by NAC treatment. CONCLUSION: H_2O_2 could up-regulate the functional expression of P-gp in mitochondria of D407 cells, while antioxidants might suppress oxidative-stress-induced over-expression of functional P-gp. It is indicative that limiting the mitochondrial P-gp transport in retinal pigment epithelium cells would be to improve the effect of mitochondria-targeted antioxidant therapy in age-related macular degeneration-like retinopathy.
基金Supported by Shandong Provincial Natural Science Foundation,China(No.ZR2014HM029)
文摘AIM:To investigate the effect of anti-vascular epithelial growth factor(VEGF)agents on the expression of fibrosisrelated inflammatory mediators under normoxic and hypoxic conditions,and to further clarify the mechanism underlying fibrosis after anti-VEGF therapy. METHODS:Human retinal pigment epithelial(RPE)cells were incubated under normoxic and hypoxic conditions.For hypoxia treatment,CoCl_2 at 200μmol/L was added to the media. ARPE-19 cells were treated as following:1)control group:no treatment; 2)bevacizumab group:bevacizumab at 0.25 mg/mL was added to the media; 3)hypoxia group:CoCl_2 at 200 μmol/L was added to the media; 4)hypoxia+bevacizumab group:CoCl_2 at 200 μmol/L and bevacizumab at 0.25 mg/mL were added to the media.The expression of interleukin(IL)-1β,IL-6,IL-8 and tumor necrosis factor(TNF)-α were evaluated using real-time polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA)at 6,12,24 and 48 h. RESULTS:Both m RNA and protein levels of IL-1β,IL-6 and IL-8 were statistically significantly higher in the bevacizumab group than in the control group at each time point,and TNF-α gene and protein expression was only significantly higher only at 24 and 48h(P<0.05). Under hypoxic conditions,bevacizumab significantly increased the expression of IL-1β,IL-6,IL-8 and TNF-α at 6,12,24 and 48h(P<0.05). IL-1β,IL-8 and TNF-α peaked at 24 h and IL-6 peaked at 12 h after the bevacizumab treatment under both normoxic and hypoxic conditions. CONCLUSION:Treatment of ARPE-19 cells with bevacizumab can significantly increase the expression of fibrosis-related inflammatory mediators under bothnormoxic and hypoxic conditions. Inflammatory factors might be involved in the process of fibrosis after antiVEGF therapy,and the up-regulation of inflammatory factors induced by anti-VEGF drugs might promote the fibrosis process.
基金Supported by Major Project on Health Care and Innovation of Guangzhou,China(No.201400000003-3)Science and Technology Planning Project of Guangdong Province,China(No.2015A020212026)
文摘AIM: To explore whether resveratrol(Res) can inhibit human retinal pigment epithelial cell(ARPE-19 cell)proliferation and migration, and to research the molecular mechanisms.·METHODS: ARPE-19 cells were pretreated with various concentrations at 0, 50, 100, 150, 200 and 300 μmol/L of Res, and with 0 μmol/L Res as the control for 24, 48 and72 h. The cell proliferation, apoptosis and migration were measured with cell counting kit-8(CCK-8), flow cytometry, and wound-healing and Transwell assays,respectively. The expression of proliferating cell nuclear antigen(PCNA), P21 and P27, as well as matrix metalloproteinase-9(MMP-9) and p38 mitogen-activated protein kinases(p38MAPK) was identified by Western blot.·RESULTS: Cell proliferation was effectively inhibited by Res(P <0.05). When pretreated with Res, cells arrested in S-phase increased remarkably(P <0.05), but the apoptosis ratios showed no significant difference between the treatment and control groups(P >0.05). Cell migration was suppressed by Res both in wound-healing assay and Transwell migration assay(P <0.05). Decreases of PCNA, MMP-9 and p38 MAPK, as well as increases of P21 and P27 were detected by Western blot(P <0.05).·CONCLUSION: Res can inhibit APRE-19 cell proliferation and migration in a concentration-dependent manner with up-regulation of the expression of P21 and P27, and down-regulation of PCNA, MMP-9 and p38 MAPK.
基金Supported by Shandong Provincial Natural Science Foundation,China(No.ZR2012HQ004)the Research Fund for Fundamental Research Project of Qingdao(No.13-1-4-180-jch)+1 种基金the Scientific Research Fund of Huangdao District of Qingdao City(No.2014-1-74)the Young People Scientific Research Fund of Affiliated Hospital,Qingdao University(No.QDFY134)
文摘AIM: To evaluate whether protein tyrosine phosphatase1B(PTP1B) contributed to initiate human retinal pigment epithelium cells(ARPE)-19 migration and investigate the signaling pathways involved in this process.·METHODS: ARPE-19 cells were cultured and treated with the si RNA-PTP1 B. Expression of PTP1 B was confirmed by quantitative reverse transcriptase-polymerase chain reaction(q RT-PCR). AG1478 [a selective inhibitor of epidermal growth factor receptor(EGFR)] and PD98059(a specific inhibitor of the activation of mitogen-activated protein kinase) were used to help to determine the PTP1 B signaling mechanism.Western blot analysis verified expression of EGFR and extracellular signal-regulated kinase(ERK) in ARPE-19 cells. The effect of si RNA-PTP1 B on cell differentiation was confirmed by immunostaining for α-smooth muscle actin(α-SMA) and q RT-PCR. Cell migration ability was analyzed by transwell chamber assay.·RESULTS: The m RNA levels of PTP1 B were reduced by si RNA-PTP1 B as determined by q RT-PCR assay.Si RNA-PTP1 B activated EGFR and ERK phosphorylation.α-SMA staining and q RT-PCR assay demonstrated that si RNA-PTP1 B induced retinal pigment epithelium(RPE)cells to differentiate toward better contractility and motility. Transwell chamber assay proved that PTP1 B inhibition improved migration activity of RPE cells.Treatment with AG1478 and PD98059 abolished si RNA-PTP1B-induced activation of EGFR and ERK, α-SMA expression and cell migration.·CONCLUSION: PTP1 B inhibition promoted myofibroblast differentiation and migration of ARPE-19 cells, and EGFR/ERK signaling pathway played important role in migration process.
基金Supported by grants from Basic Science Research Program through the National Research Foundation of Korea(NRF)funded by the ministry of Education,Science,and Technology(No.2016R1A2B4008376Seoul,Republic of Korea)partially supported by the Soonchunhyang University Research Fund
文摘AIM: To investigate the effects of high glucose levels and anti-vascular endothelial growth factor(VEGF) agents(bevacizumab,ranibizumab and aflibercept) on retinal pigment epithelium(RPE) cells.METHODS: ARPE-19 cells were cultured at different glucose levels(5.5 mmol/L,25 mmol/L,and 75 mmol/L).Cell viability was evaluated by MTT assay at 3d after treatment with D-glucose.Cell migration ability was measured by wound healing assay at 3d.A cell death detection kit was used to assess apoptosis at 3 and 14 d.Cell proliferation was assessed by EdU assay at 3d.The culture medium was treated with anti-VEGF agents at clinically relevant concentrations.The experiment was then repeated at a different glucose level.RESULTS: The viability and migration of ARPE-19 cells were significantly decreased in the presence of 75 mmol/L as compared to 5.5 mmol/L glucose.The percentage of TUNEL-positive cells was significantly increased and the proliferative potential was decreased with 75 mmol/L compared to 5.5 mmol/L glucose.There were no significant differences in the results between 25 mmol/L and 5.5 mmol/L glucose.In the presence of 75 mmol/L glucose,the groups treated with anti-VEGF showed decreased cell viability and proliferation and increased apoptosis.However,there were no significant differences between the anti-VEGF groups.CONCLUSION: High glucose level decreases the viability,wound healing ability,and proliferation of RPE cells,while increasing apoptosis.Furthermore,anti-VEGF agents interfered with the physiological functions of RPE cells under high-glucose conditions,accompanied by decreases in cell viability and proliferation.
文摘AIM:To study the effects of cytokeratin 17(CK17) on sodium iodate(NalO_3) induced rat retinal pigment epithelium(RPE) degeneration,laser induced rat choroidal neovascularization(CNV),and oxidative stress of human retinal pigment epithelium cells(ARPE-19) and human umbilical vein endothelial cell(HUVEC).· METHODS:Thirty 8-week-old male Brown Norway rats were randomly divided into 3 groups,10 rats in control group treated with solvent alone;10 rats in NalO_3 group treated with solvent and 35 mg/kg NalO_3 injection through hypoglossal vein and 10 rats in CK17+NalO_3group treated with 1%CK17 eye drop 3 times a day for1 wk before and 4wk after NalO_3 injection.RPE function was measured with c-wave of electroretinogram(ERG).Another 20 rats were randomly divided into 2 groups.Of them 10 rats in CK17 group were anesthetized to receive Nd:YAG laser and given 1%CK17 eye drop before same as above;10 rats in control were received Nd:YAG and treated with solvent.The development of choroidal neovascularization(CNV) was determined by fundus fluorescein angiography(FFA) performed on 4wk after laser.Methylthiazoly tetrazolium(MTT) assay was used to study effect of CK17 on various oxidants induced injury in ARPE-19 and HUVEC in vitro.· RESULTS:Four weeks after NalO_3 injection,the cwave amplitude of ERG was 0.393±0.02 V in the control group,0.184 ±0.018 V in NalO_3 group and 0.3±0.01 V in CK17+NalO_3 group.There was a significant reversal of the c-wave by CK17 as compared to NalO_3 group(P<0.01).Four weeks after laser,the size of the CNV lesion was2.57±0.27 mm^2 in control group and 1.64±0.08 mm_2 in CK17 group.The lesion size significantly diminished in CK17 group(P<0.01).The in vitro results showed CK17 also reversed the various oxidants induced injuries in ARPE-19 at the dose of 100 μg/mL and enhanced the injury in HUVECs at different concentrations.CONCLUSION:CK17 can significantly protect RPE from NalO_3 induced degeneration in vivo and in vitro and also could reverse the various oxidants induced injuries in vitro.It inhibits the development of CNV in rat model,interfered with vascular endothelial cell proliferation in vitro.
基金Supported by the National Natural Science Foundation of China(No.81100670)the Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry of China
文摘Age-related macular degeneration(AMD) is the leading cause of vision loss in the elderly throughout the world. Treatment of AMD utilizing retinal pigment epithelium(RPE) transplantation represents a promising therapy. However, simplex RPE transplantation can only replace the diseased RPE cells, but has no abilities to stop the development of AMD. It has been indicated that oxidization triggers the development of AMD by inducing the dysfunction and degeneration of RPE cells, which results in the upregulation of local monocyte chemotactic protein-1(MCP-1) expression. MCP-1 induces macrophage recruiment which triggers local inflammation. As a result, the expression of vascular endothelial growth factor(VEGF) is upregulated by MCP-1mediated inflammation and results in the formation of choroidal neovascularization(CNV). We accordingly propose a targeted therapy of AMD by subretinal transplanting the compound of RPE cell, MCP-1 antibody, and VEGF antibody and using a magnetic system to guide RPE cell compounds conjugated with superparamagnetic iron oxide nanoparticles(SPIONs). Furthermore, SPION-labelled RPE cells can be tracked and detected in vivo by non-invasive magnetic resonance imaging(MRI). This novel RPE cell transplantation methodology seems very promising to provide a new therapeutic approach for the treatment of AMD.
基金Supported by the National Natural Science Foundation of China(No.81271025No.81271023)
文摘AIM:To investigate the effect of Htr A1 on the proliferation,migration and apoptosis of human retinal pigment epithelium(RPE) cells in the light injured model,as well as the expression of the apoptosis related molecules.METHODS:The human RPE cell line ARPE-19 was exposed to blue light to establish the light injured model.The cells were transfected with Htr A1 si RNA to knockdown Htr A1 expression.Subsequent expression of Htr A1 was determined by real-time polymerase chain reaction(RTPCR) and Western blot,respectively.Changes in cell proliferation,migration and apoptosis were assessed by cell counting kit-8(CCK-8),Transwell assay and flow cytometry respectively,as well as changes in the m RNA and protein levels of Bax,Caspase-3 and Bcl-2 expression.RESULTS:Htr A1 was highly expressed in ARPE-19 cells after blue light irradiation.Knockdown of Htr A1 expression inhibited the proliferation,migration and apoptosis of the blue-light-irradiated ARPE-19 cells(P<0.05).Bax and Caspase-3 expression were significantly reduced both at m RNA and protein levels(P<0.05) after si RNA treatment.Bcl-2 expression significantly increased in blue-lightirradiated ARPE-19 cells after si RNA interference(P<0.05).CONCLUSION:Silence of Htr A1 may inhibit the proliferation,migration and apoptosis of ARPE-19 cells in light injured model.Moreover,Htr A1 suppression in blue-lightirradiated ARPE-19 cells may ameliorate cell apoptosis through down-regulation of Bax and Caspase-3,and upregulation of Bcl-2 expression.
基金Supported by National Natural Science Foundation of China(No.81160118,81400372)Clinical Medicine Research Special-purpose Foundation of China(No.L2012052)+5 种基金Jiangxi Province Sailing Engineering(No.2014022)Science Technology Foundation of Jiangxi Province(No.20151BBG70223)Youth Science Foundation of Jiangxi Province(No.20151BAB215016)Education Department Youth Scientific Research Foundation(No.GJJ14170)Health Development Planning Commission Science Foundation of Jiangxi Province(NO:20155154)Health Department Tradition Chinese Medicine Science and Technology Foundation(No.2010A015,2012A139,2013A073)
文摘AIM: To determine the effect of different concentrations of the acetylcholinesterase(ACh E)inhibitors tacrine and donepezil on retinal protection in AChE+/- mice(ACh E knockout mice) of various ages.·METHODS: Cultured ARPE-19 cells were treated with hydrogen peroxide(H2O2) at concentrations of 0, 250,500, 1000 and 2000 μmol/L and protein levels were measured using Western blot. Intraperitoneal injections of tacrine and donepezil( 0. 1 mg / m L, 0. 2 mg / m L and0.4 mg/m L) were respectively given to AChE+/- mice aged2 mo and 4mo and wild-type S129 mice for 7d; phosphate buffered saline(PBS) was administered to the control group. The mice were sacrificed after 30 d by in vitro cardiac perfusion and retinal samples were taken. ACh Edeficient mice were identified by polymerase chain reaction(PCR) analysis using specific genotyping protocols obtained from the Jackson Laboratory website.H&E staining, immunofluorescence and Western blot were performed to observe ACh E protein expression changes in the retinal pigment epithelial(RPE) cell layer.·RESULTS: Different concentrations of H2O2 induced ACh E expression during RPE cell apoptosis. AChE+/- mice retina were thinner than those in wild-type mice(P <0.05); the retinal structure was still intact at 2mo but became thinner with increasing age(P <0.05); furthermore,AChE+/- mice developed more slowly than wild-type mice(P <0.05). Increased concentrations of tacrine and donepezil did not significantly improve the protection of the retina function and morphology(P >0.05).·CONCLUSION: In vivo, tacrine and donepezil can inhibit the expression of ACh E; the decrease of ACh E expression in the retina is beneficial for the development of the retina.
基金Supported by the National Natural Science Foundation of China(No.81641057)the Natural Science Foundation of Liaoning Province(No.201602292No.201602298)
文摘AIM: To investigate the roles of PKC-α/ezrin signals in phagocytosis crisis of retinal pigment epithelium(RPE) cells in light damage model. METHODS: Light induced mice RPE injury model was established by continuously irradiating cool white light at different exposure time(0, 4, 8h light intensity: 4.18×10-6 J/cm2). In vitro, human ARPE-19 cells treated with the doses and intensity(1.57×10-6 J/cm2) of laser irradiation. Histology analysis was evaluated by hematoxylin and eosin(HE) staining. In vivo RPE phagocytosis was quantified by measuring the accumulation of photoreceptor outer segments in the sub-retinal space. In vitro RPE phagocytosis was assessed by calculating the relative fluorescence intensity of FITC-labeled microspheres in ARPE-19 cells. To further investigate the molecular mechanism, the activation of PKC-α/ezrin signal was evaluated by Western blot in vivo and in vitro.RESULTS: HE staining revealed that the thickness of outer nuclear layer decreased significantly after 4 and 8h light exposure. By immunostaining with rhodopsin, a significant greater accumulation of photoreceptor outer segment was noticed after light injury. In vitro, light injuredRPE cells had less phagocytic activity in a dose dependent manner than that of the normal control(P<0.01). Western blot suggested the activation of PKC-α/ezrin signaling was down-regulated in a dose-dependent manner after light exposure. CONCLUSION: Our data suggest that light induced phagocytic crisis of RPE cells may result from the downregulation of PKC-α/ezrin signaling.
文摘AIM:To identify the effects of interleukin(IL)-13 on retinal pigment epithelial(RPE) cells and the IL-13 level in aqueous humor of age-related macular degeneration(AMD) patients.METHODS:IL-13 levels in aqueous humor specimens from AMD patients were detected with enzyme-linked immunosorbent assay(ELISA).ARPE-19 cells were treated with 10 ng/m L IL-13 for 12,24,and 48 h.The cell proliferaton was evaluated by the MTS method.The m RNA and protein levels of α-SMA and ZO-1 were evaluated with quantitative real-time polymerase chain reaction(q RT-PCR) and Western blot respectively.The expression of tumor necrosis factor-α(TNF-α),transforming growth factor-β(TGF-β) and vascular endothelial growth factor(VEGF) were assessed by ELISA.RESU LTS:IL-13 levels in the aqueous humor of patients with AMD were significantly higher than those in the control(167.33±17.64 vs 27.12±5.65 pg/m L;P<0.01).In vit ro,IL-13 of high concentrations(10,15,and 20 ng/m L) inhibited ARPE-19 cell proliferation.α-SMA m RNA in ARPE-19 cell were increased(1.017±0.112 vs 1.476±0.168;P<0.001) and ZO-1 decreased(1.051±0.136 vs 0.702±0.069;P<0.001) after treated with 10 ng/m L IL-13 for 48 h.The protein expression of α-SMA and ZO-1 also showed the same tendency(α-SMA:P=0.038;ZO-1:P=0.008).IL-13 significantly reduced the level of TNF-α(44.70±1.67 vs 31.79±3.53 pg/m L;P=0.005) at 48 h,but the le vel of TGF-β2 was significantly increased from 34.44±2.92 to 57.61±6.31 pg/m L at 24h(P=0.004) and from 61.26±1.11 to 86.91±3.59 pg/m L at 48h(P<0.001).While expressions of VEGF didn't change after IL-13 treatment.CONCLUSION:IL-13 in vitr o inhibit ARPE-19 cell proliferation and expression in the aqueous may be associated with AMD.
