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AIM: To investigate the effect of inhibiting Ca2+/calmodulin-dependent protein kinase Ⅱ(CAMKⅡ)expression in adult retinal pigment epithelial cell line-19(ARPE-19)cells on the migration, invasion, and tube formation of human umbilical vein endothelial cells(HUVECs)in a non-contact co-culture system.METHODS: RNA sequencing was performed on ARPE-19 cells overexpressing CAMKⅡ-δ, and bioinformatics was used to analyze the biological functions of the differentially expressed genes. Transwell inserts was used to construct a non-contact co-culture system of ARPE-19 and HUVECs. The experimental groups included: blank group: only HUVECs were inoculated without ARPE-19 cells; control group: ARPE-19 and HUVECs cells were co-cultured with complete medium; AIP group(CAMKⅡ inhibition group): ARPE-19 cells in AIP(160 nmol/L)were co-cultured with HUVECs in complete medium. The migration, invasion and tube formation abilities of HUVECs were detected. The protein expression levels of CAMKⅡ/AMPK/mTOR/VEGFA were detected by Western blotting.RESULTS:Bioinformatics analysis found that the differentially expressed genes could affect biological processes such as cell growth and death and cell movement. The scratch test and transwell migration test showed that the relative mobility of HUVECs in the AIP group was significantly lower than that in the control group(all P<0.05). However, the invasion and tube formation assay showed that the relative invasion rate and tube formation rate of the AIP group were not significantly different from those of the control group(both P>0.05). Western blotting results showed that the expression levels of CAMKⅡ, P-mTOR, and VEGFA proteins in the AIP group were significantly lower than those in the control group, while the expression level of the P-AMPK protein was significantly higher than that in the control group(all P<0.05).CONCLUSION:In the non-contact co-culture system, inhibition of CAMKⅡ expression in ARPE-19 cells significantly reduced the migration ability of HUVECs, but it cannot change the invasion and tube formation ability, which may be achieved by AMPK/mTOR/VEGFA.
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Objective:To investigate the antagonistic effect and potential mechanism of specific AKT activator SC79 on the apoptosis of human retinal pigment epithelial (ARPE)-19 cells induced by high glucose in vitro. Methods:The ARPE-19 cells were cultured in high glucose medium (containing 30 mmol/L glucose) plus 5, 10 or 20 μg/ml SC79, respectively.After 6-, 12- and 24-hour culture, the optimal experimental concentration and timing were determined according to cell proliferation rate.Then ARPE-19 cells were divided into four groups, normal control group cultured in normal medium containing 5.6 mmol/L glucose for 48 hours, mannitol group cultured in medium containing 5.6 mmol/L glucose and 24.4 mmol/L mannitol for 48 hours, high glucose group cultured in high glucose medium for 48 hours, and high glucose+ SC79 group cultured in normal medium containing 10 μg/ml SC79 for 12 hours plus in high glucose medium for 36 hours.The proliferation rate of APRE-19 cells was detected by MTS assay.The apoptosis rate was measured by flow cytometry.The relative expression levels of phosphorylated protein kinase B (p-Akt), X-linked inhibitor of apoptosis protein (XIAP), caspase-9, caspase-3 and its active fragments (active-caspase-3) were assayed by Western blot.The ARPE-19 cells were divided into Neg-shRNA group, AKT shRNA group and blank control group and were treated with the corresponding transfection complex and serum-free medium.The AKT mRNA expression was detected by real-time PCR.The transfected ARPE-19 cells were divided into Neg-shRNA+ SC79 group and AKT shRNA+ SC79 group and were cultured according to the culturing method of high-glucose+ SC79 group.The apoptosis rate of the two groups was tested by flow cytometry.Results:Among different concentrations of SC79 and treatment times, the proliferation rate of cells treated with 10 μg/ml SC79 for 12 hours was the highest.The proliferation rate of ARPE-19 cells in high-glucose group was significantly lower than that in normal control group, mannitol group and high-glucose+ SC79 group, and the differences were statistically significant (all at P<0.01). The apoptosis rate of cells in the high-glucose group was (52.27±3.21)%, which was significantly higher than (3.90±0.71)% in normal control group and (20.70±3.62)% in high-glucose+ SC79 group (both at P<0.01). The relative expression levels of p-Akt, XIAP, caspase-9 and caspase-3 were significantly lower and the relative expression level of active-caspase-3 was significantly higher in high glucose group than those in normal control group and high-glucose+ SC79 group (all at P<0.05). The relative expression level of AKT mRNA in normal control group, Neg-shRNA group and AKT shRNA group was 0.60±0.07, 0.59±0.03 and 0.11±0.10, respectively, showing a statistically significant difference among the groups ( F=30.44, P<0.01). The apoptosis rate of cells in the AKT shRNA+ SC79 group was significantly higher than that in high-glucose+ SC79 group and Neg-shRNA+ SC79 group (both at P<0.001). Conclusions:SC79 can partially antagonize the apoptosis of ARPE-19 cells induced by high glucose, which is related to the activation of AKT/XIAP pathway and the inhibition of the caspase family.
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@#AIM: To investigate the synthesis method of curcumin nanoparticles grafted with deoxycholic acid and the effect of curcumin nanoparticles on human retinal pigment epithelial(hRPE)cells. <p>METHODS: The synthesis and performance analysis of Cur/Chit-DC. The relationship between FITC/Chit-DC and hRPE cells was observed under an inverted fluorescence microscope after treating hRPE cells with FITC(FITC/Chit-DC)and Cur/Chit-DC(FITC/Cur/Chit-DC)for 24h, keeping them in dark for 1, 3 and 5d respectively.<p>RESULTS: By mixing Cur and Chit-DC, the nanoparticles containing chitosan derivatives were light yellow. The drug release from the nanoparticles reached equilibrium after 96h, and the cumulative drug release amount was 31.6%. After FITC/Chit-DC was treated with hRPE cells for 1d, most of Chit-DC nanoparticles were still located near the cell membrane. After 3d, the nanoparticles gradually converged towards the nucleus and most of them were located around the nucleus. After 5d, it was observed that Chit-DC nanoparticles had entered the nucleus and were partially degraded under the action of intracellular lysosomes. The relationship between Cur/Chit-DC and cellular action is roughly the same as the relationship between Chit-DC and cellular action. <p>CONCLUSION: Cur can be continuously released from Cur/Chit-DC nanoparticle, which has long-lasting sustained-release function.
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Age-related macular degeneration (AMD) is one of the main causes of vision loss among middle-aged and elderly people worldwide. The prevention and treatment of AMD is a current topic of interest in ophthalmology but remains challenging. Oxidative stress-induced retinal pigment epithelial cell autophagic dysfunction, cellular senescence, and an abnormal immune inflammatory response are key pathogenic factors for AMD. Many bioactive ingredients of traditional Chinese medicine not only exert anti-oxidative, anti-inflammatory, anti-aging, and anti-apoptotic effects, but also prevent/block the occurrence of AMD through different pathways. This review summarizes our current understanding of the pathogenesis of AMD, the types of natural bioactive ingredients capable of treating AMD, as well as the known mechanisms by which these agents act, and may provide new strategies for the prevention and treatment of AMD.
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@#AIM: To investigate the effects of different wavelength of blue light on human retinal pigment epithelial(RPE)cells. <p>METHODS: ARPE-19 cells cultured <i>in vitro</i> were randomly divided into four groups, which were control group, 447nm blue light group, 456nm blue light group and 468nm blue light group. The cells in control group were cultured under normal conditions whereas the cells in blue light group were irradiated with different wavelengths of OLED blue light with the illumination intensity of 200Lux for 72h. Live/Dead staining assay, CCK-8 assay and real-time PCR were performed to compare the effects of different wavelengths of blue light on the morphology, cell viability, proliferation capacity, mRNA expression level of visual cycle biomarkers and inflammatory biomarkers of ARPE-19 cells, respectively.<p>RESULTS: After blue light irradiation, the abnormal morphology and the decrease of cell confluence of ARPE-19 cells were observed. Furthermore, with the decrease in the wavelength of blue light, the inhibition effect of blue light on RPE proliferation was enhanced, and the mRNA expression of the proliferation marker Ki-67 and visual cycle biomarkers LRAT, CRALBP, RDH and IRBP decreased. In addition, the mRNA expression levels of inflammatory factors MCP-1 and IL-6 in RPE cells were up-regulated with the decrease in the wavelength of blue light.<p>CONCLUSION: Our data demonstrated that blue light in different wavelengths exerted detrimental effects on RPE cells. The shorter the wavelength of blue light was, the more severe damage it caused on the RPE cells.
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The present study was designed to analyze the metabolites of all-frans-retinal (atRal) and compare the cytotoxicity of atRal versus its derivative all-frans-retinoic acid (atRA) in human retinal pigment epithelial (RPE) cells. We confirmed that atRA was produced in normal pig neural retina and RPE. The amount of all-trans-retinol (atROL) converted from atRal was about 2.7 times that of atRal-derived atRA after incubating RPE cells with 10 µmol/L atRal for 24 h, whereas atRA in medium supernatant is more plentiful (91 vs. 29 pmol/mL), suggesting that atRA conversion facilitates elimination of excess atRal in the retina. Moreover, we found that mRNA expression of retinoic acid-specific hydroxylase CYP26b1 was dose-dependently up-regulated by atRal exposure in RPE cells, indicating that atRA inactivation may be also initiated in atRal-accumulated RPE cells. Our data show that atRA-caused viability inhibition was evidently reduced compared with the equal concentration of its precursor atRal. Excess accumulation of atRal provoked intracellular reactive oxygen species (ROS) overproduction, heme oxygenase-1 (HO-1) expression, and increased cleaved poly(ADP-ribose) polymerase 1 (PARP1) expression in RPE cells. In contrast, comparable dosage of atRA-induced oxidative stress was much weaker, and it could not activate apoptosis in RPE cells. These results suggest that atRA generation is an antidotal metabolism pathway for atRal in the retina. Moreover, we found that in the eyes of ABCA4−/−RDH8−/− mice, a mouse model with atRal accumulation in the retina, the atRA content was almost the same as that in the wild type. It is possible that atRal accumulation simultaneously and equally promotes atRA synthesis and clearance in eyes of ABCA4−/−RDH8−/− mice, thus inhibiting the further increase of atRA in the retina. Our present study provides further insights into atRal clearance in the retina.
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@#【Objective】To investigate the effects of up-regulating RA signal and inhibiting AP-1 transcriptional activity on TGF-β2 secretion by RPEs and its possible pathways.【Methods】① To investigate the effects of ATRA treat⁃ ment，human retinal pigment epithelial cell line ARPE-19 cells were divided into 5 groups：control group and 4 interven⁃ tion groups（6 h，12 h，24 h and 48 h after RA treatment）. Western blot，RT-qPCR and immunofluorescence staining were carried out to analyze RARβ and c-Fos expression. ②To investigate the effects of RARβ inhibitor LE540 treatment on expression of RARβ and c-Fos that were induced by ATRA，ARPE-19 cells were divided into 4 groups：control group，ATRA group，LE540 group and ATRA+LE540 group. RARβ and c-Fos expression was assessed by western blot and RT-qPCR. ③ To investigate the effects of AP-1 inhibitor T-5224 treatment，ARPE-19 cells were divided into 4 groups：control group and treatment groups（12 h，24 h and 48 h after T-5224 treatment）. EMSA was carried out to ana⁃ lyze the AP-1 DNA binding activity. ④To investigate the effects of LE540 and T-5224 administration on ATRA- induced TGF-β2 secretion，ARPE-19 cells were divided into 4 groups：control group，ATRA group，ATRA+LE540 group and ATRA+LE540 group. Western blot and ELISA were carried out to analyze TGF-β2 secretion in ARPE-19 cells.【Results】 RARβ level in ARPE-19 cells was significantly higher in treatment group than in control group after being treated with ATRA for 24 and 48 hours（P＜0.05）. C-Fos level was first up-regulated and then decreased. After treatment with ATRA for 6 and 12 hours，c-Fos expression were significantly upregulated（P＜0.01），but at 48 h after treatment，their expression were significantly decreased to the level which had no statistical difference compared with the control group （P＞0.05）. The AP-1 DNA binding activity was significantly decreased in ARPE-19 cells after being treated with T-5224 for 24 and 48 hours（P＜0.01）. Compared with ATRA group，TGF-β2 secretion was statistically down-regulated after being treated with LE540 and T-5224 for 48 hours（P＜0.05）.【Conclusion】ATRA can induce TGF-β2 secretion in RPE cells through affecting RARβ expression and AP-1 transcriptional activity.
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The present study was designed to analyze the metabolites of all-trans-retinal (atRal) and compare the cytotoxicity of atRal versus its derivative all-trans-retinoic acid (atRA) in human retinal pigment epithelial (RPE) cells. We confirmed that atRA was produced in normal pig neural retina and RPE. The amount of all-trans-retinol (atROL) converted from atRal was about 2.7 times that of atRal-derived atRA after incubating RPE cells with 10 μmol/L atRal for 24 h, whereas atRA in medium supernatant is more plentiful (91 vs. 29 pmol/mL), suggesting that atRA conversion facilitates elimination of excess atRal in the retina. Moreover, we found that mRNA expression of retinoic acid-specific hydroxylase CYP26b1 was dose-dependently up-regulated by atRal exposure in RPE cells, indicating that atRA inactivation may be also initiated in atRal-accumulated RPE cells. Our data show that atRA-caused viability inhibition was evidently reduced compared with the equal concentration of its precursor atRal. Excess accumulation of atRal provoked intracellular reactive oxygen species (ROS) overproduction, heme oxygenase-1 (HO-1) expression, and increased cleaved poly(ADP-ribose) polymerase 1 (PARP1) expression in RPE cells. In contrast, comparable dosage of atRA-induced oxidative stress was much weaker, and it could not activate apoptosis in RPE cells. These results suggest that atRA generation is an antidotal metabolism pathway for atRal in the retina. Moreover, we found that in the eyes of ABCA4-/-RDH8-/- mice, a mouse model with atRal accumulation in the retina, the atRA content was almost the same as that in the wild type. It is possible that atRal accumulation simultaneously and equally promotes atRA synthesis and clearance in eyes of ABCA4-/-RDH8-/- mice, thus inhibiting the further increase of atRA in the retina. Our present study provides further insights into atRal clearance in the retina.
Subject(s)
Animals , Humans , Mice , ATP-Binding Cassette Transporters/physiology , Alcohol Oxidoreductases/physiology , Cell Survival/drug effects , Cells, Cultured , Inactivation, Metabolic , Retina/metabolism , Retinal Pigment Epithelium/metabolism , Swine , Tretinoin/pharmacologyABSTRACT
·AIM:To investigate the effect of epidermal growth factor (EGF) on proliferation and migration of retinal pigment epithelial (RPE) cells. ·METHODS:Human RPE cell lines(ARPE-19 cell) were treated with different doses of EGF. The methyl thiazolyl tetrazolium (MTT) assay was used to detect cell viability. The 5 - bromodeoxyuridine ( BrdU ) incorporation assay was used to detect cell proliferation and the"scratch-wound assay"was used to detect cell migration ability. The epidermal growth factor receptor (EGFR) and protein kinase B (AKT) proteins were detected by Western blot. · RESULTS: The MTT assay results showed that treatment with 50 and 100ng/mL EGF for 12h increased ARPE-19 cell viability. The BrdU incorporation assay and the"scratch-wound assay"showed that treatment with 100ng/mL EGF for 24h increased ARPE - 19 cell proliferation and migration. The Western blot results showed that treatment with 10-100ng/mL EGF for 12h or 100ng/mL EGF for 15-180min increased phosphorylation levels of EGFR and decreased total levels of EGFR. Similarly, treatment with 10-100ng/mL EGF for 12h or 100ng/mL EGF for 15-180min increased phosphorylation levels of AKT,but not affected total levels of AKT. ·CONCLUSION: EGF affects ARPE- 19 cell viability, proliferation and migration through inducing phosphorylation of the EGFR/AKT signaling pathway. The EGFR/AKT signaling pathway might play an important role in abnormal proliferation and migration of RPE cells in proliferative vitreoretinopathy.
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Objective To investigate the expression level of miRNA-146a (miR-146a) in retinal pigment epithelial (RPE) cells aging and age-related macular degeneration (AMD),and discuss its regulation mechanism of AMD by repressing VEGF-A.Methods The expressions of miR-146 and VEGF-A were examined by qRT-PCR in RPE cells in mice aged 2 months,8 months,12 months,18 months or 24 months,and in RPE cells from 75 years old AMD patients.The protein level of VEGF-A was also detected by Western Blotting.Finally,the effects of overexpression of miR-146a in APRE-19 cell line on expression of VEGF-A was checked.Results MiR-146 was up-regulated to 8 times or 24 times at 18 months or 24 months aged mice,and the expression of VEGFA was down-regulated in aging RPE from 1.5 times to 0.8 times.However,the expression of miR-146 decreased to 14.5 times and VEGF-A increased in RPE cells of AMD.In cultured cells,overexpression of miR-146a inhibited the expression of VEGF-A.Conclusion Up-regulation of miR-146a in aging RPE cells and its down-regulation in AMD suggest a potential of miR-146a as molecular marker.MiR-146a overexpression inhibits the expression of VEGF-A,supporting a potential clinical treatment of miR-146 in AMD.
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Background The transdifferentiation of retinal pigment epithelial (RPE) cell to myofibroblast is a central event in the development and progression of proliferative vitreoretinopathy (PVR).N-acetylcysteine (NAC) can inhibit the transdifferentiation of multiple cells to myofibroblasts via suppressing the production of reactive oxygen species (ROS) and the phosphorylation of MAPK induced by transforming growth factor-β1 (TGF-β1),but the effect of NAC on the TGF-β1-induced transdifferentiation of human RPE cell line ARPE-19 cell to myofibroblast and its underlying molecular mechanism is still unclear.Objective This study was to investigate the effect of NAC on the TGF-β1-induced transdifferentiation of ARPE-19 cell to myofibroblast and its underlying mechanisms.Methods Human ARPE-19 cells were divided into control group,TGF-β1 treatment group,NAC intervention group and simple NAC group.Cells in the control group were not disposed,10 ng/ml TGF-β1,10 ng/ml TGF-β1 +5 mmol/L NAC and 5 mmol/L NAC were used to interfere the ARPE-19 cells for 48 hours in the other 3 groups.The morphological changes of the cells were observed by phase contrast microscopy.Real-time quantitative PCR,Western blot,immunofluorescence and ELISA were used to detect the expression of transdifferentiation marker genes-α-smoot h muscle actin (α-SMA),fibronectin and type Ⅰ collagen.The expression level of intracellular ROS induced by TGF-β1 in the presence or absence of NAC was detected using the non-fluorescent probe DCFH-DA performed by flow cytometry.The phosphorylation level of p38MAPK,ERK1/2 and SAPK/JNK were detected by Western blot.The cell counting kit-8 (CCK-8) assay was used to investigate the effect of NAC on the viability of ARPE-19 cells.Results The expressions of α-SMA,fibronectin and type Ⅰ collagen mRNA in the TGF-β1 treatment group were significantly increased,and were (2.15±0.29),(9.54 ± 1.08),(25.78 ±0.66) times higher than those of the control group,with significant differences between the two groups (all at P＜0.05).The expressions of α-SMA,fibronectin and type Ⅰ collagen protein were significantly increased,and were (8.49± 0.32),(2.53 ± 0.69),(4.45 ± 1.05) times higher than those of the control group,respectively,with significant differences between the two groups (all at P＜0.05).The expressions of α-SMA,fibronectin and type Ⅰ collagen mRNA in the NAC intervention group were (66.70± 12.57)％,(66.11±8.35)％ and (33.19±6.90)％ lower than those of the TGF-β1 treatment group,and the expressions of protein were (52.30±4.83)％,(55.03 ±2.58)％ and (56.08 ±3.65)％ lower than those of the TGF-β1 treatment group,respectively,with significant differences between the two groups (all at P＜0.05).Flow cytometry results showed that the expression level of intracellular ROS in the TGF-β1 treatment group was (2.12±0.20) times higher than that of the control group,and the intracellular ROS in the NAC intervention group was (57.41 ±9.45) ％ lower than that of the TGF-β1 treatment group,with significant differences between the groups (both at P＜0.01).Western blot results showed that the phosphorylation levels of p38MAPK,SAPK/JNK and ERK1/2 in the TGF-β1 treatment group were (9.18±1.00),(4.87±0.81) and (4.20±0.69) times higher than those of the control group,respectively,showing significant differences between the two groups (all at P＜0.05).The phosphorylation level of p38MAPK,SAPK/JNK and ERK1/2 in the NAC intervention group were (48.16± 14.82) ％,(67.90±2.90) ％ and (74.52±4.00)％ lower than those of the TGF-β1 treatment group,respectively,showing significant differences between the two groups (all at P＜0.05).Conclusions NAC inhibits the TGF-β1-induced transdifferentiation of ARPE-19 cells possibly via suppressing the production of intraeellular ROS and the phosphorylation of p38MAPK,SAPK/JNK and ERK1/2.
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?AIM:To investigate the effects of Bevacizumab on the proliferation and the expression of E -Cadherin and fibronectin in human retinal pigment epithelial cell ( ARPE-19) in vitro.?METHODS: Different concentrations (0, 0.625, 1.25, 2.5, 5.0mg/mL) of bevacizumab were exposed to ARPE-19 cells, then cell viability was analyzed by CCK-8, cell cycle was determined by flow cytometry, and the expression of E-Cadherin and fibornectin was detected by Western blot and RT-PCR.?RESULTS:The concentration as 2.5mg/mL or 5.0mg/mL of bevacizumab was shown to effectively suppress the proliferation and cell cycle of ARPE-19 cell (P<0.05). In addition, 2.5mg/mL or 5.0mg/mL of bevacizumab could downregulate the expression of E-cadherin and promote the transcription of fibronection gene (P<0.05).?CONCLUSION:High concentration of bevacizumab was able to inhibit ARPE-19 proliferation, downregulate E-Cadherin expression and promote fibronectin expression, indicating epithelial-mesenchymal transition induced by bevacizumab in ARPE-19 cell.
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PURPOSE: To evaluate the effects of bevacizumab on expression of B-cell leukemia/lymphoma (Bcl)-2 and apoptosis in retinal pigment epithelial (RPE) cells under oxidative stress conditions. METHODS: RPE cells were treated with H2O2 (0, 100, 200, 300, and 400 microM) and bevacizumab at or above the doses normally used in clinical practice (0, 0.33, 0.67, 1.33, and 2.67 mg/mL). Cell apoptosis was measured using flow cytometry with annexin V-fluorescein isothiocyanate. The expression of Bcl-2 mRNA was determined using reverse transcription polymerase chain reaction. RESULTS: Under low oxidative stress conditions (H2O2 100 microM), cell apoptosis was not significantly different at any concentration of bevacizumab, but Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL). Under moderate oxidative stress conditions (H2O2 200 microM), Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL), but cell apoptosis increased only at 2.67 mg/mL of bevacizumab. Under high oxidative stress (300 microM) conditions, cell apoptosis increased at high concentrations of bevacizumab (1.33 and 2.67 mg/mL), but it did not correlate with Bcl-2 expression. CONCLUSIONS: Withdrawal of vascular endothelial growth factor can lead to RPE cell apoptosis and influences the expression of anti-apoptotic genes such as Bcl-2 under oxidative stress conditions. Since oxidative stress levels of each patient are unknown, repeated injections of intravitreal bevacizumab, as in eyes with age-related macular degeneration, might influence RPE cell survival.
Subject(s)
Humans , Angiogenesis Inhibitors/pharmacology , Apoptosis/drug effects , Bevacizumab/pharmacology , Cell Line , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Expression Regulation/physiology , Hydrogen Peroxide/toxicity , Oxidative Stress/drug effects , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Retinal Pigment Epithelium/drug effects , Vascular Endothelial Growth Factor A/antagonists & inhibitorsABSTRACT
Background Sphingosine-l-phospate (S1P) is a bioactive lipid and important messenger molecule in cells.It participates in the regulation of many biological processes,such as cell proliferation,migration,survival,differentiation,apoptosis,etc.Hypoxia is a trigger factor of choriod neovascularization (CNV) and pathological basis of many diseases,and retinal pigment epithelial (RPE) cells are involved in formation of CNV.However,the effects of S1P on proliferation and apoptosis of RPE cells are below understood.Objective This study was to investigate the influence of S1P on proliferation and apoptosis of human RPE cells under hypoxic conditions.Methods Human RPE cells line-D407 cells were cultured and passaged and generation 3-5 cells were used and divided into 6 groups.The cells were regularly cultured in the blank control group using DMEM containing 10％ fetal bovine serum.CoCl2(200.00 μmol/L) was added into the colture medium for 2 hours in the hypooxic group.S1P of different concentrations (0.01,0.10,1.00,10.00 μmol/L) were added in culture medium 2 hours after the affection of 200.00 μmol/L CoCl2.The proliferative values of the cells were detected using WST-1 method as the absorbance (A value) and the proliferative rate of different groups were calculated.The apoptosis of the cells was assayed by Hoechst staining.The results were compared among different groups.Results Cultured cells showed the round-like in shape with clear nuclei and pigment.The proliferative values (A value) was 0.91 ±0.08,0.37±0.09,0.46±0.08,0.52±0.09,0.61 ±0.06,0.70±0.10 in the blank control group,hypoxic group and 0.01,0.10,1.00,10.00 μmol/L S1P groups,respectively,with a significant difference among the groups (F=21.104,P=0.000),and A values in various S1P groups were higher than those in the hypoxiac group (all at P＜0.05).The proliferative rate was gradually raised with the increase of dose of S1P.Hoechst staining exhibited a few apoptosis cells in the blank control group,but in the hypoxic group,a lots of apoptosis cells were seen with the light-blue nuclei and condensable chromatin.However,the number of apoptosis cells was significantly decreased in various concentrations of S 1 P groups.The apoptosis rates were (1.21 ±0.08) ％,(8.99 ±0.09) ％,(6.60 ±0.08) ％,(5.95 ±0.09) ％,(4.81 ± 0.06)％ and (3.96±0.10)％ in the blank control group,hypoxic group and the 0.01,0.10,1.00,10.00 μmol/L S1P groups,respectively,with a significant difference among the groups (F =25.070,P =0.000).Compared with the hypoxia group,the cellular apoptosis rates of various S1P groups were lower (all at P＜0.05).Conclusions Under the hypoxia condition,S1P can promote the proliferation of human RPE cells and inhibit apoptosis.
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To observe the expression of N - cadherin and fibronectin in retinal pigment epithelium ( RPE) cells in vitro under high glucose conditions, furthermore, to explore the effects of high glucose on epithelial -mesenchymal transition (EMT) in RPE cells. ●METHODS: Human RPE (hRPE) cells were cultured in vitro. Containing a final concentration of 60mmol/ L glucose was used for high glucose treatment. The cells were divided into normal glucose group (5. 5mmol/ L, NG) and high glucose group (24, 48 and 72h) respectively. The expression of N - cadherin and fibronectin in hRPE cells were evaluated by immunofluorescence and real -time PCR. ●RESULTS:RPE cells became disorganized and swollen over time under high glucose conditions, especially in 72h subgroup. lmmunohistochemical analysis revealed that the expression of N - cadherin in RPE cells under high glucose conditions was decreased compared with that in the control group, while the expression of fibronectin was increased. Real - time PCR results showed that the expression of N - cadherin mRNA in high glucose group was decreased at 24h compared with that in the control group, and declined markedly at 72h ( F = 12. 252, P =0. 000). There were no significant differences between the control group and the high glucose group at 24h, while the differences between the control group and the high glucose group (48 and 72h) were significant respectively (P ●CONCLUSlON: The expression of epithelium marker N-cadherin is down - regulated under high glucose conditions in hRPE cells in vitro. Meanwhile, the expression of mesenchymal maker fibronectin is induced and appeared to EMT changes. Results of this study will enrich our growing understanding in proliferative diabetic retinopathy and hopefully lead to novel insights for the pathogenesis and therapeutic treatments.
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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.
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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.
ABSTRACT
Background Insulin can promote the occurrence of myopia.It has been proven that insulin receptor exists in human retinal pigment epithelial (RPE) cells and can promote RPE cells to secrete transforming growth factor-β2(TGF-β2),which is one of the most important myopic signal molecules.Objective This study was to investigate if PI3K/Akt mediates the promotive effects of insulin on proliferation of human RPE cells and secretion of TGF-β2.Methods Human RPE cell line,ARPE-19 cells,were regularly cultured using DMEM containing 10％ fetal bovine serum,and 10× 103 U/ml insulin,LY294002,10× 103 U/ml insulin+LY294002,Wortmanin,10× 103 U/ml insulin+Wortmanin were added into the medium respectively for 48 hours,and the regularly cultured cells served as blank controls.The proliferation value (absorbance,A) of the cells was evaluated by MTS,and the TGF-β2 level in the cell supernatant was detected by ELISA.The relative expression of TGF-β2 mRNA in the cells was assayed using reverse transcription PCR (RT-PCT) 1 hour and 2 hours after the addition of reagents.Results MTS showed that the proliferation value of the cells in the insulin+LY294002 group was 0.75±0.03,which was significantly lower than that in the insulin group (0.98± 0.04).No significant difference was seen in the proliferative value between the insulin+Wortmanin group and the insulin group (0.97±0.07 versus 0.98± 0.04,P＞0.05).ELISA revealed that the content of TGF-β2 in the the cell supernatant was (11.59±2.85) pg/ml and (49.16± 10.94) pg/ml in the insulin + LY294002 group and the insulin + Wortmanin group,respectively,showing a significant decline in comparison with (548.50±35.18) pg/ml in the insulin group (both at P＜0.05).A significant difference was found in the TGF-β2 content between the insulin+LY294002 group and the insulin+Wortmanin group (t =8.131,P =0.000).The RT-PCR showed that 1 hour and 2 hours after addition of the reagents,the expression levels of TGF-β2 mRNA in the cells were lower in both insulin+LY294002 group and insulin+Wortmanin group than those in the insulin group (P＜0.05).The decline range of TGF-β2 mRNA expression level was more significant in the insulin+LY294002 group than that in the insulin+Wortmanin group at 1 hour (t=4.176,P=0.014) rather than at 2 hours (t=0.756,P=0.492).Conclusions Insulin can promote the proliferation of human RPE cells and secretion of TGF-β2 through PI3K/Akt pathway.This may be one of the mechanisms of insulin causes myopia.
ABSTRACT
Background Hypoxia can increase the secretion of vascular endothelial growth factor-A (VEGF A) by retinal pigment epithelium (RPE) cells and initiate choroidal neovascularization (CNV) consequently.Two VEGF-A isoforms,the angiogenic VEGF-A family (VEGFxxx) and the anti-angiogenic VEGF-A family (VEGFxxxb),are found and formed by alternative splicing.However,the expressing changes and its effect in human RPE cells under the normoxia and hypoxia are unclear.Objective This study was to investigate the expression of VEGFxxx b in human RPE cells under hypoxia.Methods ARPE-19,a human RPE cell line,were cultured.CoCl2 150 μmol/L was added into the medium to mimic the hypoxia environment for 0 hour (normoxia group),3,12 and 24 hours to induce the hypoxia cells.The cells or suspension was harvested at various time points.The expressions of VEGFxxx mRNA and VEGFxxxb mRNA in the cells were detected,and the expressions of the total VEGF-A protein and VEGFxxxb protein in the cells were assayed by Western blot.ELISA was used to determine the contents of total VEGF-A and VEGFxxxb proteins in suspension.Results VEGFxxx mRNA and VEGFxxxb mRNA were expressed in the ARPE-19 cells in both normoxia and hypoxia,and multiple VEGFxxx mRNA isoforms appeared,including VEGF121 mRNA,VEGF165 mRNA and extremely faint VEGF189 mRNA,but only VEGF165b band was seen in VEGFxxxb mRNA.With the prolong of hypoxia culture,the expression of VEGFxxx mRNA showed gradual increase,while VEGFxxx b mRNA appeared gradual decrease.Western blot exhibited that VEGFxxxb was expressed in the cells cultured by normoxia and hypoxia with the strongest expression in VEGF165b.The VEGFxxxb content in suspension of medium was (166.82± 2.55) pg/ml under the normoxia environment and (125.35 ±2.10) pg/ml in 24 hours under the hypoxia,and the total VEGF-A protein elevated from (294.27 ± 11.97) pg/ml under the normoxia environment to (582.26 ± 12.98) pg/ml in 24 hours under the hypoxia.In addition,the proportion of VEGFxxxb to total VEGF-A in the cells lowed from (56.71 ± 1.02) ％ under the normoxia environment to (21.53 ±0.08) ％ in 24 hours under the hypoxia.Conclusions VEGFxxxb isoforms are expressed in both normoxia and hypoxia ARPE-19 cells.VEGFxxxb isoform is a predominant isoform in normoxia ARPE-19 cells.In hypoxia ARPE-19 cells,however,the expression of VEGF A is significantly stronger than that of VEGFxxxb.
ABSTRACT
BackgroundRetinal pigment epithelial(RPE) cells can secrete platelet-derived growth factor (PDGF) and PDGF receptor(PDGFR).Studies have shown that PDGF plays a key role in the formation of proliferative vitreous retinopathy(PVR). ObjectiveThis study was to investigate the proliferation and apoptosis changes of RPE after blockage of the PDGFR-α expression by antisense oligonucleotide ( ASODN ) in vitro. Methods Human RPE cells strain was cultured in low glucose DMEM with 10％ fetal bovine serum.Logarithmic phase cells were collected and incubated in 96-well plate at the density of 5 × 105 cells/hole.PDGFR-α ASODN was transfected into RPE cells at different concentrations for 48 hours.The cells of the blank control group were regularly cultured without any transfection.The changes of PDGFR-α expression were detected by reverse transcription-polymerase chain reaction(RT-PCR),and the proliferation of RPE was detected by MTT as the A490 value.Hoechst 33258 fluorescence staining was used to determine the apoptosis of RPE.Flow cytometry method (FCM) was applied to detect the change of cell cycle and apoptosis rate of RPE cells. ResultsThe A490 values of RPE cells were 1.45±0.12,1.07±0.06,0.65±0.05 in blank control group,1.0 μmol/L Lipo-ASODN group and 2.0 μmol/L Lipo-ASODN group with the significant difference(P=0.00 ),and that of 1.0 μmol/L Lipo-ASODN group and 2.0 μ mol/L Lipo-ASODN group were significantly lower than the blank control group ( P =0.00,0.00).Hoechst 33258 staining showed that the apoptosis cells were obviously more in Lipo-ASODN group compared with blank control group.PDGFR-α ASODN transfection induced an increase of percentage of RPE cells in G0/G1 phase( F =206.70,P =0.00),and the apoptosis rates in 1.0 μmol/L Lipo-ASODN group and 2.0 μmol/L Lipo-ASODN group were significantly enhanced in comparison with blank control group ( 37.8 ± 1.3 vs 10.5 ± 0.1,61.2 ± 1.9 vs 10.5 ± 0.1 ) ( F =1808.90,P =0.00 ).Expression intensity of PDGFR-α mRNA in RPE cells in Lipo-ASODN groups was lower. ConclusionsBlocking the PDGFR-α expression with ASODN technology can suppress proliferation and induce apoptosis of RPE cells.Intensity of PDGFR-α mRNA expression in RPE cells is ASODN dose-dependent.ASODN targeted to PDGFR-α offers an experimental basis of the gene therapy for PVR.