ABSTRACT
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.
ABSTRACT
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 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.
ABSTRACT
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.
ABSTRACT
Objective To observe the effects of fructose,fibroblast growth supplement(FGS) and ethylamine sulfonic acid on the total number,the survival rate and the survival number of Human Retinal Pigment Epithelial(hRPE) Cell.Methods Microencapsulated hRPE cells were plated and cultured in four kinds of mediums,which contained fructose,fibroblast growth supplement(FGS),ethylamine sulfonic acid or no extra ingredient respectively.The total cell number,survival rate and viable cell number of the microencapsulated hRPE cell on day 0th,1st,3rd,7th were calculated.Results After 7days of culture,the lowest cell survival rate of microencapsulated hRPE cells in the four groups was(75.00±3.00)%,but there were no significantly differences(Ps＞0.05) among the groups.The total number of cells in the fibroblast growth factor group([8.00±0.46]×104) and ethylamine sulfonic acid group([7.20±0.36]×104) were significantly higher than the blank group(([6.10±0.56]×104),Ps＜0.05),while no statistical difference was observed in the comparison between the fructose group([6.00±0.46]×104) and blank control(P＞0.05).Conclusion The FGS and ethylamine sulfonic acid can promote the proliferation of the microencapsulated hRPE cells.
ABSTRACT
This study evaluated the effects of glucose in human retinal pigment epithelial (RPE) cells to investigate the cause of diabetic retinal complications. Human RPE cells were cultured in media containing 5.5 mM, 11.0 mM, and 16.5 mM D-glucose. The present study performed proliferation and migration assays, and conducted western blotting for the protein expression, as well as RT-PCR for the mRNA expression, of MMP-2 and -9, and TIMP-1 and -2. The results of the western blotting analysis showed that increasing glucose concentration significantly increased the expression of MMP-2 and -9, but significantly decreased the expression of TIMP-1 and -2. Moreover, the RT-PCR results indicated significant increases in the mRNA expression of MMP-2 and -9, as well as of TIMP-1 and -2, by raising glucose concentration. This study provides fundamental data for future research on the mechanism of retinal complication in diabetic patients.
Subject(s)
Humans , Blotting, Western , Cell Movement , Cell Proliferation , Cells, Cultured , Comparative Study , Dose-Response Relationship, Drug , Glucose/pharmacology , In Vitro Techniques , Matrix Metalloproteinases/genetics , Pigment Epithelium of Eye/drug effects , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Tissue Inhibitor of Metalloproteinases/geneticsABSTRACT
PURPOSE: Oxidative stress is the well known causative factor for retinal damage. This study investigated the effects of glutathione on reactive oxygen species(ROS) induced injury in human retinal pigment epithelial(HRPE) cells. OBJECTS AND METHODS: HRPE cells (ATCC:CRL-2302) were cultured with DMEM media and exposed to oxidative stress (paraquat, hydrogen peroxide) and/or glutathione modulator[(buthionine sulfoximine (BSO), glutathione (GSH), 2-oxo 4-thiazolidine carboxylic acid (OTC)] for 2 days. The cell viability was determined by measuring the amount of reduced 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). RESULTS: The rate of MTT reduction of HRPE cells decreased by either paraquat or hydrogen peroxide treatment. BSO as a inhibitor of glutathione biosynthesis potentiated paraquat- or hydrogen peroxideinduced HRPE cells injury. On the other hand GSH or OTC reduced the rate of decrement of MTT reduction in HRPE cells by paraquat and hydrogen peroxide. CONCLUSIONS: Glutathione seemed to play some role in prevention of ROS-induced HRPE cells injury and OTC may be used as an agents for prevention of free radical induced HRPE cell injury.