Simvastatin inhibits HIF-1α and VEGF expression in RPE cells under hypoxia conditions / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the effects of simvastatin(Sim)on human retinal pigment epithelial cells(RPE-19)and the possible mechanisms <i>in vitro</i> under hypoxia. <p>METHODS: RPE-19 cells were divided into three group: control group, hypoxia group(the final concentration of CoCl2 in the medium was 125 μmol/L), and Sim treatment group(3 μmol/L Sim was added in the RPE cells' medium which contain 125 μmol/L CoCl2). After 24h, the morphology of RPE-19 cells were observed, the proliferation of cells were calculated by MTT, the secretion levels and protein expression of hypoxia-inducible factor 1-Alpha(HIF-1α)and vascular endothelial growth factor(VEGF)were detected by enzyme-linked immunosorbent assay(ELISA)and Western blotting. The expression level of autophagy protein was detected by Western blot and apoptosis was detected by TUNEL.<p>RESULTS: The morphology and activity of RPE-19 cells showed an apparent change under hypoxia. The expression of HIF-1α and VEGF protein were increased obviously in the hypoxia group and then significantly decreased after Sim treatment. Beclin1, and LC3B proteins were decreased in the CoCl2+Sim group, and the expression levels were lower than the control and CoCl2 group. Under hypoxia, Sim inhibited RPE cells' proliferation and promoted the apoptosis.<p>CONCLUSION:Sim inhibits RPE cells' proliferation, decreases HIF-1α and VEGF protein, and promotes apoptosis under hypoxia. Our results suggested that the mechanism by which Sim promoted apoptosis in RPE cells may be related to its inhibition of autophagy.

Research progress on the mechanism of epithelial mesenchymal transformation in RPE cells and the treatment of PVR / 国际眼科杂志(Guoji Yanke Zazhi)

@#Proliferative vitreoretinopathy(PVR)is a serious complication arisen from ocular trauma, diabetic retinopathy, vascular retinopathy, inflammatory retinopathy and other ocular diseases. It is also the most important reason for the failure of rhegmatogenous retinal detachment surgery, which is a great threat of visual function. A large number of studies have proved that the main risk factor for PVR is the damage of blood-retinal barrier, in which retinal pigment epithelial(RPE)cells are stimulated by cytokines in the vitreous cavity. RPE cells underwent epithelial-mesenchymal transition(EMT), which transformed into fibroblasts. The cell morphology changed, the tight junctions between cells disappeared, the cell polarity lost, and the proliferation, migration, and invasion abilities were enhanced. A contractile fibrous proliferative membrane is formed on the anterior surface or under the retina. The fibrous proliferative membrane will lead to the retina folds, pull the retina and lead to retinal detachment, which will eventually lead to vision loss or even blindness. Nowadays, plenty of studies investigating the prevention and treatment of PVR have been carried out at home and abroad. In this review, we briefly illustrated the signaling pathways related to epithelial-mesenchymal transformation in RPE cells and the treatment of PVR.

[Ca2+]i homeostasis and caspase-3 gene expression in verapamil-induced retinal pigment epithelium cells apoptosis in vitro / 国际眼科杂志(Guoji Yanke Zazhi)

(Ver)-induced human retinal pigment epithelium (RPE) cells apoptosis.hours to induce RPE cells apoptosis.The expression of apoptotic effector gene caspase-3 was assessed by reverse transcription polymerase chain reaction (RT-PCR).Single cell was measured using fluorescence indicator Fura-3/AM with MetaFluo4.5/coolsnapfx/IX70 intracellular Ca2+ fluorescence imaging system.RPE cells and it significantly increased after co-cultured with Ver.The fluorescence in resting RPE cells was strong and distributed throughout the cells.The nucleus appeared more fluorescent than the cytoplasm.Calcium fluorescence of RPE cells attenuated after co-cultured with Ver.[Ca2+]i homeostasis might play pivotal roles in Ver-induced RPE cells apoptosis.

Role of HGF/c-Met in Serum-Starved ARPE-19 Cells

PURPOSE: Hepatocyte growth factor (HGF) and its receptor (HGFR/c-Met) regulate motility, mitogenesis, and morphogenesis in a cell type-dependent fashion. We report the role of HGF and c-Met on stress-induced ARPE-19 human retinal pigment epithelial (RPE) cells in this study. METHODS: The cells were cultured either with or without serum. Southern and Western blot analyses were done to determine the expression patterns of HGF/c-Met in serum-starved ARPE-19 cells. The cell proliferation pattern in serum-starved condition was analyzed using MTS assay. Inhibition level of cell proliferation was analyzed using a neutralizing monoclonal antibody against c-Met (2 microgram/ml). RESULTS: Abnormal cell proliferation and scattering of ARPE-19 cells was observed under serum starvation. HGF/c-Met were expressed in serum-starved ARPE-19 cells. ARPE-19 cell proliferation was also enhanced with recombinant HGF treatment. Neutralization against c-Met inhibited the proliferation of serum-deprived ARPE-19 by 64.5% (n=9, S.D. 5.5%). Serum starvation appears to induce epithelial-mesenchymal transition of ARPE-19 cells, resulting in scatter, and the expression of alpha-smooth muscle actin (alpha-SMA), a marker for fibrosis. CONCLUSIONS: In conclusion, c-Met induced under non-physiologic conditions has significant effects on the activation of RPE cells.

Improved Culture Method of Retinal Pigment Epithelial Cells and Functional-morphological Characteristics In Vitro

To study the isolation and purification and proliferation of the cell in cell culture system, and to develop an improved culture method by a modified cell isolation technique and modified culture medium. The RPE cells were cultured in 3 different mediums: type I(MEM medium with 20% FCS) type II(F-10 medium with 20% FCS) and type III(DMEM medium with 10% FCS, EGF, hydrocortisone, insulin, ethanolamine, phosphoethanolamine, chorea toxin, triiodotyronine, adenine, transferrin and BPE). We compared population doubling(P.D.), population doubling time(P.D.T), morphologic changes and phagocytic activity during a 7week period. Rapid proliferation and high purity of retinal pigment epithelial cells(RPE cells) showed in type III culture medium. Type III culture medium presented the best results in P.D., P.D.T. and cell purification. In type III culture medium, single RPE cells produced about 6 X 10(7) RPE cells in the 7week period and morphology and phagocytic activity were well maintained, when UV-B irradiation at RPE was used to produce melanin, it had no effect, but the RPE cell was inhibited by UV-B irradiation. This improved culture method for RPE cells will provide a good in-vitro model for the studies of biochemistry, cellular function of the RPE cell, as well as its clinical application in eye disease.

The Effect of Vitreous on Proliferation of Retinal Pigment Epithelial CelIs

Several studies have indicated that retinal pigment epithelial(RPE) cells migrate from their normal location into the vitreous cavity where they then undergo proliferation and membrane formation in proliferative vitreoretinopathy(PVR). Little attention has been given to the role of vitreous on cellular proliferation. Our study is to determine the effect of vitreous on RPE cell proliferation and to examine the morphology of cultured RPE cells on vitreous explants. The vitreous from pigmented rabbit was extracted and added to the cultured media RPE cells proliferated rapidly along the margin of the vitreous as fibrocyte like cells and were less invasive into the virtrous gel. Liquified vitreous with media stimulated the proliferation of RPE cells, but vitreous alone showed the decrease of inoculated RPE cells.

A Study of Lipofuscin on Cultured Human Retinal Pigment Epithelium

One consistent finding in the aging human RPE is the intracellular accumulation of lipofuscin Lipofuscin was applied to the cell culture technique to clarify the relationship between age related macular degeneration and lipofuscin accumulation in the human RPE. The results obtained in this study are as follows: 1. The initial suspensions of dissociated RPE cells consisted of densely pigmented and hexagonal cells. Rapid cell proliferation and confluency of a culture occured from 2 weeks after seeding. No pigment granule was observed in subcultured cells of 3rd passage. 2. When RPE cultures were subcultured the cells gradually de pigmented due to a redistribution of pigment granules. Quantitative analysis demonstrated that intensity of fluorescence for a certain number of cells reduced proportionally as the cell division proceeded. 3. Cultured human retinal pigment epithelial cells readily ingested lipofuscin isolated from human RPE cells. The amount of lipofuscin phagocytized by cultured RPE cells were greater than that phagocytized by aged cells. Accumulation and/or saturation of lipofuscin in the RPE cells at least in cell culture did not necessarily affect RPE cell survival.