Diurnal changes of lysosomes including ultrastructural changes of phagosomes and acid phosphatase reactions in phagosomes, as well as diurnal biochemical changes in cathepsin D activity, were studied in the retinal pigment epithelium (RPE) of the rabbit. The rabbit was maintained on a natural light-dark cycle over seven days in fall and was sacrificed at various times during the day and night. The number of lysosomes or phagosomes in the RPE was the highest at 1.5 hours after exposure to sunlight (8:00 AM), and thereafter decreased with time. Three types of phagosomes were observed and acid phosphatase reactions were different in each type of phagosome; the fresh phagosomes were negative or positive, lamellar bodies positive, and dense bodies partially positive. The biochemical activity of cathepsin D was the highest at 8:00 AM, and this was consistent with the time of peak in phagocytic activity in the RPE. This report shows that phagocytic activity in the RPE occurred in the early stage after exposure to sunlight, and that fresh phagosomes were sequentially degraded to lamellar or dense bodies. Cathepsin D activity also increased, and this was consistent with the phagocytic activity in the RPE.
Acid Phosphatase/metabolism ; Animals ; Cathepsin D/metabolism ; Cell Count ; Choroid/metabolism/ultrastructure ; Circadian Rhythm/*physiology ; Lysosomes/*metabolism/ultrastructure ; Phagosomes/*metabolism/ultrastructure ; Pigment Epithelium of Eye/*metabolism/ultrastructure ; Rabbits

BACKGROUNDThe cell layer of the ciliary epithelium is responsible for aqueous humor secretion and maintenance. Ion channels play an important role in these processes. The main aim of this study was to determine whether the well-characterized members of the Kv1 family (Kv1.3) contribute to the Kv currents in ciliary epithelium.

METHODSNew Zealand White rabbits were maintained in a 12 hours light/dark cycle. Ciliary epithelium samples were isolated from the rabbits. We used Western blotting and immunocytochemistry to identify the expression and location of a voltage-gated potassium channel Kv1.3 in ciliary body epithelium. Membrane potential change after adding of Kv1.3 inhibitor margatoxin (MgTX) was observed with a fluorescence method.

RESULTSWestern blotting and immunocytochemical studies showed that the Kv1.3 protein expressed in pigment ciliary epithelium and nonpigment ciliary epithelium, however it seemed to express more in the apical membrane of the nonpigmented epithelial cells. One nmol/L margatoxin, a specific inhibitor of Kv1.3 channels caused depolarization of the cultured nonpigmented epithelium (NPE) membrane potential. The cytosolic calcium increased after NPE cell depolarization, this increase of cytosolic calcium was partially blocked by 12.5 micromol/L dantrolene and 10 micromol/L nifedipine. These observations suggest that Kv1.3 channels modulate ciliary epithelium potential and effect calcium dependent mechanisms.

CONCLUSIONKv1.3 channels contribute to K+ efflux at the membrane of rabbit ciliary epithelium.

Animals ; Blotting, Western ; Calcium ; metabolism ; Ciliary Body ; cytology ; metabolism ; physiology ; Immunohistochemistry ; In Vitro Techniques ; Kv1.3 Potassium Channel ; metabolism ; physiology ; Membrane Potentials ; physiology ; Pigment Epithelium of Eye ; cytology ; metabolism ; physiology ; Rabbits

The loss of retinal pigment epithelium (RPE) with aging is related to age-related macular degeneration (AMD). This study was conducted to investigate the mechanism of hydrogen peroxide (H2O2) induced cell death in a human retinal pigment epithelial cell line, ARPE-19. Hydrogen peroxide was added at different concentrations to ARPE-19 cells and cultured. The cytotoxicity was assayed by mitochondrial function using 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) testing. The patterns of cell damage were assessed using an acridine orange-ethidium bromide differential staining method, in situ end labeling (ISEL) assay and transmission electron microscopy (TEM). Catalase, a major antioxidant, was used to prevent cell death. The cleavage of procaspase 3 and poly (ADP-ribose) polymerase (PARP) was determined by western blot analysis. Hydrogen peroxide significantly induced cell death in ARPE-19 cells, whereas pretreatment of the cells with catalase prevented cell death. Application of the ISEL assay and acridine orange/ethidium bromide staining demonstrated that the H2O2-induced cell death occurred by an apoptotic mechanism at lower concentrations of H2O2 (400, 500, 600 microM), whereas higher concentrations of H2O2 induced necrosis rather than apoptosis. Caspase 3 was associated with the apoptotic pathway in human RPE cell death. Western blot analysis confirmed caspase 3 activation and cleavage of substrate proteins in ARPE-19 cells treated with an H2O2 concentration of 600 microM. These results indicate that treatment with H2O2 induces apoptotic and necrotic cell death in ARPE-19, and that caspase 3 is associated with apoptotic cell death. Therefore, H2O2 may induce the destruction of RPE cells in AMD by the combined effects of apoptosis and necrosis.
Apoptosis ; Caspases/metabolism ; Catalase/pharmacology ; Cell Line ; Cell Survival/drug effects ; Enzyme Activation ; Human ; Hydrogen Peroxide/*pharmacology ; Necrosis ; Pigment Epithelium of Eye/*drug effects/enzymology/pathology/*physiology

To clarify the effects of zinc on the proliferation and apoptosis of cultured human retinal pigment epithelia (RPE) and the expression of caspase-3 in RPE cells. The effect of Zinc on the proliferation of RPE were examined with MTT method. TUNEL method was used to detect the apoptosis of RPE cells. Caspase-3 was detected by immunohistochemistry. A concentration of zinc higher than 0.001 microM could inhibit the proliferation of RPE. And the relationship between concentration of zinc higher than 10 microM and growth prohibition rate of RPE cells was dose-dependent. All concentrations of zinc including 0.001 microM enhanced the expression of caspase-3 of RPE. But only the concentration of zinc higher than 0.01 microM could induce apoptosis of RPE. It is concluded that zinc could enhance the expression of caspase-3 of RPE cells and induce apoptosis of RPE cells. Caution should be taken when using zinc supplements for the treatment of ARMD patients without deficiency of zinc.
Apoptosis ; drug effects ; Caspase 3 ; Caspases ; metabolism ; Cell Division ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Humans ; Pigment Epithelium of Eye ; cytology ; Zinc ; pharmacology

Blotting, Western ; Cells, Cultured ; Humans ; Pigment Epithelium of Eye ; metabolism ; Plasmids ; RNA Interference ; RNA, Small Interfering ; pharmacology ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; genetics

To clarify the effects of zinc on the proliferation and apoptosis of cultured human retinal pigment epithelia (RPE) and the expression of caspase-3 in RPE cells. The effect of Zinc on the proliferation of RPE were examined with MTT method. TUNEL method was used to detect the apoptosis of RPE cells. Caspase-3 was detected by immunohistochemistry. A concentration of zinc higher than 0.001 microM could inhibit the proliferation of RPE. And the relationship between concentration of zinc higher than 10 microM and growth prohibition rate of RPE cells was dose-dependent. All concentrations of zinc including 0.001 microM enhanced the expression of caspase-3 of RPE. But only the concentration of zinc higher than 0.01 microM could induce apoptosis of RPE. It is concluded that zinc could enhance the expression of caspase-3 of RPE cells and induce apoptosis of RPE cells. Caution should be taken when using zinc supplements for the treatment of ARMD patients without deficiency of zinc.
Apoptosis/*drug effects ; Caspase 3 ; Caspases/metabolism ; Cell Division/drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Pigment Epithelium of Eye/*cytology ; Zinc/*pharmacology

Small hairpin RNA (shRNA) was used to silence the HIF1alpha gene in human retinal pigment epithelial cells (RPE) under hypoxia in order to observe the effect of gene silencing on the expression of matrix metalloproteinase tissue inhibitor 1 (TIMP1). By using chemical hypoxic inducer CoCl2, to mimic RPE hypoxic environment, shRNA against the targeting region of HIF1alpha mRNA sequence was synthesized by a method of in vitro transcription, and the HIF1alpha was interfered in RPE cultured under hypoxia (induced by 150 micromol/L CoCl2). RT-PCR was employed to detect the expression of HIF1alpha and TIMP1. The expression levels of HIF1alpha and TIMP1 were measured by using Western blotting. The results showed that after the RPE were transfected with specific shRNA against HIF1alpha mRNA, RT-PCR revealed that under hypoxia, the efficacy of HIF1alpha gene silencing in RPE was 83.4%. Western blotting revealed that the expression levels of HIF1alpha protein was dramatically dropped. In addition. RT-PCR results demonstrated that the expression of TIMP1 mRNA was decreased by 28.9%, and the expression levels of TIMP1 protein were also significantly reduced by Western blotting. It was suggested that shRNA targeted against HIF1alpha mRNA could effectively silence the HIF1alpha gene, subsequently effectively inhibit the hypoxia-induced up-regulation of TIMP1.
Cell Hypoxia ; Cells, Cultured ; Gene Silencing ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics ; Pigment Epithelium of Eye/cytology ; Pigment Epithelium of Eye/*metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; RNA, Small Interfering/*pharmacology ; Retina/cytology ; Retina/*metabolism ; Tissue Inhibitor of Metalloproteinase-1/*biosynthesis ; Tissue Inhibitor of Metalloproteinase-1/genetics

BACKGROUNDRetinal pigment epithelial (RPE) cells play an important role in the occurrence of choroidal neovascularization (CNV). Vascular endothelial growth factor (VEGF) as a positive regulatory growth factor is produced by the RPE in an autocrine or paracrine manner, promoting CNV development. Duplexes of 21 nt RNAs, known as short interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference when introduced into mammalian cells. We searched for an efficient siRNA to interfere with VEGF expression in RPE cells and shed light on the treatment of CNV.

METHODSHuman primary RPE (hRPE) cells were cultured and identified. Three pairs of siRNAs were designed according to the sequence of VEGF 1-5 extrons and synthesized by T7 RNA polymerase transcription in vitro. To evaluate the inhibitory activity of T7-siRNAs, hRPE cells were transfected via siPORT Amine. The interfering effect of T7-siRNAs in hRPE cells was examined by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence.

RESULTSThree pairs of T7-siRNAs synthesized by in vitro transcription with T7 RNA polymerase suppressed VEGF gene expression with efficiency from 65% to 90%. T7-siRNA (B), targeted region at 207 nt to 228 nt and double stranded for 21 nt with 2 nt UU 3' overhangs, was the most effective sequence tested for inhibition of VEGF expression in hRPE cells. Compared with nontransfected cells, the mean fluorescence in hRPE cells transfected with T7-sRNAs was significantly less (P < 0.01). siRNA with a single-base mismatch and ssRNA(+) did not show suppressing effect. Furthermore, it was found that siRNAs had a dose dependent inhibitory effect (5 to 10 pmol).

CONCLUSIONT7-siRNA can effectively and specifically suppress VEGF expression in hRPE cells and may be a new way to treat CNV.

Base Sequence ; Cells, Cultured ; Choroidal Neovascularization ; therapy ; DNA-Directed RNA Polymerases ; metabolism ; Humans ; Molecular Sequence Data ; Pigment Epithelium of Eye ; cytology ; metabolism ; RNA Interference ; RNA, Small Interfering ; biosynthesis ; pharmacology ; Transcription, Genetic ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; genetics ; Viral Proteins ; metabolism

OBJECTIVETo study the difference in stress and apoptosis related genes transcription between hTERT-RPE1 cells exposed to simulated microwave radiation and the cells with heat water bath, and the effects of microwave on gene transcription in cultured human retina pigment epithelial cells.

METHODScDNA microarray technique was used to detect the mRNA isolated from hTERT-RPE1 cells exposed to 2 450 MHz simulated microwave radiation and with heat water bath, respectively.

RESULTSAmong the 97 related aim genes, there were seven genes up-regulating its transcription, i.e., M31166 (2.52fold), L24123 (2.66fold), AF039704 (2.22fold), U67156 (2.07fold), AF040958 (2.13fold), NM-001423 (2.63fold) and NM-005346 (3.68fold). But, no notably down-regulating gene in transcription was detected.

CONCLUSIONSMicrowave could induce up-regulating in multiple stress and apoptosis related genes transcription in cultured human retina pigment epithelial cells, hTERT-RPE1 cells. Microwave radiation has unique effect itself in addition to its heat effect.

Gene Expression Profiling ; Gene Expression Regulation ; radiation effects ; Humans ; Microwaves ; Oligonucleotide Array Sequence Analysis ; methods ; Pigment Epithelium of Eye ; cytology ; metabolism ; radiation effects ; Retina ; cytology ; metabolism ; radiation effects ; Time Factors

The generation of functional retinal pigment epithelium (RPE) is of great therapeutic interest to the field of regenerative medicine and may provide possible cures for retinal degenerative diseases, including age-related macular degeneration (AMD). Although RPE cells can be produced from either embryonic stem cells or induced pluripotent stem cells, direct cell reprogramming driven by lineage-determining transcription factors provides an immediate route to their generation. By monitoring a human RPE specific Best1::GFP reporter, we report the conversion of human fibroblasts into RPE lineage using defined sets of transcription factors. We found that Best1::GFP positive cells formed colonies and exhibited morphological and molecular features of early stage RPE cells. Moreover, they were able to obtain pigmentation upon activation of Retinoic acid (RA) and Sonic Hedgehog (SHH) signaling pathways. Our study not only established an ideal platform to investigate the transcriptional network regulating the RPE cell fate determination, but also provided an alternative strategy to generate functional RPE cells that complement the use of pluripotent stem cells for disease modeling, drug screening, and cell therapy of retinal degeneration.
Animals ; Bestrophins ; Cell Differentiation ; Cell Line ; Cell Lineage ; Chloride Channels ; genetics ; metabolism ; Embryonic Stem Cells ; cytology ; metabolism ; Eye Proteins ; genetics ; metabolism ; Fibroblasts ; cytology ; metabolism ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Mice ; Pigmentation ; Retinal Pigment Epithelium ; cytology ; metabolism ; Transcription Factors ; metabolism