Animals ; Cell Transplantation ; methods ; Humans ; Parkinson Disease ; surgery ; Pigment Epithelium of Eye ; cytology ; physiology ; Positron-Emission Tomography

It is known that CD95 (APO-1/Fas) is expressed on the cell surface, and apoptotic cell death can be induced by the CD95 ligation in the cultured, proliferating human retinal pigment epithelial (RPE) cells. However, little is known about CD95 on the non-proliferating RPE cells. In this study, human RPE cells were cultured up to 4 weeks after they reached the confluence, to simulate the non-proliferating RPE cells in situ. There was no significant difference in CD95 expression on the cell surface between the predominantly proliferating, preconfluent cells and predominantly non-proliferating, postconfluent cells in flow cytometric assays. However, unlike proliferating cells, no cellular death occurred in the predominantly non-proliferating cells after the treatment of agonistic anti-CD95 antibody with cycloheximide, pretreated with interferon-gamma. Our results suggest that the CD95/CD95L system probably plays a physiologic role in vivo to remove the abnormal, proliferating RPE cells, and factors other than the surface expression of CD95 may determine the sensitivity to the CD95 signals.
Antigens, CD95/*pharmacology ; Apoptosis/*physiology ; Cells, Cultured ; Human ; Pigment Epithelium of Eye/cytology/*drug effects/*physiology ; Sensitivity and Specificity

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

AlamarBlue is used to induce color and fluorescence in the microenvironment of activated cells. The alamarBlue assay was studied to determine if it could be used as a method of evaluating the number of retinal pigment epithelial (RPE) cells. A series of two-fold dilutions of RPE cells were placed into 96-well culture plates. The alamarBlue was added to the culture media after attaching the cells. The absorbance and fluorescence were measured consecutively at various intervals over a period of 24 hr. Cell viability were evaluated by means of the trypan blue exclusion method and flow cytometry using a combination of propidium iodide and annexin V was done to prove the safety of alamarBlue assay to the cells. Both the absorbance and the fluorescence had a linear relationship with the number of RPE cells. Exposing the RPE cells to alamarBlue was not detrimental to the cells. In conclusion, the alamarBlue assay constitutes a one-step, extremely simple, reproducible, economical and non-toxic procedure for evaluating the number of viable RPE cells.
Cell Count ; Cell Division ; Cell Survival ; *Dyes ; Flow Cytometry ; Fluorescence ; Human ; Pigment Epithelium of Eye/*cytology/physiology ; Support, Non-U.S. Gov't ; Trypan Blue

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

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

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 present study investigated the effects of transforming growth factor (TGF)-beta on retinal pigment epithelial (RPE) transformation in a simplified model and also whether or not TGF-beta exhibits similar proliferation effects on transformed RPE cells that it has on primary RPE cells. Furthermore, we examined the cell proliferation effects of RPE-conditioned medium (CM). A vertical wound measuring 2 mm in diameter was made on primary RPE monolayers. The expression of alpha- smooth muscle actin (SMA) by the cells located at the wound edges was observed using a confocal microscope under immunofluorescent staining. Cell proliferation was measured by incorporating 3H-thymidine into DNA. The presence of alpha- SMA was observed in the cells within the wound after treatment with TGF-beta2, while negative expression was observed in control cells. TGF-betas inhibited the proliferation of the primary cultures of RPE cells in a dose-dependent manner, but the spindle-shaped late-passaged RPE cells were not inhibited by these growth factors. The medium conditioned by RPE cells stimulated the proliferation of subconjunctival fibroblasts and inhibited the proliferation of primary RPE cells, in a manner similar to TGF-beta. These findings demonstrate that TGF-beta-stimulated RPE cells may evoke proliferative vitreoretinopathy through mesenchymal transformation and cell proliferation.
Actins/analysis ; Animal ; Cell Division/drug effects ; Cells, Cultured ; Culture Media, Conditioned ; DNA/biosynthesis ; Mesoderm/*cytology ; Pigment Epithelium of Eye/*cytology ; Rabbits ; Swine ; Transforming Growth Factor beta/*physiology ; Vitreoretinopathy, Proliferative/etiology

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