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

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

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.
Blotting, Southern ; Blotting, Western ; Cell Movement/physiology ; Cell Proliferation ; Cells, Cultured ; Culture Media, Serum-Free ; *Gene Expression ; Hepatocyte Growth Factor/biosynthesis/*genetics ; Humans ; Mitosis/physiology ; Pigment Epithelium of Eye/cytology/*metabolism ; Polymerase Chain Reaction ; Proto-Oncogene Proteins c-met/biosynthesis/*genetics ; RNA/*genetics