Melatonin inhibits the proliferation of retinal pigment epithelial (RPE) cells in vitro

Summary The possible antiproliferative effect of melatonin on retinal pigment epithelial (RPE) cells in vitro was investigated. Bovine RPE cells cultured in Ham’s F12 medium supplemented with 10% fetal bovine serum had a nuclear density of 73.6 ± 6.1 nuclei/mm² at 72 h after seeding. The nuclear density at this time-point was doubled if either 50 or 100 ng/ml human epidermal growth factors (hEGF) was added to the culture medium. When these hEGF-stimulated cells were treated with melatonin from 10 to 500 pg/ml, the proliferation was suppressed with a dose-response relationship. At 250 and 500 pg/ml melatonin, the nuclear densities of the melatonin-treated cells were similar to those of the control cells. Using mitotically active SV-40 transformed human fetal RPE cells cultured in a serum-free medium, melatonin was also shown to be antiproliferative. In the presence of 500 pg/ml melatonin, the proliferation of these cells was inhibited to 77% as compared to the control. These results were further supported by the reduced [H³]thymidine uptake in the melatonin-treated cells. We propose that melatonin, at physiologic concentrations, has an antiproliferative effect, and that cultured RPE cells stimulated to proliferate by either hEGF treatment or SV-40 transfection are responsive to melatonin. Melatonin may either inhibit mitosis in actively dividing cells or modulate hEGF action.     

Actin-dependent,retrograde motility of surface-attached beads and aggregating pigment granules in dissociated teleost retinal pigment epithelial cells

Teleost retinal pigment epithelial (RPE) cells contain pigment granules within apical projections which undergo actin-dependent, bi-directional motility. Dissociated RPE cells in culture attach to the substrate and extend apical projections in a radial array from the central cell body. Pigment granules within projections can be triggered to aggregate or disperse by the presence or absence of 1 mM cAMP. Aminated, fluorescent latex beads attached to the dorsal surface of apical projections and moved in the retrograde direction, towards the cell body. Bead rates on RPE cells with aggregating or fully aggregated pigment granules were 2.2 +/- 0.5 and 2.6 +/- 0.2 microm/min (mean +/- SEM), respectively, similar to rates of aggregating (retrograde) pigment granule movement (2.0 +/- 0.4 microm/min). Bead rates were slightly slower on cells with fully dispersed or dispersing pigment granules (1.5 +/- 0.1 and 1.5 +/- 0.4 microm/min). Movements of surface-attached beads and aggregating pigment granules were closely correlated in the distal portions of apical projections, but were more independent of each other in proximal regions of the projections. The actin disrupting drug, cytochalasin D (CD), reversibly halted retrograde bead movements, suggesting that motility of surface-attached particles is actin-dependent. In contrast, the microtubule depolymerizing drug, nocodazole, had no effect on retrograde bead motility. The similar characteristics and actin-dependence of retrograde bead movements and aggregating pigment granules suggest a correlation between these two processes.     

Microtubules, microfilaments and adhesion patterns in differentiating chick retinal pigment epithelial (RPE) cells in vitro

The distribution of microtubules (MT), microfilaments (MF), and patterns of cell-to-substratum adhesion were studied by tubulin antibody labeling, NBD-phallacidin staining and by reflection interference contrast (RIC) microscopy respectively in colonies of differentiating RPE cells obtained from explants after 10 days in culture. In each colony three zones could be identified: a central zone of packed well-differentiated cuboidal cells (zone 1), an intermediate zone of more flattened, pleomorphic cells (zone 2) and a peripheral zone of very spread cells at the edge of the colony (zone 3). As visualized with antibodies to tubulin, the MT distribution in cells of each zone was distinctly different and correlated well with differences in cell shape. Changes in the distribution of MF were more striking. In the cuboidal well-differentiated cells of zone 1, prominent cortical bands but no stress fibers were observed after staining with NBD-phallacidin and RIC microscopy showed that the cells lacked strong adhesion to the substratum. Stress fibers, in addition to cortical bands of MF, were seen in the more spread, less differentiated cells of zone 2 and focal contacts were observed when these cells were examined by RIC microscopy. The flattened least differentiated cells in zone 3 lacked cortical bands but had prominent stress fibers. These cells displayed a variety of adhesion forms ranging from a mosaic of far and close contacts to numerous focal contacts and broad focal adhesions. Our results show that as the RPE cells display less differentiated morphologies, i.e. are more flattened and less densely packed towards the edge of the colony, there is a gradual decrease in the cortical bands of MF and an increase in the number and prominence of stress fibers. This increase in numbers of stress fibers is correlated with an increase in the cell adhesiveness to the substratum, as estimated by RIC microscopy. These results strongly support the general observation that normal epithelial cells in colonies tend to adhere to the substratum more strongly by marginal cells than by the more differentiated centrally located cuboidal cells which have well developed intercellular contacts.     

Effects of the actin-stabilizing drug, jasplakinolide, on pigment granule motility in isolated retinal pigment epithelial (RPE) cells of green sunfish, Lepomis cyanellus

The retinal pigment epithelium (RPE) of teleosts contains pigment granules that migrate in response to changes in light condition. Dissociated, cultured RPE cells in vitro can be triggered to aggregate or disperse pigment granules by the application of cAMP or dopamine, respectively. Previous research using the actin-disrupting drug, cytochalasin D, suggested that pigment granule motility is actin dependent. To further examine the role of actin in pigment granule motility, we tested the effects of the actin-stabilizing drug, jasplakinolide, on pigment granule motility. Pigment granules in previously dispersed RPE cells remained dispersed after jasplakinolide exposure (0.1-1 microM), but the drug halted movement of most pigment granules and stimulated rapid bi-directional movements in a small subset of granules. Jasplakinolide also blocked net pigment granule aggregation and interfered with the maintenance of full aggregation. Although jasplakinolide did not block pigment granule dispersion, it did alter the motility of dispersing granules compared to control cells; rather than the normal saltatory, primarily centrifugal movements, granules of jasplakinolide-treated cells demonstrated slow, creeping centrifugal movements and more rapid bi-directional movements. Jasplakinolide also altered cell morphology; the length and thickness of apical projections increased, and enlarged, paddle-like structures, which contained F-actin appeared at the tips of projections. Actin antibody labeling of jasplakinolide-treated cells revealed a more reticulated network of actin compared to antibody-labeled control cells. These results indicate that jasplakinolide-induced disruption of the actin network compromises normal pigment granule dispersion and aggregation in isolated RPE cells, thus providing further evidence that these movements are actin dependent.     

In vitro phototoxicity of rhodopsin photobleaching products in the retinal pigment epithelium (RPE)

Although the primary biological function of retinal photoreceptors is to absorb light and provide visual information, extensive exposure to intense light could increase the risk of phototoxic reactions mediated by products of rhodopsin bleaching that might accumulate in photoreceptor outer segments (POS). The phototoxicity of POS, isolated from bovine retinas, was examined in cultured retinal pigment epithelium cells (ARPE-19) containing phagocytised POS and in selected model systems by determining POS ability to photogenerate singlet oxygen, and photoinduce oxidation of cholesterol and serum albumin. Bleaching of rhodopsin-rich POS with green light resulted in the formation of retinoid products exhibiting distinct absorption spectra in the near-UV. Irradiation of POS-fed ARPE-19 cells with blue light reduced their survival in a dose-dependent manner with the effect being stronger for cells containing prebleached POS. The specific and non-specific phagocytic activity of ARPE-19 cells was inhibited by sub-lethal photic stress mediated by phagocytised POS. The oxidising ability of POS photobleaching products was demonstrated both in a model system consisting of serum albumin and in ARPE-19 cells. Distinct photooxidation of proteins, mediated by POS, was observed using coumarin boronic acid as a sensitive probe of protein hydroperoxides. Irradiation of POS with blue light also induced oxidation of liposomal cholesterol as determined by HPLC-EC(Hg). Time-resolved singlet oxygen phosphorescence demonstrated the efficiency of retinoids, extracted from POS by chloroform-methanol treatment, to photogenerate singlet oxygen. The results indicate that photic stress mediated by POS photobleaching products could inhibit phagocytic efficiency of RPE cells and, ultimately, compromise their important biological functions.     

Rat retinal pigment epithelial cells show specificity of phagocytosis in vitro

The retinal pigment epithelial (RPE) cell of the eye normally phagocytozes only retinal rod outer segments (ROS). The specificity of this phagocytic process was examined by incubating RPE cells with a variety of particle types. Confluent RPE cell cultures were incubated for 3 h at 37 degrees C in the presence of rat ROS, rat red blood cells (RBC), algae, bacteria, or yeast. Other cell cultures were incubated with equal numbers of ROS and one other particle type. Quantitative scanning electron microscopy was used to determine the numbers and morphology of particles bound to RPE cells, while double immunofluorescence labeling (Chaitin, M. H., and M. O. Hall, 1983, Invest. Ophthalmol. Vis. Sci., 24:812-820) was used to quantitate particle binding and ingestion. Both assays demonstrated phagocytosis to be a highly specific process. RPE cells bound 40-250 X more ROS than RBC, 30 X more ROS than algae, and 5 X more ROS than bacteria or yeast. Ingestion was more specific than binding; RPE cells ingested 970 X more ROS than RBC, 140 X more ROS than bacteria, and 35 X more ROS than yeast. The phagocytic preference for ROS was maintained in competition experiments with other particle types. Serum was found to be essential for phagocytosis. This study demonstrates that both the binding and ingestion phases of phagocytosis are highly specific processes.     

Polyplex-mediated gene transfer into human retinal pigment epithelial cells in vitro

The human retinal pigment epithelium (RPE) is a potential target tissue for directed transfer of candidate genes to treat age-related macular degeneration (AMD). The RPE is uniquely suited to gene therapy protocols that use liposome-mediated DNA transfer because of its high intrinsic phagocytic function in vivo. In these studies, we examined the efficacy of human RPE cell uptake and expression of the green fluorescent protein (GFP) and neomycin resistance marker genes by polyplex-mediated gene transfer in vitro. The effects of varying DNA and polyplex concentration and ratios on GFP transgene expression were examined. A narrow range of experimental conditions were found to maximize transgene expression; most important were the DNA concentration and the DNA:polyplex ratio. The transfection efficiency for human RPE cells was reproducibly 20% in vitro by this method and reached a maximum level of expression after 48 h. There was a rapid decline in gene expression over 2 weeks following polyplex-mediated gene transfer, but stable integration does occur at low frequencies with and without selection.     

Single-channel recordings from cultured human retinal pigment epithelial cells

We have applied patch-clamp techniques to on-cell and excised-membrane patches from human retinal pigment epithelial cells in tissue culture. Single-channel currents from at least four ion channel types were observed: three or more potassium-selective channels with single-channel slope conductances near 100, 45, and 25 pS as measured in on-cell patches with physiological saline in the pipette, and a relatively nonselective channel with subconductance states, which has a main-state conductance of approximately 300 pS at physiological ion concentrations. The permeability ratios, PK/PNa, measured in excised patches were 21 for the 100-pS channels, 3 for the 25-pS channels, and 0.8 for the 300-pS nonselective channel. The 45-pS channels appeared to be of at least two types, with PK/PNa's of approximately 41 for one type and 3 for the other. The potassium-selective channels were spontaneously active at all potentials examined. The average open time for these channels ranged from a few milliseconds to many tens of milliseconds. No consistent trend relating potassium-selective channel kinetics to membrane potential was apparent, which suggests that channel activity was not regulated by the membrane potential. In contrast to the potassium-selective channels, the activity of the nonselective channel was voltage dependent: the open probability of this channel declined to low values at large positive or negative membrane potentials and was maximal near zero. Single-channel conductances observed at several symmetrical KCl concentrations have been fitted with Michaelis-Menten curves in order to estimate maximum channel conductances and ion-binding constants for the different channel types. The channels we have recorded are probably responsible for the previously observed potassium permeability of the retinal pigment epithelium apical membrane.     

Erythropoietin protects retinal pigment epithelial cells from oxidative damage

Oxidative damage from reactive oxygen species (ROS) has been implicated in many diseases, including age-related macular degeneration, in which the retinal pigment epithelium (RPE) is considered a primary target. The aim of this study was to determine whether erythropoietin (EPO) protects cultured human RPE cells against oxidative damage and to identify the pathways that may mediate protection. EPO (1 IU/ml) significantly increased the viability of oxidant-treated RPE cells, decreased the release of the inflammatory cytokines tumor necrosis factor-α and interleukin-1β, recovered the RPE cells' barrier integrity disrupted by oxidative stress, prevented oxidant-induced cell DNA fragmentation and membrane phosphatidylserine exposure, and also reduced the levels of oxidant-induced intracellular ROS and restored cellular antioxidant potential, total antioxidant capacity, glutathione peroxidase, and superoxide dismutase and decreased malondialdehyde, the end product of lipid peroxidation. EPO inhibited caspase-3-like activity. Protection by EPO was partly dependent on the activation of Akt1 and the maintenance of the mitochondrial membrane potential. No enhanced or synergistic protection was observed during application of Z-DEVD-FMK (caspase-3 inhibitor) combined with EPO compared with cultures exposed to EPO and H₂O₂ alone. Together, these results suggest that EPO could protect against oxidative injury-induced cell death and mitochondrial dysfunction in RPE cells through modulation of Akt1 phosphorylation, mitochondrial membrane potential, and cysteine protease activity.     

Rapamycin reduces VEGF expression in retinal pigment epithelium (RPE) and inhibits RPE-induced sprouting angiogenesis in vitro

Anti-VEGF treatment has become accepted first-line treatment for choroidal neovascularisation (CNV) in age-related macular degeneration. However, VEGF-inhibition does not always lead to sustained CNV-reduction. In this study, the effect of rapamycin was superior to VEGF-inhibition in a co-culture assay of endothelial cells (ECs) and retinal pigment epithelium (RPE). Rapamycin reduced EC sprouting in groups that did not respond to anti-VEGF treatment. Rapamycin did not induce EC apoptosis, but reduced both VEGF-production in RPE and the responsiveness of ECs to stimulation. Rapamycin might therefore be a therapeutic option for CNV patients that do not respond sufficiently to the established anti-VEGF treatments.     

The effect of oxygen on melanin precursors released from retinal pigment epithelial cells in vitro.

The autoxidation of dopa to melanin in culture media causes toxicity to retinal pigment epithelial (RPE) cells and endothelial cells. The damage is specific to cell type and to the ambient oxygen concentration. To determine whether RPE cells influence the oxidation of dopa to media, we compared light absorbing dopa derivatives in the media exposed to cells with those found in the media incubated without cells. Dopa was extensively oxidized in the presence of RPE cells, and more light absorbing substances were generated with higher dopa and oxygen concentrations. However, an increase in ambient oxygen concentration decreased the quantity of several dopa derivatives which had been formed. The data provided evidence that RPE modulated dopa metabolism. Quinolic derivatives produced from a tyrosinase reaction and dopa-melanin formation moved the peak absorbance wavelength of dopa into the visible range. The spectrum between the dopa-derived compounds in the media has an absorbance at 240-275 nm and a maximum around 300 nm with a shoulder near 375 nm. Gaussian analysis (peak separation) resolved these spectra into five components: a sharp band at 248 nm, a band at 295 nm, a large band at 359 nm, and two broad bands at 459 and 585 nm.     

In vitro differentiation of retinal pigment epithelium from adult retinal stem cells

One of the limitations in molecular and functional studies of the retinal pigment epithelium (RPE) has been the lack of an in vitro system retaining all the features of in vivo RPE cells. Retinal pigment epithelium cell lines do not show characteristics typical of a functional RPE, such as pigmentation and expression of specific markers. The present study was aimed at the development of culture conditions to differentiate, in vitro, retinal stem cells (RSC), derived from the adult ciliary body, into a functional RPE. Retinal stem cells were purified from murine eyes, grown as pigmented neurospheres and induced to differentiate into RPE on an extracellular matrix substrate using specific culture conditions. After 7-15 days of culture, pigmented cells with an epithelial morphology showed a polarized organization and a capacity for phagocytosis. We detected different stages of melanogenesis in cells at 7 days of differentiation, whereas RPE at 15 days contained only mature melanosomes. These data suggest that our protocol to differentiate RPE in vitro can provide a useful model for molecular and functional studies.     

Calcium uptake, release and ryanodine binding in melanosomes from retinal pigment epithelium

High levels of calcium have been reported in pigmented tissues of the vertebrate eye, such as retinal pigment epithelium (RPE). Melanin granules also have high calcium concentrations, suggesting that melanin granules may be a calcium reservoir. Here we characterized the uptake and release of calcium in a pure melanosomal fraction obtained from frog RPE. Melanosomes take up 45Ca by a saturable system with an apparent KM of 0.5 mM. About 40% of 45Ca accumulation was insensitive to low temperature. 45Ca uptake was not affected by verapamil, nifedipine, dantrolene, vanadate, thapsigargin or cyclopiazonic acid, but it was reduced by 50% by ruthenium red, and increased by the ionophore A23187 and nigericin. Release of 45Ca-loaded was stimulated by caffeine and inositol 1,4,5 trisphosphate (IP3). Caffeine stimulated release of calcium was blocked by either ryanodine or ruthenium red, but calcium released by IP3 was not affected by heparin. No binding of 3H-IP3 was observed. The 3H-ryanodine binding sites exhibited a KB of 1.3 nM and a Bmax of 12.1 fmol/mg protein. Thus, our results suggest that melanosomes may function as intracellular organelles that regulate calcium concentration in RPE.     

Analysis of the linkage of MYRIP and MYO7A to melanosomes by RAB27A in retinal pigment epithelial cells

The apical region of the retinal pigment epithelium (RPE) typically contains melanosomes. Their apical distribution is dependent on RAB27A and the unconventional myosin, MYO7A. Evidence from studies using in vitro binding assays, melanocyte transfection, and immunolocalization have indicated that the exophilin, MYRIP, links RAB27A on melanosomes to MYO7A, analogous to the manner that melanophilin links RAB27A on melanocyte melanosomes to MYO5A. To test the functionality of this hypothesis in RPE cells, we have examined the relationship among MYRIP, RAB27A and MYO7A with studies of RPE cells in primary culture (including live-cell imaging), analyses of mutant mouse retinas, and RPE cell fractionation experiments. Our results indicate that the retinal distribution of MYRIP is limited to the RPE, mainly the apical region. In RPE cells, RAB27A, MYRIP, and MYO7A were all associated with melanosomes, undergoing both slow and rapid movements. Analyses of mutant mice provide genetic evidence that MYRIP is linked to melanosomes via RAB27A, but show that recruitment of MYRIP to apical RPE is independent of melanosomes and RAB27A. RAB27A and MYRIP also associated with motile small vesicles of unknown origin. The present results provide evidence from live RPE cells that the RAB27A-MYRIP-MYO7A complex functions in melanosome motility. They also demonstrate that RAB27A provides an essential link to the melanosome.     

Proteins from melanosomes of mouse and chick pigment cells

Purified subcellular fractions containing melanosomes from B-16 mouse melanoma were treated with 2% sodium dodecyl sulfate or 0.5 M sodium hydroxide to dissolve protein. Quantitative measurements indicate that each melanosome contains 0.065×10^?10 of protein or about 19% by weight. SDS acrylamide gel electrophoresis of proteins from purified melanosomes resolved six polypeptide bands of major density and about 15 minor bands. These results indicate that the melanosome may be more complex than previous genetic, biochemical or morphological evidence had suggested.     

Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells

Human induced pluripotent stem cells (hiPSCs) possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs). These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay): soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×10? RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research.     

Polyplex‐mediated gene transfer into human retinal pigment epithelial cells in vitro

The human retinal pigment epithelium (RPE) is a potential target tissue for directed transfer of candidate genes to treat age‐related macular degeneration (AMD). The RPE is uniquely suited to gene therapy protocols that use liposome‐mediated DNA transfer because of its high intrinsic phagocytic function in vivo. In these studies, we examined the efficacy of human RPE cell uptake and expression of the green fluorescent protein (GFP) and neomycin resistance marker genes by polyplex‐mediated gene transfer in vitro. The effects of varying DNA and polyplex concentration and ratios on GFP transgene expression were examined. A narrow range of experimental conditions were found to maximize transgene expression; most important were the DNA concentration and the DNA:polyplex ratio. The transfection efficiency for human RPE cells was reproducibly 20\% in vitro by this method and reached a maximum level of expression after 48 h. There was a rapid decline in gene expression over 2 weeks following polyplex‐mediated gene transfer, but stable integration does occur at low frequencies with and without selection. J. Cell. Biochem. 76:153–160, 1999. © 1999 Wiley‐Liss, Inc.     

Copper mediates mitochondrial biogenesis in retinal pigment epithelial cells

Age related macular degeneration (AMD) is a multifactorial disease with genetic, biochemical and environmental risk factors. We observed a significant increase in copper levels in choroid-RPE from donor eyeballs with AMD. Adult retinal pigment epithelial cells (ARPE19 cells) exposed to copper in-vitro showed a 2-fold increase in copper influx transporter CTR1 and copper uptake at 50 μM concentration. Further there was 2-fold increase in cytochrome C oxidase activity and a 2-fold increase in the mRNA expression of NRF 2 with copper treatment. There was a significant increase in mitochondrial biogenesis markers PGC1β and TFAM which was confirmed by mitochondrial mass and copy number. On the contrary, in AMD choroid-RPE, the CTR1 mRNA was found to be significantly down-regulated compared to its respective controls. SCO1 and PGC1β mRNA showed an increase in choroid–RPE. Our study proposes copper to play an important role in mitochondrial biogenesis in RPE cells.