Choroidal melanoma-associated retinal and retinal pigment epithelial changes

Many clinical findings associated with choroidal melanoma are the result of secondary changes in the adjacent tissues. There may be alterations in the choriocapillaris, the RPE, the Bruch's membrane, and the sensory retina. Proper identification of these changes is essential for diagnosis and management.     

Minimum number of adult human retinal pigment epithelial cells required to establish a confluent monolayer in vitro

PURPOSE: To determine the minimum number of cells required to establish a confluent monolayer of retinal pigment epithelium (RPE) with an epitheloid morphology in vitro. METHODS: Primary or passaged human RPE were harvested by trypsinization from 6 donors and plated onto bovine corneal endothelium extracellular matrix-coated tissue culture plastic in 96-well plates. Plating densities ranged from 1 to 66,000 viable cells/well (0.03-2062 viable cells/mm2) for primary cells or 1 to 100,000 viable cells/well (0.03-3112 viable cells/mm2) for passaged cells. The time required to reach confluence was determined by monitoring the cultures daily until they reached confluence. Mean cell area and circularity index at confluence was calculated to determine the effect of different plating densities on final RPE morphology. RESULTS: Primary RPE plated at densities above 10 viable cells/mm2 (320 cells/well) and passaged RPE plated above 2 viable cells/mm2 (64 cells/well) reached confluence on every occasion. There was a negative correlation between the plating density and time required to reach confluence. Plating densities above 3 viable cells/mm2 (96 cells/well) and 50 viable cells/mm2 (1600 cells/well) yielded smaller, rounder cells at confluence for primary and passaged RPE, respectively. CONCLUSIONS: As few as 96 primary RPE cells and 1600 passaged RPE are required to obtain a confluent, 6mm (4-disc diameter) patch of RPE in vitro. This suggests that autologous RPE grafts can be prepared with high efficiency for subsequent transplantation into the subretinal space in vivo.     

Serum-free media for culturing and serial-passaging of adult human retinal pigment epithelium

The ability of a chemically-defined serum-free culture medium to support the attachment, growth and serial passaging of primary adult human retinal pigment epithelial (RPE) cells was studied. Primary cultures of adult human RPE were established in a chemically-defined serum-free culture medium on both bare or bovine corneal endothelial extracellular matrix-coated tissue-culture plastic. Confluent cells were serially passaged in chemically-defined serum-free culture medium three times by trypsinization, and trypsin activity was quenched with aprotinin. First passage RPE cells were plated onto tissue-culture plastic precoated with bovine corneal endothelial extracellular matrix or uncoated tissue-culture plastic in 24 well plates at a density of 50 viable cells mm-2. Cells were maintained either in chemically-defined serum-free culture medium, DMEM without serum, or DMEM with 15% fetal bovine serum. For each medium plating, efficiencies were determined 24 hours after plating, and growth rates were determined on the first, third and seventh days after plating. Morphometric image analysis was performed on cells cultured for up to 6 weeks and three serial passages. Seeding efficiency on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and treated tissue-culture plastic were higher for chemically-defined serum-free culture medium (88.9+/-2.7% and 47.1+/-4.1%, respectively) and DMEM with serum (87.2+/-5.6% and 52.9+/-10.5%, respectively) than DMEM without serum (59.2+/-5.6% and 33.1+/-6.9%, respectively; P<0.01). The RPE proliferation rate in chemically-defined serum-free culture medium was comparable to DMEM with serum on both substrates within the first 3 days, although cells in DMEM with serum had a higher proliferation rate on day 7. Cells cultured in DMEM without serum, eventually decreased in number. RPE maintained in chemically-defined serum-free culture medium maintained a consistent proliferation rate, reached confluence, and retained an epitheloid morphology on either extracellular matrix or tissue-culture plastic for up to 6 weeks and three serial passages. Primary RPE reached confluence at 12+/-3 days on bovine corneal endothelial extracellular matrix-coated tissue-culture plastic and 21+/-5 days on treated tissue-culture plastic. Confluent cultures were composed of small hexagonal cells with epitheloid morphology on both substrates. We concluded that primary adult human RPE can be cultured in this chemically-defined serum-free culture medium. RPE will proliferate, reach confluence, retain their epitheloid morphology and can be serially passaged in the absence of serum.     

Culture of human retinal pigment epithelial cells from peripheral scleral flap biopsies

PURPOSE: We studied various methods for harvesting retinal pigment epithelium (RPE) biopsies from cadaver human eyes of donors over age 60 years. Our goal was to harvest cells for possible autologous RPE cell transplantation in patients with age-related macular degeneration and to test the viability of the RPE after isolation by evaluating explant growth in culture. METHODS: Choroid-RPE biopsies were excised from enucleated human eyes. The RPE was separated from the choroid by treatment with type IV collagenase. RPE patches were cultured. After 100-500 cells had grown out from the explant, the primary cultures were passaged. RESULTS: There was no clear effect of donor age on the ability to establish primary RPE cultures with good morphology from biopsies 2 x 2-10 x 10 mm2 in size. Biopsies 6 x 6 mm2 or larger produced satisfactory primary cultures more than 70% of the time. The number of viable RPE cells (defined as the number of cells adherent to the culture dish 24 h after plating) obtained after enzymatic separation of the RPE and choroid was an important determinant of our ability to establish primary cultures and passage the cells. Primary cultures with good cellular morphology were obtained 100% of the time when RPE explants > 4 mm2 in size were obtained from the biopsy specimen. Seventy-three percent of the biopsies yielding explants > 4 mm2 in size were successfully passaged. CONCLUSIONS: These results suggest that peripheral scleral flap biopsies in aging donors can be used to establish RPE explant primary cultures. These cultures may be suitable as a source for autologous RPE transplantation in patients.     

Inwardly rectifying K+ currents in isolated human retinal pigment epithelial cells

PURPOSE: K+ channels in the retinal pigment epithelium (RPE) play a number of important roles, including the establishment of membrane potential, the transport of K+ between the subretinal space and choroid, and the generation of the c-wave of the electroretinogram. Previous studies on amphibian RPE demonstrated that these functions are likely served by an inwardly rectifying K+ channel. The aim of this study was to characterize inwardly rectifying K+ channels in cultured and freshly isolated adult human RPE (hRPE) cells. METHODS: Single cells were dispersed enzymatically from primary cultures of adult hRPE or from fresh adult hRPE-choroid. Ionic currents were recorded using either the perforated-patch or whole-cell configuration of the patch-clamp technique. RESULTS: In 5 mM external K+, roughly 20% of cultured hRPE cells exhibited a strong inwardly rectifying K+ conductance that passed inward but little outward current. This conductance increased when [K+]o was increased and exhibited a voltage-dependent block by external Na+ at negative potentials. In contrast, all freshly isolated hRPE cells exhibited a mild inwardly rectifying K+ conductance that mediated substantial outward current at physiological voltages. This conductance decreased when [K+]o was increased and showed no voltage-dependent block by external Na+. CONCLUSION: The authors conclude that fresh hRPE cells express a mild inwardly rectifying K+ conductance. The operation of this conductance at physiological voltages makes it a likely candidate for the resting K+ conductances of the apical and basolateral membranes. Cultured hRPE cells express a functionally different channel type that may reflect a change in phenotype.     

Glutamate-stimulated proliferation of rat retinal pigment epithelial cells

We investigated the effects of glutamate on cell proliferation and the expression of basic fibroblast growth factor (bFGF) and its receptor (FGF-R1) mRNA in cultured rat retinal pigment epithelial (RPE) cells. The number of primary RPE cells was significantly higher after treatment with 0.2 to 1.0 mM glutamate (maximum at 1.0 mM) for 7 days than in controls. Glutamate-stimulated cell proliferation was abolished by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), but not by 6,7-dinitroquinoxaline-2,3-dione or L(+)-2-amino-3-phosphonopropionic acid. Proliferation was increased to a similar extent by N-methyl-D-aspartate (NMDA), but not by kainate, alpha-amino-3-hydroxy-3-methyl-4-isoxazolepropionic acid or trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid. NMDA-receptor-like immunoreactivity was detected in most cells cultured. Treatment of cells with glutamate increased the level of bFGF mRNA and, to a lesser extent, that of FGF-R1 mRNA, which peaked 2 and 4 days, respectively, after glutamate was added. The increase in bFGF mRNA induced by glutamate was inhibited by MK-801. These findings suggest that glutamate might stimulate proliferation of RPE cells through activation of NMDA receptors and expression of bFGF and further suggest that glutamate may be involved in the proliferative changes of RPE cells in retinal wound healing.     

Retinoic acid metabolism in cultured retinal pigment epithelial cells

PURPOSE: To examine the stability of retinoic acid administered to cultured bovine retinal pigment epithelial (RPE) cells and to determine if RPE cells metabolize retinoic acid by a cytochrome P-450 mechanism. METHODS: Retinoic acid metabolism was examined in cultured RPE cells and subcellular fractions quantitatively by a thin-layer chromatography procedure and qualitatively by normal and reverse phase high-performance liquid chromatography. RESULTS: Cultured bovine RPE cells were found to have an activity that converts retinoic acid into more polar metabolites rapidly released from the cell. The highest specific activity for this process is found in the post-mitochondrial pellet (100,000g), is induced by retinoic acid, and is inhibited by ketoconazole. The major product of the RPE cell-mediated metabolism of retinoic acid is 4-oxo-retinoic acid, a known P-450 monooxygenase product of retinoic acid. The retinoic acid metabolizing activity is greatest in primary RPE cultures and decreases with aging in culture. CONCLUSIONS: These data suggest that a cytochrome P-450 monooxygenase is involved in the metabolism of retinoic acid in RPE cells, and this is similar to the findings of other investigators using other cells and tissues. The authors' findings suggest that the RPE may be important in the deactivation of this biologically potent retinoid in the retina.     

Evaluation of oxidative processes in human pigment epithelial cells associated with retinal outer segment phagocytosis

To investigate the nature of the oxidative event that occurs during phagocytosis of retinal outer segments (ROS) by cultured human retinal pigment epithelial (RPE) cells, cells were incubated with isolated bovine ROS labeled with either the fluorescence probe carboxy-SNAFL-2 or the nonfluorescent, oxidizable probe 2',7'-dichlorodihydrofluorescein (H2DCF). The increase in fluorescence following phagocytosis was measured by a flow cytometer. Other measurements included: oxygen consumption using a Clark-type oxygen electrode, extracellular superoxide release by superoxide dismutase inhibitable lucigenin chemiluminescence, intracellular hydrogen peroxide (H2O2) production, and the effect of catalase inhibition on cellular thiobarbituric acid-reactive substances (TBARS) caused by phagocytosis. The activities of the enzymes NADPH oxidase and palmitoyl-CoA oxidase were also measured. H2DCF attached to bovine ROS was oxidized during phagocytosis with a time course suggesting oxidation subsequent to ROS uptake. Measurements of oxygen consumption showed a time-dependent increase of 10%, 4 h after ROS feeding, attributable to a doubling of the cyanide-resistant oxygen consumption. Intracellular H2O2 production also doubled 4 h after ROS phagocytosis. ROS uptake by RPE cells produced no significant extracellular superoxide, while extracellular superoxide production was readily demonstrated in a control macrophage cell line. Enzyme activity measurements showed that incubation of RPE cells with ROS doubled catalase activity without affecting superoxide dismutase or glutathione peroxidase activities. Inhibition of catalase during ROS uptake increased TBARS by 66%. Other enzyme activity measurements showed that human RPE cells possess both NADPH oxidase and palmitoyl-CoA oxidase activities. We conclude that ROS phagocytosis subjects RPE cells to an oxidative event on the same order of magnitude as measured in a macrophage. The event is not an extracellular macrophage-type respiratory burst and may be due to intracellular H2O2 resulting from an NADPH oxidase in the phagosome or from beta-oxidation of ROS lipids in peroxisomes. Irrespective of case, the enzyme catalase appears to be essential in protecting the RPE cell against reactive oxygen species produced during phagocytosis.     

Influence of viral infection on expression of cell surface antigens in human retinal pigment epithelial cells

BACKGROUND: Subacute viral infection is known to change the phenotype of infected cells, thereby causing immune-mediated tissue damage. The aim of this study was to investigate the expression of different cell surface molecules on human retinal pigment epithelial cells (RPEC) following viral infection, with special emphasis on those having immune-regulatory functions. METHODS: Cultured RPEC were infected with cytomegalovirus (CMV), coxsackie-virus B3 (CVB) or herpes simplex virus type I (HSV). Double-staining fluorescence technique was used for visualization of virus infection and cell surface markers in the same cells by laser microscopy. RESULTS: CMV downregulated MHC class I antigens on RPEC, whereas CVB and HSV did not alter MHC class I antigen expression. No induction of class II antigens was observed in RPEC infected with CVB, HSV or CMV. The intercellular adhesion molecule ICAM-1 (CD54) was strongly expressed in uninfected RPEC, and a slight increase was observed after virus infection. Vascular cell adhesion molecule 1 (VCAM-1) was expressed in low amounts in both uninfected and infected RPEC. No expression of intercellular adhesion molecule 2 (ICAM-2), E-selectin ELAM-1 or lymphocyte-function-associated antigen 1 (LFA-1) was observed on RPEC before or after virus infection. CONCLUSION: Downmodulation of immune-regulating cell surface antigens has been suggested to provide a means of long-term survival of viruses in the infected cell, favoring establishment of persistent infection. Our observation in cultured human RPEC indicates that this mechanism might indeed contribute to the development of disease affecting retinal tissue.     

Giant pigment epithelial tear and exudative retinal detachment complicating choroidal effusions

PURPOSE: To report a case of a giant pigment epithelial tear and transient exudative retinal detachment occurring in a patient with hypotonic choroidal effusions after trabeculectomy. METHODS: Case report and brief literature review. RESULT: The retinal and choroidal detachments settled, disclosing an extremely large crescentic pigment epithelial defect in the temporal midperiphery. CONCLUSIONS: An exudative retinal detachment secondary to a peripheral pigment epithelial rip may complicate choroidal effusions. Recognition of this association may prevent unnecessary investigation or surgery.     

Soluble tumor necrosis factor receptors are present in human vitreous and shed by retinal pigment epithelial cells

Tumor necrosis factor-alpha (TNF) has been implicated in the pathogenesis of several retinal diseases. Soluble forms of the TNF receptors, p55 (55 kDa) and p75 (75 kDa), have recently been identified in biological fluids and may regulate TNF activity. The potential biological significance of these receptors for the human retina was examined by determining their presence in human vitreous and their release from eye cup explants in which the retina has been removed leaving an intact retinal pigment epithelium (HRPE). Normal human vitreous and conditioned medium from eye-cup HRPE explants demonstrated the presence of soluble p55 and p75. Soluble p55 was significantly more abundant than p75 in all vitreous samples (P < 0.03). Conditioned medium from eye-cup HRPE explants contained significantly more soluble p55 than p75 (P < 0.00002). Enzyme-linked immunosorbent assay showed the presence of soluble p55, and not p75, in conditioned medium from primary cultured HRPE cells. Activation of the protein kinase C pathway in these cells with the phorbol ester PMA significantly increased the release of soluble p55 (P < or = 0.001); whereas, pharmacological inhibition of protein kinase C with calphostin-C significantly decreased the shedding of p55 (P < or = 0.001). The results indicate that primary cultured HRPE cells shed p55 and regulate this shedding in part through the protein kinase C pathway. The presence of soluble TNF receptors within normal human vitreous and within conditioned medium from the eye-cup HRPE explant model suggests that these soluble receptors may have a biological function in the eye.     

Stimulation of retinal pigment epithelial cell growth by neuropeptides in vitro

PURPOSE: The proliferation of many cell types are regulated by cytokines and neuropeptides by autocrine and paracrine mechanisms. Retinal pigment epithelial (RPE) cells are also regulated by cytokines. But RPE cells are very close to the neural retina which has some neuropeptides. The present study was to investigate the effects of neuropeptides on the growth of RPE cells. METHODS: RPE cells were obtained from the eyes of 11 day old chick embryos and cultured in Dulbecco's modified Eagle's culture medium containing 10% fetal calf serum. The growth of RPE cells was evaluated by [3H]-thymidine uptake. RESULTS: Substance P, beta-endorphin and calcitonin gene-related peptide markedly stimulated the growth of RPE cells. The effects of methionine-enkephalin, somatostatin and vasoactive intestinal peptide were intermediate. The strongest effects of substance P, beta-endorphin and calcitonin gene-related peptide were observed at 10(-6) to 10(-7) M. The stimulation of RPE cells with beta-endorphin was inhibited by naloxone, suggesting that the stimulation with beta-endorphin is mediated by an opioid receptor. beta-endorphin and substance P induced RPE cell growth stimulating activity. Leucine-enkephalin and neuropeptide Y did not affect the growth of RPE cells. CONCLUSIONS: These results suggest that neuropeptides play an important role in the regulation of RPE cell growth.     

Cultured human retinal pigment epithelial cell produced and secreted epidermal growth factor: its bioactivity and clinical significance

The epidermal growth factor bioactivity from cultured primary human and monkey retinal pigment epithelial (RPE) cell systems was detected by using the radioreceptor assay. We report that the cultured human and monkey RPE cells produce and secrete the EGF bioactively to the media as demonstrated by radioreceptor binding assay. The EGF bioactivities secreted by human and monkey RPE cells were at peak of 48 hours (human RPE cells secreted 2.11 +/- 0.46 ng/ml vs monkey RPE was 1.56 +/- 0.12 ng/ml) in the serum-free media. The results indicate that the RPE is one of important sources for EGF in the eye. The RPE cells may play much important roles in the development of proliferative retinal diseases through the autocrine or paracrine mechanism. This new discovery will be helpful to elucidate the pathogenesis of proliferative retinal diseases and also provide an important basis for the treatment of such diseases.     

Transplantation of retinal pigment epithelial cells and immune response in the subretinal space

Theoretical x-ray spectra calculated by four different codes for the same tube parameters are compared by calculating and measuring doses to computed tomography (CT) body and head phantoms. The effect on the 120 kV spectrum, and hence on the calculated dose, of varying the anode angle, tube voltage, and total filtration of the x-ray tube is investigated. Codes used were those of Nowotny and H?fer (XCOMP), Boone, Iles, and Tucker et al. The code based on the work of Tucker et al. produced calculated doses noticeably lower than the other codes and compared best to the measured value. The variation in calculated dose between the Tucker code and the others is of the same order as the variation introduced by uncertainties in total filtration of about 20%, in peak tube voltage of +/- 4 kV, and in change of anode angle from 7 degrees to 13 degrees.