Lipofuscin accumulation in cultured retinal pigment epithelial cells reduces their phagocytic capacity

PURPOSE: Retinal pigment epithelial (RPE) cells slowly accumulate lipofuscin pigment within their acidic vacuolar apparatus as a result of extra- and/or intralysosomal oxidative alterations of phagocytosed photoreceptor outer segments (POS) with consequent imperfect degradation of these structures. In old age, lipofuscin accumulation may become quite substantial. It has been suggested that pronounced accumulation of lipofuscin is related to decreased RPE function and, possibly, to age-related macular degeneration. The aim of the present investigation was to study whether heavy loading with lipofuscin of RPE acidic lysosomes would affect the further phagocytic ability of the cells. METHODS: In the first section of the investigation, cultures of rabbit RPE cells were exposed daily to bovine UV-irradiated POS (artificial lipofuscin) for 4 weeks, resulting in a pronounced lipofuscin accumulation of the cells. Fluorescent latex beads (labelled with a red fluorophore) were added to unloaded control cultures at O and 4 weeks after their establishment, and to lipofuscin loaded cells after 4 weeks of feeding with artificial lipofuscin. Cellular amounts of lipofuscin, and their phagocytotic activity, were quantified by static fluorometry measuring lipofuscin-specific and red bead-specific fluorescence, respectively. The intracellular location of the beads was verified by confocal laser scanning microscopy. RESULTS: Unloaded, and thus almost lipofuscin-free, control cells exposed to latex beads showed numerous cytoplasmic particles emitting reddish fluorescence, while few particles were taken up by cells initially loaded with artificial, POS-derived, lipofuscin. Measurement of the latex bead-specific fluorescence showed significantly (p < 0.001) higher levels in unloaded control cells than in lipofuscin-loaded ones. In the second part of the investigation, unloaded control cultures and lipofuscin-loaded cultures were exposed to native bovine Texas Red-X-labelled POS 4 weeks after the establishment of the cultures. Unloaded control cells showed a large number of cytoplasmic POS emitting reddish fluorescence, while fewer POS were phagocytosed by cells loaded with artificial lipofuscin. Measurement of the Texas Red-X-specific fluorescence, thus quantifying the phagocytic ability of the cells, showed significantly (p < 0.001) higher levels in control cells than in lipofuscin-loaded ones. CONCLUSIONS: Severe lipofuscin accumulation of RPE cells appears to result in a greatly decreased phagocytic capacity. The resulting reduction in ability to cope with the needs of the overlying photoreceptor cells, in order to eliminate the obsolete tips of their POS, may well be of significance in the development of age-related macular degeneration.     

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.     

Cytokines cause cultured retinal pigment epithelial cells to secrete metalloproteinases and to contract collagen gels

PURPOSE: Because retinal pigment epithelial cells in epiretinal membranes remodel and contract their surrounding extracellular matrix, investigations were performed to determine if these cells can produce matrix metalloproteinases and contract collagen gels in vitro in the presence of serum or cytokines. METHODS: Cells were grown on collagen gels and their production of metalloproteinases was measured using zymography. RESULTS: Cells grown in a three-dimensional collagen gel culture system produce several latent metalloproteinases that are secreted into the gel and the surrounding medium. These include molecules of 49, 56, 66, and 100 kD. In addition, an enzyme that is probably the active form of the 66 kD enzyme is present. When interleukin 1 beta is added to the cultures, latent 49 kD and 100 kD gelatinase production is greatly stimulated and an active form of both enzymes is also observed in the medium. In contrast, transforming growth factor beta has no stimulatory effect. The cells contract the collagen gel but this is small without cytokines; however, contraction is greatly enhanced in the presence of serum or interleukin 1 beta plus transforming growth factor beta. Contraction is unlikely to be the result of metalloproteinase action on the underlying extracellular matrix because complete inhibition of these enzymes has little effect. CONCLUSIONS: These results show that cytokines can cause cultured retinal pigment epithelial cells to produce metalloproteinases that can, when activated, degrade most collagens and other structural molecules in extracellular matrix. In addition, they can stimulate the contraction of extracellular matrix constituents but there is not a simple causal relationship between matrix remodeling and contraction.     

Effects of tamoxifen, toremifene and chloroquine on the lysosomal enzymes in cultured retinal pigment epithelial cells

Retinal pigment epithelial cells carry out phagocytosis and digestion of material shed from the photoreceptor outer segments. In this process, the integrity of lysosomal enzymes is of major importance. In the present study the effects of tamoxifen, toremifene and chloroquine on the activity of two lysosomal enzymes (cathepsin D and N-acetyl-beta-D-glucosaminidase) in the retinal pigment epithelial cells were studied. Retinal pigment epithelial cells from pig eyes were cultured for two weeks in Dulbecco's Modified Eagle Medium, after which the cells were exposed to 1-40 microM concentrations of tamoxifen citrate, toremifene citrate and chloroquine diphosphate. To eliminate possible medium-borne oestrogenic mechanisms, the test was repeated using phenol red-free medium with charcoal-stripped fetal calf serum. The exposure time was one week, after which the lysosomal enzymes cathepsin D and N-acetyl-beta-glucosaminidase were determined. Cellular injuries were assessed by quantifying the leakage of lactate dehydrogenase into the culture medium. Cathepsin D and N-acetyl-beta-D-glucosaminidase showed different sensitivities to tamoxifen, toremifene and chloroquine. The main lysosomal protease cathepsin D was more sensitive than N-acetyl-beta-D-glucosaminidase to the effects of tamoxifen and toremifene, possibly due to their antioestrogenic properties. The phenol red-free medium with charcoal-stripped serum seemed to make the drugs more effective than the reference medium. Chloroquine had only a minor effect on the lysosomal protease cathepsin D, but a clearer effect could be seen on N-acetyl-beta-glucosaminidase.     

The effects of dopa and oxygen on RNA concentrations in cultured chick embryonal retinal pigment epithelial cells

We measured RNA and DNA concentrations in cultured chick embryonal retinal pigment epithelial cells to investigate the effects of dopa and oxygen on DNA and RNA synthesis. RNA/DNA ratios were decreased by addition of 250 microM dopa. Decrease of RNA/DNA ratios was suppressed when the oxygen concentrations were reduced from 20% to 10%. Incubation with medium containing 100 microM dopa increased RNA/DNA ratios in 10% oxygen. Exposure of retinal pigment epithelial cells to 250 microM dopa caused the decrease of RNA concentrations in the retinal pigment epithelial cells, which was ameliorated by lowering oxygen concentrations. However, the addition of 100 microM dopa in 10% oxygen stimulated retinal pigment epithelial cells and seemed to increase RNA concentrations.     

Vitronectin is responsible for serum-stimulated uptake of rod outer segments by cultured retinal pigment epithelial cells

PURPOSE: To examine whether the vitronectin (VN) in serum is responsible for the serum stimulation of phagocytosis in the rod outer segment (ROS) by cultured retinal pigment epithelial (RPE) cells. METHODS: Vitronectin was removed from fetal bovine serum by heparin-agarose affinity chromatography. Concentrations in normal and depleted serum were determined by enzyme-linked immunosorbent assay, using a polyclonal antibody against bovine VN and commercially prepared human VN as a standard. A monoclonal antibody against human alpha v beta 5 was used in localization and in blocking experiments. Rod outer segment phagocytosis was measured using a flow cytometric assay. RESULTS: Affinity chromatography removed 95% of the VN from serum as determined by enzyme-linked immunosorbent assay. Vitronectin-depleted serum did not stimulate ROS phagocytosis by RPE cells. Commercially prepared VN added to serum-free medium stimulated ROS phagocytosis in a dose-dependent manner. Pretreatment of RPE cells with an antibody against alpha v beta 5, an integrin receptor for VN, had no effect on phagocytosis in the absence of serum but completely blocked the serum stimulation of ROS phagocytosis. Antibody against alpha v beta 5 demonstrated a variable labeling pattern on the cultured RPE cell surface with morphologically distinct cell clusters exhibiting less labeling. Those cell clusters exhibiting less receptor labeling also showed less uptake of fluorescent-labeled ROS. CONCLUSIONS: Vitronectin is the component responsible for serum stimulation of ROS uptake, and this uptake appears to be mediated by an alpha v beta 5 integrin. Although clearly important in vitro, a role for VN in ROS uptake by RPE cells in situ remains to be determined.     

Excitatory amino acid receptors in membranes from cultured human retinal pigment epithelium

The presence of specific, saturable receptor sites for excitatory amino acids (EAA) in membranes from cultured human retinal pigment epithelium (RPE) was established through the binding of [3H]L-glutamate (L-Glu). The age of the donors ranged from 6 days to 33 years. The affinity of the binding (KB) sites was between 1.2 and 1.5 microM, and did not change with the age of the donor, whereas the Bmax was slightly increased (8.6 to 13.0 pmol/mg) in membranes from the 33 year-old compared to the 29 day-old donor. The efficacy profile of agonists and antagonists acting at EAA receptors for displacing [3H]L-Glu was L-Glu = L-Aspartate > 2-amino-4-phosphonovalerate (AP5) > N-methyl-D-Aspartate (NMDA) > 1-aminocyclopentane-1,3 dicarboxylate (trans-ACPD) > 2-amino-3-phosphonopropionate (AP3). These data suggest the presence of either an NMDA-receptor sensitive to the metabotropic agonist trans-ACPD or alternatively, the presence of two different populations of receptors with similar affinity for the agonist: NMDA and metabotropic. Glycine highly stimulated Glu-binding; this effect was inversely related to the age of the donor. Taurine and to a lesser extent GABA, mimicked this effect. Stimulation by glycine was dose-dependent, insensitive to strychnine and 80% inhibited by 7-chlorokynurenate. This effect was also present in human RPE-derived fibroblasts, human scleral fibroblasts and the human lymphoblastoid cell line NB76, all continuously dividing cells. The results further support the possibility of the participation of EAA receptors in the regulation of phagocytosis in RPE.     

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.     

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.     

Transplanted and repopulated retinal pigment epithelial cells on damaged Bruch's membrane in rabbits

AIMS: The authors studied how artificially damaged Bruch's membrane influenced growth and differentiation of transplanted embryonic retinal pigment epithelial (RPE) cells and of host RPE cells in rabbits. METHODS: Embryonic RPE cells obtained from pigmented rabbits were transplanted into the subretinal space of adult albino rabbits. The host RPE was removed with a silicone cannula, and Bruch's membrane was damaged by scratching with a microhooked 27 gauge needle under the detached retina in closed vitrectomy. The transplantation sites were examined 3, 7, and 14 days after surgery by light and electron microscopy. RESULTS: Varying degrees of damage in Bruch's membrane were observed. Pigmented and hypopigmented RPE cells showed a normal polarity and tight junctions were seen at the sites of mild to moderate damage 3-7 days after the surgery. In contrast, fibroblast-like cells with no such features of RPE cells formed multiple layers at the sites of severe damage involving the full thickness of Bruch's membrane and the choriocapillaris even 14 days after the surgery. Without transplantation, host RPE cells repopulated the damaged areas in the same way as transplanted RPE cells. CONCLUSIONS: Transplanted embryonic RPE cells as well as host RPE cells grew and differentiated on the moderately damaged Bruch's membrane, while the severely damaged Bruch's membrane did not allow differentiation of RPE cells although these cells could grow and cover the damaged areas.     

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.     

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.     

Regulation of L-type calcium channels by protein tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells

The effect of protein tyrosine kinases (PTKs) on L-type calcium channel currents was studied in cultured rat and human retinal pigment epithelial cells. Barium currents through L-type channels were measured in the perforated patch-clamp technique and identified by using the L-type calcium channel opener Bay K8644 (10(-6) M). Application of the PTK blockers genistein (5 x 10(-6) M) or lavendustin A (5 x 10(-6) M) led to a decrease of L-type currents. The inactive genistein analog daidzein (10(-5) M) showed no effect on calcium channels. Intracellular application of pp60(c-src) (30 U/ml) via the patch-pipette during the conventional whole-cell configuration led to an increase of L-type currents. The protein kinase A and protein kinase G blocker H9 (10(-6) M) showed no effect on L-type currents; genistein reduced the current in the presence of H9. The protein kinase C (PKC) blocker chelerythrine (10(-5) M) reduced the L-type current; additional inhibition of PTK by lavendustin showed an additional reduction of currents. Intracellular application of myristoylated PKC substrate (5 x 10(-5) M) for PKC inhibition led to a fast rundown of L-type current amplitudes. Intracellularly applied myristoylated PKC substrate (10(-4) M) together with pp60(c-src) showed no effect on L-type current. Up-regulation of PKC by 10(-6) M phorbol-12-myristate-13-acetate (PMA) had no effect on the L-type current amplitude. However, genistein in cells pretreated with PMA led to an increase of the L-type currents. Intracellular application of pp60(c-src) in PMA-treated cells led to a reduction of L-type currents. We conclude that in the resting cell, PTK and PKC regulate L-type calcium channels in an additive manner. L-type channels appeared as a site of integration of PTK activation and of PKC-dependent pathways. The activity of PKC determines whether PTK decreases or increases L-type channel activity.     

Polarity of 11-cis retinal release from cultured retinal pigment epithelium

Moyamoya disease is characterized by bilateral stenosis and/or occlusion of the terminal portion of the internal carotid artery. Moyamoya disease is prevalent among patients <10 years of age. Although most cases appear to be sporadic, approximately 10% occur as familial cases. The incidence of familial cases has been increasing because noninvasive diagnostic equipment, such as magnetic-resonance imaging and magnetic-resonance angiography, can detect the disease in almost all affected patients, including asymptomatic patients, during screening studies. In this study, we performed a total genome search to identify the location of a familial moyamoya disease gene in 16 families, assuming an unknown mode of inheritance. A linkage was found between the disease and markers located at 3p24.2-26. A maximum NPL score of 3.46 was obtained with marker D3S3050. This is the first genetic locus found to be involved in the molecular pathogenesis of familial moyamoya disease.     

PEDF (pigment epithelium-derived factor) promotes increase and maturation of pigment granules in pigment epithelial cells in neonatal albino rat retinal cultures

AIMS: To clarify the association of p53 and CD34 expression with development of malignant solitary fibrous tumour we have studied 10 cases of solitary fibrous tumour arising in the pleura, retroperitoneum and pelvic cavity with clinicopathological features of malignancy. METHODS AND RESULTS: Tumours were localized solid masses with or without necrosis in eight and they nearly totally occupied the pleural cavity in two. Basic histology of the tumours was the proliferation of spindle cells arranged in 'patternless' pattern or in interlacing bundles with nuclear atypia and mitotic activities of various degree. In two, high-grade foci were present within low or intermediate-grade tumours. Recurrent tumours also showed more atypical features than primary tumours in two. Immunohistochemical studies showed CD34 positivity in seven, but three of them showed marked diminution or complete loss of CD34 expression in high-grade foci or a recurrent tumour. Three high-grade cases showed totally negative staining for CD34. p53 was strongly expressed in cases with fatal outcome, clinical recurrence, nuclear atypia, high mitotic activity or local invasion, whereas almost negative in benign tumours. CONCLUSIONS: Malignant solitary fibrous tumours may occur de novo or by transformation within benign or low-grade tumours and may be associated with p53 mutation. Although CD34 is a useful marker in the diagnosis of solitary fibrous tumour, one should bear in mind that its expression can be lost in high-grade tumours.