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Premelanosomes are presumed to be essential for melanogenesis in melanocytes and pre-natal retinal pigment epithelium (RPE) cells. We analysed melanin synthesis in adenoviral-transduced tyrosinase-gene-expressing amelanotic RPE (ARPE) 19 cells to determine whether premelanosome formation is needed for post-natal melanogenesis. The synthesis of melanogenic proteins and melanin granules was investigated by immunocytochemistry and light and electron microscopy. The occurrence of tyrosinase was analysed ultrastructurally by dihydroxyphenylalanine histochemistry. The viability of transduced cell cultures was examined via MTT assay. We found active tyrosinase in small granule-like vesicles throughout the cytoplasm and in the endoplasmic reticulum and nuclear membrane. Tyrosinase was also associated with multi-vesicular and multi-lamellar organelles. Typical premelanosomes, structural protein PMEL17, tyrosinase-related protein 1 and classic melanosomal stages I-IV were not detected. Instead, melanogenesis took place inside multi-vesicular and multi-lamellar bodies of unknown origin. Viability was not affected up to 10 days after transduction. We thus demonstrate a pathway of melanin formation lacking typical hallmarks of melanogenesis.

Purpose: To characterise ocular pigment abnormalities associated with iris atrophy in DBA/2J mice as a model for human pigment dispersion syndrome., Methods: Immunohistochemistry, electron and light microscopy were performed to examine the eyes of DBA/2J mice ranging in age from 2.5 to 18 months old. The focus of our study was the description of the ultrastructural modifications in the irides of DBA/2J mice., Results: The DBA/2J mice presented modifications in the melanosomes in all the pigmented parts of the eye, including the retinal pigment epithelial cells and choroidal melanocytes of the ciliary pigment epithelium. The extracellular matrix of the iris stroma disappeared with ageing. Pigmented cells detached from the iris and migrated into the trabecular meshwork exclusively on the anterior iris surface. These cells were identified as macrophages by immunohistochemistry and electron microscopy. There was no evidence that melanocytes or iris pigment epithelial cells migrated into the trabecular meshwork, but they became more and more depigmented. The aqueous outflow was blocked by pigment-laden cells, but not by cellular debris or melanosomes. No substantial amount of extracellular melanosomes was observed., Conclusion: The morphology of melanosomes is aberrant in all pigment cells in the eyes of DBA/2J mice. We conclude that the disease process begins with the transfer of both immature melanosomes from the iris pigment epithelium (IPE) and melanocytes to macrophages, which subsequently migrate into the trabecular meshwork. Accumulating macrophages cause a blockade of the chamber angle. As the disease progresses, the IPE, melanocytes and iris stroma, including blood vessels, disappear, leading to iris atrophy. It is speculated that the loss of these pigment cells is partly caused by reduction of the iris stroma.

To investigate the effects of zinc supplementation on human amelanotic (ARPE-19) and native pigmented retinal pigment epithelial cells (hRPE) under normal light conditions and after ultraviolet A light exposure. hRPE cells, containing both melanin and lipofuscin granules, were prepared from human donor eyes of 60-70 year old patients. Cells of the amelanotic ARPE-19 cell line and pigmented hRPE cells were treated with zinc chloride and subjected to oxidative stress by UV-A irradiation. Intracellular H(2)O(2) formation was measured using a fluorescence oxidation assay. Additionally, apoptosis and viability assays were performed. Control cells were treated identically except for irradiation and zinc supplementation. Under normal light conditions, zinc treated hRPE cells produced less H(2)O(2) than unsupplemented hRPE cells. Viability and apoptosis events did not change. After UV-A irradiation, ARPE and hRPE cells were greatly impaired in all tests performed compared to the non-irradiated controls. No differences were found after zinc supplementation. hRPE cells showed a higher apoptosis and mortality rate than non-pigmented cells when stressed by UV-A light. ARPE cells never showed any zinc related effects. In contrast, without irradiation, zinc supplementation reduced H(2)O(2) production in pigmented hRPE cells slightly. We did not find any zinc effect in irradiated hRPE cells. After UV light exposure, pigmented cells showed a higher apoptosis and mortality than cells lacking any pigmentation. We conclude that cells with pigmentation consisting of melanin and lipofuscin granules have more prooxidative than antioxidative capacity when stressed by UV light exposure compared to cells lacking any pigmentation.

Aim: To compare the antiproliferative and cytotoxic properties of bevacizumab (Avastin), pegaptanib (Macugen) and ranibizumab (Lucentis) on human retinal pigment epithelium (ARPE19) cells, rat retinal ganglion cells (RGC5) and pig choroidal endothelial cells (CEC)., Methods: Monolayer cultures of ARPE19, RGC5 and CEC were used. Bevacizumab (0.1-0.3 mg/ml), pegaptanib (0.025-0.08 mg/ml) or ranibizumab (0.04-0.125 mg/ml) diluted in culture medium were added to the cells. Expression of VEGF-receptors (VEGFR1 and VEGFR2) and von Willebrand factor (a marker for endothelial cells) were analysed by immunohistochemistry. CEC cells were stimulated with VEGF. Cellular proliferative activity was monitored by BrdU-incorporation into cellular DNA. For cytotoxicity assays cells were grown to confluence and then cultured in a serum-depleted medium to ensure a static milieu. MTT-test was performed after one day., Results: CEC and ARPE19 cells stained positively for VEGFR1 and VEGFR2. More than 95% of the CEC cells were positive for von Willebrand factor. Ranibizumab reduced CEC cell proliferation by 44.1%, bevacizumab by 38.2% and pegaptanib by 35.1% when the drugs were used at their established clinical doses. The differences, however, between the three drugs in respect to cell growth inhibition were not statistically significant. Only a mild antiproliferative effect of bevacizumab or pegaptanib on ARPE19 cells could be observed. Ranibizumab did not alter ARPE19 cell proliferation. No cytotoxicity on RGC5, CEC and ARPE19 cells could be seen., Conclusions: Bevacizumab, pegaptanib and ranibizumab significantly suppress choroidal endothelial cell proliferation. However, when used at the currently established doses none of the drugs was superior over the others in respect to endothelial cell growth inhibition. The biocompatibility of all three drugs--including the off-label bevacizumab--seems to be excellent when used at the currently recommended intravitreal dose.

Background: Tyrosinase (EC 1.14.18.1) is the key enzyme of melanin pigment formation and it is unclear whether it is synthesized in human postnatal retinal pigment epithelium (RPE). In this study, we investigated if phagocytosis of rod outer segments (ROS) can increase tyrosinase expression in vitro., Methods: Primary cultures of human RPE cells were fed with isolated ROS from cattle and with latex particles. After phagocytosis, RPE cells were tested for tyrosinase presence and activity with several independent methods: (1) immunocytochemistry with anti-tyrosinase antibodies and (2) ultrastructural as well as light microscopic DOPA histochemistry; (3) mRNA was isolated from human RPE before incubation with ROS and 5, 20 and 40 h after feeding with ROS. The amount of tyrosinase mRNA was determined quantitatively by real-time reverse transcription polymerase chain reaction (RT-PCR), and the tyrosinase activity was investigated by measuring tyrosine hydroxylase activity using [(3)H]tyrosine., Results: Tyrosinase was found in fed RPE cells using these methods, but was absent without feeding. Furthermore, we showed co-localization of rhodopsin and tyrosinase in the fed RPE cells. Contrary to tyrosinase activity, the mRNA for tyrosinase was clearly present in the cultured RPE cells which had not been exposed to ROS, decreased significantly from 5 h after exposure to ROS and returned to its original non-fed level 40 h after ROS feeding., Conclusion: Our study does not present new evidence that de novo melanogenesis takes place in the adult differentiated RPE. However, in contrast to the classic hypothesis, which states that tyrosinase is only detected in embryos, we provide evidence with several independent methods that the expression of tyrosinase and its enzymatic activity are induced in cultured human adult RPE by phagocytosis of ROS.

Many medications that are administered systemically for nonocular conditions may evoke ocular toxicological complications. Therefore, the eye is routinely investigated histopathologically in preclinical in vivo toxicity studies. The retinal pigment epithelium is a likely target for systemically administered compounds, since the underlying choroid is highly vascularized. The specialized pigment epithelium has numerous functions that all maintain the integrity and function of photoreceptors. Consequently, toxic effects on the pigment epithelium will eventually affect the neural retina. The potential of pigment epithelial cells to respond to toxic injury is limited, but a standardized terminology to describe its morphological changes does not exist in the scientific literature. Detailed morphologic analysis, however, might allow early detection of retinotoxicity and may provide evidence on the underlying pathomechanism. We here review toxic effects on the pigment epithelium focusing in particular on the morphology of toxic cell injury. Morphological changes comprise hypertrophy, intracytoplasmic accumulation of cellular components, loss of cell polarity, degeneration, metaplasia, and formation of subretinal membranes. Some of these changes are reversible whereas others are permanent, leading to impaired function of the pigment epithelium and eventually to photoreceptor loss and retinal atrophy.

Purpose: To determine the potential of adenovirally transduced bone marrow stromal cells (BMSCs) to differentiate into retinal pigment epithelial-like cells and to evaluabe possible rescue effects after transplantation into the retinas of Royal College of Surgeons (RCS) rats., Methods: Through a high-capacity adenoviral vector expressing either green fluorescent protein (GFP) or pigment epithelial-derived factor (PEDF), rat MSCs were transduced in vitro before subretinal transplantation into Wistar rats or, alternatively, RCS rats. Two months after cell injection, the rats were killed and the eyes enucleated. The eyes were then investigated light microscopically or processed for electron microscopic investigations. Cell differentiation and integration were analyzed immunocytochemically using antibodies against cytokeratin and the tight junction protein ZO-1. Electroretinography was performed 16 days after injection of cells, to check whether a functional rescue could be detected., Results: In vitro experiments in cocultured human MSCs and human RPE cells showed that MSCs adopted RPE-like characteristics. In grafting experiments, some rat MSCs integrate into the host RPE cell layer of Wistar and RCS rats, indicated by their hexagonal morphology. Subretinally transplanted cells express the epithelial marker cytokeratin and establish tight junctions with the host RPE cells. Furthermore, rescue effects can be demonstrated after grafting of vector-transduced and nontransduced MSCs in semithin sections of dystrophic retinas. Ultrastructurally, MSCs can be detected on top of host RPE and in close contact with photoreceptor outer segments phagocytosing rod outer segments., Conclusions: Taken together, these results raise the possibility that MSCs have the potency to replace diseased RPE cells and deliver therapeutic proteins into the subretinal space to protect photoreceptor cells from degeneration.

Tyrosinase (EC 1.14.18.1) is the rate limiting enzyme of melanogenesis and it is unclear whether it is synthesized in postnatal retinal pigment epithelium (RPE). Cultured RPE cells from cattle were fed with isolated rod outer segments (ROS). After phagocytosis, RPE cells were tested for tyrosinase presence and activity with three independent methods: (1) ultrastructural DOPA (l-3,4-dihydroxyphenylalanine) histochemistry (2) immunocytochemistry with anti-tyrosinase antibodies (3) measuring tyrosine hydroxylase activity using [(3)H]tyrosine. With all three methods tyrosinase was found in RPE cells after ROS-feeding but was absent without feeding. In contrast to the classical hypothesis, we demonstrated with three independent methods that the expression of tyrosinase and its enzymatic activity are induced in cultured adult RPE by phagocytosis of rod outer segments (ROS) in vitro.

Purpose: To analyze chloroquine-induced morphological changes in the retinal pigment epithelium (RPE) and Bruch's membrane (BM)., Methods: Retina-choroid complexes of chloroquine-treated Long-Evans rats were analyzed by electron microscopy., Results: Intercellular spaces between the RPE cells and BM were enlarged. Residual material from phagosomes was released into these enlarged spaces. Debris accumulated within BM and encircled choriocapillaris endothelial cells., Conclusion: There is a release of undegraded phagocytic material (rod outer segments) into the extracellular space between BM and RPE cells, following inhibition of lysosomal degradation. Electron-dense deposits in BM and choriocapillaris may lead to reduced oxygen and nutrition flow.

Background: Age-related macular degeneration (AMD) is associated with lower melanin pigmentation and is more prevalent among the elderly Caucasian population than among Africans. A correlation between light iris colour, fundus pigmentation and the incidence of AMD is reported. Moreover, melanin represents the main storage of zinc in the eye. Zinc enhances antioxidant capacity through its function as a cofactor of important enzymes or by influencing gene expression of regulatory elements in the eye. In this study, we investigated the uptake and storage of zinc in the human choroid/retinal pigment epithelium (RPE) complexes in dependence on the fundus pigmentation as judged by the iris colour., Material and Methods: Choroid/RPE complexes of blue and brown human eyes were used. Tissues without any substitution served as controls. Specimens from choroid/RPE complexes were incubated with 100 microM zinc chloride for 24 h. After incubation, pieces of the complexes were stored to investigate the uptake of zinc. The rest of the tissues were kept for 3 and 7 days in culture medium (DMEM) for storage examination. The concentration of zinc was measured by inductively coupled plasma mass spectrometry., Results: After 24 h of zinc treatment the concentration of zinc in the choroid/RPE complexes of blue eyes was not significantly increased. The concentration of zinc in highly pigmented tissues (brown eyes) was increased by the factor 5.1 after 24 h and remained at high levels after 3 days (factor 4.4) and 7 days (factor 2.8)., Conclusions: Zinc uptake in the choroid/RPE complex correlates to the iris colour. Alterations of the degree of iris pigmentation result in differences of zinc uptake and storage in the choroids. A potential protective role of zinc may be more prominent in dark- than in light-coloured eyes.

For the treatment of neurodegenerative disorders such as Parkinson's disease cell or gene therapeutical options are increasingly verified. For such approaches, neural stem cells or astrocytes are discussed as possible cell candidates. As also fetal retinal pigment epithelial cells have been successfully tested for such therapeutical options, we investigated the potential of iris pigment epithelial cells as an autologous source for future cell replacement therapies. Using the ELISA technique, we looked for the secretion of neurotrophic factors under basal and stimulated conditions by iris pigment epithelial cells (IPE) cells and compared them with the secretion of retinal pigment epithelial cells (RPE) cells. As iron plays a causative role in cell death during Parkinson's disease, the iron-binding capacity by IPE cells was investigated. Furthermore, we checked the integrative capacity of IPE cells after transplantation into the striatum of adult rats. Our data reveal that IPE cells produce and secrete a variety of neurotrophic factors which can be stimulated after treatment with cytokines. Following transplantation, the cells can be easily detected by their pigmentation, survive for at least 8 weeks and as shown by electron microscopy integrate within the host tissue. Moreover, cells can be transduced with high efficiency using a third generation adenoviral vector, making them promising vehicles to locally deliver therapeutic proteins for the treatment of neurodegenerative diseases in a combined cell and gene therapeutical approach.

Age-related macular degeneration (ARMD) is the leading cause for visual impairment and blindness in the elder population. Laser photocoagulation, photodynamic therapy and excision of neovascular membranes have met with limited success. Submacular transplantation of autologous iris pigment epithelial (IPE) cells has been proposed to replace the damaged retinal pigment epithelium following surgical removal of the membranes. We tested our hypothesis that the subretinal transplantation of genetically modified autologous IPE cells expressing biological therapeutics might be a promising strategy for the treatment of ARMD and other retinal disorders. Pigment epithelium-derived factor (PEDF) has strong antiangiogenic and neuroprotective activities in the eye. Subretinal transplantation of PEDF expressing IPE cells inhibited pathological choroidal neovascularization in rat models of laser-induced rupture of Bruch's membrane and of oxygen induced ischemic retinopathy. PEDF expressing IPE transplants also increased the survival and preserved rhodopsin expression of photoreceptor cells in the RCS rat, a model of retinal degeneration. These findings suggest a promising concept for the treatment of ARMD and other retinal disorders.

Purpose: To analyze the differential gene expression in cultured human retinal pigment epithelial (RPE) cells after treatment with vitreous., Methods: Cultured human RPE cells were incubated for 48 hours with 25% human vitreous from donor eyes. Total RNA from treated and untreated cells was extracted. The gene expression was analyzed by differential expression analysis (DEmRNA-PCR). The differentially expressed genes were identified by gene bank searches. Differential expression was verified by a quantitative real-time RT-PCR fluorescent nucleic acid staining system. The in vivo mRNA expression of these genes in RPE cells was shown by gene-specific RT-PCR., Results: Vitreous treatment of human RPE cells resulted in the reduced expression of NFIB2, KE03 (NY-REN-25ag), PIG-B, DKFZp564BC462, LKHA, G3BP, PAM, UEV-1, and MAP1B calibrated to the expression of GAPDH when compared with their expression in untreated cells. The reduced expression after vitreous treatment was quantified by gene-specific quantitative real-time RT-PCR and varied from 0.69 to 0.17 compared with untreated cells. The mRNA expression of UDP-GalNac mRNA remained constant. The mRNA expression of eight of these genes was demonstrated in this study for the first time in human RPE cells., Conclusions: Vitreous treatment of cultured RPE cells induces the differential expression of a variety of genes with functions in transcription, mediation of signal transduction and inflammation, glycosylation, ubiquitination and protein-protein interaction. Further examination of these genes may locate additional targets for treatment of diseases caused by contact of RPE cells with vitreous, typical in proliferative vitreoretinopathy.

Purpose: To analyze duration of gene expression in the retinal pigment epithelium (RPE) in immunocompetent animals after gene transfer with a high-capacity adenoviral (HC-Ad) vector., Methods: An HC-Ad vector was constructed to express the enhanced green fluorescence protein (EGFP) from the human CMV promoter. This vector (HC-AdFK7) was used to transduce rat RPE cells in cell culture and after subretinal injection in vivo in adult immunocompetent Wistar rats. In cell culture, expression of EGFP was analyzed by fluorescence microscopy. In vivo expression was monitored by scanning laser ophthalmoscopy and stereo fluorescence microscopy. After enucleation of the eyes, immunohistochemical and morphologic analyses by fluorescence light microscopy and electron microscopy were performed., Results: In vitro, RPE cells were efficiently transduced with HC-AdFK7. Expression persisted for the observation time of 8 weeks. In vivo, the RPE was efficiently transduced with low doses of HC-AdFK7. EGFP synthesis was confirmed by antibody staining and found to be stable for the complete study period of 6 months. The neuroretina was well preserved over areas of subretinal vector administration, and significant morphologic changes were not detected. There was no accumulation of inflammatory T cells or macrophages., Conclusions: In contrast to previous results with earlier generation adenoviral vectors, subretinal injection of an HC-Ad vector in immunocompetent rats resulted in long-term transgene expression without evidence of adverse immune reactions or significant toxicity, probably because of the absence of expression of the viral gene from this vector. Thus, HC-Ad vectors are suitable for the treatment of eye disorders that require durable gene expression in the RPE.

Background: Iris pigment epithelial (IPE) cells have mainly been investigated in the past for their proposed potential to rescue or even replace degenerated retinal pigment epithelial (RPE) cells after subretinal transplantation in patients with age-related macular degeneration (AMD). More recent reports have characterised the IPE cell as a potent source of trophic factors and cytokines. In our study we investigated the spatial distribution of IPE cells that were injected into the vitreous instead of being injected subretinally., Methods: IPE cells from Long Evans rats were isolated and injected into the vitreous cavity of Wistar rats without preculturing. Free melanin granules were injected into the vitreous in the same manner. After a period of 2 months, eyes were prepared for histological analysis. Localisation of the injected IPE cells was defined by topographical mapping of the analysed sections., Results: PVR was not observed in any eye. In 8 of 10 injected eyes, IPE cells had accumulated in the prepapillary region. In 2 of 10 eyes, no IPE cells could be detected. The injected melanin granules also accumulated at the optic nerve head, indicating that this is most likely a passive process. In sections of the papillary region containing retinal vessels, the IPE cells seemed to have migrated into the superficial tissue of the optic nerve head., Conclusion: Our results demonstrate a way to access the optic nerve head easily and securely without the danger of damaging its fragile structure. This could have important implications for new therapeutic strategies in ocular neurodegenerative diseases like glaucoma. New prospects in gene therapy will require further characterisation of the potential of the IPE cell to produce neuroprotective trophic factors at the optic nerve head.

Melanin granules in the retinal pigment epithelium (RPE) have many important functions which are not yet completely understood. Melanin in the RPE protects the cell from damage caused by oxidative stress. This pigment acts as a free radical sink and diminishes cytotoxic lipid peroxidation. Thus, melanin protects against light toxicity and against cytotoxic effects caused by ocular inflammation. Many enzymes, e.g. superoxide dismutase or carboanhydrase, are only activated in the presence of zinc. Melanin can store zinc and release it when required. The absence of melanin in patients with oculocutaneous albinism is accompanied by photophobia, poor visual acuity and nystagmus. Furthermore melanin is said to protect against damaging lipofuscin accumulation in the RPE. Melanosomes are involved in the lysosomal degradation pathways and possibly take part in the degradation of rod outer segments (ROS) in the RPE.

Purpose: The goal of this study is the characterization of the strong yellow fluorescence of oxidized melanin in the retinal pigment epithelium (RPE) and the choroid., Methods: Naturally occurring melanin in the human retina and choroid was oxidized by exposing fixed and plastic-embedded sections of a human eye to light and hydrogen peroxide. Synthetic melanin was also oxidized in vitro by exposure to light and hydrogen peroxide. The fluorescence of oxidized melanin was examined by absorption spectroscopy, fluorescence spectroscopy, and fluorescence microscopy., Results: Naturally occurring melanin oxidized in situ exhibited a lipofuscin-like yellow fluorescence. Oxidation of melanin in vitro degraded the melanin polymer, resulting in a fluorescent solution. Fluorescence spectroscopy gave an excitation maximum at approximately 470 nm and an emission maximum at approximately 540 nm for both natural and synthetic melanin. Increasing the time of exposure to light or hydrogen peroxide increased melanin fluorescence., Conclusions: The results indicate that the strong yellow fluorescence of melanin in the RPE and choroid in situ is a property of oxidized melanin and is not due to contamination of the melanin by proteinaceous or lipid materials. The data presented allow a reinterpretation of the results obtained from fluorescence investigations of melanin-containing tissue and suggest a link between melanin degradation and lipofuscin formation.

Background: Trophic factors [e.g. basic fibroblast growth factor (bFGF)] released by transplanted retinal pigment epithelial (RPE) cells are able to slow down the hereditary degeneration of the retina in the Royal College of Surgeons rat in sites distant from the site of transplantation where rod outer segment (ROS) phagocytic activity is not reconstituted by the transplants., Methods: To investigate whether iris pigmented epithelial (IPE) cells are also able to generate this rescue by trophic factors, we transplanted IPE cells from Long-Evans rats into the choroid and subretinal space of 17 young RCS rats. The eyes were enucleated after 6 months and prepared for light microscopy. Six age-matched RCS rats served as controls. Light microscope sections from the whole choroid, healthy choriocapillaris, transplanted cells and the maximum thickness of the choroid, and outer nuclear layer parameters were analyzed by computer-assisted morphometry., Results: In transplanted animals photoreceptor cells were rescued from degeneration although the majority of the transplanted IPE cells were located in the choroid. In the non-transplanted group photoreceptors were absent., Conclusions: Transplantation of IPE cells slows down degeneration of the photoreceptors in the RCS rat. This photoreceptor-sparing effect by the IPE cells was observed even when the transplants were predominantly located within the choroid. The beneficial effect observed may be related to trophic factors possibly secreted by the transplanted IPE cells.

Background: Transplantation of autologous iris pigment epithelium (IPE) into the subretinal space has been suggested as one approach for the treatment of age-related macular degeneration, as well as for other conditions in which loss of retinal pigment epithelium (RPE) occurs. Surgical removal of choroidal neovascular membranes is associated with traumatic loss of the RPE cell layer, disruption of the integrity of the photoreceptor-RPE complex, and limited visual outcome., Objective: To examine whether IPE cells can substitute for RPE cells to be transplanted to the subretinal space of patients with either RPE degenerative disease or traumatic loss of the RPE cell layer after subretinal surgery., Methods: Autologous IPE cells were transplanted to the subretinal space in 20 consecutive patients undergoing removal of subretinal fibrovascular membranes using pars plana vitrectomy. Autologous IPE cells were harvested by iridectomy, isolated, and transplanted directly to the subretinal spaces. Transplants were evaluated for 6 to 11 months by funduscopy, fluorescein angiography, and scanning laser ophthalmoscopic (SLO) microperimetry., Results: For the entire follow-up period, no evidence of any immunologic response was observed. Revisional surgery was necessary in 3 patients because of complications (rhegmatogenous retinal detachment [n = 1]; proliferative vitreoretinopathy [n = 1]; and macular pucker [n = 1]); 1 patient did not receive IPE cells. Five of 19 phakic eyes underwent cataract surgery; in 1 case this was combined with the vitrectomy. Five patients showed improved visual acuity of 3 to 4 lines, 13 patients had stable visual acuity (+/-2 lines), and 2 patients had reduced visual acuity of 6 lines., Conclusions: In this pilot study, the transplantation of autologous IPE cells was done as an addition to conventional surgical excision of choroidal neovascular membranes. Transplanted cells were well tolerated in the subretinal space and did not adversely affect the function of the photoreceptors, since improvement or stable visual acuity was observed in 18 patients after IPE transplantation. These results suggest that autologous IPE cells may be used as a substitute for autologous RPE cells to transplant to the subretinal space to treat age-related macular degeneration.

Our study was performed to test the hypothesis that subretinally injected protein can induce melanogenesis in the retinal pigment epithelium (RPE). Rod outer segments (ROS) were isolated from cattle eyes and injected into the subretinal space of Long Evans rats. Five days after surgery, the injected eyes were investigated by electron microscopy. The number of early-stage melanosomes and small melanin granules was compared in injected and noninjected eyes. It was found that the injected ROS were phagocytized by the RPE cells, and the number of early-stage melanosomes in the RPE was significantly increased in injected eyes compared to eyes without injection. The ROS-containing endosomes fused with melanolysosomes in which melanogenesis took place. The increased number of early-stage melanosomes indicates new formation of melanin., (Copyright 2000 S. Karger AG, Basel)

Background: To investigate in iris pigment epithelium (IPE) the expression of mRNA for proteins involved in retinol metabolism we used a semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) technique., Methods: RNA was prepared from freshly isolated bovine IPE and retinal pigment epithelium (RPE) cells and reverse transcribed. The expression of mRNA for cellular retinaldehyde binding protein (CRALBP), p63 (RPE63), the presumed retinal pigment epithelial membrane receptor for retinoids, and 11-cis-dehydrogenase (11cisRDH) was determined by RT-PCR using specific primers. Semi-quantitative expression data were obtained by using a series of fivefold dilution of each cDNA with a fixed number of PCR cycles., Results: Bovine IPE and RPE cells express mRNA for CRALBP, 11cisRDH, and RPE63. The mRNA expression for CRALBP and 11cisRDH is high and equal in both cell types. However, RPE63 mRNA expression in IPE cells is relatively low compared with the expression in RPE cells., Conclusions: The presence of mRNA for CRALBP, RPE63, and 11cisRDH suggests that IPE cells may be able to metabolize retinol.

Background: The fate of indigestible material injected into the subretinal space of rats was investigated., Methods: The non-toxic dye Monastral Blue (MB), which cannot be digested within the lysosomal compartment, was injected transsclerally into the subretinal space of Long Evans and Wistar rats. After 5 and 12 days respectively the eyes were enucleated and examined by light and electron microscopy. Cryo sections were made of eyes 5 days after MB injection for the application of immunohistochemical techniques using markers for epithelial cells (cytokeratin) and macrophages (ED 1)., Results: Retina, choroid and sclera were not altered in their morphology in the circumference of the MB-containing bubble generated by subretinal injection. After both 5 and 12 days no injected material was found extracellularly in the subretinal space. Especially high amounts of MB were found, in particular 5 days after injection, in lysosomes and melanosomes of RPE cells as well as in cells between choroidal melanocytes. Cells containing MB were seen in contact with choroidal and scleral blood vessels. These MB-containing cells in the choroid and in the sclera were positive for macrophage antibodies., Conclusion: Subretinal injection was confirmed as a suitable method for placing fluids into the subretinal space without affecting the morphology of the retina. Subretinal injected material was shown to be incorporated into lysosomes and melanosomes of RPE cells. The injected material was subsequently transported through Bruch's membrane to be finally removed from the eye via choroidal and scleral veins, the process involving macrophages.

Background: The aim of this study was to compare the degradation of rod outer segments (ROS) in porcine iris pigment epithelial cells (IPE) and retinal pigment epithelial (RPE) cells by measuring the increase of lipofuscin-like fluorescence., Methods: We measured the development of autofluorescence of lipofuscin-like material in living cells over a period of 4 weeks using an image-analyzing system comprising a light microscope, a filter set with an appropriate wavelength for the detection of lipofuscin-like autofluorescence and a silicon-intensified target camera connected to a computer. The lipofuscin-like fluorescence was quantified as the mean gray value of pixels over a defined area in the cell. In addition, ultrastructural examination of the cells was performed using transmission electron microscopy., Results: We found that while both cell types had increased autofluorescence over time, the increase of lipofuscin-like fluorescence was significantly higher in IPE cells than in RPE cells. The ultrastructure of both cell types was similar and no accumulation of lipofuscin-like granules was observed., Conclusion: These findings suggest that although IPE cells are able to phagocytize ROS, their ability to degrade them may be lower than in RPE cells. The increase of lipofuscin-like fluorescence is not due to the accumulation of lipofuscin-like granules.

Background: A new method for the ultrastructural localization of lipid peroxides as benzidine-reactive substances (BRS) was recently developed in our laboratory. The aim of the present study was to localize BRS in the eye after intense light exposure. The light protocol was chosen to either hamper disc shedding or to induce a shedding peak., Methods: Long-Evans rats were either kept under constant irradiation to enhance lipid peroxidation or under physiological light conditions. The light-induced peroxidation was carried out either by constant irradiation for 24 h or by constant irradiation for 20 h followed by a dark period of 4 h. The eye cups were fixed by glutaraldehyde, incubated with or without tetramethylbenzidine and embedded for electron microscopy., Results: After constant irradiation for 24 h smooth nonlamellar BRS appear exclusively intracellularly over the complete rod outer segments (ROS). After the initiation of disc shedding smooth BRS are localized in the extracellular space of the ROS and extracellularly in the basal labyrinth of the retinal pigment epithelium (RPE). Fine-lamellar BRS emerge in vacuoles of the RPE, in the basal labyrinth and in the lumen of choroidal capillaries., Conclusion: Light conditions that trigger the disc shedding possibly activate a mechanism to extrude peroxidative damaged material over the complete ROS into the extracellular space to diminish peroxidative damage inside the ROS. Indigestible residual material from the ROS degradation in the phagosomes consists of membranous lipids associated with peroxidative damaged proteins. The residual material seems to be transported through Bruch's membrane into the choriocapillaris.

Cultured human retinal pigment epithelial (RPE) cells of different passages (P0 and P3) were used as a model system to examine changes in gene expression in proliferating RPE cells by polymerase chain reaction (PCR)-based differential expressed mRNA analysis (DEmRNA-PCR). DEmRNA-PCR showed enhanced expression of a specific RNA in P3 compared with P0. Sequence alignment displayed its identity with the 3'-end of the coding sequence of the human RAS-GTPase activating protein (GAP)-binding protein (G3BP). Confirmation of the induced expression of G3BP was performed by gene-specific reverse transcription-polymerase chain reaction (RT-PCR) of freshly prepared human RPE cells and of cultured cells of P0, P3 and P8 and by immunohistochemistry of cultivated retinal pigment epithelial cells in an artificial lesion assay. The expression of G3BP mRNA increased with the number of passages. G3BP protein expression increased in cells repopulating the artificial lesion. DEmRNA-PCR in RPE cells with subsequent sequence analysis led to the characterization of dedifferentiation- and proliferation-dependent expression of a previously undetected gene product in cultured RPE cells with a possible role in modifying signal transduction responses that may have implications on the treatment of proliferative vitreoretinopathy.

Retinal pigment epithelium (RPE) is a monolayer of cuboidal cells that is strategically placed between the rod and cone photoreceptors and the vascular bed of the choriocapillaris. It has many important functions, such as phagocytic uptake and breakdown of the shedded photoreceptor membranes, uptake, processing, transport and release of vitamin A (retinol), setting up the ion gradients within the interphotoreceptor matrix, building up the blood-retina barrier, and providing all transport from blood to the retina and back. This short review focuses on the role of the pigment granules in RPE. Although the biology of the pigment granules has been neglected in the past, they do seem to be involved in many important functions, such as protection from oxidative stress, detoxification of peroxides, and binding of zinc and drugs, and, therefore, serve as a versatile partner of the RPE cell. Melanin plays a role in the development of the fovea and routing of optic nerves. New findings show that the melanin granules are connected to the lysosomal degradation pathway. Most of these functions are not yet understood. Deficit of melanin pigment is associated with age-related macula degeneration, the leading cause of blindness.

Background: Transplantation of autologous iris pigment epithelium (IPE) into the subretinal space has been suggested as one approach for the treatment of age-related macular degeneration. Autologous rabbit IPE cells were transplanted to the subretinal space to define the technique of transplantation and examine the survival of the transplanted cells., Methods: Autologous IPE cells were harvested by iridectomy and transplanted directly to the subretinal space of the fellow eye in 25 rabbits, using the parsplana approach. Animals were killed over a period of 5 months, and the retinas were examined morphologically by light and electron microscopy., Results: Autologous IPE cells survived and formed a polarized monolayer above the retinal pigment epithelium in the subretinal space, with apical microvilli adjacent to photoreceptors. Fragments of phagocytosed photoreceptor rod outer segments were observed in phagosomes in the cytoplasm of IPE cells. Adjacent rod outer segments remained healthy throughout the experimental period. No signs of a cell-mediated immunologic response were observed., Conclusions: Our results show that in rabbits, autologous IPE cells transplanted to the subretinal space survive and do not adversely affect the photoreceptors. These results suggest that in humans, IPE cells might provide a substitute for retinal pigment epithelium cells as autologous transplants for the treatment of age-related macular degeneration.

Purpose: The aim of this study was to investigate the possible rescue effect of subretinal iris pigment epithelial (IPE) cell transplantation in Royal College of Surgeons (RCS) rats by light and electron microscopic histology., Methods: IPE cells were harvested from 20- to 26-day-old Long-Evans rats and were directly trans planted transsclerally into the subretinal space of 32 16- to 20-day-old RCS rats using a 32-gauge Hamilton syringe. Specimens of transplanted eyes were embedded for electron microscopy after 8 weeks. Specimens from the iris and retinal pigment epithelium (RPE) of Long-Evans rats and RPE from RCS rats without surgical treatment were also embedded. Sham surgery was also performed in 8 eyes., Results: The IPE cells transplanted into the subretinal space were localized between host RPE and retina, had round cell shapes without polar organization, and contained phagosomes resulting from rod outer segment (ROS) uptake. The underlying host RPE cells were heavily pigmented. RPE cells from RCS rats revealed fragmentation of endoplasmic reticulum, which distinguishes them ultrastructurally from pigment epithelial cells of Long-Evans rats. Ultrastructural alterations were observed in the cytoplasm of transplanted cells. Melanin granules in the IPE cells were found in large vacuoles, which also contained phagosomes originating from ROS uptake. In 13 eyes, 1 to 4 rows and 5 to 8 rows of saved photoreceptors were detected facing transplanted IPE cells in 6 (46%) and 4 (31%) eyes, respectively, 2 months after surgery. However, in 10 (53%) and 7 (37%) of 19 eyes, 1 to 4 rows and 5 to 8 rows, respectively, were also found at sites without IPE cells in the plane of section. ROS directed toward transplanted IPE cells were seen in one case, but these rods were shortened and disorganized. At most sites between transplanted cells and inner segments of photoreceptors, outer segments and cellular debris were absent. In eyes without transplanted cells no photoreceptor cells were alive at the age of 2 months. After sham surgery 6 (75%) eyes had 1 to 4 rows and 2 (25%) 5 to 8 rows of photoreceptors., Conclusions: Transplanted IPE cells can take up and degrade ROS in vivo in RCS rats. Uptake of ROS alters the morphology of pigment granules in transplanted IPE cells. Pigmentation is an uncertain marker for identifying transplanted pigment cells. IPE transplants are not as good as RPE transplants in rescuing photoreceptors. However, there is a significant difference between transplanted eyes and nontreated eyes. The rescue effect of IPE cells was not significantly different from that of sham surgery.

The mechanism of the toxicity of light on the retina remains unclear despite a large number of investigations. The purpose of this study is to identify and localize the ultrastructural changes and the site of the earliest damage after intense light exposure. Nine adult Syrian golden hamsters (Mesocricetus auratus) have been maintained under constant illumination with a high-pressure mercury lamp (HQJ R 80 W Deluxe, Osram, Berlin, light intensity 1000 lx) for 12 h, followed by an additional 3 h in the dark. Light damage is assessed by light and electron microscopy. Morphological evaluation reveals focal damage to the retinal pigment epithelial (RPE) cells in close proximity to less-affected RPE cells and normal photoreceptors. Collagen fibers in Bruch's membrane lose their parallel orientation. Occasionally, fusion of cell membranes of neighboring rod outer segments (ROS) is also observed. Continuous, 12 h exposure of hamsters to intense light results in initial focal damage to some RPE cells, such that severely damaged RPE cells are found adjacent to intact RPE cells. Only slight damage to the photoreceptors is evident, suggesting that the sequence of the pathological changes resulting from light begins with damage to the RPE cells and associated Bruch's membrane.

Melanosomes are closely related to lysosomes and lipofuscin granules. This paper indicates the potential involvement of lysosomal degradation processes in retinal pigment epithelial (RPE) cells. RPE cells cultured on Bruch's membrane and choroid were fed with indigestible latex beads. The RPE cells from this preparation were treated with chloroquine to investigate whether membrane swelling, typical for lysosomes under this condition, can be induced in melanosomes. To investigate the fate of indigestible material associated with rod outer segments (ROS), gold-labeled ROS were injected transsclerally into the subretinal space of Long Evans rats using a 32 gauge Hamilton syringe. The degradation of labeled ROS was observed after 5 and 12 days by electron microscopy. The following results were observed. Latex particles fuse with the melanin granules of the RPE. Following chloroquine treatment, the membranes of melanin granules fused, and formed large clusters and vacuoles. Gold granules were detected inside both early stage melanosomes and mature melanin granules of the RPE cells 5 and 12 days following subretinal injection of the labeled ROS. Higher numbers of gold granules were predominantly found in immature melanosomes containing still melanofilaments and in small fused mature melanin granules. In conclusion the effect of chloroquine clearly demonstrates that the melanosomes possess active proton pumps which is typical for lysosomes. In RPE cells stressed by overload with rod outer segments or by ingestion of undegradable material (latex beads, gold particles), fusion of these phagosomes with melanosomes of different maturity is more a general rule than an exception. Therefore, melanosomes are connected to lysosomal pathways in RPE cells., (Copyright 1999 Academic Press.)

Background: Many successful pigment epithelium transplantation studies involving pink-eyed Royal College of Surgeons (RCS) dystrophic rats showed highly pigmented transplanted cells forming a double layer with slightly pigmented cells, attached to Bruch's membrane. Since it is not clear whether transplanted pigmented cells can displace retinal pigment epithelial (RPE) host cells from Bruch's membrane, we suggested that RPE cells of RCS dystrophic rats can phagocytize melanin granules, possibly derived from perished transplanted cells., Methods: In a series of three experiments, RPE cells of nine pink-eyed, 2 1/2-month-old RCS dystrophic rats were isolated by trypsinization and mechanical dissection and cultivated in Dulbecco's modified Eagles' medium. These cells were then fed with melanin granules, isolated from bovine RPE cells, double-trypsinized after phagocytosis and viewed by light and electron microscopy. We also transplanted iris pigment epithelial (IPE) cells of 20-day-old Long-Evans rats into the subretinal space of pink-eyed RCS dystrophic rats of the same age, shown in light-microscopic photography after 42 days., Results: Living RPE cells were heavily pigmented after feeding with isolated melanin granules in all three experiments as viewed by light microscopy. In addition, we identified melanin granules phagocytized by dystrophic RPE cells in electron microscopy. After transplantation of pigmented IPE cells into the subretinal space of pink-eyed RCS dystrophic rats' eyes, a layer of slightly pigmented cells was seen on Bruch's membrane below the transplanted IPE cells, shown in light microscopy., Conclusion: We have shown by phagocytosis assay that dystrophic RPE cells can take up melanin granules in vitro. Our results assume that pigmented cells in transplantation studies, found as a monolayer, attached to Bruch's membrane, cannot automatically be identified as transplanted cells. Instead, the possibility of perished transplanted cells serving as melanin donors for RPE host cells must be taken into consideration.

It has been suggested that human iris pigment epithelial (IPE) cells isolated from iridectomized tissue could be used as autologous cells for transplantation into the subretinal space in diseases with dysfunctional retinal pigment epithelium (RPE). RPE cells synthesize a number of cytokines and their receptors which are important for its proper function. Nearly nothing is known about the capacity of IPE to synthesize cytokines or responding to them. To compare the mRNA expression of 36 cytokines or their receptors in cultured adult IPE cells and RPE cells we used semi-quantitative reverse transcription polymerase chain reactions (RT-PCR). Included in our assay were cytokines with known expression in RPE to get a broad basis for comparing IPE cells: basic fibroblast growth factor (bFGF or FGF-2), and one of its receptor (FGFR-1), epidermal growth factor (EGF), and its receptor EGF-R, transforming growth factor beta(TGFbeta), and its type III receptor TGFbeta-R3, the platelet-derived growth factors and receptors (PDGF A, PDGF B, PDGF-Ralpha, PDGF-Rbeta), tumor necrosis factor alpha(TNFalpha), and two receptors TNF-R1 and TNF-R2, insulin (INS) with receptor INS-R, insulin-like growth factors (IGF1, IGF2), and receptors (IGF1-R, IGF2-R), vascular endothelial growth factor (VEGF), and two receptors (VEGF-R1 or FLT-1 and VEGF-R2 or FLK-1), the receptor for VEGF-C: VEGF-R3 or FLK-4, interleukin 6 (IL6), and its receptor (IL6-R), nerve growth factor (NGF), interleukin 1alpha(IL1alpha), and a receptor (IL1-R). In addition, cytokines or their receptors not known to be expressed in RPE were included to widen our picture of cytokine gene expression in the eye: stem cell factor (SCF), its receptor (SCF-R), low-affinity nerve growth factor receptor p75 (p75(NGF-R), ciliary neutrothropic factor (CNTF), and its receptor (CNTF-R), glycoprotein 130 interleukin 6 transducer (gp130 (IL6-SD), leukemia inhibitory factor (LIF), and its receptor (LIF-R). Semi-quantitative expression data were obtained using series of fivefold dilutions of each cDNA and a fixed number of PCR cycles. The expression of RPE 65, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta2-microglobulin (B2MG) was used as a control for cellular origin, RNA quality and PCR conditions. With the exception of insulin and tumor necrosis factor alphaall other cytokines analysed and their receptors were expressed in both IPE and RPE cells, even though the levels varied. No qualitative or quantitative difference were observed in the mRNA expression level of 34 (94%) of the cytokines or receptors between IPE and RPE. In contrast, the mRNA expression level of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 [VEGF-RS (FLK-1)] was lower in IPE than in RPE cells. As an increased expression of VEGF in the RPE in maculae with age-related macular disease could be involved in its pathogenesis, a decreased expression of angiogenic growth factors in IPE cells could possibly be beneficial for the therapy of age-related maculopathy if indeed other tasks of non-functional RPE cells could be performed by IPE cells. The similarity of the mRNA expression pattern in 94% of the cytokines analyzed supports the assumption that IPE cells potentially can perform functions of RPE cells in the appropriate environment., (Copyright 1998 Academic Press.)

Purpose: The correct orientation of retinal pigment epithelium (RPE) cells is necessary for the integrity and proper function of the retina. For transplantation of RPE/iris pigment epithelium (IPE) grafts to the subretinal space in age-related macular degeneration, this cellular orientation is most effectively provided by a membranous support. The goal of this study was to establish an autologous or homologous membrane as a substratum for the growth of RPE/IPE., Methods: Porcine and bovine RPE and IPE were placed in primary culture on a dissected sheet (5 x 5 mm) of autologous porcine and bovine Descemet's membrane in slide chambers and grown to confluence., Results: RPE and IPE cells cultured on Descemet's membrane form an intact monolayer. Light and electron microscopy showed the formation of both an intact monolayer and microvilli in both cell types., Conclusion: Since the slow host-graft rejection appears to play an important role in the failure of RPE transplantation in the subretinal space, it is critical to be able to transplant autologous materials. The techniques presented here establish a novel means to culture RPE or IPE cells on autologous Descemet's membrane where they form a "cell monolayer patch," consisting of a fragment of Descemet's membrane with cultured RPE or IPE, which can be easily manipulated and transplanted, using an established glass pipette method.

Background: This study was carried out to examine the biological activity of contraction promoters produced by dedifferentiating retinal pigment epithelial cells (RPE) and to evaluate the importance of autocrine and paracrine effects within a semi-closed environment like the vitreal cavity., Methods: RPE at different stages of dedifferentiation in culture were examined for their ability (a) to generate tractional forces in vitro, with and without serum stimulation, and (b) to produce and release contraction-stimulating proteins. Autocrine versus paracrine effects of cell-secreted promoters were tested by using RPE or human dermal fibroblasts (HDF) as target cells. The contraction-stimulating activity of the cell-secreted promoters was partially characterized and compared to the activity of defined promoters., Results: Our study confirmed that RPE can synthesize and secrete cell-contraction-promoting factor(s) active in stimulating the development of tractional forces by RPE as well as HDF. The quantity of biological activity secreted per cell decreases with progressive dedifferentiation, yet the responsiveness of the cell to contraction promoters increases. The contraction promoter(s) synthesized by RPE is partially distinct from the promoters in serum, TGF-beta 1 and beta 2, IGF-1, ET-1 and PDGF. The contraction-promoting effects of the RPE product(s) can be completely blocked by staurosporine., Conclusion: De-differentiation of RPE is characterized by increasing capacity to generate tractional forces and decreasing synthetic capacity. RPE within a semi-closed system like the vitreal cavity can, theoretically, act both as promoting and active component of traction-related events (tractional retinal detachment).

The targeted induction of apoptosis is a novel therapeutic approach to control the unlimited growth of proliferating cells. Since massive proliferation of cells at the vitreoretinal interface is a key feature of proliferative vitreoretinal disorders, we sought to identify apoptosis in epiretinal membranes from patients with proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR) and macular pucker. Further, we evaluated the possible induction of apoptosis of retinal pigment epithelial (RPE) cells by transforming growth factor-beta(TGF-beta). Apoptotic cells were identified by in situ DNA end labeling and acridine orange staining on paraffin-embedded tissue sections from epiretinal membranes of patients with all vitreoretinal disorders examined. Labeled nuclei or condensed chromatin were scattered throughout the membranes or occurred in clusters. Most apoptotic cells were RPE-derived, as assessed by cytokeratin immunochemistry. No apoptotic glial cells were detected. In PVR, proliferative activity, as confirmed by Ki-67 immunochemistry, was associated with short history and rapid disease progression. Apoptotic nuclei were observed more frequently in long-standing PVR or slow progression towards traction retinal detachment. TGF-beta was detected in all control vitreous samples by bioassay at concentrations below 20 ng ml-1. TGF-beta levels increased up to 20-fold in pathological vitreous. Marked heterogeneity was observed in all patient groups. The degree of TGF-beta activation was significantly higher in PVR than in PDR. Proapoptotic effects of TGF-beta were demonstrated in cultured human RPE cells by electron microscopy, in situ DNA end labeling, comet assay and a photometric enzyme immunoassay for histone-associated DNA fragments. Apoptosis appears to be a key regulatory mechanism of growth control of specific cell populations in proliferative vitreoretinal disorders. Administration of proapoptotic growth factors such as TGF-beta may provide a novel approach to inhibit cellular proliferation at the vitreoretinal interface., (Copyright 1997 Academic Press Limited.)

This study investigates the ability of iris epithelial cells (IPE) to ingest rod outer segments (ROS) and compares the amount of phagocytosis of porcine RPE and IPE cells by the use of a pH sensitive fluorescent dye (carboxy SNAFL) at the light microscopic level. The dye allowed investigation of ingestion separately from binding of rod outer segments. In a second set of experiments, after exposing ferritin-labeled ROS to the cultured cells, phagosomes were also counted in electron microscopic sections. Additionally immunocytochemical staining was performed with IPE and RPE cells. Both cell types stained positive with polyclonal NaK-ATPase antibodies against the alpha 1 subunit from rat brain and kidney. The epithelial nature of the cultured cells was determined by monoclonal anti-human-cytokeratin antibodies. Moreover, the ultrastructure of the cells revealed high amounts of phagosomes smaller than 1 micron in diameter present in both RPE and IPE cells. The iron label of the phagosomes was determined by EELS spectra taken from individual phagosomes. Electron and light microscopic quantification shows that cultured IPE cells have 64% of the phagocytic capacity of the RPE with respect to phagosomes larger than 1 micron in diameter.

According to a recent hypothesis the melanin granules in the retinal pigment epithelium of mammals originate from photosensory membrane degradation. To test this hypothesis the retinal pigment epithelium of cattle was kept in tissue culture and exposed to gold-labelled rod outer segments. Gold granules were later detected inside phagosomes, melanosomes and mature melanin granules. Tyrosinase, the key enzyme in melanogenesis, was additionally localized inside phagosomes. These results indicate that in cultured retinal pigment epithelium the matrix of the melanosome can originate from phagosomes. Therefore, the melanosome is a specialized lysosome.

1. The ultrastructure of the retinal pigment epithelium (RPE) of adult Syrian golden hamsters and cattle was examined with respect to pigment granules and phagosomes involved in degradation of disk membranes from rod outer segments. 2. In the RPE of cattle, phagosomes were found that contained an electron-dense melanin-like material that was not autofluorescent and therefore not lipofuscin. 3. Disk membranes of rods are about 4 nm thick and become enlarged (7-20 nm) and electron-dense during degradation in the RPE. 4. Additionally electron-dense vesiculo-globular bodies (10-100 nm) were found in phagosomes during disk membrane degradation and in mature melanin granules. 5. In the RPE of adult hamsters that had been exposed to intense light, premelanosomes containing unmelanised filaments with a striated periodicity were found in the cytoplasm or in association with mature melanin granules. Early and late stage melanosomes were also present. Phagosomes in the RPE contained degraded disk membranes, melanin-like material and melanofilaments. 6. Dopa oxidase was detected ultrastructurally within shed disk membranes that were in close contact with the microvilli of the RPE. 7. The possibility of melanogenesis within phagosomes during disk membrane degradation is discussed.

1. Endogenous peroxidase was localized by electron microscopy within the retinal tissue of the golden Syrian hamster. 2. Peroxidase activity was found in the apical parts of rod outer segments, in the cytoplasm and phagosomes of the retinal pigment epithelium (RPE), and extracellularly between the basal infoldings of the RPE. 3. No reaction product was detected when either H2O2 or tetramethyl benzidine were omitted from the reaction medium. 4. The same was true when the tissue was osmicated before the incubation in the reaction medium or treated with 100 mM sodium azide. 5. These results indicate that the peroxidatic activity was enzymatically mediated.