Embryonic Retinal Pigment Epithelium Research Articles

Transcriptome Profiling of Embryonic Retinal Pigment Epithelium Reprogramming.

The plasticity of human retinal pigment epithelium (RPE) has been observed during proliferative vitreoretinopathy, a defective repair process during which injured RPE gives rise to fibrosis. In contrast, following injury, the RPE of the embryonic chicken can be reprogrammed to regenerate neural retina in a fibroblast growth factor 2 (FGF2)-dependent manner. To better explore the mechanisms underlying embryonic RPE reprogramming, we used laser capture microdissection to isolate RNA from (1) intact RPE, (2) transiently reprogrammed RPE (t-rRPE) 6 h post-retinectomy, and (3) reprogrammed RPE (rRPE) 6 h post-retinectomy with FGF2 treatment. Using RNA-seq, we observed the acute repression of genes related to cell cycle progression in the injured t-rRPE, as well as up-regulation of genes associated with injury. In contrast, the rRPE was strongly enriched for mitogen-activated protein kinase (MAPK)-responsive genes and retina development factors, confirming that FGF2 and the downstream MAPK cascade are the main drivers of embryonic RPE reprogramming. Clustering and pathway enrichment analysis was used to create an integrated network of the core processes associated with RPE reprogramming, including key terms pertaining to injury response, migration, actin dynamics, and cell cycle progression. Finally, we employed gene set enrichment analysis to suggest a previously uncovered role for epithelial-mesenchymal transition (EMT) machinery in the initiation of embryonic chick RPE reprogramming. The EMT program is accompanied by extensive, coordinated regulation of extracellular matrix (ECM) associated factors, and these observations together suggest an early role for ECM and EMT-like dynamics during reprogramming. Our study provides for the first time an in-depth transcriptomic analysis of embryonic RPE reprogramming and will prove useful in guiding future efforts to understand proliferative disorders of the RPE and to promote retinal regeneration.

Tsg101 Is Necessary for the Establishment and Maintenance of Mouse Retinal Pigment Epithelial Cell Polarity

The retinal pigment epithelium (RPE) forms a monolayer sheet separating the retina and choroid in vertebrate eyes. The polarized nature of RPE is maintained by distributing membrane proteins differentially along apico-basal axis. We found the distributions of these proteins differ in embryonic, post-natal, and mature mouse RPE, suggesting developmental regulation of protein trafficking. Thus, we deleted tumor susceptibility gene 101 (Tsg101), a key component of endosomal sorting complexes required for transport (ESCRT), in embryonic and mature RPE to determine whether ESCRT-mediated endocytic protein trafficking correlated with the establishment and maintenance of RPE polarity. Loss of Tsg101 severely disturbed the polarity of RPE, which forms irregular aggregates exhibiting non-polarized distribution of cell adhesion proteins and activation of epidermal growth factor receptor signaling. These findings suggest that ESCRT-mediated protein trafficking is essential for the development and maintenance of RPE cell polarity.

Generation of an inducible RPE-specific Cre transgenic-mouse line.

The retinal pigment epithelium (RPE) is an epithelial monolayer in the back of the vertebrate eye. RPE dysfunction is associated with retinal degeneration and blindness. In order to fully understand how dysregulation affects visual function, RPE-specific gene knockouts are indispensable. Since the currently available RPE-specific Cre recombinases show lack of specificity or poor recombination, we sought to generate an alternative. We generated a tamoxifen-inducible RPE-specific Cre transgenic mouse line under transcriptional control of an RPE-specific Tyrosinase enhancer. We characterized the Cre-mediated recombinant expression by crossing our RPE-Tyrosinase-CreErT2 mouse line with the tdTomato reporter line, Ai14. Detected fluorescence was quantified via high-content image analysis. Recombination was predominantly observed in the RPE and adjacent ciliary body. RPE flatmount preparations revealed a high level of recombination in adult mice (47.25–69.48%). Regional analysis of dorsal, ventral, nasal and temporal areas did not show significant changes in recombination. However, recombination was higher in the central RPE compared to the periphery. Higher levels of Cre-mediated recombinant expression was observed in embryonic RPE (~83%). Compared to other RPE-specific Cre transgenic mouse lines, this newly generated RPE-Tyrosinase-CreErT2 line shows a more uniform and higher level of recombination with the advantage to initiate recombination in both, prenatal and postnatal animals. This line can serve as a valuable tool for researches exploring the role of individual gene functions, in both developing and differentiated RPE.

RPE65 has an additional function as the lutein to meso-zeaxanthin isomerase in the vertebrate eye

Carotenoids are plant-derived pigment molecules that vertebrates cannot synthesize de novo that protect the fovea of the primate retina from oxidative stress and light damage. meso-Zeaxanthin is an ocular-specific carotenoid for which there are no common dietary sources. It is one of the three major carotenoids present at the foveal center, but the mechanism by which it is produced in the eye is unknown. An isomerase enzyme is thought to be responsible for the transformation of lutein to meso-zeaxanthin by a double-bond shift mechanism, but its identity has been elusive. We previously found that meso-zeaxanthin is produced in a developmentally regulated manner in chicken embryonic retinal pigment epithelium (RPE)/choroid in the absence of light. In the present study, we show that RPE65, the isomerohydrolase enzyme of the vertebrate visual cycle that catalyzes the isomerization of all-trans-retinyl esters to 11-cis-retinol, is also the isomerase enzyme responsible for the production of meso-zeaxanthin in vertebrates. Its RNA is up-regulated 23-fold at the time of meso-zeaxanthin production during chicken eye development, and we present evidence that overexpression of either chicken or human RPE65 in cell culture leads to the production of meso-zeaxanthin from lutein. Pharmacologic inhibition of RPE65 function resulted in significant inhibition of meso-zeaxanthin biosynthesis during chicken eye development. Structural docking experiments revealed that the epsilon ring of lutein fits into the active site of RPE65 close to the nonheme iron center. This report describes a previously unrecognized additional activity of RPE65 in ocular carotenoid metabolism.

Subretinal implantation of human stem cell‐derived RPE on ultrathin carriers in rabbits

SummaryTransplantation of retinal pigment epithelium (RPE) is being developed as a cell replacement therapy for age‐related macular degeneration (AMD). Human embryonic and adult human RPE stem cells (hESC and hRPESC, resp.) are known potential sources that are currently being pursued towards the clinic. Polarized monolayers of SC‐derived RPE were shown to grow on biostable polyester (PET) membranes. They were found to be similar to fetal hRPE monolayers and to have near‐native characteristics. Stamped pieces of RPE monolayers on the carrier were loaded into a custom‐designed surgical instrument and transplanted subretinally in the rabbit, a large‐eyed animal model. Compared to fetal and hRPESC derived RPE, hESC‐RPE xenografts showed better preservation of the neural retina overlying the implant. Histology obtained 4 weeks after implantation confirmed a continuous polarized human RPE monolayer on PET. We demonstrate that the xeno‐RPE monolayer implant survived well and retained its polarization. Moreover, our initial data suggest a distinctly advantageous tolerance of hESC derived RPE xenografts in rabbit subretinal space.

Both PAX6 and MITF are required for pigment epithelium development in vivo

Both PAX6 and MITF are required for pigment epithelium development <i>in vivo</i>

Efflux protein expression in human stem cell-derived retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP), the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC) and RPE derived from the hESC (hESC-RPE). Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE -derived diseases, drug testing and targeted drug therapy.

Effects of different wave-length lights on proliferation and secretion of growth factors in human retinal pigment epithelium cells

Background The study of myopia development is always the hotspot worldwide. Recently,scientist found that some growth factor secreted by retinal nerve epithelium cells and retinal pigment epithelium(RPE)cells are associated with the development of myopia. Whenever, the absorption of RPE cells to different wave-length lights is different. ObjectiveThis study was to investigate the effects of different wave-lengths lights on the proliferation of human RPE cells, and explore the influence of different wave-lengths lights on RPE cells secreting hepatocyte growth factor(HGF) ,basic fibroblast growth factor(bFGF) and transforming growth factor-β(TGF-β).Methods The fourth to fifth passages of human embryonic RPE cells were exposed to blue light( λ =480 nm),red light( λ =775 nm) and white light. The cells of control group were harvested in normal condition. The proliferation and growth of RPE cells were assayed by MTT,and the ultrastructure of cells was examined under the transmission electron microscopy at 48 hours after light exposure of RPE cells. Enzyme linked immunosorbent assay(ELISA) was adopted to determine the concentrations of HGF,bFGF and TGF-β in the culture medium in 12,24,48,72 hours. The expression of HGF mRNA in RPE cells was detected by RT-PCR. This study was approved by Ethic committee of Fudan University. Results The A490 values of the cells exposed to blue light,red light,white light and white light were 0. 0218±0. 0014 ;0. 0353±0. 0025 ;0. 0371 ±0. 0024 and 0. 0445 +0. 0046 respectively with the significant difference among 4 groups ( F =12. 579, P＜0.05 ), and A490 value in blue light group, red light group were significantly lower than that of the control group ( t =2.043 ; t =2.024, P＜0.05 ). ELISA showed that the concentrations of HGF and TGF-β in culture medium were evidently elevated as the prolongation of light exposure in various light exposure groups in 72 hours(HGF) and 48 hours(TGF-β) compared with 12 hours with a predominating rise in the control group. The statistically significant differences were found in the concentrations of HGF and TGF-β between control group and blue light group or red light group in the( all P＜0. 05 ). The bFGF level was decreased with the time increase of various light exposure with the significant differences in 72 hours compared with 12 hours( P＜0.05 ). RT-PCR revealed the considerable difference about expression of HGF mRNA in RPE cells among these four groups( P＜0. 05 ), and the lest expression in HGF mRNA was in the blue light group compared with control group( t =3. 972,P＜0.05 ). Thinning of the chromatin, decreasing of organelle and loss of cellular membrane were seen in the blue light group, but no obvious change of ultrastructure of human embryo RPE cells was found in the ret and white light groups. Conclusions The irradiation of different wave-length light can effect the growth and proliferation and secretion of HGF,bFGF and TGFβ in human RPE cells in vitro,implying myopia formation is associated to exposure of different wave-length light. Key words: Myopia; Retinal pigment epithelial cell; Hepatocyte growth factor; Basic fibroblast growth factor; Transforming growth factor-β ; Light exposure

Rescue and repair during photoreceptor cell renewal mediated by docosahexaenoic acid-derived neuroprotectin D1

Retinal degenerative diseases result in retinal pigment epithelial (RPE) and photoreceptor cell loss. These cells are continuously exposed to the environment (light) and to potentially pro-oxidative conditions, as the retina's oxygen consumption is very high. There is also a high flux of docosahexaenoic acid (DHA), a PUFA that moves through the blood stream toward photoreceptors and between them and RPE cells. Photoreceptor outer segment shedding and phagocytosis intermittently renews photoreceptor membranes. DHA is converted through 15-lipoxygenase-1 into neuroprotectin D1 (NPD1), a potent mediator that evokes counteracting cell-protective, anti-inflammatory, pro-survival repair signaling, including the induction of anti-apoptotic proteins and inhibition of pro-apoptotic proteins. Thus, NPD1 triggers activation of signaling pathway/s that modulate/s pro-apoptotic signals, promoting cell survival. This review provides an overview of DHA in photoreceptors and describes the ability of RPE cells to synthesize NPD1 from DHA. It also describes the role of neurotrophins as agonists of NPD1 synthesis and how photoreceptor phagocytosis induces refractoriness to oxidative stress in RPE cells, with concomitant NPD1 synthesis.

Ontogenetic expression of the Otx2 and Crx homeobox genes in the retina of the rat

Otx2 and Crx are vertebrate orthologs of the orthodenticle family of homeobox genes, which are involved in retinal development. In this study, the temporal expression patterns of Otx2 and Crx in the rat retina during embryonic and postnatal stages of development were analyzed in detail. This confirmed the presence of Otx2 mRNA in both the embryonic retinal pigment epithelium and the developing neural retina. During development, the expression of Otx2 persists in the pigment epithelium, whereas Otx2 expression of the neural retina becomes progressively restricted to the outer nuclear layer and the outer part of the inner nuclear layer. Immunohistochemistry revealed that Otx2 protein is also present in cell bodies of the ganglion cell layer, which does not contain the Otx2 transcript, suggesting that Otx2 protein is synthesized in cell bodies of the bipolar neurons and then transported to and taken up by cells in the ganglion cell layer. Crx is also highly expressed in the outer nuclear layer starting at E17 and postnatally in the inner nuclear layer. The onset of expression of Crx lags behind that of Otx2 consistent with evidence that Otx2 activates Crx transcription. These expression patterns are consistent with evidence that Otx2 and Crx function during retinal development and extend the period of probable functionality to the adult. In this regard, these results provide an enhanced and expanded temporal and spatial framework for understanding the multiple roles of Otx2 and Crx in the developing and mature mammalian retina.

Suppression of Mitf by small interfering RNA induces dedifferentiation of chick embryonic retinal pigment epithelium

Recent studies indicate a key role of Mitf (microphthalmia-associated transcription factor) in the differentiation of the retinal pigment epithelium (RPE). To explore transdifferentiation processes, we used small interfering RNA (siRNA) targeting Mitf. Transfection of embryonic chick RPE cells with a non-silencing fluorescein-labelled control siRNA demonstrated a high (84%) transfection efficiency. Transfection of Mitf siRNA reduced Mitf synthesis at the mRNA and protein levels as analysed by real-time RT-PCR and Western blot. Mitf siRNA suppressed the expression of Mitf RNA to 25.0% of the level in control cells ( P<0.005). The expression of melanosomal matrix glycoprotein 115 (MMP115), a marker of differentiated pigment cells, was also markedly suppressed (to 52.2±6.6%) ( P<0.0005). Moreover, the expression of Pax6 was increased by Mitf siRNA (to 143.5±18.0%) ( P<0.0005), and induced dedifferentiation of the RPE cells. These data suggest siRNA can be an effective gene silencing approach in RPE, and reduction of Mitf expression is essential for the dedifferentiation of RPE cells.

VIP enhances the differentiation of retinal pigment epithelium in culture: from cAMP and pp60(c-src) to melanogenesis and development of fluid transport capacity.

The retinal pigment epithelium (RPE) is a single cell layer juxtaposed between the neural retina and the choroid and functions as a blood-retina barrier. The RPE performs functions essential for photoreceptor (PR) survival. Although the regulation of these functions has remained unknown, it is a distinct possibility that the RPE is under constant regulation by signaling molecules coming from the choroid and the retina. Vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide present in the retina and in the choroid, has been shown to promote the growth and differentiation of a variety of cells in tissue and organ cultures. In cultured RPE cells, VIP is the one most effective stimulator of the cAMP signaling pathway among a long list of neurotransmitters and modulators tested. For example, VIP, at 1 microM, stimulates the intracellular cAMP to 80-100- and 20-fold in 3 min in RPE cells cultured from chick embryos and adult human donor eyes, respectively. In cultured chick embryonic RPE, VIP is also shown to be a potent and effective modulator of pp60(c-src), the non-receptor tyrosine kinase present in differentiating and terminally differentiated cells. VIP stimulates both overall phosphorylation at unknown sites and phosphotyrosine dephosphorylation in pp60(c-src). A 190-kDa microtubule-associated protein is known to be one of the downstream targets in VIP-modulated signaling pathways. At the cellular level, VIP stimulates cell proliferation modestly and melanogenesis pronouncedly in growing chick embryonic RPE cultures. Ultimately, the differentiation goal of RPE cells in vivo is to perform functions that are essential for photoreceptor survival. On bare permeable supports (that is, without biological material coating), the chick embryonic RPE cells grow to become RPE sheets with a cytoarchitecture that allows the display of two of the RPE functions. These cultures demonstrate structural polarity and are functionally polarized, allowing for proper macromolecule secretion and fluid transport. VIP is shown to stimulate macromolecule secretion at the apical surface (retina facing) and the development of the capacity for fluid transport from the apical to the basal surface of the RPE sheet. In conclusion, studies in our laboratory indicate that VIP is a differentiation promotor during the development of a functional RPE. Recent advances in the molecular biology of melanogenesis and the fluid transport-linked Na-K-2Cl cotransporter in other cells will allow future studies of VIP modulated events in the RPE at the molecular level. Finally, identification of RPE differentiation factors may prove essential for the ultimate success of RPE transplantation, thus promoting the rescue of photoreceptor cells in retinal degeneration.

Expression of an array of photoreceptor genes in chick embryonic retinal pigment epithelium cell cultures under the induction of neuroD

Coaxing plastic, non-neuronal cells to transdifferentiate into a particular type of neurons might have clinical applications. Previously we reported that neuroD induces transdifferentiation of retinal pigment epithelium (RPE) cells derived from day-6 chick embryos into cells that resemble young photoreceptor cells. These cells also express visinin, a gene expressed early during cone photoreceptor differentiation. Further characterization showed that the transdifferentiated cells express a number of photoreceptor genes, including interphotoreceptor retinoid binding protein, the α-subunit of phosphodiesterase, and opsin genes encoding rhodopsin, the red, the green, and the blue visual pigments. Our data demonstrate that neuroD can reprogram RPE to become photoreceptor cells with substantial differentiation, and suggest the possibility of generating photoreceptor cells from RPE using neuroD as a molecular trigger.

Tyrosinase-Related Protein 2 Promoter Targets Transgene Expression to Ocular and Neural Crest-Derived Tissues

In an effort to identify a promoter suitable for studying early ocular development, we generated transgenic mice carrying the lacZ reporter gene linked to the tyrosinase-related protein 2 (TRP2) promoter. TRP2-lacZ was expressed in early retinal pigment epithelium (RPE) and early neural crest cells in embryos. The promoter activity was robust and consistent in independent transgenic lines. The transgene was also expressed in the optic nerve and neural crest-derived neuronal cells in which the endogenous TRP2 gene is not expressed. This suggests that repressor elements may be missing in the promoter used in this study. To test whether this promoter can be used to study melanocyte development, we cross-mated TRP2-lacZ transgenic mice with mice heterozygous for the Patch (Ph) mutation. The pattern of β-galactosidase activity in the embryos correlates well with the pigmentation phenotype in postnatal and adult Ph/+ mice. We also generated transgenic mice expressing fibroblast growth factor 9 (FGF9) directed by the TRP2 promoter and examined the effect on ocular development. Ectopic expression of FGF9 in the early embryonic RPE switched its differentiation pathway to a neuronal fate, resulting in formation of a duplicated neural retina in transgenic mice. These studies demonstrate that the TRP2 promoter is valuable for transgenic studies of ocular differentiation and development of neural crest cells.

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

AIMSThe 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.METHODSEmbryonic RPE cells obtained...

Transdifferentiation of chick embryonic retinal pigment epithelial cells to lentoid structure in suspension culture.

To test transdifferentiation of retinal pigment epithelial (RPE) cells in suspension culture, chick embryonic RPE sheets and dissociated RPE cells were cultured for two months in a non-adherent dish for suspension culture. RPE cells, isolated as a sheet, aggregated immediately and remained the same size with their differentiated characteristics for two months. The presence of basic fibroblast growth factor (bFGF) at concentrations of 10 ng/ml or higher induced the formation of a spherical lentoid structure which was positive for crystallin and bFGF receptor. In contrast, dissociated RPE cells did reaggregate but did not develop the lentoid structure even in the presence of bFGF. The transdifferentiation of RPE cells to the lentoid structure in this study was in contrast to their transdifferentiation to the retina, as reported in a previous study.

Role of Mitf in Differentiation and Transdifferentiation of Chicken Pigmented Epithelial Cell

Mitfencodes a basic helix-loop-helix-leucine-zipper (bHLHzip) protein that is known to function in the development of melanocytes, pigmented epithelial cells (PECs), osteoclasts, and mast cells. In this paper, we report on the isolation, expression, and overexpression of the chickenMitfand discuss the role of its protein product in the differentiation and transdifferentiation of PECs. Northern blotting showed that chickenMitfis predominantly expressed in embryonic retinal pigmented epithelium (PE), but is expressed at low levels in other tissues. A 5′ RACE analysis revealed differences in the 5′ region ofMitfmRNA in PE and other tissues. Immunological analysis revealed that Mitf, the protein encoded byMitf,is first detected in the nuclei of the optic vesicle cells at embryonic stage 13 in a restricted region covered with mesenchymal cells. From stage 14 to 24, the specific staining is observable in the PE and precursor of the PE, the outer layer of the optic cup. In embryos at stages later than stage 29 the signals for Mitf in the future iris, ciliary body, and posterior retinal regions become faint. These results show that expression of Mitf starts at the optic vesicle stage at which no other marker genes for PECs such asmmp115andtyrosinaseare expressed. Dedifferentiation of cultured retinal PECs (rPECs) was induced by phenylthiourea and testicular hyaluronidase, bFGF, or TGF-β.Mitfexpression was inhibited by these factors and reactivated during redifferentiation of the dedifferentiated cells into rPECs, showing the correlation betweenMitfexpression and rPEC differentiation. Retrovirus-mediated overexpression ofMitfinhibited bFGF-induced dedifferentiation and transdifferentiation of rPECs to both lens and neural cells. These findings showed that downregulation ofMitfexpression is essential for the transdifferentiation of rPEC.Mitfoverexpression caused hyperpigmentation in cultured rPECs and suppressed the changes in gene expression induced by bFGF.Mitfoverexpression promoted expression ofmmp115andtyrosinasein bFGF-treated rPECs suggesting a critical role for Mitf in rPEC differentiation.Mitfoverexpression, however, did not promote expression of another rPEC-specific gene,pP344,in bFGF-treated rPECs. This result suggests the presence of other regulatory genes promoting rPEC differentiation. The expression patterns ofpax6andMitfare complementary bothin vivoandin vitro.Overexpression ofMitfinhibited expression ofpax6in cultured rPECs. These observations suggest that Mitf regulatespax6expression negatively.

Expression of 17β-Hydroxysteroid Dehydrogenase Type IV in Chick Retinal Pigment Epithelium

Retinal pigment epithelium (RPE) has essential roles that maintain functions of the neural retina. Previously, we described that the 3b5 monoclonal antibody (3b5 mAb) recognized the RPE cells in the chick embryonic eye. In the present study, a gene coding for the 3b5 antigen has been cloned and sequenced. A 10-day-old embryonic RPE cDNA library was constructed in lambda Uni-ZAP XR, and screened with the 3b5 mAb as a probe. The complete nucleotide sequence of the antigen recognized by the 3b5 mAb was obtained. The cDNA encodes a 735 amino acid protein with a calculated molecular mass of 80147 Da. The deduced amino acid sequence shares 72% identity with the human 17β-hydroxysteroid dehydrogenase (17β-HSD) type IV. Northern blot analysis detected a 2.9 kb transcript. Immunohistochemical and in-situ hybridization studies revealed that 17β-HSD type IV was preferentially expressed in the RPE cells. The expression of steroid metabolizing enzymes in the RPE cells is able to create estrogenic environment, which may have a role of maintenance of the neural retina.

Phagosome Number and Distribution in Retinal Pigment Epithelial Cells of Vitiligo Mutant Mice

The vitiligo mutant mouse has a disorder affecting the interaction of retinal pigment epithelium (RPE) and photoreceptor cells of the neural retina. Among the phenotypic features are patches of hyper- and hypopigmentation in the embryonic RPE, increased RPE cell production neonatally, and a later onset of progressive photoreceptor cell degeneration that continues for more than one year until all photoreceptor cells are gone. Failure of RPE microvilli to intertwine with rod outer segments (ROS) at any age, the accumulation of ROS membranous fragments in the subretinal space, and a relatively early retinal separation from the RPE suggested analysis of whether RPE phagocytosis might be impaired. Post-natal day 23 (P23) and P36 mutant and congenic control wild-type mice were kept in darkness overnight and eyes were examined by light and transmission electron microscopy 0.5hr before, 1.5hr after and 10.5hr after lights turned on at 0700hr. At these ages ROS have not yet degenerated, though they are shorter than normal and somewhat misoriented. The number of phagosomes per RPE cell was markedly reduced in mutants compared to controls at both ages and all time points. Nonetheless, the highest counts were obtained 1.5hr after the lights turned on in mutant and control specimens. In the mutant eyes, the proportion of phagosomes in the microvillous zone of the RPE cells was consistently lower than in any other cellular compartment. Phagosome distribution in the apical and basal zones of the RPE cell cytoplasm was within normal limits. Macrophage-like cells become numerous in the subretinal space at older ages, but were already present at P23 and P36, and contained phagosomes in their cytoplasm. The hypothesis is proposed that binding of ROS to RPE cells might be defective in vitiligo mice, in contrast to the rdyrat, where the work of others indicates that binding is normal and the subsequent ingestion of phagosomes is impaired.

Effects of Superoxide Dismutase and Catalase on Growth of Retinal Pigment Epithelial Cells in vitro following Addition of Linoleic Acid or Linoleic Acid Hydroperoxide

The rod outer segments of the retina that are phagocytized by retinal pigment epithelial (RPE) cells are susceptible to lipid peroxidation because of their high content of polyunsaturated fatty acids. Linoleic hydroperoxides (LHP), synthesized by peroxidation of linoleic acids (LA), produce greater damage to retinal function than does LA. We compared the effects of LHP and LA on the growth of cultured chick embryonic RPE cells and analyzed a model of data sets using multiple linear regression for the number of cells as a function of time. The spectrum of LA had a sharp peak at 205 nm and a broad spectrum at 235 nm, while LHP had only a broad spectrum at 235 nm. Exposure to LA and LHP caused dose-dependent damage of chick embryonic RPE cells: they were significantly more affected by the addition of LHP than LA. The antioxidative enzymes catalase and superoxide dismutase minimized damage to the RPE cells caused by LHP in proportion to the enzyme concentration. However, RPE cells incubated with LA were more affected by the enzymes than when no enzymes were added.