Retinal Pigment Epithelium Cultures Research Articles

Subretinal Delivery of Ultrathin Rigid-Elastic Cell Carriers Using a Metallic Shooter Instrument and Biodegradable Hydrogel Encapsulation

To develop a surgical technique for the subretinal implantation of cell carriers suitable for the transplantation of cultured retinal pigment epithelium (RPE) in a preclinical animal model. Cell carriers were porous 10-μm-thick polyester membranes. A custom-made shooter instrument consisted of a 20-gauge metallic nozzle with a nonstick plunger. Fetal human RPE cultures were used for vitality assessment during instrument handling. Transvitreal subretinal implantation of carriers without RPE was performed in 31 rabbits after vitrectomy. Fourteen of 31 implants were encapsulated in gelatin. Fluid turbulence over the implantation site was minimized using a novel infusion cannula. Six rabbits had intravitreal plasmin injections before surgery. SD-OCT in vivo images were obtained after 3, 7, and 14 days, followed by perfusion-fixed histology. Gelatin encapsulation of RPE/polyester implants made cell loss during handling reproducible, compared with 40% of controls showing random, large damage zones. Gelatin implants were ejected smoothly in 12 of 14 surgeries (86%), whereas "naked" implants frequently became trapped with the instrument, which reduced success to 9 of 17 cases (53%). Vitreous remnants after vitrectomy alone complicated subretinal placement of encapsulated and naked implants in 7 of 25 cases (28%). Plasmin-assisted vitrectomy resulted in implant ejection unperturbed by vitreous adhesions in six experiments. SD-OCT and histology demonstrated atraumatic subretinal implant delivery after uncomplicated surgery. A novel shooter instrument design allows for safe and atraumatic transvitreal delivery of hydrogel-encapsulated, ultrathin, rigid-elastic carriers into the subretinal space. The procedure may be used in the future to deliver cultured RPE.

Complementing apolipoprotein secretion by cultured retinal pigment epithelium

Age-related macular degeneration (AMD) is a major cause of vision loss in the elderly, and it has two times the prevalence of Alzheimer disease in the United States. The underlying metabolic/vascular disease, with secondary neurodegeneration of photoreceptors, is still poorly understood, despite clinical success in treating neovascular complications. Most enigmatic have been drusen, the characteristic extracellular lesions that develop posterior to a support cell layer, the retinal pigment epithelium (RPE). Drusen are established ocular risk factors for progression to sight-threatening stages of disease. A major impediment to understanding drusen has been the scarcity of suitable experimental systems. In PNAS, the work by Johnson et al. (1) describes an RPE culture system exhibiting secretion of druse component apolipoprotein E, a cholesterol transporter, and activation of systemically derived complement, a pathway fingered in AMD by multiple genetic association studies.

The effect of vascular endothelial growth factor and hypoxia on the secretion of opticin in retinal pigment epithelium cells

To evaluate the role of vascular endothelial growth factor or hypoxia on the secretion of opticin in retinal pigment epithelium cells. Human RPE cells were cultured, the third to sixth passage of retinal pigment epithelium (RPE) cells were placed in 6-well culture plates at a density of 4 × 10(4)/well. For hypoxia experiment, the cells were cultured under hypoxic condition for different times. For vascular endothelial growth factor (VEGF) experiment, the media was changed to DMEM containing different concentration VEGF (1, 10, 50, 100 µg/L) for 24 h respectively. VEGF mRNA levels were determined by RT-RCR method. The protein content of opticin in RPE cells or culture media was detected by Western blot. Matrix metalloproteinase activity in culture media was analysis by zymography. One way ANOVA was used to test the comparisons between experimental groups and control group. Western blot experiment showed the opticin expression was not changed in RPE cells after hypoxia treatment, however was significantly decreased in culture media. Compared with control group (0.21 ± 0.03). The relative density of VEGF mRNA levels (0.81 ± 0.04, 0.67 ± 0.07) in RPE cells were increased after 12 h or 24 h hypoxia treatment (F = 483.60, P < 0.05). Opticin expression in RPE cells was also remain unchanged after vary concentration VEGF addition treatment (F = 2.16, P > 0.05), the relative density of opticin expression in VEGF conditioned culture medium were 0.65 ± 0.02, 0.52 ± 0.04, 0.23 ± 0.03, 0.30 ± 0.03 respectively, and the difference in culture media was significant compared to control group (0.73 ± 0.04) (F = 141.38, P < 0.05). Zymography indicate a matrix metalloproteinases type 2 digest band, the activities were enhanced with VEGF increasing. The decrease of opticin in culture media after VEGF treatment could be inhibited by low condition of EDTA. VEGF and hypoxia have an effect on the on the secretion of opiticin in RPE cells, it may be contributed to the increasing levels of matrix metalloproteinases type 2.

Mature Peripheral RPE Cells Have an Intrinsic Capacity to Proliferate; A Potential Regulatory Mechanism for Age-Related Cell Loss

BackgroundMammalian peripheral retinal pigmented epithelium (RPE) cells proliferate throughout life, while central cells are senescent. It is thought that some peripheral cells migrate centrally to correct age-related central RPE loss.Methodology/Principal FindingsWe ask whether this proliferative capacity is intrinsic to such cells and whether cells located centrally produce diffusible signals imposing senescence upon the former once migrated. We also ask whether there are regional differences in expression patterns of key genes involved in these features between the centre and the periphery in vivo and in vitro. Low density RPE cultures obtained from adult mice revealed significantly greater levels of proliferation when derived from peripheral compared to central tissue, but this significance declined with increasing culture density. Further, exposure to centrally conditioned media had no influence on proliferation in peripheral RPE cell cultures at the concentrations examined. Central cells expressed significantly higher levels of E-Cadherin revealing a tighter cell adhesion than in the peripheral regions. Fluorescence-labelled staining for E-Cadherin, F-actin and ZO-1 in vivo revealed different patterns with significantly increased expression on central RPE cells than those in the periphery or differences in junctional morphology. A range of other genes were investigated both in vivo and in vitro associated with RPE proliferation in order to identify gene expression differences between the centre and the periphery. Specifically, the cell cycle inhibitor p27Kip1 was significantly elevated in central senescent regions in vivo and mTOR, associated with RPE cell senescence, was significantly elevated in the centre in comparison to the periphery.ConclusionsThese data show that the proliferative capacity of peripheral RPE cells is intrinsic and cell-autonomous in adult mice. These differences between centre and periphery are reflected in distinct patterns in junctional markers. The regional proliferation differences may be inversely dependent to cell-cell contact.

Evaluation of RPE65, CRALBP, VEGF, CD68, and Tyrosinase Gene Expression in Human Retinal Pigment Epithelial Cells Cultured on Amniotic Membrane

The retinal pigment epithelium (RPE) plays a key role in the maintenance of the normal functions of the retina. Tissue engineering using amniotic membrane as a substrate to culture RPE cells may provide a promising new strategy to replace damaged RPE. We established a method of culturing RPE cells over the amniotic membrane as a support for their growth and transplantation. The transcription of specific genes involved in cellular function of native RPE, including RPE65, CRALBP, VEGF, CD68, and tyrosinase, were then measured using quantitative real-time PCR. Data showed a considerable increase in transcription of RPE65, CD68, and VEGF in RPE cells cultured on amniotic membrane. The amounts of CRALBP and tyrosinase transcripts were not affected. This may simply indicate that amniotic membrane restricted dedifferentiation of RPE cells in culture. The results suggest that amniotic membrane may be considered as an elective biological substrate for RPE cell culture.

The neonatal Fc receptor is expressed by human retinal pigment epithelial cells and is downregulated by tumour necrosis factor-alpha

Background/aimsThe neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from catabolism, controls its transport between cell layers and extends its serum half-life. In the human, vitreous IgG can be found,...

A Nanofibrillar Surface Promotes Superior Growth Characteristics in Cultured Human Retinal Pigment Epithelium

Background: To evaluate the influence of surface topography on the proliferation of the retinal pigment epithelium (RPE) by comparing nanofibrillar and smooth substrates. Methods: Electrospun polyamide nanofibers (EPN) are an engineered surface mimicking native basement membranes. Commonly used plastic (polystyrene, PS) and glass substrates have a smooth topography. All were analyzed by scanning electron microscopy. RPE cultures were established from fetal and adult donors. Growth curves were established on the above substrates. Cell cycle and growth fractions were analyzed with 5-ethynyl-2′-deoxyuridine (EdU) and 4′,6-diamidino-2-phenylindole (DAPI). Results: At a magnification of ×5,000, EPN showed randomly overlapping fibers and pores. The surface of glass was slightly studded yet regular, in contrast to ideally smooth PS. Polygonal cells grew on nanofibers in a colony-like distribution, while randomly spread spindle-shaped cell morphologies were seen on smooth surfaces. This was observed at all donor ages. Initial proliferation rates were higher on EPN, and similar final cell densities were reached in all age groups, compared to an age-related decline on PS. EdU/DAPI revealed faster cell cycles on EPN. Growth fractions were higher and maintained longer on EPN. Observed substrate differences in growth behavior were statistically significant. Conclusion: Surface topography appears to induce distinct RPE proliferation characteristics.

αB Crystallin Is Apically Secreted within Exosomes by Polarized Human Retinal Pigment Epithelium and Provides Neuroprotection to Adjacent Cells

αB Crystallin is a chaperone protein with anti-apoptotic and anti-inflammatory functions and has been identified as a biomarker in age-related macular degeneration. The purpose of this study was to determine whether αB crystallin is secreted from retinal pigment epithelial (RPE) cells, the mechanism of this secretory pathway and to determine whether extracellular αB crystallin can be taken up by adjacent retinal cells and provide protection from oxidant stress. We used human RPE cells to establish that αB crystallin is secreted by a non-classical pathway that involves exosomes. Evidence for the release of exosomes by RPE and localization of αB crystallin within the exosomes was achieved by immunoblot, immunofluorescence, and electron microscopic analyses. Inhibition of lipid rafts or exosomes significantly reduced αB crystallin secretion, while inhibitors of classic secretory pathways had no effect. In highly polarized RPE monolayers, αB crystallin was selectively secreted towards the apical, photoreceptor-facing side. In support, confocal microscopy established that αB crystallin was localized predominantly in the apical compartment of RPE monolayers, where it co-localized in part with exosomal marker CD63. Severe oxidative stress resulted in barrier breakdown and release of αB crystallin to the basolateral side. In normal mouse retinal sections, αB crystallin was identified in the interphotoreceptor matrix. An increased uptake of exogenous αB crystallin and protection from apoptosis by inhibition of caspase 3 and PARP activation were observed in stressed RPE cultures. αB Crystallin was taken up by photoreceptors in mouse retinal explants exposed to oxidative stress. These results demonstrate an important role for αB crystallin in maintaining and facilitating a neuroprotective outer retinal environment and may also explain the accumulation of αB crystallin in extracellular sub-RPE deposits in the stressed microenvironment in age-related macular degeneration. Thus evidence from our studies supports a neuroprotective role for αB crystallin in ocular diseases.

Attainment of polarity promotes growth factor secretion by retinal pigment epithelial cells: Relevance to age-related macular degeneration

The antiangiogenic and neurotrophic growth factor, pigment epithelial derived factor (PEDF), and the proangiogenic growth factor, vascular endothelial growth factor-A (VEGF), are released from retinal pigment epithelial (RPE) cells where they play a critical role in the pathogenesis of age-related macular degeneration (AMD). Since RPE polarity may be altered in advanced AMD, we studied the effect of polarization of differentiated, human RPE monolayer cultures on expression and secretion of PEDF and VEGF. Polarized RPE demonstrated apical microvilli, expression of tight junction proteins, apical localization of Na/K- ATPase, and high transepithelial resistance (490 +/- 17 Omega x cm(2)). PEDF secretion was about 1000 fold greater than that for VEGF in both polarized and non-polarized cultures. Polarization of the RPE monolayer increased PEDF secretion, which was predominantly apical, by 34 fold (p<0.02) and VEGF secretion, which was predominantly basolateral, by 5.7 fold (p<0.02). Treatment of non-polarized RPE cultures with bone morphogenetic protein-4 (BMP-4) had no effect on PEDF or VEGF secretion, but resulted in a dose-dependent >2-fold increase in basolateral VEGF secretion (p<0.05) in polarized cultures. Our data show that polarity is an important determinant of the level of PEDF and VEGF secretion in RPE and support the contention that loss of polarity of RPE in AMD results in marked loss of neurotrophic and vascular support for the retina potentially leading to photoreceptor loss and blindness.

IFNγ regulates retinal pigment epithelial fluid transport

The present experiments show that IFNgamma receptors are mainly localized to the basolateral membrane of human retinal pigment epithelium (RPE). Activation of these receptors in primary cultures of human fetal RPE inhibited cell proliferation and migration, decreased RPE mitochondrial membrane potential, altered transepithelial potential and resistance, and significantly increased transepithelial fluid absorption. These effects are mediated through JAK-STAT and p38 MAPK signaling pathways. Second messenger signaling through cAMP-PKA pathway- and interferon regulatory factor-1-dependent production of nitric oxide/cGMP stimulated the CFTR at the basolateral membrane and increased transepithelial fluid absorption. In vivo experiments using a rat model of retinal reattachment showed that IFNgamma applied to the anterior surface of the eye can remove extra fluid deposited in the extracellular or subretinal space between the retinal photoreceptors and RPE. Removal of this extra fluid was blocked by a combination of PKA and JAK-STAT pathway inhibitors injected into the subretinal space. These results demonstrate a protective role for IFNgamma in regulating retinal hydration across the outer blood-retinal barrier in inflammatory disease processes and provide the basis for possible therapeutic interventions.

Expression and regulation of enzymes in the ceramide metabolic pathway in human retinal pigment epithelial cells and their relevance to retinal degeneration

Ceramide and its metabolic derivatives are important modulators of cellular apoptosis and proliferation. Dysregulation or imbalance of their metabolic pathways may promote the development of retinal degeneration. The aim of this study was to identify the expression and regulation of key enzymes of the ceramide pathway in retinal pigment epithelial (RPE) cells. RT-PCR was used to screen the enzymes involved in ceramide metabolism that are expressed in RPE. Over-expression of neutral sphingomyelinase-2 (SMPD3) or sphingosine kinase 1 (Sphk1) in ARPE-19 cells was achieved by transient transfection of SMPD3 or Sphk1 cDNA subcloned into an expression vector. The number of apoptotic or proliferating cells was determined using TUNEL and BrdU assays, respectively. Neutral sphingomyelinase-1, neutral sphingomyelinase-2, acidic ceramidase, ceramide kinase, SphK1 and Sphk2 were expressed in both ARPE-19 and early passage human fetal RPE (fRPE) cells, while alkaline ceramidase 2 was only expressed in fRPE cells. Over-expression of SMPD3 decreased RPE cell proliferation and increased cell apoptosis. The percentage of apoptotic cells increased proportionally with the amount of transfected SMPD3 DNA. Over-expression of SphK1 promoted cell proliferation and protected ARPE-19 cells from ceramide-induced apoptosis. The effect of C 2 ceramide on induction of apoptosis was evaluated in polarized vs. non-polarized RPE cultures; polarization of RPE was associated with much reduced apoptosis in response to ceramide. In conclusion, RPE cells possess the synthetic machinery for the production of ceramide, sphingosine, ceramide-1-phosphate (C1P), and sphingosine-1-phosphate (S1P). Over-expression of SMPD3 may increase cellular ceramide levels, leading to enhanced cell death and arrested cell proliferation. The selective induction of apoptosis in non-polarized RPE cultures by C 2 ceramide suggests that increased ceramide levels will preferentially affect non-polarized RPE, as are found in late age-related macular degeneration lesions, and may spare the normal RPE monolayer. SphK1 over-expression increased cellular S1P, which promoted cell proliferation and protected RPE from ceramide-induced apoptosis. Understanding the relationship between the metabolism of sphingolipids and their effects in RPE cell survival/death may help us to develop effective and efficient therapies for retinal degeneration.

Expression of the iron-regulatory protein haemojuvelin in retina and its regulation during cytomegalovirus infection

Haemochromatosis is a genetic disorder of iron overload resulting from loss-of-function mutations in genes coding for the iron-regulatory proteins HFE [HLA-like protein involved in iron (Fe) homoeostasis], transferrin receptor 2, ferroportin, hepcidin and HJV (haemojuvelin). Expression of the first four genes coding for these proteins in retina has been established. Here we report on the expression of HJV. Since infection of retina with CMV (cytomegalovirus) causes blindness, we also investigated the expression of HJV and other iron-regulatory proteins in retina during CMV infection. HJV (HJV gene) mRNA was expressed in RPE (retinal pigment epithelium)/eyecup and neural retina in mouse. In situ hybridization and immunohistochemistry confirmed the presence of HJV mRNA and protein in RPE, outer and inner nuclear layers, and ganglion cell layer. Immunocytochemistry with cell lines and primary cell cultures showed HJV expression in RPE and Müller cells. In RPE, the expression was restricted to apical membrane. Infection of primary cultures of mouse RPE with CMV increased HJV mRNA and protein levels. Under similar conditions, HFE (HFE gene) mRNA levels were not altered, but HFE protein was decreased. Hepcidin expression was, however, not altered. These findings were demonstrable in vivo with CMV-infected mouse retina. The CMV-induced up-regulation of HJV in RPE was independent of changes in HFE because the phenomenon was also seen in HFE-null RPE cells. CMV-infected primary RPE cells showed evidence of iron accumulation and oxidative stress, as indicated by increased levels of ferritin and hydroxynonenal. The observed changes in HJV expression and iron status during CMV infection in retina may have significance in the pathophysiology of CMV retinitis.

Bacterial endotoxin activates retinal pigment epithelial cells and induces their degeneration through IL-6 and IL-8 autocrine signaling

Inflammation is a major contributing factor to many blinding disorders including uveitis, diabetic retinopathy, and age-related macular degeneration. Here we examined the response of the retinal pigment epithelium (RPE) to physiological levels of lipopolysaccharide (LPS) to understand the role of this epithelium in inflammatory retinal conditions. Expression of a group of inflammatory mediators was identified by gene array analysis and confirmed by PCR and immunocytochemistry in primary human RPE cultures and ARPE19. The effects of LPS on the expression of these cytokines and RPE survival were examined by PCR, Luminex bead, and MTT assays. RPE cells express many cytokine receptors including IL-1R, -4R, -6R, -8RA, IFNAR1, IFNGR1/2 and secrete a range of pro- and anti-inflammatory cytokines including IL-4, -6, -8, -10, -17, IFN-γ, MCP-1, and VEGF. LPS increases IL-13RA1 and IFNAR1, and decreases IL-7R receptor expression. It also increases RPE secretion of IL-4, -6, -8, -10, IFN-γ and MCP-1, and is toxic to RPE cells at LC50=17.7μg/ml. LPS toxicity is mediated by IL-6 and IL-8 through an autocrine feedback loop. Silencing IL-6R and IL-8RA gene expression by siRNA blocks death by their respective ligands or LPS. These findings imply that RPE cells are acutely sensitive to inflammatory stress and that over secretion of IL-6 and IL-8 by this epithelium during inflammatory stimulus may be an underlying factor in the progression of some retinal pathologies.

Biologically Active Fibronectin Fragments Stimulate Release of MCP-1 and Catabolic Cytokines from Murine Retinal Pigment Epithelium

High-temperature requirement serine protease (HTRA1) was identified as a candidate age-related macular degeneration gene in multiple genetic studies in humans. To date, no functional studies have shown a mechanism for HTRA1 to instigate ocular tissue abnormalities. In the present study, the authors focused on a substrate of HTRA1, fibronectin, because fibronectin fragments (Fnfs) stimulate biochemical events in other age-related degenerative diseases that are analogous to changes associated with age-related macular degeneration (AMD). The purpose of the study was to determine whether Fnfs stimulate the release of proinflammatory and catabolic cytokines from murine retinal pigment epithelium (RPE). Fibronectin was purified from murine serum by gelatin cross-linked agarose chromatography and subsequently was enzymatically digested with alpha-chymotrypsin. The bioactivity of Fnfs was verified by measuring levels of IL-6 and TNF-alpha in Fnf-exposed murine splenocytes. To analyze the effect of Fnfs on RPE, cytokine and chemokine levels in RPE culture supernatants were assayed by ELISA. IL-6 and TNF-alpha proinflammatory cytokines were released from primary murine splenocytes in proportion to the dose and length of Fnf treatment, indicating that alpha-chymotryptic digests of fibronectin are biologically active. Fnf treatment of murine RPE cells stimulated the release of microgram and nanogram levels of IL-6, MMP-3, MMP-9, and MCP-1, whereas only picogram levels were detected in untreated cells. Fnfs stimulate the release of proinflammatory cytokines, matrix metalloproteinases, and monocyte chemoattractant protein from murine RPE cells. This observation indicated that Fnfs could contribute to ocular abnormalities by promoting inflammation, catabolism, and monocyte chemoattraction.

Up-regulation of complement factor B in retinal pigment epithelial cells is accompanied by complement activation in the aged retina

Complement activation is involved in the pathogenesis of age-related macular degeneration. How complement is activated in the retina is not known. Previously we have shown that complement factor H (CFH) is constitutively expressed by retinal pigment epithelial (RPE) cells and the production of CFH is negatively regulated by inflammatory cytokines and oxidative insults. Here we investigated the production and regulation of complement factor B (CFB) in RPE cells. Immunohistochemistry showed that CFB is expressed at low levels on the apical portion of the RPE cells in normal physiological conditions. With age, CFB expression increases and extends to the basal part of RPE cells. Confocal microscopy and real-time PCR of RPE cultures indicated that the production of CFB by RPE cells is positively regulated by TNF-α, IFN-γ and long-term (30 days) photoreceptor outer segments treatments. Increased CFB expression in RPE cells in vivo is accompanied by the accumulation of complement C3 and C3a deposition at the Bruch's membrane and the basal layer of RPE cells. Our results suggest that RPE cells play important roles in regulating complement activation in the retina. Increased complement activation in the aged retina may be important for retinal homeostasis in the context of accumulating photoreceptor waste products.

Epithelial phenotype and the RPE: Is the answer blowing in the Wnt?

Cells of the human retinal pigment epithelium (RPE) have a regular epithelial cell shape within the tissue in situ, but for reasons that remain elusive the RPE shows an incomplete and variable ability to re-develop an epithelial phenotype after propagation in vitro. In other epithelial cell cultures, formation of an adherens junction (AJ) composed of E-cadherin plays an important early inductive role in epithelial morphogenesis, but E-cadherin is largely absent from the RPE. In this review, the contribution of cadherins, both minor (E-cadherin) and major (N-cadherin), to RPE phenotype development is discussed. Emphasis is placed on the importance for future studies of actin cytoskeletal remodeling during assembly of the AJ, which in epithelial cells results in an actin organization that is characteristically zonular. Other markers of RPE phenotype that are used to gauge the maturation state of RPE cultures including tissue-specific protein expression, protein polarity, and pigmentation are described. An argument is made that RPE epithelial phenotype, cadherin-based cell-cell adhesion and melanization are linked by a common signaling pathway: the Wnt/beta-catenin pathway. Analyzing this pathway and its intersecting signaling networks is suggested as a useful framework for dissecting the steps in RPE morphogenesis. Also discussed is the effect of aging on RPE phenotype. Preliminary evidence is provided to suggest that light-induced sub-lethal oxidative stress to cultured ARPE-19 cells impairs organelle motility. Organelle translocation, which is mediated by stress-susceptible cytoskeletal scaffolds, is an essential process in cell phenotype development and retention. The observation of impaired organelle motility therefore raises the possibility that low levels of stress, which are believed to accompany RPE aging, may produce subtle disruptions of cell phenotype. Over time these would be expected to diminish the support functions performed by the RPE on behalf of photoreceptors, theoretically contributing to aging retinal disease such as age-related macular degeneration (AMD). Analyzing sub-lethal stress that produces declines in RPE functional efficiency rather than overt cell death is suggested as a useful future direction for understanding the effects of age on RPE organization and physiology. As for phenotype and pigmentation, a role for the Wnt/beta-catenin pathway is also suggested in regulating the RPE response to oxidative stress. Exploration of this pathway in the RPE therefore may provide a unifying strategy for advancing our understanding of both RPE phenotype and the consequences of mild oxidative stress on RPE structure and function.

Novel organotypic culture model of adult mammalian neurosensory retina in co-culture with retinal pigment epithelium

PurposeThe purpose of this study was to assess survival of adult mammalian neurosensory retina cultured in contact with the layer of a choroid–retinal pigment epithelium (RPE) explant. MethodsThe entire adult porcine neurosensory retina and RPE–choroid layer were placed in tissue culture by juxtaposing both tissues in their original orientation. Culture of the neurosensory retina alone and freshly prepared retina were used as control. After 3 days in culture retinal explants were fixed and processed for immunohistochemistry and TUNEL technique. ResultsWe observed limited nuclei loss and significant reduction in apoptotic cells in nuclear cell layers (GCL, INL, and ONL) and decreased Muller cell hypertrophy in retina–RPE cultures compared to retinal cultures alone. In addition, cultures were characterized by reduced upregulation of GFAP, vimentin as well as S100 and increased glutamine synthetase expression. ConclusionsAs any tissue culture model, retinal tissue culture is a short-term system and since degenerative processes begin quite early it may be a good model to investigate degenerative processes in the retina. However, our model of culture of retina adjacent to the RPE–choroid layer improves the maintenance of neural retina as evidenced by reduced apoptosis in nuclear cell layers (GCL, INL, and ONL) and reduced gliosis as indicated by the diminished expression of glial-specific proteins and increased glutamine synthetase compared to cultures of retina alone. Thus the retina–RPE–choroid culture system can enable the evaluation of interactions between RPE and neural retina, the role of signaling molecules as well the effect of pharmaceuticals on retinal biology.

A Novel Rabbit Model for Studying RPE Transplantation

The goal of this project was to develop a model of retinal pigment epithelium (RPE) transplantation that permits extensive and reliable analysis of the transplants. Cultures of newborn rabbit RPE were evaluated by morphology, electrophysiology, and the expression of zonula occludens-1, cytokeratin, and the melanocyte marker S-100. Cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) were transplanted into the subretinal space of rabbits with a 30-gauge needle without making a conjunctival flap or sclerotomy. The transplants were examined by fundus photography, confocal scanning laser ophthalmoscopy (cSLO), optical coherence tomography (OCT), and angiography. At 2 months, the retina was examined histochemically. A 1-minute incubation at 37 degrees C with 20 muM CFDA-SE did not affect morphology or the expression of marker proteins. In coculture, the labeled cells integrated into monolayers that developed a normal transepithelial electrical resistance of 400 to 450 Omega . cm(-2). Dye was not transferred from labeled to nonlabeled RPE cells. Transplanted RPE was detectable for at least 2 months. Angiography demonstrated an intact blood-retinal barrier. The normal morphology of the retina and lack of debris in the subretinal space suggested that the transplanted RPE was functional. Primary cultures of newborn rabbit RPE were highly differentiated, even when labeled with CFDA-SE. Labeled cells were observed long-term in vitro and in vivo. This model can be used to examine how culture and transplantation protocols affect the reformation of a functional RPE monolayer. The similar size of rabbit and human eyes will facilitate the translation of these protocols to the bedside.

N-(4-hydroxyphenyl) Retinamide Augments Laser-Induced Choroidal Neovascularization in Mice

To evaluate the effect of N-4-hydroxyphenyl retinamide (4-HPR) on experimental laser-induced choroidal neovascularization (CNV) and on the expression and secretion of relevant growth factors by cultured human retinal pigment epithelial (RPE) cells. CNV was induced by laser photocoagulation in C57BL/6 mice. 4-HPR (0.2 or 1 mg) or vehicle, was injected intraperitoneally twice daily for 14 days. Plasma and tissue levels of 4-HPR were measured by HPLC. CNV was evaluated by fluorescein angiography, histology, and quantitative confocal analysis of isolectin B4 histochemistry on days 7 and 14. Induction of apoptosis and expression and secretion of growth factors was studied in 4-HPR-treated RPE cultures. Mice treated with 4-HPR exhibited time- and dose-dependent increases in plasma and tissue 4-HPR levels. CNV lesions showed increased volume with increased vascular leakage and contained fewer lesion-associated RPE in treated versus untreated mice. Treatment of nonpolarized RPE cultures with 4-HPR in the presence of serum resulted in RPE apoptosis; however, apoptosis was minimal in similarly treated highly polarized RPE. Treatment of RPE cells with 4-HPR resulted in the upregulation of VEGF-A and -C (P < 0.05) and Ang-1 (P < 0.01) mRNA and increased secretion of VEGF-A and -C (P < 0.05), whereas pigment epithelium-derived growth factor (PEDF) and thrombospondin (TSP)-1 mRNA expression and secretion were downregulated (P < 0.05). 4-HPR increases lesion size and leakage in laser-induced CNV and is associated with the upregulation of key proangiogenic factors and the downregulation of antiangiogenic factors. Consistent with the preferential loss of RPE in CNV lesions in vivo, 4-HPR induces apoptosis of nonpolarized RPE in the presence of serum.

Constancy of ERp29 Expression in Cultured Retinal Pigment Epithelial Cells in the Ccl2/Cx3cr1 Deficient Mouse Model of Age-Related Macular Degeneration

Purpose: Given the critical role of the retinal pigment epithelium (RPE) in the pathogenesis of age-related macular generation (AMD) and the links drawn between chaperone proteins and neurodegenerative disease, we aimed to culture RPE from the Ccl2−/−/Cx3cr1−/− mouse model of AMD and evaluate expression of chaperone protein ERp29. Materials and Methods: Murine RPE cells were surgically and chemically isolated and cultured. ERp29 mRNA and protein expression were evaluated by real-time RT-PCR, immunohistochemistry, and Western blot. Results: Ccl2−/−/Cx3cr1−/− RPE was successfully isolated and cultured. They presented a decreased but statistically insignificant difference in ERp29 transcript and protein expression compared to C57B6/L wild type mouse RPE. Conclusions: The effective murine RPE culture described here enables future investigation into RPE biology and AMD pathogenesis. Although we found a decrease in ERp29 expression in the Ccl2−/−/Cx3cr1−/− RPE, the difference was less than we previously observed in the whole retina. This suggests that the RPE may not contribute to the greater differential expression and ERp29 might have a more significant role in the neuroretina than in the RPE during AMD pathogenesis.