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Sample records for renders retinal pigment

  1. Neoplasia versus hyperplasia of the retinal pigment epithelium

    DEFF Research Database (Denmark)

    Heegaard, Steffen; Larsen, J.N.B.; Fledelius, Hans C.

    2001-01-01

    ophthalmology, retinal pigment epithelium, adenoma, tumor-like hyperplasia, histology, immunohistochemistry, tumor, neoplasm, ultrasonography......ophthalmology, retinal pigment epithelium, adenoma, tumor-like hyperplasia, histology, immunohistochemistry, tumor, neoplasm, ultrasonography...

  2. Comparison of antioxidation systems of retinal pigment epithelium of pigmented and albino animals

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    Sakina, N.L.; Dontsov, A.E.; Ostrovskiy, M.A.

    1985-01-01

    The effectiveness of the lipid peroxidation inhibition process by tissue homogenates of retinal pigment epithelium of pigmented rabbits is higher than that of albino rabbits. The superoxide dismutase and glutathione perioxidase activity is nearly the same in both tissues of the pigment epithelium, the ..cap alpha..-tocopherol content is higher in retinal pigment epithelium tissue of albino animals, and the oxidizability of the lipid fraction of pigment epithelium tissue is higher in pigmented animals than in albinos. It is concluded that the higher resistance of the pigment epithelium of pigmented animals to the effects of prooxidant systems is due to the presence of melanoprotein granules in the pigment epithelium.

  3. Growth of cultured porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Wiencke, A.K.; Kiilgaard, Jens Folke; Nicolini, Jair;

    2003-01-01

    To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation.......To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation....

  4. Functional annotation of the human retinal pigment epithelium transcriptome

    NARCIS (Netherlands)

    J.C. Booij (Judith); S. van Soest (Simone); S.M.A. Swagemakers (Sigrid); A.H.W. Essing (Anke); J.H.M. Verkerk (Annemieke); P.J. van der Spek (Peter); T.G.M.F. Gorgels (Theo); A.A.B. Bergen (Arthur)

    2009-01-01

    textabstractBackground: To determine level, variability and functional annotation of gene expression of the human retinal pigment epithelium (RPE), the key tissue involved in retinal diseases like age-related macular degeneration and retinitis pigmentosa. Macular RPE cells from six selected healthy

  5. Functional annotation of the human retinal pigment epithelium transcriptome

    NARCIS (Netherlands)

    Booij, J.C.; van Soest, S.; Swagemakers, S.M.A.; Essing, A.H.W.; Verkerk, A.J.M.H.; van der Spek, P.J.; Gorgels, T.G.M.F.; Bergen, A.A.B.

    2009-01-01

    ABSTRACT: BACKGROUND: To determine level, variability and functional annotation of gene expression of the human retinal pigment epithelium (RPE), the key tissue involved in retinal diseases like age-related macular degeneration and retinitis pigmentosa. Macular RPE cells from six selected healthy hu

  6. Glucose metabolism in rat retinal pigment epithelium.

    Science.gov (United States)

    Coffe, Víctor; Carbajal, Raymundo C; Salceda, Rocío

    2006-01-01

    The retinal pigment epithelium (RPE) is the major transport pathway for exchange of metabolites and ions between choroidal blood supply and the neural retina. To gain insight into the mechanisms controlling glucose metabolism in RPE and its possible relationship to retinopathy, we studied the influence of different glucose concentrations on glycogen and lactate levels and CO(2) production in RPE from normal and streptozotocin-treated diabetic rats. Incubation of normal RPE in the absence of glucose caused a decrease in lactate production and glycogen content. In normal RPE, increasing glucose concentrations from 5.6 mM to 30 mM caused a four-fold increase in glucose accumulation and CO(2) yield, as well as reduction in lactate and glycogen production. In RPE from diabetic rats glucose accumulation did not increase in the presence of high glucose substrate, but it showed a four- and a seven-fold increase in CO(2) production through the mitochondrial and pentose phosphate pathways, respectively. We found high glycogen levels in RPE which can be used as an energy reserve for RPE itself and/or neural retina. Findings further show that the RPE possesses a high oxidative capacity. The large increase in glucose shunting to the pentose phosphate pathway in diabetic retina exposed to high glucose suggests a need for reducing capacity, consistent with increased oxidative stress.

  7. Culturing of retinal pigment epithelium cells.

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    Valtink, Monika; Engelmann, Katrin

    2009-01-01

    The retinal pigment epithelium (RPE) is a monolayer of cells adjacent to the photoreceptors of the retina. It plays a crucial role in maintaining photoreceptor health and survival. Degeneration or dysfunction of the RPE can lead to photoreceptor degeneration and as a consequence to visual impairment. The most common diseased state of the RPE becomes manifest in age-related macular degeneration, an increasing cause of blindness in the elderly. RPE cells are therefore of great interest to researchers working in the field of tissue engineering and cell transplantation. In fact, studies in animal models have proven that the transplantation of RPE cells can delay the course of photoreceptor degenerative diseases. Although first attempts to transplant RPE cells into the subretinal space in human individuals suffering from age-related macular degeneration were less successful, RPE cell transplantation is still favored as a future therapeutic option, and much work is done to develop and design cell transplants. Cell banking is a prerequisite to have well-differentiated and characterized cells at hand when needed for research purposes, but also for therapeutic approaches. In this chapter the authors will describe methods to isolate, culture and preserve adult human RPE cells for the purpose of RPE cell banking. Copyright 2009 S. Karger AG, Basel.

  8. Rapid Retinal Release from a Cone Visual Pigment Following Photoactivation*

    Science.gov (United States)

    Chen, Min-Hsuan; Kuemmel, Colleen; Birge, Robert R.; Knox, Barry E.

    2012-01-01

    As part of the visual cycle, the retinal chromophore in both rod and cone visual pigments undergoes reversible Schiff base hydrolysis and dissociation following photobleaching. We characterized light-activated retinal release from a short-wavelength sensitive cone pigment (VCOP) in 0.1% dodecyl maltoside using fluorescence spectroscopy. The half-time (t1/2) of retinal release from VCOP was 7.1 s, 250-fold faster than rhodopsin. VCOP exhibited pH-dependent release kinetics, with the t1/2 decreasing from 23 s to 4 s with pH 4.1 to 8, respectively. However, the Arrhenius activation energy (Ea) for VCOP derived from kinetic measurements between 4° and 20°C was 17.4 kcal/mol, similar to 18.5 kcal/mol for rhodopsin. There was a small kinetic isotope (D2O) effect in VCOP, but less than that observed in rhodopsin. Mutation of the primary Schiff base counterion (VCOPD108A) produced a pigment with an unprotonated chromophore (⌊max = 360 nm) and dramatically slowed (t1/2 ~ 6.8 min) light-dependent retinal release. Using homology modeling, a VCOP mutant with two substitutions (S85D/ D108A) was designed to move the counterion one alpha helical turn into the transmembrane region from the native position. This double mutant had a UV-visible absorption spectrum consistent with a protonated Schiff base (⌊max = 420 nm). Moreover, VCOPS85D/D108A mutant had retinal release kinetics (t1/2 = 7 s) and Ea (18 kcal/mol) similar to the native pigment exhibiting no pH-dependence. By contrast, the single mutant VCOPS85D had a ~3-fold decrease in retinal release rate compared to the native pigment. Photoactivated VCOPD108A had kinetics comparable to a rhodopsin counterion mutant, RhoE113Q, both requiring hydroxylamine to fully release retinal. These results demonstrate that the primary counterion of cone visual pigments is necessary for efficient Schiff base hydrolysis. We discuss how the large differences in retinal release rates between rod and cone visual pigments arise, not from

  9. Detachments of the retinal pigment epithelium at the posterior pole.

    Science.gov (United States)

    Noble, K G; Levitzky, M J; Carr, R E

    1976-08-01

    Multiple vitelliform cysts of the retina, a disorder of unknown cause in which there are multiple detachments of the retinal pigment epithelium at the posterior pole, occurred in five patients. In four patients all lesions were located outside the parafoveal area while one patient showed bilateral foveal elevations associated with more eccentric detachments. Several patients showed slow resolution of some of the detachments with mild disturbances of the pigment epithelium.

  10. Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill

    Science.gov (United States)

    González, Alfredo; Crittenden, Elizabeth L; García, Dana M

    2004-01-01

    Background In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. Results The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl) carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Conclusions Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion. PMID:15251036

  11. Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill

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    Crittenden Elizabeth L

    2004-07-01

    Full Text Available Abstract Background In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. Results The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Conclusions Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

  12. Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations

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    Aaron H Fronk

    2016-07-01

    Full Text Available The retinal pigment epithelium is an important part of the vertebrate eye, particularly in studying the causes and possible treatment of age-related macular degeneration. The retinal pigment epithelium is difficult to access in vivo due to its location at the back of the eye, making experimentation with age-related macular degeneration treatments problematic. An alternative to in vivo experimentation is cultivating the retinal pigment epithelium in vitro, a practice that has been going on since the 1970s, providing a wide range of retinal pigment epithelial culture protocols, each producing cells and tissue of varying degrees of similarity to natural retinal pigment epithelium. The purpose of this review is to provide researchers with a ready list of retinal pigment epithelial protocols, their effects on cultured tissue, and their specific possible applications. Protocols using human and animal retinal pigment epithelium cells, derived from tissue or cell lines, are discussed, and recommendations for future researchers included.

  13. In vitro differentiation of retinal pigment epithelium from adult retinal stem cells.

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    Aruta, Claudia; Giordano, Francesca; De Marzo, Anna; Comitato, Antonella; Raposo, Graça; Nandrot, Emeline F; Marigo, Valeria

    2011-02-01

    One of the limitations in molecular and functional studies of the retinal pigment epithelium (RPE) has been the lack of an in vitro system retaining all the features of in vivo RPE cells. Retinal pigment epithelium cell lines do not show characteristics typical of a functional RPE, such as pigmentation and expression of specific markers. The present study was aimed at the development of culture conditions to differentiate, in vitro, retinal stem cells (RSC), derived from the adult ciliary body, into a functional RPE. Retinal stem cells were purified from murine eyes, grown as pigmented neurospheres and induced to differentiate into RPE on an extracellular matrix substrate using specific culture conditions. After 7-15 days of culture, pigmented cells with an epithelial morphology showed a polarized organization and a capacity for phagocytosis. We detected different stages of melanogenesis in cells at 7 days of differentiation, whereas RPE at 15 days contained only mature melanosomes. These data suggest that our protocol to differentiate RPE in vitro can provide a useful model for molecular and functional studies.

  14. [Classification signs of end-stage pigmented retinitis].

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    Zhukova, S I; Shchuko, A G; Malyshev, V V

    2007-01-01

    Identification of objective criteria for early-stage pigmented retinitis (PR) remains urgent today. Visual system changes reflecting retinal metabolic and structural disturbances (a change in the time and amplitude parameters of ERG, an increase in dark adaptation, changes in the color palette and the thickness of layers of photoreceptors and pigment epithelium of the retina on the OST scans) were detected in 31% of the examined relatives of patients with PR. The authors show the diagnostic value of retinal optic coherent tomography in the diagnosis of PR and the expediency of its use for objective estimation of retinal structural changes along with functional studies. The statistical studies including descriptive, regression, and discriminant analyses have provided evidence that the characteristics of the visual system in patients with end-stage PR differ from those in the controls. Studies that can determine differences in the state of the visual system of the groups under study and significantly discriminate persons with the normally functioning visual system from patients with PR have been identified.

  15. Aquaporin-1 Expression in Retinal Pigment Epithelial Cells Overlying Retinal Drusen

    DEFF Research Database (Denmark)

    Tran, Thuy Linh; Bek, Toke; la Cour, Morten

    2016-01-01

    PURPOSE: In the outer retina, age-related macular degeneration (AMD) results in reduced hydraulic conductivity in Bruch's membrane, possibly leading to altered water transport in retinal pigment epithelial (RPE) cells. We hypothesize that RPE cells may express aquaporin-1 (AQP1) to compensate...

  16. Optical modulation of transgene expression in retinal pigment epithelium

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    Palanker, D.; Lavinsky, D.; Chalberg, T.; Mandel, Y.; Huie, P.; Dalal, R.; Marmor, M.

    2013-03-01

    Over a million people in US alone are visually impaired due to the neovascular form of age-related macular degeneration (AMD). The current treatment is monthly intravitreal injections of a protein which inhibits Vascular Endothelial Growth Factor, thereby slowing progression of the disease. The immense financial and logistical burden of millions of intravitreal injections signifies an urgent need to develop more long-lasting and cost-effective treatments for this and other retinal diseases. Viral transfection of ocular cells allows creation of a "biofactory" that secretes therapeutic proteins. This technique has been proven successful in non-human primates, and is now being evaluated in clinical trials for wet AMD. However, there is a critical need to down-regulate gene expression in the case of total resolution of retinal condition, or if patient has adverse reaction to the trans-gene products. The site for genetic therapy of AMD and many other retinal diseases is the retinal pigment epithelium (RPE). We developed and tested in pigmented rabbits, an optical method to down-regulate transgene expression in RPE following vector delivery, without retinal damage. Microsecond exposures produced by a rapidly scanning laser vaporize melanosomes and destroy a predetermined fraction of the RPE cells selectively. RPE continuity is restored within days by migration and proliferation of adjacent RPE, but since the transgene is not integrated into the nucleus it is not replicated. Thus, the decrease in transgene expression can be precisely determined by the laser pattern density and further reduced by repeated treatment without affecting retinal structure and function.

  17. Druse-Induced Morphology Evolution in Retinal Pigment Epithelium

    CERN Document Server

    Mazzitello, K I; Chrenek, M A; Family, F; Grossniklaus, H E; Nickerson, J M; Jiang, Y

    2016-01-01

    The retinal pigment epithelium (RPE) is a key site of pathogenesis for many retina diseases. The formation of drusen in the retina is characteristic of retinal degeneration. We investigate morphological changes in the RPE in the presence of soft drusen using an integrated experimental and modeling approach. We collect RPE flat mount images from donated human eyes and develop 1) statistical tools to quantify the images and 2) a cell-based model to simulate the morphology evolution. We compare three different mechanisms of RPE repair evolution, cell apoptosis, cell fusion, and expansion, and Simulations of our RPE morphogenesis model quantitatively reproduce deformations of human RPE morphology due to drusen, suggesting that a purse-string mechanism is sufficient to explain how RPE heals cell loss caused by drusen-damage. We found that drusen beneath tissue promote cell death in a number that far exceeds the cell numbers covering the drusen. Tissue deformations are studied using area distributions, Voronoi doma...

  18. Retinal Pigment Epithelium Cell Alignment on Nanostructured Collagen Matrices

    OpenAIRE

    Ulbrich, Stefan; Friedrichs, Jens; Valtink, Monika; Murovski, Simo; Franz, Clemens M.; Müller, Daniel J.; Richard H. W. Funk; Engelmann, Katrin

    2014-01-01

    We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α2 were examined b...

  19. Enhanced generation of retinal progenitor cells from human retinal pigment epithelial cells induced by amniotic fluid

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    Sanie-Jahromi Fatemeh

    2012-04-01

    Full Text Available Abstract Background Retinal progenitor cells are a convenient source of cell replacement therapy in retinal degenerative disorders. The purpose of this study was to evaluate the expression patterns of the homeobox genes PAX6 and CHX10 (retinal progenitor markers during treatment of human retinal pigment epithelium (RPE cells with amniotic fluid (AF, RPE cells harvested from neonatal cadaver globes were cultured in a mixture of DMEM and Ham's F12 supplemented with 10% FBS. At different passages, cells were trypsinized and co-cultured with 30% AF obtained from normal fetuses of 1416 weeks gestational age. Results Compared to FBS-treated controls, AF-treated cultures exhibited special morphological changes in culture, including appearance of spheroid colonies, improved initial cell adhesion and ordered cell alignment. Cell proliferation assays indicated a remarkable increase in the proliferation rate of RPE cells cultivated in 30% AF-supplemented medium, compared with those grown in the absence of AF. Immunocytochemical analyses exhibited nuclear localization of retinal progenitor markers at a ratio of 33% and 27% for CHX10 and PAX6, respectively. This indicated a 3-fold increase in retinal progenitor markers in AF-treated cultures compared to FBS-treated controls. Real-time PCR data of retinal progenitor genes (PAX6, CHX10 and VSX-1 confirmed these results and demonstrated AF's capacity for promoting retinal progenitor cell generation. Conclusion Taken together, the results suggest that AF significantly promotes the rate of retinal progenitor cell generation, indicating that AF can be used as an enriched supplement for serum-free media used for the in vitro propagation of human progenitor cells.

  20. Transport of protons and lactate in cultured human fetal retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Hamann, Steffen; Cour, Morten la; Ming Lui, Ge

    2000-01-01

    Electron microscopy, intracellular pH, monocarboxylate transport, pigment epithelium of eye, proton-lactate cotransport, retinal metabolism, sodium/proton exchange......Electron microscopy, intracellular pH, monocarboxylate transport, pigment epithelium of eye, proton-lactate cotransport, retinal metabolism, sodium/proton exchange...

  1. Magnetic Nanoparticles as Intraocular Drug Delivery System to Target Retinal Pigmented Epithelium (RPE

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    Martina Giannaccini

    2014-01-01

    Full Text Available One of the most challenging efforts in drug delivery is the targeting of the eye. The eye structure and barriers render this organ poorly permeable to drugs. Quite recently the entrance of nanoscience in ocular drug delivery has improved the penetration and half-life of drugs, especially in the anterior eye chamber, while targeting the posterior chamber is still an open issue. The retina and the retinal pigment epithelium/choroid tissues, located in the posterior eye chamber, are responsible for the majority of blindness both in childhood and adulthood. In the present study, we used magnetic nanoparticles (MNPs as a nanotool for ocular drug delivery that is capable of specific localization in the retinal pigmented epithelium (RPE layer. We demonstrate that, following intraocular injection in Xenopus embryos, MNPs localize specifically in RPE where they are retained for several days. The specificity of the localization did not depend on particle size and surface properties of the MNPs used in this work. Moreover, through similar experiments in zebrafish, we demonstrated that the targeting of RPE by the nanoparticles is not specific for the Xenopus species.

  2. Yap and Taz regulate retinal pigment epithelial cell fate

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    Miesfeld, Joel B.; Gestri, Gaia; Clark, Brian S.; Flinn, Michael A.; Poole, Richard J.; Bader, Jason R.; Besharse, Joseph C.; Wilson, Stephen W.; Link, Brian A.

    2015-01-01

    The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Tead-responsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yap-dependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosage-dependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson's chorioretinal atrophy and congenital retinal coloboma. PMID:26209646

  3. Cytotoxic effects of curcumin in human retinal pigment epithelial cells.

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    Margrit Hollborn

    Full Text Available BACKGROUND: Curcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE cells in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Cellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM and delayed apoptosis (above 1 µM. The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein. CONCLUSION: It is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (∼10 µM has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as

  4. Cytotoxic Effects of Curcumin in Human Retinal Pigment Epithelial Cells

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    Hollborn, Margrit; Chen, Rui; Wiedemann, Peter; Reichenbach, Andreas; Bringmann, Andreas; Kohen, Leon

    2013-01-01

    Backround Curcumin from turmeric is an ingredient in curry powders. Due to its antiinflammatory, antioxidant and anticarcinogenic effects, curcumin is a promising drug for the treatment of cancer and retinal diseases. We investigated whether curcumin alters the viability and physiological properties of human retinal pigment epithelial (RPE) cells in vitro. Methodology/Principal Findings Cellular proliferation was investigated with a bromodeoxy-uridine immunoassay, and chemotaxis was investigated with a Boyden chamber assay. Cell viability was determined by trypan blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation ELISA. Gene expression was determined by real-time PCR, and secretion of VEGF and bFGF was examined with ELISA. The phosphorylation level of proteins was revealed by Western blotting. The proliferation of RPE cells was slightly increased by curcumin at 10 µM and strongly reduced by curcumin above 50 µM. Curcumin at 50 µM increased slightly the chemotaxis of the cells. Curcumin reduced the expression and secretion of VEGF under control conditions and abolished the VEGF secretion induced by PDGF and chemical hypoxia. Whereas low concentrations of curcumin stimulated the expression of bFGF and HGF, high concentrations caused downregulation of both factors. Curcumin decreased dose-dependently the viability of RPE cells via induction of early necrosis (above 10 µM) and delayed apoptosis (above 1 µM). The cytotoxic effect of curcumin involved activation of caspase-3 and calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress, increased phosphorylation of p38 MAPK and decreased phosphorylation of Akt protein. Conclusion It is concluded that curcumin at concentrations described to be effective in the treatment of tumor cells and in inhibiting death of retinal neurons (∼10 µM) has adverse effects on RPE cells. It is suggested that, during the intake of curcumin as concomitant therapy of

  5. Functional annotation of the human retinal pigment epithelium transcriptome

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    Gorgels Theo GMF

    2009-04-01

    Full Text Available Abstract Background To determine level, variability and functional annotation of gene expression of the human retinal pigment epithelium (RPE, the key tissue involved in retinal diseases like age-related macular degeneration and retinitis pigmentosa. Macular RPE cells from six selected healthy human donor eyes (aged 63–78 years were laser dissected and used for 22k microarray studies (Agilent technologies. Data were analyzed with Rosetta Resolver, the web tool DAVID and Ingenuity software. Results In total, we identified 19,746 array entries with significant expression in the RPE. Gene expression was analyzed according to expression levels, interindividual variability and functionality. A group of highly (n = 2,194 expressed RPE genes showed an overrepresentation of genes of the oxidative phosphorylation, ATP synthesis and ribosome pathways. In the group of moderately expressed genes (n = 8,776 genes of the phosphatidylinositol signaling system and aminosugars metabolism were overrepresented. As expected, the top 10 percent (n = 2,194 of genes with the highest interindividual differences in expression showed functional overrepresentation of the complement cascade, essential in inflammation in age-related macular degeneration, and other signaling pathways. Surprisingly, this same category also includes the genes involved in Bruch's membrane (BM composition. Among the top 10 percent of genes with low interindividual differences, there was an overrepresentation of genes involved in local glycosaminoglycan turnover. Conclusion Our study expands current knowledge of the RPE transcriptome by assigning new genes, and adding data about expression level and interindividual variation. Functional annotation suggests that the RPE has high levels of protein synthesis, strong energy demands, and is exposed to high levels of oxidative stress and a variable degree of inflammation. Our data sheds new light on the molecular composition of BM, adjacent to the

  6. Evaluation of ultraviolet light toxicity on cultured retinal pigment epithelial and retinal ganglion cells

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    Sankarathi Balaiya

    2010-01-01

    Full Text Available Sankarathi Balaiya, Ravi K Murthy, Vikram S Brar, Kakarla V ChalamDepartment of Ophthalmology, University of Florida College of Medicine, Jacksonville, FL, USAPurpose: Our study is aimed at evaluating the role of UVB light in inducing cytotoxicity in an in vitro model.Methods: RGC-5 and ARPE-19 cells were exposed to different time periods of UVB light: 0, 15, 30, and 45 min. They were subsequently examined for changes in cell morphology, cell viability (neutral red uptake assay, generation of reactive oxygen species (ROS, expression of bax, bcl-2 and cytochome C by reverse transcriptase polymerase chain reaction and western blot, respectively.Results: Dose-dependent reduction in cell viability to UVB light was demonstrated with parallel increase in ROS. Increased duration of exposure (>15 minutes, was associated with increased expression of bax and cytochrome C, and absence of bcl-2 expression.Conclusion: UVB light exposure results in cell cytotoxicity. The concomitant generation of ROS and expression of apoptotic markers suggests the role of oxidative stress in UVB-mediated apoptosis in an in vitro model of retinal ganglion and pigment epithelial cells.Keywords: ultraviolet light, retinal pigment epithelium, retinal ganglion cell, reactive oxygen species, cytochrome C

  7. Trafficking of osteonectin by retinal pigment epithelial cells: evidence for basolateral secretion.

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    Ratnayaka, Arjuna; Paraoan, Luminita; Nelson, Glyn; Spiller, Dave G; White, Michael R H; Hiscott, Paul

    2007-01-01

    Osteonectin is a glycoprotein that modulates several aspects of cellular behaviour including proliferation and adhesion. The retinal pigment epithelium forms a continuous monolayer of polarised cells immediately bellow the neuroretina, and is integral to the homeostasis of photoreceptor cells. While osteonectin is expressed by normal retinal pigment epithelium in situ, its expression is significantly increased in retinal pigment epithelial cells associated with several common retinal diseases. This pattern of expression implies an important role for osteonectin in the biology of retinal pigment epithelial cells. However, the trafficking, processing, and eventual fate of osteonectin in these cells is not clear at present. Although the theoretical report of a leader sequence within the osteonectin open reading frame and its extracellular presence in some tissues indirectly support secretion of the protein, there is no direct experimental demonstration of the secretion route to date. As a first step towards understanding the role of osteonectin in retinal pigment epithelium, we studied the intracellular distribution and trafficking of the protein in living cells. Here, we present experimental evidence that a precursor osteonectin fusion protein is targeted to the endoplasmic reticulum/Golgi pathway, with a likely basal secretion in retinal pigment epithelial cells. In addition, we show that the precursor osteonectin protein having the leader sequence masked fails to undergo secretion leading to cell death, a phenotype which may be of relevance not only for retinal pathology, but also for other diseases such as the bone disorder known as pseudoachondroplasia that is associated with a lack of osteonectin secretion.

  8. Effect of curcumin on aging retinal pigment epithelial cells

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    Zhu W

    2015-09-01

    Full Text Available Wei Zhu,1,* Yan Wu,2,* Yi-Fang Meng,1 Jin-Yu Wang,1 Ming Xu,1 Jian-Jun Tao,1 Jiong Lu1 1Department of Ophthalmology, Changshu No 2 People’s Hospital, Changshu, 2Department of Ophthalmology, The First People’s Hospital of Kunshan Affiliated with Jiangsu University, Suzhou, People’s Republic of China *These authors contributed equally to this work Abstract: Age-related macular degeneration (AMD is now one of the leading causes of blindness in the elderly population. The antioxidative effects of curcumin on aging retinal pigment epithelial (RPE cells are still unclear. We conducted an in vitro study to investigate the effects of curcumin on aging RPE cells. A pulsed H2O2 exposure aging model was adopted. Aging RPE cells were treated with curcumin 20 µM, 40 µM, and 80 µM. Apoptosis of RPE cells was analyzed by flow cytometry. The intracellular reactive oxygen species concentration was detected using a specific probe and apoptosis-associated proteins were detected by Western blot. Expression of oxidative biomarkers, including superoxide dismutase, maleic dialdehyde, and glutathione, was detected commercially available assay kits. Compared with normal cells, lower cell viability, higher apoptosis rates, and more severe oxidation status were identified in the aging RPE cell model. Curcumin improved cell viability and decreased apoptosis and oxidative stress. Further, curcumin had a significant influence on expression of apoptosis-associated proteins and oxidative stress biomarkers. In conclusion, treatment with curcumin was able to regulate proliferation, oxidative stress, and apoptosis in aging RPE cells. Accordingly, application of curcumin may be a novel strategy to protect against age-related change in AMD. Keywords: curcumin, retinal pigment epithelium, apoptosis, age-related macular degeneration

  9. Yap and Taz regulate retinal pigment epithelial cell fate.

    Science.gov (United States)

    Miesfeld, Joel B; Gestri, Gaia; Clark, Brian S; Flinn, Michael A; Poole, Richard J; Bader, Jason R; Besharse, Joseph C; Wilson, Stephen W; Link, Brian A

    2015-09-01

    The optic vesicle comprises a pool of bi-potential progenitor cells from which the retinal pigment epithelium (RPE) and neural retina fates segregate during ocular morphogenesis. Several transcription factors and signaling pathways have been shown to be important for RPE maintenance and differentiation, but an understanding of the initial fate specification and determination of this ocular cell type is lacking. We show that Yap/Taz-Tead activity is necessary and sufficient for optic vesicle progenitors to adopt RPE identity in zebrafish. A Tead-responsive transgene is expressed within the domain of the optic cup from which RPE arises, and Yap immunoreactivity localizes to the nuclei of prospective RPE cells. yap (yap1) mutants lack a subset of RPE cells and/or exhibit coloboma. Loss of RPE in yap mutants is exacerbated in combination with taz (wwtr1) mutant alleles such that, when Yap and Taz are both absent, optic vesicle progenitor cells completely lose their ability to form RPE. The mechanism of Yap-dependent RPE cell type determination is reliant on both nuclear localization of Yap and interaction with a Tead co-factor. In contrast to loss of Yap and Taz, overexpression of either protein within optic vesicle progenitors leads to ectopic pigmentation in a dosage-dependent manner. Overall, this study identifies Yap and Taz as key early regulators of RPE genesis and provides a mechanistic framework for understanding the congenital ocular defects of Sveinsson's chorioretinal atrophy and congenital retinal coloboma. © 2015. Published by The Company of Biologists Ltd.

  10. The retinal pigment epithelium utilizes fatty acids for ketogenesis.

    Science.gov (United States)

    Adijanto, Jeffrey; Du, Jianhai; Moffat, Cynthia; Seifert, Erin L; Hurle, James B; Philp, Nancy J

    2014-07-25

    Every day, shortly after light onset, photoreceptor cells shed approximately a tenth of their outer segment. The adjacent retinal pigment epithelial (RPE) cells phagocytize and digest shed photoreceptor outer segment, which provides a rich source of fatty acids that could be utilized as an energy substrate. From a microarray analysis, we found that RPE cells express particularly high levels of the mitochondrial HMG-CoA synthase 2 (Hmgcs2) compared with all other tissues (except the liver and colon), leading to the hypothesis that RPE cells, like hepatocytes, can produce β-hydroxybutyrate (β-HB) from fatty acids. Using primary human fetal RPE (hfRPE) cells cultured on Transwell filters with separate apical and basal chambers, we demonstrate that hfRPE cells can metabolize palmitate, a saturated fatty acid that constitutes .15% of all lipids in the photoreceptor outer segment, to produce β-HB. Importantly, we found that hfRPE cells preferentially release β-HB into the apical chamber and that this process is mediated primarily by monocarboxylate transporter isoform 1 (MCT1). Using a GC-MS analysis of (13)C-labeled metabolites, we showed that retinal cells can take up and metabolize (13)C-labeled β-HB into various TCA cycle intermediates and amino acids. Collectively, our data support a novel mechanism of RPE-retina metabolic coupling in which RPE cells metabolize fatty acids to produce β-HB, which is transported to the retina for use as a metabolic substrate.

  11. Zinc deficiency leads to lipofuscin accumulation in the retinal pigment epithelium of pigmented rats.

    Directory of Open Access Journals (Sweden)

    Sylvie Julien

    Full Text Available BACKGROUND: Age-related macular degeneration (AMD is associated with lipofuscin accumulation whereas the content of melanosomes decreases. Melanosomes are the main storage of zinc in the pigmented tissues. Since the elderly population, as the most affected group for AMD, is prone to zinc deficit, we investigated the chemical and ultrastructural effects of zinc deficiency in pigmented rat eyes after a six-month zinc penury diet. METHODOLOGY/PRINCIPAL FINDINGS: Adult Long Evans (LE rats were investigated. The control animals were fed with a normal alimentation whereas the zinc-deficiency rats (ZD-LE were fed with a zinc deficient diet for six months. Quantitative Energy Dispersive X-ray (EDX microanalysis yielded the zinc mole fractions of melanosomes in the retinal pigment epithelium (RPE. The lateral resolution of the analysis was 100 nm. The zinc mole fractions of melanosomes were significantly smaller in the RPE of ZD-LE rats as compared to the LE control rats. Light, fluorescence and electron microscopy, as well as immunohistochemistry were performed. The numbers of lipofuscin granules in the RPE and of infiltrated cells (Ø>3 µm found in the choroid were quantified. The number of lipofuscin granules significantly increased in ZD-LE as compared to control rats. Infiltrated cells bigger than 3 µm were only detected in the choroid of ZD-LE animals. Moreover, the thickness of the Bruch's membrane of ZD-LE rats varied between 0.4-3 µm and thin, rangy ED1 positive macrophages were found attached at these sites of Bruch's membrane or even inside it. CONCLUSIONS/SIGNIFICANCE: In pigmented rats, zinc deficiency yielded an accumulation of lipofuscin in the RPE and of large pigmented macrophages in the choroids as well as the appearance of thin, rangy macrophages at Bruch's membrane. Moreover, we showed that a zinc diet reduced the zinc mole fraction of melanosomes in the RPE and modulated the thickness of the Bruch's membrane.

  12. Multifocal central serous chorioretinopathy with photoreceptor-retinal pigment epithelium diastasis in heritable pulmonary arterial hypertension

    DEFF Research Database (Denmark)

    Li, Xiao Qiang; Pryds, Anders; Carlsen, Jørn;

    2015-01-01

    with clinical examination, enhanced depth optical coherence tomography, fluorescein and indocyanine green angiography, and fundus photography. RESULTS: At presentation, atypical central serous chorioretinopathy with multiple retinal pigment epithelial detachments, a thick subfoveal choroid, and dilated...

  13. Puerarin antagonizes peroxyntrite-induced injury in retinal pigment epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Lina Hao; Xudong Zhang; Tao Yang; Junling Ma

    2012-01-01

    A rat model of diabetes mellitus was established by intraperitoneal injection of streptozotocin. Three days later, the rats were intraperitoneally administered 140 mg puerarin/kg daily, for a total of 60 successive days. DNA ladder results showed increased apoptosis over time in retinal pigment epithelial cells from rats with streptozotocin-induced diabetes mellitus. Western blot analysis, Reverse transcription-PCR, immunohistochemistry, and flow cytometry results showed increased expression of 3-nitrotyrosine, a peroxyntrite marker, as well as inducible nitric synthase and Fas/FasL, in retinal pigment epithelial cells. Puerarin reversed these changes, and results demonstrated that puerarin inhibited Fas/FasL expression and alleviated peroxyntrite injury to retinal pigment epithelial cells. These results suggested that puerarin inhibited production of inducible nitric oxide synthase and directly antagonized peroxyntrite injury in retinal pigment epithelial cells.

  14. Retinoic acid from retinal pigment epithelium induces T regulatory cells.

    Science.gov (United States)

    Kawazoe, Yuko; Sugita, Sunao; Keino, Hiroshi; Yamada, Yukiko; Imai, Ayano; Horie, Shintaro; Mochizuki, Manabu

    2012-01-01

    Primary cultured retinal pigment epithelial (RPE) cells can convert T cells into T regulatory cells (Tregs) through inhibitory factor(s) including transforming growth factor β (TGFβ) in vitro. Retinoic acid (RA) enhances induction of CD4(+) Tregs in the presence of TGFβ. We investigated whether RA produced by RPE cells can promote generation of Tregs. We found that in vitro, RA-treated T cells expressed high levels of Foxp3 in the presence of recombinant TGFβ. In GeneChip analysis, cultured RPE cells constitutively expressed RA-associated molecules such as RA-binding proteins, enzymes, and receptors. RPE from normal mice, but not vitamin A-deficient mice, contained significant levels of TGFβ. RPE-induced Tregs from vitamin A-deficient mice failed to suppress activation of target T cells. Only a few Foxp3(+) T cells were found in intraocular cells from vitamin A-deficient experimental autoimmune uveitis (EAU) mice, whereas expression was higher in cells from normal EAU mice. RA receptor antagonist-pretreated or RA-binding protein-siRNA-transfected RPE cells failed to convert CD4(+) T cells into Tregs. Our data support the hypothesis that RPE cells produce RA, thereby enabling bystander T cells to be converted into Tregs through TGFβ promotion, which can then participate in the establishment of immune tolerance in the eye.

  15. Surgical treatment in combined hamartoma of the retina and retinal pigment epithelium.

    Science.gov (United States)

    Sánchez-Vicente, J L; Rueda-Rueda, T; Llerena-Manzorro, L; Molina-Socola, F E; Contreras-Díaz, M; Szewc, M; Vital-Berral, C; Alfaro-Juárez, A; Medina-Tapia, A; López-Herrero, F; González-García, L; Muñoz-Morales, A

    2017-03-01

    The case is presented of a 39 year-old man with a combined hamartoma of the retina and retinal pigment epithelium, who experienced progressive visual loss and worsening of metamorphopsia. The patient underwent vitrectomy and epiretinal component peeling, with improvement in visual acuity, metamorphopsia, and retinal architecture, assessed by optical coherence tomography. Selected patients with combined hamartomas of the retina and retinal pigment epithelium may benefit from surgical management. Copyright © 2016 Sociedad Española de Oftalmología. Publicado por Elsevier España, S.L.U. All rights reserved.

  16. Retinal pigment epithelium cell alignment on nanostructured collagen matrices.

    Science.gov (United States)

    Ulbrich, Stefan; Friedrichs, Jens; Valtink, Monika; Murovski, Simo; Franz, Clemens M; Müller, Daniel J; Funk, Richard H W; Engelmann, Katrin

    2011-01-01

    We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α(2) were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α(2) was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α(2) expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α(2)-mediated matrix binding was verified by preincubation with an α(2)-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriers.

  17. Controlled exosome release from the retinal pigment epithelium in situ.

    Science.gov (United States)

    Locke, Christina J; Congrove, Nicole R; Dismuke, W Michael; Bowen, Trent J; Stamer, W Daniel; McKay, Brian S

    2014-12-01

    Retinal Pigment Epithelial cells (RPE) express both GPR143 and myocilin, which interact in a signal transduction-dependent manner. In heterologous systems, activation of GPR143 with ligand causes transient recruitment of myocilin to internalized receptors, which appears to be the entry point of myocilin to the endocytic pathway. In some but not all cells, myocilin also traffics through the multivesicular body (MVB) and is released on the surface of exosomes in a signal transduction-dependent fashion. Little is known regarding the role of exosomes in RPE, but they likely serve as a mode of communication between the RPE and the outer retina. In this study, we used posterior poles with retina removed from fresh human donor eyes as a model to test the relationship between GPR143, myocilin, and exosomes in an endogenous system. We isolated exosomes released by RPE using differential centrifugation of media conditioned by the RPE for 25 min, and then characterized the exosomes using nanoparticle tracking to determine the number and size of the exosomes. Next, we tested whether ligand stimulation of GPR143 using l-DOPA altered RPE exosome release. Finally, we investigated whether myocilin was present on the exosomes released by RPE and whether l-DOPA stimulation of GPR143 caused recruitment of myocilin to the endocytic pathway, as we have previously observed using cultured cells. Activation of GPR143 halted RPE exosome release, while simultaneously recruiting myocilin to the endocytic compartment. Together, our results indicate that GPR143 and myocilin function in a signal transduction system that can control exosome release from RPE.

  18. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M;

    1998-01-01

    PURPOSE: The immune privilege of the eye has been thought to be dependent on physical barriers and absence of lymphatic vessels. However, the immune privilege may also involve active immunologic processes, as recent studies have indicated. The purpose of the present study was to investigate whether...... human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...

  19. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  20. Derivation of Neural Progenitors and Retinal Pigment Epithelium from Common Marmoset and Human Pluripotent Stem Cells

    Directory of Open Access Journals (Sweden)

    Laughing Bear Torrez

    2012-01-01

    Full Text Available Embryonic and induced pluripotent stem cells (IPSCs derived from mammalian species are valuable tools for modeling human disease, including retinal degenerative eye diseases that result in visual loss. Restoration of vision has focused on transplantation of neural progenitor cells (NPCs and retinal pigmented epithelium (RPE to the retina. Here we used transgenic common marmoset (Callithrix jacchus and human pluripotent stem cells carrying the enhanced green fluorescent protein (eGFP reporter as a model system for retinal differentiation. Using suspension and subsequent adherent differentiation cultures, we observed spontaneous in vitro differentiation that included NPCs and cells with pigment granules characteristic of differentiated RPE. Retinal cells derived from human and common marmoset pluripotent stem cells provide potentially unlimited cell sources for testing safety and immune compatibility following autologous or allogeneic transplantation using nonhuman primates in early translational applications.

  1. Infection of Human Retinal Pigment Epithelium with Chlamydia trachomatis.

    Directory of Open Access Journals (Sweden)

    Ernest Boiko

    Full Text Available Little is known about the susceptibility of posterior segment tissues, particularly the human retinal pigment epithelium (hRPE, to Chlamydia trachomatis. The purpose of the study was to investigate the possibility of infecting the hRPE with Chlamydia trachomatis, and to examine the infectivity of different Chlamydia trachomatis clinical isolates for hRPE cells and the hRPE cell response to the infection.Cultured hRPE and McCoy cells were inoculated with eight Chlamydia trachomatis (serovar E clinical isolates at multiplicity of infection (MOI of 2.0 or 0.3. To detect Chlamydia trachomatis, samples were stained immunohistochemically with anti-major outer membrane protein antibodies at 24h, 48h, and 72h postinoculation (PI. The changes in the expression of signaling molecules and proteins of cytoskeleton and extracellular matrix in hRPE cells were examined immunohistochemically.All eight clinical isolates demonstrated ability to infect hRPE cells. At equal MOI of 0.3, the infectivity of Chlamydia trachomatis clinical isolates for RPE culture was found to be at least as high as that for McCoy cell culture. At 24h PI, the percentage of inclusion-containing cells varied from 1.5 ± 0.52 to 14.6 ± 3.3% in hRPE cell culture infected at MOI of 2.0 against 0.37 ± 0.34 to 8.9 ± 0.2% in McCoy cell culture infected at MOI of 0.3. Collagen type I, collagen type IV, basic fibroblast growth factor, transforming growth factor-beta and interleukin-8 expression at 48h PI were maximally increased, by 2.1-, 1.3-, 1.5-, 1.5- and 1.6-fold, respectively, in the Chlamydia trachomatis-infected compared with control hRPE cell culture specimens (P < 0.05.This study, for the first time, proved the possibility of infecting hRPE cultured cells with Chlamydia trachomatis, which leads to proproliferative and proinflammatory changes in the expression of signaling molecules and extracellular matrix components.

  2. Low aqueous solubility of 11-cis-retinal limits the rate of pigment formation and dark adaptation in salamander rods.

    Science.gov (United States)

    Frederiksen, Rikard; Boyer, Nicholas P; Nickle, Benjamin; Chakrabarti, Kalyan S; Koutalos, Yiannis; Crouch, Rosalie K; Oprian, Daniel; Cornwall, M Carter

    2012-06-01

    We report experiments designed to test the hypothesis that the aqueous solubility of 11-cis-retinoids plays a significant role in the rate of visual pigment regeneration. Therefore, we have compared the aqueous solubility and the partition coefficients in photoreceptor membranes of native 11-cis-retinal and an analogue retinoid, 11-cis 4-OH retinal, which has a significantly higher solubility in aqueous medium. We have then correlated these parameters with the rates of pigment regeneration and sensitivity recovery that are observed when bleached intact salamander rod photoreceptors are treated with physiological solutions containing these retinoids. We report the following results: (a) 11-cis 4-OH retinal is more soluble in aqueous buffer than 11-cis-retinal. (b) Both 11-cis-retinal and 11-cis 4-OH retinal have extremely high partition coefficients in photoreceptor membranes, though the partition coefficient of 11-cis-retinal is roughly 50-fold greater than that of 11-cis 4-OH retinal. (c) Intact bleached isolated rods treated with solutions containing equimolar amounts of 11-cis-retinal or 11-cis 4-OH retinal form functional visual pigments that promote full recovery of dark current, sensitivity, and response kinetics. However, rods treated with 11-cis 4-OH retinal regenerated on average fivefold faster than rods treated with 11-cis-retinal. (d) Pigment regeneration from recombinant and wild-type opsin in solution is slower when treated with 11-cis 4-OH retinal than with 11-cis-retinal. Based on these observations, we propose a model in which aqueous solubility of cis-retinoids within the photoreceptor cytosol can place a limit on the rate of visual pigment regeneration in vertebrate photoreceptors. We conclude that the cytosolic gap between the plasma membrane and the disk membranes presents a bottleneck for retinoid flux that results in slowed pigment regeneration and dark adaptation in rod photoreceptors.

  3. Retinal β-ionone ring-salinixanthin interactions in xanthorhodopsin: a study using artificial pigments.

    Science.gov (United States)

    Smolensky Koganov, Elena; Hirshfeld, Amiram; Sheves, Mordechai

    2013-02-19

    Xanthorhodopsin (xR) is a retinal protein that contains, in addition to the retinal chromophore, a carotenoid (salinixanthin) that functions as a light-harvesting antenna [Balashov, S. P., et al. (2005) Science 309, 2061-2064]. The center-center distance between the two polyene chains is 12-13 Å, but the distance between the two rings of retinal and salinixanthin is surprisingly small (~5 Å) with an angle of ~45° [Luecke, H., et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 16561-16565]. We aimed to clarify the role of the β-ionone ring in the binding of retinal to apo-xR, as well as a possible role that the β-ionone ring plays in fixation of the salinixanthin 4-keto ring. The binding of native retinal and series of synthetic retinal analogues modified in the β-ionone ring to apo-xR was monitored by absorption and circular dichroism (CD) spectroscopies. The results indicate that the β-ionone ring modification significantly affected formation of the retinal-protein covalent bond as well as the pigment absorption and CD spectra. It was observed that several retinal analogues, modified in the retinal β-ionone ring, did not bind to apo-xR and did not form the pigment. Also, none of these analogues induced the fixation of the salinixanthin 4-keto ring. In addition, we show that the native retinal within its binding site adopts exclusively the 6-s-trans ring-chain conformation.

  4. Ophthalmoscopy for congenital hypertrophy of the retinal pigment epithelium (CHRPE) in patients with sporadic colorectal carcinoma

    DEFF Research Database (Denmark)

    Hartvigsen, A; Myrhøj, T; Bülow, Steffen

    1995-01-01

    In order to investigate the frequency of congenital hypertrophy of the retinal pigment epithelium (CHRPE) in sporadic colorectal cancer, ophthalmoscopy was carried out in 34 patients with colorectal carcinoma without known familial disposition. CHRPE is one of the most frequent extracolonic...

  5. RNA interference inhibits expression of vascular endothelial growth factor (VEGF) in human retinal pigment epithelial cells

    Institute of Scientific and Technical Information of China (English)

    CAI Chun-mei; SUN Bao-chen; LIU Xu-yang; WANG Jin-jin; LI Jun-fa; HAN Song; WANG Ning-li; LU Qing-jun

    2005-01-01

    @@ Choroidal neovascularization (CNV), a major cause of vision loss, is the result of the increased vascular endothelial growth factor (VEGF) expression in human retinal pigment epithelial (RPE) cells. It is important to inhibit the expression of VEGF protein in RPE cells.

  6. Ophthalmoscopy for congenital hypertrophy of the retinal pigment epithelium (CHRPE) in patients with sporadic colorectal carcinoma

    DEFF Research Database (Denmark)

    Hartvigsen, A; Myrhøj, T; Bülow, Steffen

    1995-01-01

    In order to investigate the frequency of congenital hypertrophy of the retinal pigment epithelium (CHRPE) in sporadic colorectal cancer, ophthalmoscopy was carried out in 34 patients with colorectal carcinoma without known familial disposition. CHRPE is one of the most frequent extracolonic...

  7. Sectoral iris heterochromia and retinal pigment variation in 13q-syndrome.

    Science.gov (United States)

    Kutzbach, Beth; Mendelsohn, Nancy; Rath, Pamela; Summers, C Gail

    2007-10-01

    Chromosome 13q deletion syndrome is characterized by growth retardation, cognitive delays, and organ and musculoskeletal deformities. Typical ocular associations include retinoblastoma, microphthalmia, and colobomas. We report a case of bilateral iris heterochromia and retinal pigment abnormalities in a child with 13q-syndrome.

  8. Generation of retinal pigment epithelial cells from human embryonic stem cell-derived spherical neural masses.

    Science.gov (United States)

    Cho, Myung Soo; Kim, Sang Jin; Ku, Seung-Yup; Park, Jung Hyun; Lee, Haksup; Yoo, Dae Hoon; Park, Un Chul; Song, Seul Ae; Choi, Young Min; Yu, Hyeong Gon

    2012-09-01

    Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy.

  9. Pathological, Ultrastructural and Immunohistochemical Observations of Adenoma of Retinal Pigment Epithelium

    Institute of Scientific and Technical Information of China (English)

    Ping Zhang; Guanguang Feng; Tao Yue; Jianxian Lin; Yuzhen Yi; Youjian Pang

    2001-01-01

    Purpose: To Study the clinical, pathological, ultrastructural and immunohistchemicalcharacters of adenoma of the retinal pigment epithelium in order to offer evidence todiagnose this tumor.Methods: Routine paraffin slices HE stain, histochemistry PAS and VG stain,transmission electron microscopy, and immunohistochemistry for S-100 and vimentinwith LSAB method were used.Results: The tumor cells were oval and cuboidal in shape. Part of the tumor had atubular arrangement. Around the sheets of tumors cells there was a large amount ofuniform red stick-like substances. The above matter represented positive in PAS stain.Most of the above matter was yellow, while less of the matter showed red in VG stain.Transmission electron microscopy showed that there were tight junctions between tumorcells. Immunohistochemistry showed positive for S-100, negative for vimentin.Conclusions: The ultrastructural and immunohistochemical characters of the adenoma ofretinal pigment epithelium are consistent with the retinal pigment epithelium.

  10. Human Bone Marrow Stromal Cells can Differentiate to a Retinal Pigment Epithelial Phenotype when Co-Cultured with Pig Retinal Pigment Epithelium using a Transwell System

    Directory of Open Access Journals (Sweden)

    Ping Duan

    2013-05-01

    Full Text Available Background: There is an increasing interest in generating retinal pigment epithelial (RPE cells from stem cells for therapy against degenerative eye diseases. Human bone marrow stromal cells (hBMSCs can be induced to express retinal neuron-specific markers when co-cultured with retinal neurons, however, whether hBMSCs can differentiate into RPE-like cells in a co-culture system has not been clarified. Methods: The induction of hBMSCs into RPE-like cells was performed by combining hBMSCs and pig RPE cells in a transwell system. The biomarkers of hBMSCs-derived RPE cells were determined by quantitative RT-PCR and immunofluorescence. The function of induced cells was assayed by ELISA for secretion of neurotrophic factors. Results: Intracellular pigment granules and many RPE markers existed in hBMSCs-derived RPE cells after co-culturing with pig RPE cells for 14 days. Typical RPE functions, such as phagocytosis of photoreceptor outer segments and secretion of the trophic factors, brain-derived neurotrophic factor (BDNF and glia-derived neurotrophic factor (GDNF, were observed in these induced cells. Conclusion: hBMSCs can be induced toward functional RPE cells simply by transwell-based co-culture with RPE cells.

  11. Cotransport of H+, lactate, and H2O in porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Hamann, Steffen; Kiilgaard, Jens Folke; la Cour, Morten

    2003-01-01

    The retinal pigment epithelium (RPE) of the eye transports water and lactate ions in the direction from retina to choroid. The water transport is important in maintenance of retinal adhesion and the transport of lactate ions serves to regulate the lactate levels and pH of the subretinal space....... This study investigates by means of a non-invasive technique the mechanism of coupling between transport of H(+), lactate ion, and water in the monocarboxylate transporter (MCT1) located in the apical (retinal) membrane of a mammalian RPE. Primary cultures of porcine RPE cells were grown to confluence...... using the fluorescent dye BCECF. In lactate-free solutions, mannitol addition to the retinal bath caused intracellular acidification and cell shrinkage, given by a single osmotic water permeability of 1.2+/-0.1 x 10(-4)cmsec(-1) (osmoll(-1))(-1). In solutions containing 50 mmoll(-1) lactate, however...

  12. Bimonthly half-dose ranibizumab in large pigment epithelial detachment and retinal angiomatous proliferation with high risk of retinal pigment epithelium tear: a case report

    Directory of Open Access Journals (Sweden)

    Monés J

    2013-06-01

    Full Text Available Jordi Monés,1,2 Marc Biarnés,1 Josep Badal11Institut de la Màcula i de la Retina, Barcelona, Spain; 2Barcelona Macula Foundation, Barcelona, SpainIntroduction: The management of large pigment epithelial detachments (PEDs associated with retinal angiomatous proliferation (RAP remains a challenge due to the high risk of retinal pigment epithelial (RPE tear. We describe the successful progressive anatomical result and the maintenance of visual acuity to bimonthly, half-dose ranibizumab in a patient with this condition.Purpose: To describe the management of a large PED secondary to RAP with bimonthly, half-dose ranibizumab.Method: Case report.Patient: A 71-year-old woman presented with visual symptoms due to an enlarged PED, compared with previous visits, secondary to a RAP lesion, with a visual acuity of 20/32. To reduce the risk of an RPE tear and a significant decrease in vision, we discussed with the patient the possibility of treating the lesion in a progressive manner, with more frequent but smaller doses of ranibizumab. The patient was treated biweekly with 0.25 mg of ranibizumab until flattening of the PED.Results: The large PED flattened progressively, and visual acuity was preserved with no adverse events.Discussion: The use of half-dose antiangiogenic therapy may be useful in managing large vascularized PED associated with RAP, in an attempt to reduce the risk of RPE tear.Keywords: age-related macular degeneration, pigment epithelial detachment, ranibizumab, retinal angiomatous proliferation, RPE tear

  13. Massive Retinal Pigment Epithelial Detachment Following Acute Hypokalemic Quadriparesis in Dengue Fever.

    Science.gov (United States)

    Goel, Neha; Bhambhwani, Vishaal; Jain, Pooja; Ghosh, Basudeb

    2016-01-01

    To describe an unusual retinal manifestation of dengue fever in an endemic region. A 35 year old male presenting with acute onset decreased vision in his right eye, was found to have a massive retinal pigment epithelial detachment (PED) extending up to the vascular arcades. He had been diagnosed with acute hypokalemic quadriparesis in dengue fever in the preceding week, which had resolved following treatment. The patient was managed conservatively. At three months follow up, there was spontaneous flattening of the PEDs with improvement in visual acuity. Dengue fever complicated by acute hypokalemic quadriparesis can be associated with PED, which can be large. The condition resolves spontaneously and bears a good prognosis.

  14. Lipofuscin and A2E accumulate with age in the retinal pigment epithelium of Nrl-/- mice.

    Science.gov (United States)

    Boyer, Nicholas P; Tang, Peter H; Higbee, Daniel; Ablonczy, Zsolt; Crouch, Rosalie K; Koutalos, Yiannis

    2012-01-01

    Lipofuscin is a fluorescent material with significant phototoxic potential that accumulates with age in the retinal pigment epithelium (RPE) of the eye. It is thought to be a factor in retinal degeneration diseases. The most extensively characterized lipofuscin component, N-retinylidene-N-retinylethanolamine (A2E), has been proposed to be a byproduct of reactions involving the visual pigment chromophore. To examine the impact of the visual pigment and photoreceptor cell type on lipofuscin accumulation, we analyzed the RPE from Nrl(-/-) mice of various ages for lipofuscin fluorescence and A2E levels. The photoreceptor cells of the Nrl(-/-) retina contain only cone-like pigments, and produce cone-like responses to photostimulation. The cone-like nature of these cells was confirmed by the presence of RPE65. Lipofuscin was measured with fluorescence imaging, whereas A2E was quantified by UV/VIS absorbance spectroscopy coupled to HPLC. The identity of A2E was corroborated with tandem mass spectrometry. Lipofuscin and A2E accumulated with age, albeit to lower levels compared with wild type mice. The emission spectra of RPE lipofuscin granules from Nrl(-/-) mice were similar to those from wild type mice, with λ(max) ca 610 nm. These results demonstrate that cone visual pigments can contribute to the production of lipofuscin and A2E. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

  15. Phloroglucinol protects retinal pigment epithelium and photoreceptor against all-trans-retinal-induced toxicity and inhibits A2E formation.

    Science.gov (United States)

    Cia, David; Cubizolle, Aurélie; Crauste, Céline; Jacquemot, Nathalie; Guillou, Laurent; Vigor, Claire; Angebault, Claire; Hamel, Christian P; Vercauteren, Joseph; Brabet, Philippe

    2016-09-01

    Among retinal macular diseases, the juvenile recessive Stargardt disease and the age-related degenerative disease arise from carbonyl and oxidative stresses (COS). Both stresses originate from an accumulation of all-trans-retinal (atRAL) and are involved in bisretinoid formation by condensation of atRAL with phosphatidylethanolamine (carbonyl stress) in the photoreceptor and its transformation into lipofuscin bisretinoids (oxidative stress) in the retinal pigment epithelium (RPE). As atRAL and bisretinoid accumulation contribute to RPE and photoreceptor cell death, our goal is to select powerful chemical inhibitors of COS. Here, we describe that phloroglucinol, a natural phenolic compound having anti-COS properties, protects both rat RPE and mouse photoreceptor primary cultures from atRAL-induced cell death and reduces hydrogen peroxide (H2 O2 )-induced damage in RPE in a dose-dependent manner. Mechanistic analyses demonstrate that the protective effect encompasses decrease in atRAL-induced intracellular reactive oxygen species and free atRAL levels. Moreover, we show that phloroglucinol reacts with atRAL to form a chromene adduct which prevents bisretinoid A2E synthesis in vitro. Taken together, these data show that the protective effect of phloroglucinol correlates with its ability to trap atRAL and to prevent its further transformation into deleterious bisretinoids. Phloroglucinol might be a good basis to develop efficient therapeutic derivatives in the treatment of retinal macular diseases.

  16. Retinal pigment epithelial acid lipase activity and lipoprotein receptors: effects of dietary omega-3 fatty acids.

    OpenAIRE

    2002-01-01

    PURPOSE: To show that fish oil-derived omega-3 polyunsaturated fatty acids, delivered to the retinal pigment epithelium (RPE) by circulating low-density lipoproteins (LDL), enhance already considerable RPE lysosomal acid lipase activity, providing for more efficient hydrolysis of intralysosomal RPE lipids, an effect that may help prevent development of age-related macular degeneration (ARMD). METHODS: Colorimetric biochemical and histochemical techniques were used to demonstrate RPE acid lipa...

  17. In vitro ultraviolet–induced damage in human corneal, lens, and retinal pigment epithelial cells

    OpenAIRE

    Youn, Hyun-Yi; McCanna, David J.; Sivak, Jacob G.; Jones, Lyndon W.

    2011-01-01

    Purpose The purpose was to develop suitable in vitro methods to detect ocular epithelial cell damage when exposed to UV radiation, in an effort to evaluate UV-absorbing ophthalmic biomaterials. Methods Human corneal epithelial cells (HCEC), lens epithelial cells (HLEC), and retinal pigment epithelial cells (ARPE-19) were cultured and Ultraviolet A/Ultraviolet B (UVA/UVB) blocking filters and UVB-only blocking filters were placed between the cells and a UV light source. Cells were irradiated w...

  18. Origins and consequences of hyperosmolar stress in retinal pigmented epithelial cells.

    Science.gov (United States)

    Willermain, François; Libert, Sarah; Motulsky, Elie; Salik, Dany; Caspers, Laure; Perret, Jason; Delporte, Christine

    2014-01-01

    The retinal pigmented epithelium (RPE) is composed of retinal pigmented epithelial cells joined by tight junctions and represents the outer blood-retinal barrier (BRB). The inner BRB is made of endothelial cells joined by tight junctions and glial extensions surrounding all the retinal blood vessels. One of the functions of the RPE is to maintain an osmotic transepithelial gradient created by ionic pumps and channels, avoiding paracellular flux. Under such physiological conditions, transcellular water movement follows the osmotic gradient and flows normally from the retina to the choroid through the RPE. Several diseases, such as diabetic retinopathy, are characterized by the BRB breakdown leading to leakage of solutes, proteins, and fluid from the retina and the choroid. The prevailing hypothesis explaining macular edema formation during diabetic retinopathy incriminates the inner BRB breakdown resulting in increased osmotic pressure leading in turn to massive water accumulation that can affect vision. Under these conditions, it has been hypothesized that RPE is likely to be exposed to hyperosmolar stress at its apical side. This review summarizes the origins and consequences of osmotic stress in the RPE. Ongoing and further research advances will clarify the mechanisms, at the molecular level, involved in the response of the RPE to osmotic stress and delineate potential novel therapeutic targets and tools.

  19. Aliskiren inhibits the renin-angiotensin system in retinal pigment epithelium cells.

    Science.gov (United States)

    Simão, Sónia; Santos, Daniela F; Silva, Gabriela A

    2016-09-20

    Observations of increased angiotensin II levels and activation of the (pro)renin receptor in retinopathies support the role of ocular renin-angiotensin system (RAS) in the development of retinal diseases. While targeting RAS presents significant therapeutic potential, current RAS-based therapies are ineffective halting the progression of these diseases. A new class of drugs, the direct renin inhibitors such as aliskiren, is a potential therapeutic alternative. However, it is unclear how aliskiren acts in the retina, in particular in the retinal pigment epithelium (RPE), the structure responsible for the maintenance of retinal homeostasis whose role is deeply compromised in retinal diseases. We firstly analyzed the expression and activity of the main RAS components in RPE cells. Time- and concentration-dependent treatments with aliskiren were performed to modulate different pathways of the RAS in RPE cells. Our data demonstrate that RPE cells express the main RAS constituents. Exposure of RPE cells to aliskiren inhibited the activity of renin and consequently decreased the levels of angiotensin II. Additionally, aliskiren reduced the translocation of the (pro)renin receptor to the cellular membrane of RPE cells preventing the activation of ERK1/2. Our findings of the RPE well-defined RAS, together with the demonstration that aliskiren effectively blocks this system at different steps of the cascade, suggest that aliskiren might be an alternative and successful drug in preventing the deleterious effects derived from the overactivation of the RAS, known to contribute to the pathogenesis of different retinal diseases.

  20. The Retinome – Defining a reference transcriptome of the adult mammalian retina/retinal pigment epithelium

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    Goetz Thomas

    2004-07-01

    Full Text Available Abstract Background The mammalian retina is a valuable model system to study neuronal biology in health and disease. To obtain insight into intrinsic processes of the retina, great efforts are directed towards the identification and characterization of transcripts with functional relevance to this tissue. Results With the goal to assemble a first genome-wide reference transcriptome of the adult mammalian retina, referred to as the retinome, we have extracted 13,037 non-redundant annotated genes from nearly 500,000 published datasets on redundant retina/retinal pigment epithelium (RPE transcripts. The data were generated from 27 independent studies employing a wide range of molecular and biocomputational approaches. Comparison to known retina-/RPE-specific pathways and established retinal gene networks suggest that the reference retinome may represent up to 90% of the retinal transcripts. We show that the distribution of retinal genes along the chromosomes is not random but exhibits a higher order organization closely following the previously observed clustering of genes with increased expression. Conclusion The genome wide retinome map offers a rational basis for selecting suggestive candidate genes for hereditary as well as complex retinal diseases facilitating elaborate studies into normal and pathological pathways. To make this unique resource freely available we have built a database providing a query interface to the reference retinome 1.

  1. Overexpression of Snail in retinal pigment epithelial triggered epithelial–mesenchymal transition

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hui; Li, Min; Xu, Ding; Zhao, Chun; Liu, Guodong; Wang, Fang, E-mail: milwang_122@msn.com

    2014-03-28

    Highlights: • First reported overexpression of Snail in RPE cells could directly trigger EMT. • Further confirmed the regulator role of Snail in RPE cells EMT in vitro. • Snail may be a potential therapeutic target to prevent the fibrosis of PVR. - Abstract: Snail transcription factor has been implicated as an important regulator in epithelial–mesenchymal transition (EMT) during tumourigenesis and fibrogenesis. Our previous work showed that Snail transcription factor was activated in transforming growth factor β1 (TGF-β1) induced EMT in retinal pigment epithelial (RPE) cells and may contribute to the development of retinal fibrotic disease such as proliferative vitreoretinopathy (PVR). However, whether Snail alone has a direct role on retinal pigment epithelial–mesenchymal transition has not been investigated. Here, we analyzed the capacity of Snail to drive EMT in human RPE cells. A vector encoding Snail gene or an empty vector were transfected into human RPE cell lines ARPE-19 respectively. Snail overexpression in ARPE-19 cells resulted in EMT, which was characterized by the expected phenotypic transition from a typical epithelial morphology to mesenchymal spindle-shaped. The expression of epithelial markers E-cadherin and Zona occludin-1 (ZO-1) were down-regulated, whereas mesenchymal markers a-smooth muscle actin (a-SMA) and fibronectin were up-regulated in Snail expression vector transfected cells. In addition, ectopic expression of Snail significantly enhanced ARPE-19 cell motility and migration. The present data suggest that overexpression of Snail in ARPE-19 cells could directly trigger EMT. These results may provide novel insight into understanding the regulator role of Snail in the development of retinal pigment epithelial–mesenchymal transition.

  2. Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat.

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    Amanda-Jayne Carr

    Full Text Available Transformation of somatic cells with a set of embryonic transcription factors produces cells with the pluripotent properties of embryonic stem cells (ESCs. These induced pluripotent stem (iPS cells have the potential to differentiate into any cell type, making them a potential source from which to produce cells as a therapeutic platform for the treatment of a wide range of diseases. In many forms of human retinal disease, including age-related macular degeneration (AMD, the underlying pathogenesis resides within the support cells of the retina, the retinal pigment epithelium (RPE. As a monolayer of cells critical to photoreceptor function and survival, the RPE is an ideally accessible target for cellular therapy. Here we report the differentiation of human iPS cells into RPE. We found that differentiated iPS-RPE cells were morphologically similar to, and expressed numerous markers of developing and mature RPE cells. iPS-RPE are capable of phagocytosing photoreceptor material, in vitro and in vivo following transplantation into the Royal College of Surgeons (RCS dystrophic rat. Our results demonstrate that iPS cells can be differentiated into functional iPS-RPE and that transplantation of these cells can facilitate the short-term maintenance of photoreceptors through phagocytosis of photoreceptor outer segments. Long-term visual function is maintained in this model of retinal disease even though the xenografted cells are eventually lost, suggesting a secondary protective host cellular response. These findings have identified an alternative source of replacement tissue for use in human retinal cellular therapies, and provide a new in vitro cellular model system in which to study RPE diseases affecting human patients.

  3. MicroRNA expression profiles of human iPS cells, retinal pigment epithelium derived from iPS, and fetal retinal pigment epithelium.

    Science.gov (United States)

    Greene, Whitney A; Muñiz, Alberto; Plamper, Mark L; Kaini, Ramesh R; Wang, Heuy-Ching

    2014-06-24

    The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

  4. Acute Retinal Pigment Epitheliitis: Spectral Domain Optical Coherence Tomography, Fluorescein Angiography, and Autofluorescence Findings

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    Tuğba Aydoğan

    2015-01-01

    Full Text Available A 17-year-old presented with central and paracentral scotomas in his right eye for one week. There was no remarkable medical or ocular history. Blood analyses were within normal range. At presentation both eyes’ best-corrected visual acuities were 20/20. Slit-lamp examination result was normal. Fundus examination revealed yellow-white hypopigmented areas in the macula. Fluorescein angiography (FA showed hypofluorescence surrounded by ring of hyperfluorescence. Fundus autofluorescence (FAF was slightly increased. Spectral domain optical coherence tomography (SD-OCT showed disruption of IS/OS junction with expansion of abnormal hyperreflectivity from retinal pigment epithelium to the outer nuclear layer (ONL. One month later fundus examination showed disappearance of the lesions. FA revealed transmission hyperfluorescence. FAF showed increased autofluorescence and pigment clumping. Hyperreflective band in SD-OCT disappeared. Loss of photoreceptor segment layers was observed in some of the macular lesions. The diagnosis of acute retinal pigment epitheliitis can be challenging after disappearance of fundus findings. FA, FAF, and SD-OCT are important tests for diagnosis after resolution of the disease.

  5. Macular Pigment and Lutein Supplementation in ABCA4-associated Retinal Degenerations

    Science.gov (United States)

    Aleman, Tomas S.; Cideciyan, Artur V.; Windsor, Elizabeth A. M.; Schwartz, Sharon B.; Swider, Malgorzata; Chico, John D.; Sumaroka, Alexander; Pantelyat, Alexander Y.; Duncan, Keith G.; Gardner, Leigh M.; Emmons, Jessica M.; Steinberg, Janet D.; Stone, Edwin M.; Jacobson, Samuel G.

    2008-01-01

    PURPOSE To determine macular pigment (MP) optical density (OD) in patients with ABCA4-associated retinal degenerations (ABCA4-RD) and the response of MP and vision to supplementation with lutein. METHODS Stargardt disease or cone-rod dystrophy patients with foveal fixation and with known or suspected disease-causing mutations in the ABCA4 gene were included. MPOD profiles were measured with heterochromatic flicker photometry. Serum carotenoids, visual acuity, foveal sensitivity and retinal thickness were quantified. Changes in MPOD and central vision were determined in a subset of patients receiving oral supplementation with lutein for 6 months. RESULTS MPOD in patients ranged from normal to markedly abnormal. As a group, ABCA4-RD patients had reduced foveal MPOD and there was strong correlation with retinal thickness. Average foveal tissue concentration of MP, estimated by dividing MPOD by retinal thickness, was normal in patients whereas serum concentration of lutein and zeaxanthin was significantly lower than normal. After oral lutein supplementation for 6 months, 91% of the patients showed significant increases in serum lutein and 63% of the patient eyes showed a significant augmentation in MPOD. The retinal responders tended to be female, and have lower serum lutein and zeaxanthin, lower MPOD and greater retinal thickness at baseline. Responding eyes had significantly lower baseline MP concentration compared to non-responding eyes. Central vision was unchanged after the period of supplementation. CONCLUSIONS MP is strongly affected by the stage of ABCA4 disease leading to abnormal foveal architecture. MP could be augmented by supplemental lutein in some patients. There was no change in central vision after 6 months of lutein supplementation. Long-term influences on the natural history of this supplement on macular degenerations require further study. PMID:17325179

  6. Effects of white light-emitting diode (LED) exposure on retinal pigment epithelium in vivo.

    Science.gov (United States)

    Jaadane, Imene; Villalpando Rodriguez, Gloria Elisa; Boulenguez, Pierre; Chahory, Sabine; Carré, Samuel; Savoldelli, Michèle; Jonet, Laurent; Behar-Cohen, Francine; Martinsons, Christophe; Torriglia, Alicia

    2017-06-29

    Ageing and alteration of the functions of the retinal pigment epithelium (RPE) are at the origin of lost of vision seen in age-related macular degeneration (AMD). The RPE is known to be vulnerable to high-energy blue light. The white light-emitting diodes (LED) commercially available have relatively high content of blue light, a feature that suggest that they could be deleterious for this retinal cell layer. The aim of our study was to investigate the effects of "white LED" exposure on RPE. For this, commercially available white LEDs were used for exposure experiments on Wistar rats. Immunohistochemical stain on RPE flat mount, transmission electron microscopy and Western blot were used to exam the RPE. LED-induced RPE damage was evaluated by studying oxidative stress, stress response pathways and cell death pathways as well as the integrity of the outer blood-retinal barrier (BRB). We show that white LED light caused structural alterations leading to the disruption of the outer blood-retinal barrier. We observed an increase in oxidized molecules, disturbance of basal autophagy and cell death by necrosis. We conclude that white LEDs induced strong damages in rat RPE characterized by the breakdown of the BRB and the induction of necrotic cell death. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  7. Monte Carlo investigation on quantifying the retinal pigment epithelium melanin concentration by photoacoustic ophthalmoscopy.

    Science.gov (United States)

    Shu, Xiao; Liu, Wenzhong; Zhang, Hao F

    2015-10-01

    The retinal pigment epithelium (RPE) melanin plays an important role in maintaining normal visual functions. A decrease in the RPE melanin concentration with aging is believed to be associated with several blinding diseases, including age-related macular degeneration. Quantifying the RPE melanin noninvasively is therefore important in evaluating the retinal health and aging conditions. Photoacoustic ophthalmoscopy (PAOM), as an optical absorption-based imaging technology, can potentially be applied to measure variations in the RPE melanin if the relationship between the detected photoacoustic (PA) signal amplitudes and the RPE melanin concentrations can be established. In this work, we tested the feasibility of using PA signals from retinal blood vessels as references to measure RPE melanin variation using Monte Carlo (MC) simulation. The influences from PAOM axial resolution, the depth and diameter of the retinal blood vessel, and the RPE thickness were examined. We proposed a calibration scheme by relating detected PA signals to the RPE melanin concentrations, and we found that the scheme is robust to these tested parameters. This study suggests that PAOM has the capability of quantitatively measuring the RPE melanin in vivo.

  8. Histological and electrophysiological changes in the retinal pigment epithelium after injection of sodium iodate in the orbital venus plexus of pigmented rats

    Directory of Open Access Journals (Sweden)

    Hamid Aboutaleb Kadkhodaeian

    2016-01-01

    Conclusion: NaIO3injection into the retrobulbar venous plexus of pigmented rats can result in significant and progressive damage to the RPE and subsequently to the neuroretina of the injected eye, and may serve as a model of retinal degeneration.

  9. A method for the isolation and culture of adult rat retinal pigment epithelial (RPE cells to study retinal diseases

    Directory of Open Access Journals (Sweden)

    Janosch Peter Heller

    2015-11-01

    Full Text Available Diseases such as age-related macular degeneration (AMD affect the retinal pigment epithelium (RPE and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 minutes yielded 4 x 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases.

  10. Retinal pigment epithelial atrophy following indocyanine green dye-assisted surgery for serous macular detachment

    Directory of Open Access Journals (Sweden)

    Hussain Nazimul

    2008-01-01

    Full Text Available To report subretinal migration of indocyanine green dye (ICG and subsequent retinal pigment epithelial (RPE atrophy during macular surgery for serous macular detachment. A 65-year-old woman presented with residual epiretinal membrane and serous detachment of the macula following vitreoretinal surgery for epiretinal membrane. She underwent resurgery with ICG-assisted internal limiting membrane peeling and intraocular tamponade. Intraoperatively a large area of subretinal ICG was seen with subsequent RPE mottling and atrophy of the macula in the area involved during follow-up. This case demonstrates that subretinal migration of ICG is possible and can be toxic to RPE.

  11. Quantum-classical model of retinal photoisomerization reaction in visual pigment rhodopsin.

    Science.gov (United States)

    Lakhno, V D; Shigaev, A S; Feldman, T B; Nadtochenko, V A; Ostrovsky, M A

    2016-11-01

    A quantum-classical model of photoisomerization of the visual pigment rhodopsin chromophore is proposed. At certain (and more realistic) parameter value combinations, the model is shown to accurately reproduce a number of independent experimental data on the photoreaction dynamics: the quantum yield, the time to reach the point of conical intersection of potential energy surfaces, the termination time of the evolution of quantum subsystem, as well as the characteristic low frequencies of retinal molecular lattice fluctuations during photoisomerization. In addition, the model behavior is in good accordance with experimental data about coherence and local character of quantum transition.

  12. A regulatory loop involving PAX6, MITF, and WNT signaling controls retinal pigment epithelium development.

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    Kapil Bharti

    2012-07-01

    Full Text Available The separation of the optic neuroepithelium into future retina and retinal pigment epithelium (RPE is a critical event in early eye development in vertebrates. Here we show in mice that the transcription factor PAX6, well-known for its retina-promoting activity, also plays a crucial role in early pigment epithelium development. This role is seen, however, only in a background genetically sensitized by mutations in the pigment cell transcription factor MITF. In fact, a reduction in Pax6 gene dose exacerbates the RPE-to-retina transdifferentiation seen in embryos homozygous for an Mitf null allele, and it induces such a transdifferentiation in embryos that are either heterozygous for the Mitf null allele or homozygous for an RPE-specific hypomorphic Mitf allele generated by targeted mutation. Conversely, an increase in Pax6 gene dose interferes with transdifferentiation even in homozygous Mitf null embryos. Gene expression analyses show that, together with MITF or its paralog TFEC, PAX6 suppresses the expression of Fgf15 and Dkk3. Explant culture experiments indicate that a combination of FGF and DKK3 promote retina formation by inhibiting canonical WNT signaling and stimulating the expression of retinogenic genes, including Six6 and Vsx2. Our results demonstrate that in conjunction with Mitf/Tfec Pax6 acts as an anti-retinogenic factor, whereas in conjunction with retinogenic genes it acts as a pro-retinogenic factor. The results suggest that careful manipulation of the Pax6 regulatory circuit may facilitate the generation of retinal and pigment epithelium cells from embryonic or induced pluripotent stem cells.

  13. Gene expression analysis of zebrafish melanocytes, iridophores, and retinal pigmented epithelium reveals indicators of biological function and developmental origin.

    Science.gov (United States)

    Higdon, Charles W; Mitra, Robi D; Johnson, Stephen L

    2013-01-01

    In order to facilitate understanding of pigment cell biology, we developed a method to concomitantly purify melanocytes, iridophores, and retinal pigmented epithelium from zebrafish, and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co-enrichment in melanocytes and retinal pigmented epithelium, as well as in melanocytes and iridophores. We found 214 genes co-enriched in melanocytes and retinal pigmented epithelium, indicating the shared functions of melanin-producing cells. We found 62 genes significantly co-enriched in melanocytes and iridophores, illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine-based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate-limiting substrate for purine synthesis, phosphoribosyl pyrophosphate, thus constituting a guanine cycle. The purification procedure and expression analysis described here, along with the accompanying transcriptome-wide expression data, provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types, and will be a useful resource for further studies of pigment cell biology.

  14. Gene expression analysis of zebrafish melanocytes, iridophores, and retinal pigmented epithelium reveals indicators of biological function and developmental origin.

    Directory of Open Access Journals (Sweden)

    Charles W Higdon

    Full Text Available In order to facilitate understanding of pigment cell biology, we developed a method to concomitantly purify melanocytes, iridophores, and retinal pigmented epithelium from zebrafish, and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co-enrichment in melanocytes and retinal pigmented epithelium, as well as in melanocytes and iridophores. We found 214 genes co-enriched in melanocytes and retinal pigmented epithelium, indicating the shared functions of melanin-producing cells. We found 62 genes significantly co-enriched in melanocytes and iridophores, illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine-based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate-limiting substrate for purine synthesis, phosphoribosyl pyrophosphate, thus constituting a guanine cycle. The purification procedure and expression analysis described here, along with the accompanying transcriptome-wide expression data, provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types, and will be a useful resource for further studies of pigment cell biology.

  15. Novel localization of peripherin 2, the photoreceptor-specific retinal degeneration slow protein, in retinal pigment epithelium.

    Science.gov (United States)

    Uhl, Patrizia B; Amann, Barbara; Hauck, Stefanie M; Deeg, Cornelia A

    2015-01-26

    Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye. Since one typical feature of the autoimmune disease, equine recurrent uveitis (ERU), is the breakdown of this barrier, we recently performed comparative analysis of healthy and uveitic RPE. We identified for the first time peripherin 2, which is responsible for visual perception and retina development, to be localized in RPE. The purpose of this study was therefore to validate our findings by characterizing the expression patterns of peripherin 2 in RPE and retina. We also investigated whether peripherin 2 expression changes in ERU and if it is expressed by the RPE itself. Via immunohistochemistry, significant downregulation of peripherin 2 in uveitic RPE compared to the control was detectable, but there was no difference in healthy and uveitic retina. A further interesting finding was the clear distinction between peripherin 2 and the phagocytosis marker, rhodopsin, in healthy RPE. In conclusion, changes in the expression pattern of peripherin 2 selectively affect RPE, but not retina, in ERU. Moreover, peripherin 2 is clearly detectable in healthy RPE due to both phagocytosis and the expression by the RPE cells themselves. Our novel findings are very promising for better understanding the molecular mechanisms taking place on RPE in uveitis.

  16. Novel Localization of Peripherin 2, the Photoreceptor-Specific Retinal Degeneration Slow Protein, in Retinal Pigment Epithelium

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    Patrizia B. Uhl

    2015-01-01

    Full Text Available Retinal pigment epithelium (RPE builds the outer blood-retinal barrier of the eye. Since one typical feature of the autoimmune disease, equine recurrent uveitis (ERU, is the breakdown of this barrier, we recently performed comparative analysis of healthy and uveitic RPE. We identified for the first time peripherin 2, which is responsible for visual perception and retina development, to be localized in RPE. The purpose of this study was therefore to validate our findings by characterizing the expression patterns of peripherin 2 in RPE and retina. We also investigated whether peripherin 2 expression changes in ERU and if it is expressed by the RPE itself. Via immunohistochemistry, significant downregulation of peripherin 2 in uveitic RPE compared to the control was detectable, but there was no difference in healthy and uveitic retina. A further interesting finding was the clear distinction between peripherin 2 and the phagocytosis marker, rhodopsin, in healthy RPE. In conclusion, changes in the expression pattern of peripherin 2 selectively affect RPE, but not retina, in ERU. Moreover, peripherin 2 is clearly detectable in healthy RPE due to both phagocytosis and the expression by the RPE cells themselves. Our novel findings are very promising for better understanding the molecular mechanisms taking place on RPE in uveitis.

  17. Effects of PDTC on the Proliferation and PCNA Expression of Human Retinal Pigment Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    HU Jun; LI Guigang

    2006-01-01

    To investigate the effects of pyrrolidine dithiocarbamate (PDTC) on the proliferation and PCNA (proliferating cell nuclear antigen) expression of cultured human retinal pigment epithelium cells, human retinal pigment epithelium cells (RPE) were cultured from normal adults who died accidentally. The effects of PDTC on the proliferation of RPE cells were examined by using methyl thiazlyl tetrazolium (MTT) assay. The effects of PDTC on the PCNA expression of RPE cells were immunohistochemically examined by employing biological image analysis system (BIAS). After treatment with PDTC of various of concentration ranging from 0.062 to 1 g/L for 24 h, or concentrations ranging from 0. 031 to 1 g/L, the proliferation of RPE cells decreased in a dose-dependent manner. After treatment with PDTC of concentration varying from 0. 062 to 1 g/L for 24 h, the PCNA expression was also suppressed in a dose-dependent manner. It is concluded that PDTC can inhibit the proliferation of RPE cells in vitro in a dose-and time-dependent manner, at least in part,by down-regulating the expression of PCNA. PDTC may be used to prevent and treat the proliferative vitreoretinopathy (PVR).

  18. Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells.

    Science.gov (United States)

    Vogt, Rhonda R; Unda, Richard; Yeh, Lee-Chuan C; Vidro, Eileen K; Lee, John C; Tsin, Andrew T

    2006-08-01

    Retinal pigment epithelial (RPE) cells secrete vascular endothelial growth factor (VEGF), a cytokine known to promote angiogenesis. Results from RNase protection assays (RPAs) show that RPE from non-diabetic human donors and from adult retinal pigment epithelium-19 (ARPE-19) cells expressed significant bone morphogenetic protein-4 (BMP-4) message. In addition, ARPE-19 cells cultured in high glucose (25 mM), compared to those in physiological glucose (5.5 mM) released significantly more BMP-4 into the conditioned media (CM). However, the effect of BMP-4 on the release of VEGF by ARPE-19 cells has not been studied. Accordingly, ARPE-19 cells were treated with BMP-4 to determine VEGF secretion. BMP-4 and VEGF levels in the CM and cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). Cells treated with exogenous BMP-4 had higher VEGF in the CM and this treatment effect was dose- and time-dependent, while cell lysates had low levels of VEGF. Addition of cycloheximide (CHX) or actinomycin-D (ACT) significantly reduced VEGF secretion from cells treated with BMP-4, suggesting that the BMP-4-induced secretion of VEGF requires new RNA and protein synthesis. Our results suggest that BMP-4 may play a role in the regulation of ocular angiogenesis associated with diabetic retinopathy (DR) by stimulating VEGF release from RPE cells.

  19. Complement factor H, vitronectin, and opticin are tyrosine-sulfated proteins of the retinal pigment epithelium.

    Directory of Open Access Journals (Sweden)

    Yogita Kanan

    Full Text Available Lack of tyrosine sulfation of ocular proteins results in disorganized photoreceptor structure and drastically reduced visual function, demonstrating the importance of this post-translational modification to vision. To understand the role that tyrosine sulfation plays in the function of ocular proteins, we identified some tyrosine-sulfated proteins in the retinal pigment epithelium using two independent methods, immuno-affinity column purification with an anti-sulfotyrosine specific antibody and computer-based sequence analysis of retinal pigment epithelium secretome by means of the prediction program Sulfinator. Radioactive labeling followed by thin layer electrophoresis revealed that three proteins, vitronectin, opticin, and complement factor H (CFH, were post-translationally modified by tyrosine sulfation. The identification of vitronectin and CFH as tyrosine-sulfated proteins is significant, since both are deposited in drusen in the eyes of patients with age-related macular degeneration (AMD. Furthermore, mutations in CFH have been determined to be a major risk factor in the development of AMD. Future studies that seek to understand the role of CFH in the development of AMD should take into account the role that tyrosine sulfation plays in the interaction of this protein with its partners, and examine whether modulating sulfation provides a potential therapeutic target.

  20. Establishment of a blue light damage model of human retinal pigment epithelial cells in vitro.

    Science.gov (United States)

    Su, G; Cai, S J; Gong, X; Wang, L L; Li, H H; Wang, L M

    2016-06-24

    To establish a blue-light damage model of human retinal pigment epithelium (RPE). Fourth-generation human RPE cells were randomly divided into two groups. In group A, cells were exposed to blue light (2000 ± 500 lux) for 0 (control), 3, 6, 9, and 12 h, and cell culture was stopped after 12 h. In group B, cells were exposed to blue light at the same intensity and time periods, but cell culture was stopped after 24 h. TdT-mediated dUTP nick-end labeling (TUNEL) assay was performed to determine the most suitable illuminating time with apoptotic index. Flow cytometry was used to determine apoptotic ratio of RPEs. In group A, the apoptotic index of cells that received 6, 9 and 12 h of blue light was higher than that of control. The apoptotic index of cells receiving 9 and 12 h was higher than that of 6 h (P = 0.000). In group B, the apoptotic index and RPE cell apoptosis ratio of cells exposed to 6, 9 and 12 h of blue light were higher than that of 3 h (P = 0.000); and cells receiving 9 and 12 h had higher values than that of 6 h. This study demonstrated that the best conditions to establish a blue light damage model of human retinal pigment epithelial cells in vitro are 2000 ± 500 lux light intensity for 6 h, with 24 h of cell culture post-exposure.

  1. Lycium barbarum polysaccharides protected human retinal pigment epithelial cells against oxidative stressinduced apoptosis

    Institute of Scientific and Technical Information of China (English)

    Lian; Liu; Wei; Lao; Qing-Shan; Ji; Zhi-Hao; Yang; Guo-Cheng; Yu; Jing-Xiang; Zhong

    2015-01-01

    AIM: To investigate the protective effect and its mechanism of lycium barbarum polysaccharides(LBP)against oxidative stress-induced apoptosis in human retinal pigment epithelial cells.METHODS: ARPE-19 cells, a human retinal pigment epithelial cell lines, were exposed to different concentrations of H2O2 for 24h, then cell viability was measured by Cell Counting Kit-8(CCK-8) assay to get the properly concentration of H2O2 which can induce half apoptosis of APRE-19. With different concentrations of LBP pretreatment, the ARPE-19 cells were then exposed to appropriate concentration of H2O2, cell apoptosis was detected by flow cytometric analysis. Expression levels of Bcl-2 and Bax were measured by real time quantitative polymerase chain reaction(RT-PCR) technique.RSULTS: LBP significantly reduced the H2O2-induced ARPE-19 cells’ apoptosis. LBP inhibited the H2O2-induced down-regulation of Bcl-2 and up-regulation of Bax.CONCLUSION: LBP could protect ARPE-19 cells from H2O2-induced apoptosis. The Bcl-2 family had relationship with the protective effects of LBP.

  2. Proteomic Profiling of Cigarette Smoke Induced Changes in Retinal Pigment Epithelium Cells.

    Science.gov (United States)

    Merl-Pham, Juliane; Gruhn, Fabian; Hauck, Stefanie M

    2016-01-01

    Age-related macular degeneration (AMD) is a medical condition usually affecting older adults and resulting in a loss of vision in the macula, the center of the visual field. The dry form of this disease presents with atrophy of the retinal pigment epithelium, resulting in the detachment of the retina and loss of photoreceptors. Cigarette smoke is one main risk factor for dry AMD and increases the risk of developing the disease by three times. In order to understand the influence of cigarette smoke on retinal pigment epithelial cells, cultured human ARPE-19 cells were treated with cigarette smoke extract for 24 h. Using quantitative mass spectrometry more than 3000 proteins were identified and their respective abundances were compared between cigarette smoke-treated and untreated cells. Altogether 1932 proteins were quantified with at least two unique peptides, with 686 proteins found to be significantly differentially abundant with p > 0.05. Of these proteins the abundance of 64 proteins was at least 2-fold down-regulated after cigarette smoke treatment while 120 proteins were 2-fold up-regulated. The analysis of associated biological processes revealed an alteration of proteins involved in RNA processing and transport as well as extracellular matrix remodelling in response to cigarette smoke treatment.

  3. Effects of modified LDL and HDL on retinal pigment epithelial cells: a role in diabetic retinopathy?

    Science.gov (United States)

    Du, M; Wu, M; Fu, D; Yang, S; Chen, J; Wilson, K; Lyons, T J

    2013-10-01

    Blood-retina barrier leakage in diabetes results in extravasation of plasma lipoproteins. Intra-retinal modified LDLs have been implicated in diabetic retinopathy (DR), but their effects on retinal pigment epithelial (RPE) cells and the added effects of extravasated modified HDLs are unknown. In human retinas from individuals with and without diabetes and DR, immunohistochemistry was used to detect ApoB, ApoA1 and endoplasmic reticulum (ER) stress markers. In cell culture, human RPE cells were treated with native LDL (N-LDL) or heavily-oxidised glycated LDL (HOG-LDL) with or without pretreatment with native HDL (N-HDL) or heavily-oxidised glycated HDL (HOG-HDL). Cell viability, oxidative stress, ER stress, apoptosis and autophagy were assessed by Cell Counting Kit-8 assay, dichlorofluorescein assay, western blotting, immunofluorescence and TUNEL assay. In separate experiments, RPE cells were treated with lipid oxidation products, 7-ketocholesterol (7-KC, 5-40 μmol/l) or 4-hydroxynonenal (4-HNE, 5-80 μmol/l), with or without pretreatment with N-HDL or HOG-HDL. ApoB, ApoA1 staining and RPE ER stress were increased in the presence of DR. HOG-LDL but not N-LDL significantly decreased RPE cell viability and increased reactive oxygen species generation, ER stress, apoptosis and autophagy. Similarly, 4-HNE and 7-KC decreased viability and induced ER stress. Pretreatment with N-HDL mitigated these effects, whereas HOG-HDL was less effective by most, but not all, measures. In DR, extravascular modified LDL may promote RPE injury through oxidative stress, ER stress, autophagy and apoptosis. N-HDL has protective effects, but HOG-HDL is less effective. Extravasation and modification of HDL may modulate the injurious effects of extravasated modified LDL on the retinal pigment epithelium.

  4. Applying photoacoustics to quantification of melanin concentration in retinal pigment epithelium (Conference Presentation)

    Science.gov (United States)

    Shu, Xiao; Zhang, Hao F.; Liu, Wenzhong

    2016-03-01

    The melanin in the retinal pigment epithelium (RPE) protects retina and other ocular tissues by photo-screening and acting as antioxidant and free radical scavenger. It helps maintain normal visual functions since human eye is subjected to lifelong high oxygen stress and photon exposure. Loss of the RPE melanin weakens the protection mechanism and jeopardizes ocular health. Local decrease in the RPE melanin concentration is believed to be both a cause and a sign of early-stage age-related macular degeneration (AMD), the leading blinding disease in developed world. Current technology cannot quantitatively measure the RPE melanin concentration which might be a promising marker in early AMD screening. Photoacoustic ophthalmoscopy (PAOM), as an emerging optical absorption-based imaging technology, can potentially be applied to measure the RPE melanin concentration if the dependence of the detectable photoacoustic (PA) signal amplitudes on the RPE melanin concentrations is verified. In this study, we tested the feasibility of using PA signal ratio from RPE melanin and the nearby retinal blood vessels as an indicator of the RPE melanin variation. A novel whole eye optical model was designed and Monte Carlo modeling of light (MCML) was employed. We examined the influences on quantification from PAOM axial resolution, the depth and diameter of the retinal blood vessel, and the RPE thickness. The results show that the scheme is robust to individual histological and illumination variations. This study suggests that PAOM is capable of quantitatively measuring the RPE melanin concentration in vivo.

  5. Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

    Science.gov (United States)

    Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

    2015-03-01

    The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

  6. Beta cyclodextrins bind, stabilize, and remove lipofuscin bisretinoids from retinal pigment epithelium.

    Science.gov (United States)

    Nociari, Marcelo M; Lehmann, Guillermo L; Perez Bay, Andres E; Radu, Roxana A; Jiang, Zhichun; Goicochea, Shelby; Schreiner, Ryan; Warren, J David; Shan, Jufang; Adam de Beaumais, Ségolène; Ménand, Mickaël; Sollogoub, Matthieu; Maxfield, Frederick R; Rodriguez-Boulan, Enrique

    2014-04-08

    Accumulation of lipofuscin bisretinoids (LBs) in the retinal pigment epithelium (RPE) is the alleged cause of retinal degeneration in genetic blinding diseases (e.g., Stargardt) and a possible etiological agent for age-related macular degeneration. Currently, there are no approved treatments for these diseases; hence, agents that efficiently remove LBs from RPE would be valuable therapeutic candidates. Here, we show that beta cyclodextrins (β-CDs) bind LBs and protect them against oxidation. Computer modeling and biochemical data are consistent with the encapsulation of the retinoid arms of LBs within the hydrophobic cavity of β-CD. Importantly, β-CD treatment reduced by 73% and 48% the LB content of RPE cell cultures and of eyecups obtained from Abca4-Rdh8 double knock-out (DKO) mice, respectively. Furthermore, intravitreal administration of β-CDs reduced significantly the content of bisretinoids in the RPE of DKO animals. Thus, our results demonstrate the effectiveness of β-CDs to complex and remove LB deposits from RPE cells and provide crucial data to develop novel prophylactic approaches for retinal disorders elicited by LBs.

  7. Conditional ablation of the choroideremia gene causes age-related changes in mouse retinal pigment epithelium.

    Directory of Open Access Journals (Sweden)

    Silène T Wavre-Shapton

    Full Text Available The retinal pigment epithelium (RPE is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1. REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm(Flox, Tyr-Cre+. Transmission electron microscopy (TEM was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch's membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5-6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm(Flox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD suggest that membrane traffic defects may contribute to the pathogenesis of AMD.

  8. The effects of taurine on vigabatrin, high light intensity and mydriasis induced retinal toxicity in the pigmented rat.

    Science.gov (United States)

    Rasmussen, Allan D; Truchot, Nathalie; Pickersgill, Nigel; Thale, Zia Irene; Rosolen, Serge G; Botteron, Catherine

    2015-01-01

    The overall purpose of this study was to establish a model that may be used for examining the effect of Vigabatrin-induced retinal toxicity in pigmented rats, and subsequently examine the possible effects of taurine on the retinal toxicity. In the first part of the study, pigmented Long Evans rats were subjected to combinations of induced mydriasis, low/high light intensities (40/2000 lx) and oral administration of near-MTD (Maximum Tolerated Dose) doses (200 mg/kg/day) of Vigabatrin for up to 6 weeks. The combination of mydriasis and high light intensity applied to Long Evans rats resulted in retinal damage that was increased by the administration of Vigabatrin. In the second part of the study Long Evans rats were subjected to combinations of induced mydriasis and high/low light intensity (40/2000 lx) while being orally administered low (30 mg/kg/day) or high (200 mg/kg/day) doses of Vigabatrin for up to 6 weeks. In addition, selected groups of animals were administered taurine via the drinking water (20 mg/ml), resulting in systemic taurine concentrations of approximately threefold the endogenous concentration. The combined results of the studies demonstrate that retinal damage can be induced in pigmented animals when combining mydriasis and high light intensity. Retinal damage was functionally evaluated by electroretinography (ERG), then confirmed by histopathology. While depending on mydriasis and high light intensity, administration of Vigabatrin increased the retinal toxicity and resulted in the formation of rosette-like structures in the retina in a dose-related manner. Administration of taurine did not alleviate the Vigabatrin-induced retinal toxicity, as demonstrated either functionally by ERG or morphologically, although systemic concentrations of 3-fold the endogenous levels were reached, and it was thus not possible to demonstrate a protective effect of taurine in these pigmented animals. Copyright © 2014 Elsevier GmbH. All rights reserved.

  9. Change of morphological and functional characteristics of retinal pigment epithelium cells during cultivation of retinal pigment epithelium-choroid perfusion tissue culture.

    Science.gov (United States)

    Miura, Yoko; Klettner, Alexa; Noelle, Bernhard; Hasselbach, Heike; Roider, Johann

    2010-01-01

    To evaluate the changes of morphological and functional characteristics of the retinal pigment epithelium (RPE)-choroid perfusion culture during cultivation. PorcineRPE-choroid tissue was cultivated in a perfusion tissue culture system. After the indicated times, histology, immunolocalization of collagen IV and von Willebrand factor, RPE cell viability with calcein-AM, TUNEL assay and occludin immunolocalization of RPE cells were examined. The tissue was treated with selective RPE treatment laser after different time periods and the wound healing response was characterized. Vascular endothelial growth factor secretion was measured by enzyme-linked immunosorbent assay. On day 8, prominent morphological degenerative changes of RPE cells were observed in histology. According to the immunohistochemistry for collagen IV, the Bruch's membrane did not display any obvious decomposition until day 8. Von Willebrand factor staining decreased during cultivation, especially at the choriocapillaris. Calcein-AM staining and TUNEL assay displayed the increase of apoptotic changes in only a minority of the cells on day 4, but in many cells on day 8. Occludin delocalization was observed on day 8. Selective RPE treatment laser-produced wounds were completely closed by monolayer RPE when wounded on fresh and 3-day-old cultures, but not when wounded on 6-day-old cultures. Vascular endothelial growth factor secretion was stable between days 2 and 5, but increased after that. Under the stated culture perfusion conditions, porcine RPE-choroid tissue was suitable for experimentation up to 5 days of maintenance. Copyright 2009 S. Karger AG, Basel.

  10. Unraveling the cellular uptake of bioreducible poly(amido amine) — Gene complexes in cells of the retinal pigment epithelium

    NARCIS (Netherlands)

    Vercauteren, D.; Piest, M.; Soraj, M. Al; Jones, A.T.; Engbersen, J.F.J.; Smedt, de S.C.; Braeckmans, K.

    2010-01-01

    In vitro endocytosis of gene complexes composed of a bioreducible polyamidoamine CBA ABOL and plasmid DNA, in cells of the retinal pigment epithelium (RPE) was studied, the latter being an interesting target for ocular gene therapy. We found that cationic CBA ABOL DNA polyplexes attach to cell surfa

  11. Primary Adult Human Retinal Pigment Epithelial Cell Cultures on Human Amniotic Membranes

    Directory of Open Access Journals (Sweden)

    Singhal Shweta

    2005-01-01

    Full Text Available Purpose: Retinal pigment epithelial (RPE cells grow well on surfaces that provide an extracellular matrix. Our aim was to establish primary adult human RPE cell cultures that retain their epithelial morphology in vitro using human amniotic membrane (hAM as substrate. Materials and Methods: Human cadaver eyeballs (16 were obtained from the eye bank after corneal trephination. RPE cells were harvested by a mechanical dissection of the inner choroid surface (10, group 1 or by b enzymatic digestion using 0.25% Trypsin/0.02% EDTA (6, group 2. The cells were explanted onto de-epithelialized hAM, nourished using DMEM/HAMS F-12 media and monitored for growth under the phase contrast microscope. Cell cultures were characterised by whole mount studies and paraffin sections. Growth data in the two groups were compared using the students′ ′t′ test. Results: Eleven samples (68.75% showed positive cultures with small, hexagonal cells arising from around the explant which formed a confluent and progressively pigmented monolayer. Whole mounts showed closely placed polygonal cells with heavily pigmented cytoplasm and indistinct nuclei. The histologic sections showed monolayers of cuboidal epithelium with variable pigmentation within the cytoplasm. Growth was seen by day 6-23 (average 11.5 days in the mechanical group, significantly earlier ( P Conclusions: Primary adult human RPE cell cultures retain epithelial morphology in vitro when cultured on human amniotic membranes . Mechanical dissection of the inner choroid surface appears to be an effective method of isolating RPE cells and yields earlier growth in cultures as compared to isolation by enzymatic digestion

  12. Pattern of inner retinal layers involvement in pigmented paravenous retinochoroidal atrophy as determined by SD-OCT: case report.

    Science.gov (United States)

    Junqueira, Daniela Laura Melo; Lopes, Flavio Siqueira Santos; Biteli, Luís Gustavo; Prata, Tiago Santos

    2013-01-01

    Pigmented paravenous retinochoroidal atrophy is an ocular disease characterized by outer retina and choroidal atrophy often with overlying intraretinal bone spicule pigment deposition along the retinal veins. As a rare condition, there is scant information in the literature regarding the pattern of inner retinal layers involvement. We present a case of a 41-year-old white man initially referred for a glaucoma evaluation. Fundoscopy revealed patches of retinochoroidal atrophy and light pigmentation extending from the optic nerve head along the inferior-temporal retinal veins in both eyes. Using different spectral-domain optical coherence tomography (SD-OCT) protocols we identified a significant thinning of the inner retinal layers along the inferior-temporal veins, but with a lucid interval surrounding the optic nerve head. Standard automated perimetry revealed a superior absolute arcuate scotoma sparing the central fixation (good structure-functional correlation). This pattern of inner retinal layers involvement was not previously described. We believe SD-OCT added significantly to the anatomical description of this case. Physicians should consider these new anatomical findings and correlate them with functional status while assessing these patients.

  13. Pattern of inner retinal layers involvement in pigmented paravenous retinochoroidal atrophy as determined by SD-OCT: case report

    Directory of Open Access Journals (Sweden)

    Daniela Laura Melo Junqueira

    2013-12-01

    Full Text Available Pigmented paravenous retinochoroidal atrophy is an ocular disease characterized by outer retina and choroidal atrophy often with overlying intraretinal bone spicule pigment deposition along the retinal veins. As a rare condition, there is scant information in the literature regarding the pattern of inner retinal layers involvement. We present a case of a 41-year-old white man initially referred for a glaucoma evaluation. Fundoscopy revealed patches of retinochoroidal atrophy and light pigmentation extending from the optic nerve head along the inferior-temporal retinal veins in both eyes. Using different spectral-domain optical coherence tomography (SD-OCT protocols we identified a significant thinning of the inner retinal layers along the inferior-temporal veins, but with a lucid interval surrounding the optic nerve head. Standard automated perimetry revealed a superior absolute arcuate scotoma sparing the central fixation (good structure-functional correlation. This pattern of inner retinal layers involvement was not previously described. We believe SD-OCT added significantly to the anatomical description of this case. Physicians should consider these new anatomical findings and correlate them with functional status while assessing these patients.

  14. Modification of Isolation and Culture of Human Retinal Pigment Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    ZhengJL; GuoY

    1999-01-01

    Purpose:To modify the isolation of human retinal pigment pithelial(RPE)cells and to increase the purification and production of cultured RPE cells.Methods:The human eyecups were fixed on a fubber holder.After digestion by trypsin,RPE cells were collected,then cultured and identified by morphology,immunohistochemistry and electron microscopy.Results:The cultured RPE cells grew actively in the early stage with transparent nucleus and abundant melanin particles in cytoplasm.These cells were positive in DOPA oxidase reaction and in anti-pancytokeratin antibody staining.Cellular microvilli and tight junctions could be seen through transmission electrom microscopy.Conclusion:We developed a rubber holder to fix the eyecup.Using this holder,more and purer cultured RPE cells can be obtained.These cultured REP cells are similar to those in vivo in morphology and immunohistochemical staining.

  15. Prolactin protects retinal pigment epithelium by inhibiting sirtuin 2-dependent cell death.

    Science.gov (United States)

    Meléndez García, Rodrigo; Arredondo Zamarripa, David; Arnold, Edith; Ruiz-Herrera, Xarubet; Noguez Imm, Ramsés; Baeza Cruz, German; Adán, Norma; Binart, Nadine; Riesgo-Escovar, Juan; Goffin, Vincent; Ordaz, Benito; Peña-Ortega, Fernando; Martínez-Torres, Ataúlfo; Clapp, Carmen; Thebault, Stéphanie

    2016-05-01

    The identification of pathways necessary for retinal pigment epithelium (RPE) function is fundamental to uncover therapies for blindness. Prolactin (PRL) receptors are expressed in the retina, but nothing is known about the role of PRL in RPE. Using the adult RPE 19 (ARPE-19) human cell line and mouse RPE, we identified the presence of PRL receptors and demonstrated that PRL is necessary for RPE cell survival via anti-apoptotic and antioxidant actions. PRL promotes the antioxidant capacity of ARPE-19 cells by reducing glutathione. It also blocks the hydrogen peroxide-induced increase in deacetylase sirtuin 2 (SIRT2) expression, which inhibits the TRPM2-mediated intracellular Ca(2+) rise associated with reduced survival under oxidant conditions. RPE from PRL receptor-null (prlr(-/-)) mice showed increased levels of oxidative stress, Sirt2 expression and apoptosis, effects that were exacerbated in animals with advancing age. These observations identify PRL as a regulator of RPE homeostasis.

  16. Prolactin protects retinal pigment epithelium by inhibiting sirtuin 2-dependent cell death

    Directory of Open Access Journals (Sweden)

    Rodrigo Meléndez García

    2016-05-01

    Full Text Available The identification of pathways necessary for retinal pigment epithelium (RPE function is fundamental to uncover therapies for blindness. Prolactin (PRL receptors are expressed in the retina, but nothing is known about the role of PRL in RPE. Using the adult RPE 19 (ARPE-19 human cell line and mouse RPE, we identified the presence of PRL receptors and demonstrated that PRL is necessary for RPE cell survival via anti-apoptotic and antioxidant actions. PRL promotes the antioxidant capacity of ARPE-19 cells by reducing glutathione. It also blocks the hydrogen peroxide-induced increase in deacetylase sirtuin 2 (SIRT2 expression, which inhibits the TRPM2-mediated intracellular Ca2+ rise associated with reduced survival under oxidant conditions. RPE from PRL receptor-null (prlr−/− mice showed increased levels of oxidative stress, Sirt2 expression and apoptosis, effects that were exacerbated in animals with advancing age. These observations identify PRL as a regulator of RPE homeostasis.

  17. Blockage of Notch Signaling Inhibits the Migration and Proliferation of Retinal Pigment Epithelial Cells

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    Weiwei Liu

    2013-01-01

    Full Text Available The Notch signaling is an evolutionarily conserved cell-cell communication pathway that plays critical roles in the proliferation, survival, apoptosis, and fate determination of mammalian cells. Retinal pigment epithelial (RPE cells are responsible for supporting the function of the neural retina and maintaining vision. This study investigated the function of Notch signaling in RPE cells. We found that the members of the Notch signaling pathway components were differentially expressed in RPE cells. Furthermore, blockage of Notch signaling inhibited the migration and proliferation of RPE cells and reduced the expression levels of certain Notch signaling target genes, including HES1, MYC, HEY2, and SOX9. Our data reveal a critical role of Notch signaling in RPE cells, suggesting that targeting Notch signaling may provide a novel approach for the treatment of ophthalmic diseases related to RPE cells.

  18. Lack of FasL expression in cultured human retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Kaestel, C G; Madsen, H O; Prause, J U

    2001-01-01

    Retinal pigment epithelial (RPE) cells have been proposed to play a part in maintaining the eye as an immune privileged organ. However, our knowledge of the implicated mechanism is still sparse. Fas ligand (FasL) expression of RPE cells is generally recognized to be essential for the immune...... blotting, RT-PCR and RNase Protection assay for FasL expression. Additionally, sections of ocular tissue were stained for FasL by immunohistochemistry. None of the used methods indicated FasL expression in cultured fetal or adult RPE cells of various passages. However, RPE cells in vivo, as judged from...... tissue sections, were positive for FasL, indicating a discrepancy between RPE cells in vitro and in vivo with regard to this molecule....

  19. BMP-induced reprogramming of the neural retina into retinal pigment epithelium requires Wnt signalling

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    Jörg Steinfeld

    2017-07-01

    Full Text Available In vertebrates, the retinal pigment epithelium (RPE and photoreceptors of the neural retina (NR comprise a functional unit required for vision. During vertebrate eye development, a conversion of the RPE into NR can be induced by growth factors in vivo at optic cup stages, but the reverse process, the conversion of NR tissue into RPE, has not been reported. Here, we show that bone morphogenetic protein (BMP signalling can reprogram the NR into RPE at optic cup stages in chick. Shortly after BMP application, expression of Microphthalmia-associated transcription factor (Mitf is induced in the NR and selective cell death on the basal side of the NR induces an RPE-like morphology. The newly induced RPE differentiates and expresses Melanosomalmatrix protein 115 (Mmp115 and RPE65. BMP-induced Wnt2b expression is observed in regions of the NR that become pigmented. Loss of function studies show that conversion of the NR into RPE requires both BMP and Wnt signalling. Simultaneous to the appearance of ectopic RPE tissue, BMP application reprogrammed the proximal RPE into multi-layered retinal tissue. The newly induced NR expresses visual segment homeobox-containing gene (Vsx2, and the ganglion and photoreceptor cell markers Brn3α and Visinin are detected. Our results show that high BMP concentrations are required to induce the conversion of NR into RPE, while low BMP concentrations can still induce transdifferentiation of the RPE into NR. This knowledge may contribute to the development of efficient standardized protocols for RPE and NR generation for cell replacement therapies.

  20. The Retinal Pigment Epithelium: a Convenient Source of New Photoreceptor cells?

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    Shu-Zhen Wang

    2014-01-01

    Full Text Available Recent success in restoring visual function through photoreceptor replacement in mouse models of photoreceptor degeneration intensifies the need to generate or regenerate photoreceptor cells for the ultimate goal of using cell replacement therapy for blindness caused by photoreceptor degeneration. Current research on deriving new photoreceptors for replacement, as regenerative medicine in general, focuses on the use of embryonic stem cells and induced pluripotent stem (iPS cells to generate transplantable cells. Nonetheless, naturally occurring regeneration, such as wound healing, involves awakening cells at or near a wound site to produce new cells needed to heal the wound. Here we discuss the possibility of tweaking an ocular tissue, the retinal pigment epithelium (RPE, to produce photoreceptor cells in situ in the eye. Unlike the neural retina, the RPE in adult mammals maintains cell proliferation capability. Furthermore, progeny cells from RPE proliferation may differentiate into cells other than RPE. The combination of proliferation and plasticity opens a question of whether they could be channeled by a regulatory gene with pro-photoreceptor activity towards photoreceptor production. Studies using embryonic chick and transgenic mouse showed that indeed photoreceptor-like cells were produced in culture and in vivo in the eye using genedirected reprogramming of RPE cells, supporting the feasibility of using the RPE as a convenient source of new photoreceptor cells for in situ retinal repair without involving cell transplantation.

  1. Effects of vegetable oils on biochemical and biophysical properties of membrane retinal pigment epithelium cells.

    Science.gov (United States)

    Said, Toihiri; Tremblay-Mercier, Jennifer; Berrougui, Hicham; Rat, Patrice; Khalil, Abdelouahed

    2013-10-01

    The aim of this study was to investigate the effect of vegetable oil enrichment of retinal pigment epithelial (RPE) cells on their biochemical and biophysical properties. For this, RPE cells were incubated with 4 different vegetables oils (olive oil, corn oil, argan oil, and camelina oil). The cytotoxicity of these vegetable oils was assessed in vivo on 8-week-old mice and in vitro by using the neutral red and YO-PRO-1 tests. Membrane fluidity was evaluated by fluorescence anisotropy using the fluorescent probe diphenylhexatriene, and membrane fatty acid composition was assessed by gas chromatography. None of the oils tested displayed cytotoxic effects. In vitro, omega-3 rich oils improved membrane fluidity by 47% compared with the control cells. The omega-3 PUFA content within membranes decreased by 38% to 55% when cells were incubated separately with olive oil, corn oil, or argan oil, and increased when cells were incubated with a mixture of those oils, or with camelina oil alone (50% and 103% increase, respectively). Our results show that the fatty acids in vegetable oil incorporate into retinal cells and increase the plasma membrane fluidity.

  2. Dynamics and detection of laser induced microbubbles in the retinal pigment epithelium (RPE)

    Science.gov (United States)

    Fritz, Andreas; Ptaszynski, Lars; Stoehr, Hardo; Brinkmann, Ralf

    2007-07-01

    Selective Retina Treatment (SRT) is a new method to treat eye diseases associated with disorders of the RPE. Selective RPE cell damage is achieved by applying a train of 1.7 μs laser pulses at 527 nm. The treatment of retinal diseases as e.g. diabetic maculopathy (DMP), is currently investigated within clinical studies, however 200 ns pulse durations are under investigation. Transient micro bubbles in the retinal pigment epithelium (RPE) are expected to be the origin of cell damage due to irradiation with laser pulses shorter than 50 μs. The bubbles emerge at the strongly absorbing RPE melanosomes. Cell membrane disruption caused by the transient associated volume increase is expected to be the origin of the angiographically observed RPE leakage. We investigate micro bubble formation and dynamics in porcine RPE using pulse durations of 150 ns. A laser interferometry system at 830 nm with the aim of an online dosimetry control for SRT was developed. Bubble formation was detected interferometrically and by fast flash photography. A correlation to cell damage observed with a vitality stain is found. A bubble detection algorithm is presented.

  3. Genomic regulation of senescence and innate immunity signaling in the retinal pigment epithelium.

    Science.gov (United States)

    Chaum, Edward; Winborn, Christina S; Bhattacharya, Sujoy

    2015-06-01

    The tumor suppressor p53 is a major regulator of genes important for cell cycle arrest, senescence, apoptosis, and innate immunity, and has recently been implicated in retinal aging. In this study we sought to identify the genetic networks that regulate p53 function in the retina using quantitative trait locus (QTL) analysis. First we examined age-associated changes in the activation and expression levels of p53; known p53 target proteins and markers of innate immune system activation in primary retinal pigment epithelial (RPE) cells that were harvested from young and aged human donors. We observed increased expression of p53, activated caspase-1, CDKN1A, CDKN2A (p16INK4a), TLR4, and IFNα in aged primary RPE cell lines. We used the Hamilton Eye Institute (HEI) retinal dataset ( www.genenetwork.org ) to identify genomic loci that modulate expression of genes in the p53 pathway in recombinant inbred BXD mouse strains using a QTL systems biology-based approach. We identified a significant trans-QTL on chromosome 1 (region 172-177 Mb) that regulates the expression of Cdkn1a. Many of the genes in this QTL locus are involved in innate immune responses, including Fc receptors, interferon-inducible family genes, and formin 2. Importantly, we found an age-related increase in FCGR3A and FMN2 and a decrease in IFI16 levels in RPE cultures. There is a complex multigenic innate immunity locus that controls expression of genes in the p53 pathway in the RPE, which may play an important role in modulating age-related changes in the retina.

  4. Potential role of retinal pigment epithelial lipofuscin accumulation in age-related macular degeneration.

    Science.gov (United States)

    Katz, Martin L

    2002-01-01

    Age-related macular degeneration (AMD) is a leading cause of severe visual impairment in developed countries. The vision loss associated with AMD is the result of degenerative changes in the central region of the retina called the macula. Maintenance of normal structure and function of the macular retina, and of the remainder of the retina as well, is critically dependent on the supporting role of the adjacent retinal pigment epithelium (RPE). Impairment of normal RPE functions is known to result in retinal degeneration and loss of visual function. Thus, it has been hypothesized that the retinal degeneration that characterizes AMD is secondary to age-related deterioration in RPE support functions. Like many other postmitotic cell types, the RPE accumulates autofluorescent lysosomal storage bodies (lipofuscin) during senescence. In human eyes, lipofuscin comes to occupy a substantial fraction of the RPE cytoplasmic volume in the elderly. Does this lipofuscin accumulation contribute to the development of AMD? This question is a specific case of the broader question of whether lipofuscin accumulation in general is detrimental to cells. Unfortunately, definitive data do not exist to allow these questions to be answered. Although a correlation between RPE lipofuscin content and AMD has been reported, a cause-and-effect relationship between RPE lipofuscin accumulation and the development of this disease has not been established. It has been reported that a mutation in a gene encoding a photoreceptor-specific protein results in massive RPE lipofuscin accumulation and early-onset macular degeneration. However, again the accelerated RPE lipofuscin accumulation has not been shown to be the cause of the accompanying macular degeneration. The lack of a definitive link between RPE lipofuscin accumulation and AMD illustrates one of the biggest challenges remaining in lipofuscin research-determining whether lipofuscin accumulation per se has an impact on cell function.

  5. Noninvasive two-photon microscopy imaging of mouse retina and retinal pigment epithelium through the pupil of the eye.

    Science.gov (United States)

    Palczewska, Grazyna; Dong, Zhiqian; Golczak, Marcin; Hunter, Jennifer J; Williams, David R; Alexander, Nathan S; Palczewski, Krzysztof

    2014-07-01

    Two-photon excitation microscopy can image retinal molecular processes in vivo. Intrinsically fluorescent retinyl esters in subcellular structures called retinosomes are an integral part of the visual chromophore regeneration pathway. Fluorescent condensation products of all-trans-retinal accumulate in the eye with age and are also associated with age-related macular degeneration (AMD). Here, we report repetitive, dynamic imaging of these compounds in live mice through the pupil of the eye. By leveraging advanced adaptive optics, we developed a data acquisition algorithm that permitted the identification of retinosomes and condensation products in the retinal pigment epithelium by their characteristic localization, spectral properties and absence in genetically modified or drug-treated mice. This imaging approach has the potential to detect early molecular changes in retinoid metabolism that trigger light- and AMD-induced retinal defects and to assess the effectiveness of treatments for these conditions.

  6. Inhibition of autophagy induces retinal pigment epithelial cell damage by the lipofuscin fluorophore A2E

    Directory of Open Access Journals (Sweden)

    Khandakar A.S.M. Saadat

    2014-01-01

    Full Text Available In this study, we show augmented autophagy in the retinal pigment epithelial cell line ARPE-19 when cultured in the presence of the lipofuscin pigment A2E. A2E alone does not induce RPE cell death, but cell death was induced in the presence of A2E with the autophagy inhibitor 3-methyladenine (3MA, with a concomitant increase in the generation of mitochondrial reactive oxygen species. On the other hand, the ATP production capacity of mitochondria was decreased in the presence of A2E, and pharmacological inhibition of autophagy had no additional effects. The altered mRNA expression level of mitochondrial function markers was confirmed by real-time polymerase chain reaction, which showed that the antioxidant enzymes SOD1 and SOD2 were not reduced in the presence of A2E alone, but significantly suppressed with the addition of 3MA. Furthermore, transmission electron micrography revealed autophagic vacuole formation in the presence of A2E, and inhibition of autophagy resulted in the accumulation of abnormal mitochondria with loss of cristae. Spheroid culture of human RPE cells demonstrated debris accumulation in the presence of A2E, and this accumulation was accelerated in the presence of 3MA. These results indicate that autophagy in RPE cells is a vital cytoprotective process that prevents the accumulation of damaged cellular molecules.

  7. Retbindin is an extracellular riboflavin-binding protein found at the photoreceptor/retinal pigment epithelium interface.

    Science.gov (United States)

    Kelley, Ryan A; Al-Ubaidi, Muayyad R; Naash, Muna I

    2015-02-20

    Retbindin is a novel retina-specific protein of unknown function. In this study, we have used various approaches to evaluate protein expression, localization, biochemical properties, and function. We find that retbindin is secreted by the rod photoreceptors into the inter-photoreceptor matrix where it is maintained via electrostatic forces. Retbindin is predominantly localized at the interface between photoreceptors and retinal pigment epithelium microvilli, a region critical for retinal function and homeostasis. Interestingly, although it is associated with photoreceptor outer segments, retbindin's expression is not dependent on their presence. In vitro, retbindin is capable of binding riboflavin, thus implicating the protein as a metabolite carrier between the retina and the retinal pigment epithelium. Altogether, our data show that retbindin is a novel photoreceptor-specific protein with a unique localization and function. We hypothesize that retbindin is an excellent candidate for binding retinal flavins and possibly participating in their transport from the extracellular space to the photoreceptors. Further investigations are warranted to determine the exact function of retbindin in retinal homeostasis and disease.

  8. Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

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    Marius Ueffing

    2012-10-01

    Full Text Available The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses’ vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS, and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP and retinal pigment epithelium-specific protein 65kDa (RPE65. Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies.

  9. Structure and function of the retinal pigment epithelium, photoreceptors and cornea in the eye of Sardinella aurita (Clupeidae, Teleostei

    Directory of Open Access Journals (Sweden)

    Mostafa Ali Salem

    2016-05-01

    Full Text Available The structure of the pigment epithelium, photoreceptors and the cornea in the eye of a teleost, Sardinella aurita was examined by light and electron microscopy. The retinal pigment epithelium forms a single layer of cells joined laterally by cell junctions. Centrally in the retina these cells are columnar, while more peripherally they become cuboidal in shape. The basal (scleral border of the pigment epithelial cells is not infolded but is relatively smooth. Phagosomes containing lysosome-like bodies are also common features of the retinal pigment epithelium. Numerous melanosomes (pigment granules are abundant throughout the epithelial cells. These melanosomes probably absorb light which has passed through the photoreceptor layer. Four photoreceptor cells were identified; rods, long single cones, short single cones and double cones. The presence of these types suggests a diversity of photoreceptor function. Square mosaic pattern of cones and well-developed choroid gland are also main features of the eye. The inner segment of rods and cones were rich in organelles indicating much synthetic activity. Calycal processes projecting from cone outer segments are also observed. The cornea includes an epithelium with a complex pattern of surface microplicae, a basement membrane, dermal stroma, an iridescent layer, scleral stroma, Descemet’s membrane and endothelium. The autochthonous layer which is seen in some teleosts has not been observed in the cornea of this species. These and other observations were discussed in relation to the photic environment and habits of this fish.

  10. Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient.

    Science.gov (United States)

    Cereso, Nicolas; Pequignot, Marie O; Robert, Lorenne; Becker, Fabienne; De Luca, Valerie; Nabholz, Nicolas; Rigau, Valerie; De Vos, John; Hamel, Christian P; Kalatzis, Vasiliki

    2014-01-01

    Inherited retinal dystrophies (IRDs) comprise a large group of genetically and clinically heterogeneous diseases that lead to progressive vision loss, for which a paucity of disease-mimicking animal models renders preclinical studies difficult. We sought to develop pertinent human cellular IRD models, beginning with choroideremia, caused by mutations in the CHM gene encoding Rab escort protein 1 (REP1). We reprogrammed REP1-deficient fibroblasts from a CHM (-/y) patient into induced pluripotent stem cells (iPSCs), which we differentiated into retinal pigment epithelium (RPE). This iPSC-derived RPE is a polarized monolayer with a classic morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. We assayed a panel of adeno-associated virus (AAV) vector serotypes and showed that AAV2/5 is the most efficient at transducing the iPSC-derived RPE and that CHM gene transfer normalizes the biochemical phenotype. The high, and unmatched, in vitro transduction efficiency is likely aided by phagocytosis and mimics the scenario that an AAV vector encounters in vivo in the subretinal space. We demonstrate the superiority of AAV2/5 in the human RPE and address the potential of patient iPSC-derived RPE to provide a proof-of-concept model for gene replacement in the absence of an appropriate animal model.

  11. Taurine inhibits interleukin-6 expression and release induced by ultraviolet B exposure to human retinal pigment epithelium cells.

    Science.gov (United States)

    Dayang, Wu; Jinsong, Zhang

    2015-01-01

    The massive uptake of compatible osmolytes is a self-protective response shared by retina exposed to hypertonic stress and ultraviolet stress. This study aimed to investigate the protective effects of taurine against ultraviolet damage in human retinal pigment epithelium cells. Real-time PCR, radioimmunoassay, ELISA and immunoassay were used to measure osmolyte uptake and IL-6 expression. Compared with normotonic stress, hypertonic stress led to an induction of osmolyte uptake including betaine, myoinositol and taurine. UVB exposure upregulated osmolyte transporter mRNA expression and increased osmolyte uptake respectively. Especially, taurine suppressed UVB-induced IL-6 mRNA expression significantly. The accumulation of IL-6 in UVB-exposed human retinal pigment epithelial cells supernatant was much slower when the cells were preincubated with taurine. Moreover, taurine suppressed IL-6 concentration in aqueous humour. The effect of compatible osmolyte taurine on IL-6 expression and release may play an important role in cell resistance and adaption to UVB exposure.

  12. Imaging human retinal pigment epithelium cells using adaptive optics optical coherence tomography

    Science.gov (United States)

    Liu, Zhuolin; Kocaoglu, Omer P.; Turner, Timothy L.; Miller, Donald T.

    2016-03-01

    Retinal pigment epithelium (RPE) cells are vital to health of the outer retina, but are often compromised in ageing and major ocular diseases that lead to blindness. Early manifestation of RPE disruption occurs at the cellular level, and while biomarkers at this scale hold considerable promise, RPE cells have proven extremely challenging to image in the living human eye. We present a novel method based on optical coherence tomography (OCT) equipped with adaptive optics (AO) that overcomes the associated technical obstacles. The method takes advantage of the 3D resolution of AO-OCT, but more critically sub-cellular segmentation and registration that permit organelle motility to be used as a novel contrast mechanism. With this method, we successfully visualized RPE cells and characterized their 3D reflectance profile in every subject and retinal location (3° and 7° temporal to the fovea) imaged to date. We have quantified RPE packing geometry in terms of cell density, cone-to-RPE ratio, and number of nearest neighbors using Voronoi and power spectra analyses. RPE cell density (cells/mm2) showed no significant difference between 3° (4,892+/-691) and 7° (4,780+/-354). In contrast, cone-to- RPE ratio was significantly higher at 3° (3.88+/-0.52:1) than 7° (2.31+/- 0.23:1). Voronoi analysis also showed most RPE cells have six nearest neighbors, which was significantly larger than the next two most prevalent associations: five and seven. Averaged across the five subjects, prevalence of cells with six neighbors was 51.4+/-3.58% at 3°, and 54.58+/-3.01% at 7°. These results are consistent with histology and in vivo studies using other imaging modalities.

  13. Deletion of autophagy inducer RB1CC1 results in degeneration of the retinal pigment epithelium.

    Science.gov (United States)

    Yao, Jingyu; Jia, Lin; Khan, Naheed; Lin, Chengmao; Mitter, Sayak K; Boulton, Michael E; Dunaief, Joshua L; Klionsky, Daniel J; Guan, Jun-Lin; Thompson, Debra A; Zacks, David N

    2015-01-01

    Autophagy regulates cellular homeostasis and response to environmental stress. Within the retinal pigment epithelium (RPE) of the eye, the level of autophagy can change with both age and disease. The purpose of this study is to determine the relationship between reduced autophagy and age-related degeneration of the RPE. The gene encoding RB1CC1/FIP200 (RB1-inducible coiled-coil 1), a protein essential for induction of autophagy, was selectively knocked out in the RPE by crossing Best1-Cre mice with mice in which the Rb1cc1 gene was flanked with Lox-P sites (Rb1cc1(flox/flox)). Ex vivo and in vivo analyses, including western blot, immunohistochemistry, transmission electron microscopy, fundus photography, optical coherence tomography, fluorescein angiography, and electroretinography were performed to assess the structure and function of the retina as a function of age. Deletion of Rb1cc1 resulted in multiple autophagy defects within the RPE including decreased conversion of LC3-I to LC3-II, accumulation of autophagy-targeted precursors, and increased numbers of mitochondria. Age-dependent degeneration of the RPE occurred, with formation of atrophic patches, subretinal migration of activated microglial cells, subRPE deposition of inflammatory and oxidatively damaged proteins, subretinal drusenoid deposits, and occasional foci of choroidal neovascularization. There was secondary loss of photoreceptors overlying the degenerated RPE and reduction in the electroretinogram. These observations are consistent with a critical role of autophagy in the maintenance of normal homeostasis in the aging RPE, and indicate that disruption of autophagy leads to retinal phenotypes associated with age-related degeneration.

  14. Pharmacological protection of retinal pigmented epithelial cells by sulindac involves PPAR-α.

    Science.gov (United States)

    Sur, Arunodoy; Kesaraju, Shailaja; Prentice, Howard; Ayyanathan, Kasirajan; Baronas-Lowell, Diane; Zhu, Danhong; Hinton, David R; Blanks, Janet; Weissbach, Herbert

    2014-11-25

    The retinal pigmented epithelial (RPE) layer is one of the major ocular tissues affected by oxidative stress and is known to play an important role in the etiology of age-related macular degeneration (AMD), the major cause of blinding in the elderly. In the present study, sulindac, a nonsteroidal antiinflammatory drug (NSAID), was tested for protection against oxidative stress-induced damage in an established RPE cell line (ARPE-19). Besides its established antiinflammatory activity, sulindac has previously been shown to protect cardiac tissue against ischemia/reperfusion damage, although the exact mechanism was not elucidated. As shown here, sulindac can also protect RPE cells from chemical oxidative damage or UV light by initiating a protective mechanism similar to what is observed in ischemic preconditioning (IPC) response. The mechanism of protection appears to be triggered by reactive oxygen species (ROS) and involves known IPC signaling components such as PKG and PKC epsilon in addition to the mitochondrial ATP-sensitive K(+) channel. Sulindac induced iNOS and Hsp70, late-phase IPC markers in the RPE cells. A unique feature of the sulindac protective response is that it involves activation of the peroxisome proliferator-activated receptor alpha (PPAR-α). We have also used low-passage human fetal RPE and polarized primary fetal RPE cells to validate the basic observation that sulindac can protect retinal cells against oxidative stress. These findings indicate a mechanism for preventing oxidative stress in RPE cells and suggest that sulindac could be used therapeutically for slowing the progression of AMD.

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

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    Kati Juuti-Uusitalo

    Full Text Available 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.

  16. Recognition of mannose 6-phosphate ligands by dystrophic rat retinal pigment epithelium

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    Tarnowski, B.; Shepherd, V.; McLaughlin, B.

    1986-05-01

    Retinal pigment epithelium (RPE) phagocytize discarded rod outer segments (ROS) during normal eye function. In the dystrophic rat, an animal model for retinitis pigmentosa in humans, ROS phagocytosis is defective. Dystrophic RPE can phagocytize particles other than ROS, suggesting that the defect may be in the RPE phagocytic recognition. They are currently investigating the recognition markers on RPE in dystrophic rats. In studies using ligand-coated latex beads, no uptake of mannose-coated beads was found in dystrophic rat RPE. They found that dystrophic RPE could specifically phagocytize phosphomannan-coated beads. Studies were begun to examine the presence and function of a phosphomannan receptor (PMR) on dystrophic RPE. ..cap alpha..-Mannosidase, isolated from D. discoideum has been shown to be an efficient ligand for the PMR in fibroblasts and macrophages. It is also recognized by the macrophage mannose receptor. Dystrophic rat RPE and retina explants were placed in culture dishes (5-7/well). /sup 125/I-Labelled ..cap alpha..-mannosidase was added to each well in the presence or absence of 10 mM mannose 6-phosphate (M6P) or yeast mannan (lmg/ml). Explants were incubated at 37/sup 0/ for 2 hr., washed and bound /sup 125/I-mannosidase quantitated. Approximately 2-3% of total counts added were bound to the RPE via a M6P-inhibitable recognition process. The binding to RPE was not blocked by mannan. No mannan or M6P-specific binding was found in retina explants. These results support the findings of specific uptake of phosphomannan-coated beads and demonstrate the presence of a specific PMR on dystrophic RPE phagocytic membranes.

  17. Retinal pigment epithelial cells upregulate expression of complement factors after co-culture with activated T cells

    DEFF Research Database (Denmark)

    Juel, Helene Bæk; Kaestel, Charlotte; Folkersen, Lasse

    2011-01-01

    In this study we examined the effect of T cell-derived cytokines on retinal pigment epithelial (RPE) cells with respect to expression of complement components. We used an in vitro co-culture system in which CD3/CD28-activated human T cells were separated from the human RPE cell line (ARPE-19) by ...... of inflammatory ocular diseases such as uveitis and age-related macular degeneration. --------------------------------------------------------------------------------...

  18. From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium.

    Science.gov (United States)

    Reichman, Sacha; Terray, Angélique; Slembrouck, Amélie; Nanteau, Céline; Orieux, Gaël; Habeler, Walter; Nandrot, Emeline F; Sahel, José-Alain; Monville, Christelle; Goureau, Olivier

    2014-06-10

    Progress in retinal-cell therapy derived from human pluripotent stem cells currently faces technical challenges that require the development of easy and standardized protocols. Here, we developed a simple retinal differentiation method, based on confluent human induced pluripotent stem cells (hiPSC), bypassing embryoid body formation and the use of exogenous molecules, coating, or Matrigel. In 2 wk, we generated both retinal pigmented epithelial cells and self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs). We report sequential differentiation from RPCs to the seven neuroretinal cell types in maturated NR-like structures as floating cultures, thereby revealing the multipotency of RPCs generated from integration-free hiPSCs. Furthermore, Notch pathway inhibition boosted the generation of photoreceptor precursor cells, crucial in establishing cell therapy strategies. This innovative process proposed here provides a readily efficient and scalable approach to produce retinal cells for regenerative medicine and for drug-screening purposes, as well as an in vitro model of human retinal development and disease.

  19. Anatomical and visual outcomes of ranibizumab injections in retinal pigment epithelium tears

    Directory of Open Access Journals (Sweden)

    Muhammet Kazım Erol

    2015-06-01

    Full Text Available ABSTRACT Purpose: To report the anatomical and visual results in patients diagnosed as having retinal pigment epithelium (RPE tears after receiving ranibizumab injections. Methods: Eyes diagnosed as having RPE tears with a minimum 6-month follow-up were retrospectively evaluated. Each eye was treated with at least three doses of ranibizumab at monthly intervals. Best-corrected visual acuity (BCVA, anterior segment findings, intraocular pressure, and fundus examination results were evaluated during control visits. Color fundus photography, fundus fluorescein angiographies, fundus autofluorescence, and spectral domain optical coherence tomography (SD-OCT images were obtained. The height of pigment epithelial detachment (PED was measured by SD-OCT. Results: Twelve eyes with RPE tears were studied. Nine eyes (75% developed RPE tears during ranibizumab injections for choroidal neovascularization (eight eyes with vascularized PED and one eye with choroidal osteoma, and tears occurred in three eyes before any injections. The median number of ranibizumab injections after diagnosis of RPE tears was 3 (min 2, max 5. In the most recent follow-up visit, there was no statistically significant correlation between the grade of RPE and logMAR of BCVA (p>0.05, r=0.112. Eight of twelve eyes had PED, and seven of these had irregular PED contours before injection therapy. The mean PED height was 447 ± 122 µm. Conclusions: In this series, RPE tears developed mostly after intravitreal anti-VEGF injections for vascularized PED. Increased vertical height and irregular contours of the PEDs can be risk factors for the formation of RPE tears. The continuation of anti-VEGF therapy after tear formation is beneficial for vision improvement in eyes with RPE tears.

  20. Optical properties of photoreceptor and retinal pigment epithelium cells investigated with adaptive optics optical coherence tomography

    Science.gov (United States)

    Liu, Zhuolin

    Human vision starts when photoreceptors collect and respond to light. Photoreceptors do not function in isolation though, but share close interdependence with neighboring photoreceptors and underlying retinal pigment epithelium (RPE) cells. These cellular interactions are essential for normal function of the photoreceptor-RPE complex, but methods to assess these in the living human eye are limited. One approach that has gained increased promise is high-resolution retinal imaging that has undergone tremendous technological advances over the last two decades to probe the living retina at the cellular level. Pivotal in these advances has been adaptive optics (AO) and optical coherence tomography (OCT) that together allow unprecedented spatial resolution of retinal structures in all three dimensions. Using these high-resolution systems, cone photoreceptor are now routinely imaged in healthy and diseased retina enabling fundamental structural properties of cones to be studied such as cell spacing, packing arrangement, and alignment. Other important cell properties, however, have remained elusive to investigation as even better imaging performance is required and thus has resulted in an incomplete understanding of how cells in the photoreceptor-RPE complex interact with light. To address this technical bottleneck, we expanded the imaging capability of AO-OCT to detect and quantify more accurately and completely the optical properties of cone photoreceptor and RPE cells at the cellular level in the living human retina. The first objective of this thesis was development of a new AO-OCT method that is more precise and sensitive, thus enabling a more detailed view of the 3D optical signature of the photoreceptor-RPE complex than was previously possible (Chapter 2). Using this new system, the second objective was quantifying the waveguide properties of individual cone photoreceptor inner and outer segments across the macula (Chapter 3). The third objective extended the AO

  1. Phospholipase D1 modulates protein kinase C-epsilon in retinal pigment epithelium cells during inflammatory response.

    Science.gov (United States)

    Tenconi, Paula E; Giusto, Norma M; Salvador, Gabriela A; Mateos, Melina V

    2016-12-01

    Inflammation is a key factor in the pathogenesis of several retinal diseases. In view of the essential role of the retinal pigment epithelium in visual function, elucidating the molecular mechanisms elicited by inflammation in this tissue could provide new insights for the treatment of retinal diseases. The aim of the present work was to study protein kinase C signaling and its modulation by phospholipases D in ARPE-19 cells exposed to lipopolysaccharide. This bacterial endotoxin induced protein kinase C-α/βII phosphorylation and protein kinase-ε translocation to the plasma membrane in ARPE-19 cells. Pre-incubation with selective phospholipase D inhibitors demonstrated that protein kinase C-α phosphorylation depends on phospholipase D1 and 2 while protein kinase C-ε activation depends only on phospholipase D1. The inhibition of α and β protein kinase C isoforms with Go 6976 did not modify the reduced mitochondrial function induced by lipopolysaccharide. On the contrary, the inhibition of protein kinase C-α, β and ε with Ro 31-8220 potentiated the decrease in mitochondrial function. Moreover, inhibition of protein kinase C-ε reduced Bcl-2 expression and Akt activation and increased Caspase-3 cleavage in cells treated or not with lipopolysaccharide. Our results demonstrate that through protein kinase C-ε regulation, phospholipase D1 protects retinal pigment epithelium cells from lipopolysaccharide-induced damage.

  2. Increased vascular density and vitreo-retinal membranes accompany vascularization of the pigment epithelium in the dystrophic rat retina.

    Science.gov (United States)

    Caldwell, R B; Roque, R S; Solomon, S W

    1989-09-01

    Observations of vascularization of the retinal pigment epithelium (RPE) and formation of vitreo-retinal membranes (VRMs) in Royal College of Surgeons (RCS) rats with inherited retinal dystrophy suggest that vascular proliferation occurs in this model. To test this hypothesis, we studied the progression of vascular changes in RCS and age-matched control rats using quantitative light microscope morphometry and electron microscopy. At 2 weeks, prior to photoreceptor degeneration, the dystrophic retina is comparable with the control. By 2 months, extensive degeneration of photoreceptor cells results in significant thinning of the dystrophic retina as compared with the control. Signs of vascular degeneration are evident at the electron microscope level--"ghost" vessels consisting of acellular basal lamina surrounded by amorphous electron-dense material; degenerating endothelial cells and pericytes; and abnormal deposits of extracellular matrix (ECM) material around blood vessels. Vascular degeneration is accompanied by glial changes in the form of necrotic perivascular glial processes and abnormal ECM deposits among the altered Muller cell processes. At 2-4 months in the dystrophic retina, numbers of vessel profiles in dystrophic retinas are decreased as compared with controls. However, vascular degeneration is overshadowed by the formation of numerous capillary tufts within the RPE layer, which together with retinal thinning results in increased vessel density. Between 4-12 months, the retinal thickness diminishes further, vascularization of the RPE increases, vitreo-retinal membranes are formed, and vascular density increases. In summary, following an initial period of vascular degeneration, vascularization of the RPE is accompanied by an increase in retinal vessel density and by the formation of vitreo-retinal membranes.

  3. Hyperhomocysteinemia disrupts retinal pigment epithelial structure and function with features of age-related macular degeneration.

    Science.gov (United States)

    Ibrahim, Ahmed S; Mander, Suchreet; Hussein, Khaled A; Elsherbiny, Nehal M; Smith, Sylvia B; Al-Shabrawey, Mohamed; Tawfik, Amany

    2016-02-23

    The disruption of retinal pigment epithelial (RPE) function and the degeneration of photoreceptors are cardinal features of age related macular degeneration (AMD); however there are still gaps in our understanding of underlying biological processes. Excess homocysteine (Hcy) has been reported to be elevated in plasma of patients with AMD. This study aimed to evaluate the direct effect of hyperhomocysteinemia (HHcy) on structure and function of RPE. Initial studies in a mouse model of HHcy, in which cystathionine-β-synthase (cbs) was deficient, revealed abnormal RPE cell morphology with features similar to that of AMD upon optical coherence tomography (OCT), fluorescein angiography (FA), histological, and electron microscopic examinations. These features include atrophy, vacuolization, hypopigmentation, thickened basal laminar membrane, hyporeflective lucency, choroidal neovascularization (CNV), and disturbed RPE-photoreceptor relationship. Furthermore, intravitreal injection of Hcy per se in normal wild type (WT) mice resulted in diffuse hyper-fluorescence, albumin leakage, and CNV in the area of RPE. In vitro experiments on ARPE-19 showed that Hcy dose-dependently reduced tight junction protein expression, increased FITC dextran leakage, decreased transcellular electrical resistance, and impaired phagocytic activity. Collectively, our results demonstrated unreported effects of excess Hcy levels on RPE structure and function that lead to the development of AMD-like features.

  4. Effect of Hypericin on Confocal Imaging of Ca2+ Signaling in Cultured Human Retinal Pigment Epithelium

    Institute of Scientific and Technical Information of China (English)

    Qianying Gao; Yannian Hui; Yusheng Wang; Lin Wang

    2001-01-01

    Purpose: To investigate the mechanism of the Ca2 + signaling in cultured human retinal pigment epithelial(RPE) cells with the protein kinase C(PKC) specific inhibitor-hypericin stimulation.Methods: Cultured human RPE cells were analyzed using the fluorescence Ca2+ dye fluo-3 AM and laser scanning confocal microscope(LSCM) after stimulation with 100nM phorbol 12-myristate 13-acetate(PMA) and (or)5 concentrations of hypericin(1, 2, 3, 4 and 5 μM).Results: The normal fluorescence in RPE cells was strong and distributed throughout the cells. The nucleus appeared to be more fluorescent than the cytoplasm. After stimulation with PMA alone or 5 concentrations of hypericin, a rapid decrease in flurescence intensity was observed. There was no obvious difference in decreased curve among 5concentrations. However, after stimulation with a 24 hr preincubation of PMA and 5 concentrations of hypericin, a further decrease was not observed.Conclusion: Fluo-3 AM appears to be a good indicator of the change in Ca2+ occurring in RPE cells and hypericin is a strong inhibitor of Ca2 + influx channel. Hypericin has potential as a therapeutic drug for proliferative vitreoretinopathy(PVR), the inhibitory effect on PVR might be caused by blocking the PKC activity and inhibiting Ca2+ influxpathway.

  5. A strategy to ensure safety of stem cell-derived retinal pigment epithelium cells.

    Science.gov (United States)

    Choudhary, Parul; Whiting, Paul John

    2016-09-02

    Cell replacement and regenerative therapy using embryonic stem cell-derived material holds promise for the treatment of several pathologies. However, the safety of this approach is of prime importance given the teratogenic potential of residual stem cells, if present in the differentiated cell product. Using the example of embryonic stem cell-derived retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration, we present a novel strategy for ensuring the absence of stem cells in the RPE population. Based on an unbiased screening approach, we identify and validate the expression of CD59, a cell surface marker expressed on RPE but absent on stem cells. We further demonstrate that flow sorting on the basis of CD59 expression can effectively purify RPE and deplete stem cells, resulting in a population free from stem cell impurity. This purification helps to ensure removal of stem cells and hence increases the safety of cells that may be used for clinical transplantation. This strategy can potentially be applied to other pluripotent stem cell-derived material and help mitigate concerns of using such cells for therapy.

  6. Retinal Development and Ommin Pigment in the Cranchiid Squid Teuthowenia pellucida (Cephalopoda: Oegopsida.

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    Aaron B Evans

    Full Text Available The cranchiid Teuthowenia pellucida, like many deep-sea squid species, possesses large eyes that maximise light sensitivity in a nearly aphotic environment. To assess ontogenetic changes in the visual system, we conducted morphometric and histological analyses of the eyes using specimens from New Zealand collections. While the ratio between eye diameter and mantle length maintained a linear relationship throughout development, histological sections of the retina revealed that the outer photoreceptor layer became proportionally longer as the animal aged, coincident with a habitat shift into deeper, darker ocean strata. Other retinal layers maintained the same absolute thickness as was observed in paralarvae. Granules of the pigment ommin, normally located in the screening layer positioned at the base of the photoreceptors, were also observed at the outer end of the photoreceptor segments throughout the retina in young and mid-sized specimens. Early developmental stages of this species, dwelling in shallow waters, may therefore rely on migratory ommin to help shield photoreceptors from excess light and prevent over-stimulation. The oldest, deeper-dwelling specimens of T. pellucida examined had longer photoreceptors, and little or no migrated ommin was observed; we suggest therefore that short-term adaptive mechanisms for bright light conditions may be used primarily during epipelagic, early life stages in this species.

  7. Melissa Officinalis L. Extracts Protect Human Retinal Pigment Epithelial Cells against Oxidative Stress-Induced Apoptosis.

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    Jeung, In Cheul; Jee, Donghyun; Rho, Chang-Rae; Kang, Seungbum

    2016-01-01

    We evaluated the protective effect of ALS-L1023, an extract of Melissa officinalis L. (Labiatae; lemon balm) against oxidative stress-induced apoptosis in human retinal pigment epithelial cells (ARPE-19 cells). ARPE-19 cells were incubated with ALS-L1023 for 24 h and then treated with hydrogen peroxide (H2O2). Oxidative stress-induced apoptosis and intracellular generation of reactive oxygen species (ROS) were assessed by flow cytometry. Caspase-3/7 activation and cleaved poly ADP-ribose polymerase (PARP) were measured to investigate the protective role of ALS-L1023 against apoptosis. The protective effect of ALS-L1023 against oxidative stress through activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) was evaluated by Western blot analysis. ALS-L1023 clearly reduced H2O2-induced cell apoptosis and intracellular production of ROS. H2O2-induced oxidative stress increased caspase-3/7 activity and apoptotic PARP cleavage, which were significantly inhibited by ALS-L1023. Activation of the PI3K/Akt pathway was associated with the protective effect of ALS-L1023 on ARPE-19 cells. ALS-L1023 protected human RPE cells against oxidative damage. This suggests that ALS-L1023 has therapeutic potential for the prevention of dry age-related macular degeneration.

  8. Effects of light-emitting diode radiations on human retinal pigment epithelial cells in vitro.

    Science.gov (United States)

    Chamorro, Eva; Bonnin-Arias, Cristina; Pérez-Carrasco, María Jesús; Muñoz de Luna, Javier; Vázquez, Daniel; Sánchez-Ramos, Celia

    2013-01-01

    Human visual system is exposed to high levels of natural and artificial lights of different spectra and intensities along lifetime. Light-emitting diodes (LEDs) are the basic lighting components in screens of PCs, phones and TV sets; hence it is so important to know the implications of LED radiations on the human visual system. The aim of this study was to investigate the effect of LEDs radiations on human retinal pigment epithelial cells (HRPEpiC). They were exposed to three light-darkness (12 h/12 h) cycles, using blue-468 nm, green-525 nm, red-616 nm and white light. Cellular viability of HRPEpiC was evaluated by labeling all nuclei with DAPI; Production of reactive oxygen species (ROS) was determined by H2DCFDA staining; mitochondrial membrane potential was quantified by TMRM staining; DNA damage was determined by H2AX histone activation, and apoptosis was evaluated by caspases-3,-7 activation. It is shown that LED radiations decrease 75-99% cellular viability, and increase 66-89% cellular apoptosis. They also increase ROS production and DNA damage. Fluorescence intensity of apoptosis was 3.7% in nonirradiated cells and 88.8%, 86.1%, 83.9% and 65.5% in cells exposed to white, blue, green or red light, respectively. This study indicates three light-darkness (12 h/12 h) cycles of exposure to LED lighting affect in vitro HRPEpiC.

  9. Bacterial cellulose as a support for the growth of retinal pigment epithelium.

    Science.gov (United States)

    Gonçalves, Sara; Padrão, Jorge; Rodrigues, Inês Patrício; Silva, João Pedro; Sencadas, Vítor; Lanceros-Mendez, Senentxu; Girão, Henrique; Dourado, Fernando; Rodrigues, Lígia R

    2015-04-13

    The feasibility of bacterial cellulose (BC) as a novel substrate for retinal pigment epithelium (RPE) culture was evaluated. Thin (41.6 ± 2.2 μm of average thickness) and heat-dried BC substrates were surface-modified via acetylation and polysaccharide adsorption, using chitosan and carboxymethyl cellulose. All substrates were characterized according to their surface chemistry, wettability, energy, topography, and also regarding their permeability, dimensional stability, mechanical properties, and endotoxin content. Then, their ability to promote RPE cell adhesion and proliferation in vitro was assessed. All surface-modified BC substrates presented similar permeation coefficients with solutes of up to 300 kDa. Acetylation of BC decreased it's swelling and the amount of endotoxins. Surface modification of BC greatly enhanced the adhesion and proliferation of RPE cells. All samples showed similar stress-strain behavior; BC and acetylated BC showed the highest elastic modulus, but the latter exhibited a slightly smaller tensile strength and elongation at break as compared to pristine BC. Although similar proliferation rates were observed among the modified substrates, the acetylated ones showed higher initial cell adhesion. This difference may be mainly due to the moderately hydrophilic surface obtained after acetylation.

  10. Acetylated bacterial cellulose coated with urinary bladder matrix as a substrate for retinal pigment epithelium.

    Science.gov (United States)

    Gonçalves, Sara; Rodrigues, Inês Patrício; Padrão, Jorge; Silva, João Pedro; Sencadas, Vitor; Lanceros-Mendez, Senentxu; Girão, Henrique; Gama, Francisco M; Dourado, Fernando; Rodrigues, Lígia R

    2016-03-01

    This work evaluated the effect of acetylated bacterial cellulose (ABC) substrates coated with urinary bladder matrix (UBM) on the behavior of retinal pigment epithelium (RPE), as assessed by cell adhesion, proliferation and development of cell polarity exhibiting transepithelial resistance and polygonal shaped-cells with microvilli. Acetylation of bacterial cellulose (BC) generated a moderate hydrophobic surface (around 65°) while the adsorption of UBM onto these acetylated substrates did not affect significantly the surface hydrophobicity. The ABS substrates coated with UBM enabled the development of a cell phenotype closer to that of native RPE cells. These cells were able to express proteins essential for their cytoskeletal organization and metabolic function (ZO-1 and RPE65), while showing a polygonal shaped morphology with microvilli and a monolayer configuration. The coated ABC substrates were also characterized, exhibiting low swelling effect (between 1.5-2.0 swelling/mm(3)), high mechanical strength (2048MPa) and non-pyrogenicity (2.12EU/L). Therefore, the ABC substrates coated with UBM exhibit interesting features as potential cell carriers in RPE transplantation that ought to be further explored.

  11. Cytotoxicity and genotoxicity of bacterial magnetosomes against human retinal pigment epithelium cells

    Science.gov (United States)

    Qi, Lei; Lv, Xiujuan; Zhang, Tongwei; Jia, Peina; Yan, Ruiying; Li, Shuli; Zou, Ruitao; Xue, Yuhua; Dai, Liming

    2016-06-01

    A variety of nanomaterials have been developed for ocular diseases. The ability of these nanomaterials to pass through the blood-ocular barrier and their biocompatibility are essential characteristics that must be considered. Bacterial magnetosomes (BMs) are a type of biogenic magnetic nanomaterials synthesized by magnetotactic bacteria. Due to their unique biomolecular membrane shell and narrow size distribution of approximately 30 nm, BMs can pass through the blood-brain barrier. The similarity of the blood-ocular barrier to the blood-brain barrier suggests that BMs have great potential as treatments for ocular diseases. In this work, BMs were isolated from magnetotactic bacteria and evaluated in various cytotoxicity and genotoxicity studies in human retinal pigment epithelium (ARPE-19) cells. The BMs entered ARPE-19 cells by endocytosis after a 6-h incubation and displayed much lower cytotoxicity than chemically synthesized magnetic nanoparticles (MNPs). MNPs exhibited significantly higher genotoxicity than BMs and promoted the expression of Bax (the programmed cell death acceleration protein) and the induction of greater cell necrosis. In BM-treated cells, apoptosis tended to be suppressed via increased expression of the Bcl-2 protein. In conclusion, BMs display excellent biocompatibility and potential for use in the treatment of ocular diseases.

  12. An unusual case of bilateral multifocal retinal pigment epithelial detachment with methanol-induced optic neuritis.

    Science.gov (United States)

    Ranjan, Ratnesh; Kushwaha, Rajnath; Gupta, Ramesh Chandra; Khan, Perwez

    2014-03-01

    To describe an unusual case of methanol-induced optic neuritis with bilateral multifocal extrafoveal serous retinal pigment epithelial (RPE) detachment. Single case report. A 40-year-old male presented with acute bilateral loss of vision and history of consumption of adulterated alcohol. On examination, his vision was perception of light in the right eye and finger counting at 1-ft distance in the left eye. Pupillary reactions were sluggish. The optic discs were normal. An elevated lesion with subretinal serous fluid was present over macula adjacent to superior major vessel arcade in the right eye, which was confirmed as a large extrafoveal RPE detachment on fluorescein angiography. There were two more small RPE detachments in the right eye as well as in the left eye. All RPE detachments were extrafoveal in location. The patient was managed medically with intravenous methylprednisolone (1 g) in 500 ml of ringer lactate for three consecutive days. After three doses, visual acuity of both eyes was recorded as 20/20. We herein report an unusual case of bilateral multifocal extrafoveal serous RPE detachment in a patient of methanol-induced optic neuritis. RPE detachments may be due to the toxic effect of methanol metabolites.

  13. Choroidal neovascularization induced by immunogenic alteration of the retinal pigment epithelium in dengue Fever.

    Science.gov (United States)

    Veloso, Carlos Eduardo; Schmidt-Erfurth, Ursula; Nehemy, Márcio B

    2015-01-01

    To report the first case of choroidal neovascularization (CNV) secondary to dengue fever. A 54-year-old female was referred to our department with blurred vision and metamorphopsia in her left eye. Two weeks earlier, she had presented all of the classic symptoms of dengue fever including a positive serology. Her best-corrected visual acuity (BCVA) was 20/150 in the left eye. She underwent a fundus examination, fluorescein angiography (FA) and spectral domain optical coherence tomography. All findings were consistent with CNV secondary to dengue fever. FA revealed a classic CNV associated with focal retinal pigment epithelium (RPE) destruction and detachment. Three consecutive monthly injections of intravitreal ranibizumab resulted in functional and anatomical improvement for as long as 6 months with a BCVA of 20/25. However, CNV recurred 2 years later, again with an improvement after ranibizumab therapy, but with persistence of a fibrovascular RPE detachment, highlighting the pathomechanism of a classic CNV formation. Maculopathy in dengue fever may be followed by CNV as a result of the immunologic alteration of the RPE. Physicians should be aware of this manifestation to be able to initiate adequate treatment with excellent functional and anatomical results.

  14. Light-Induced Phosphorylation of Crystallins in the Retinal Pigment Epithelium

    Science.gov (United States)

    Lee, Hyunju; Chung, Hyewon; Lee, Sung Haeng; Jahng, Wan Jin

    2017-01-01

    Protein phosphorylations have essential regulatory roles in visual signaling. Previously, we found that phosphorylation of several proteins in the retina and the retinal pigment epithelium (RPE) is involved in anti-apoptotic signaling under oxidative stress conditions, including light exposure. In this study, we used a phosphoprotein enrichment strategy to evaluate the light-induced phosphoproteome of primary bovine RPE cells. Phosphoprotein-enriched extracts from bovine RPE cells exposed to light or dark conditions for 1 hour were separated by 2D SDS-PAGE. Serine and tyrosine phosphorylation were visualized by 2D phospho western blotting and specific phosphorylation sites were analyzed by tandem mass spectrometry. Light induced a marked increase in tyrosine phosphorylation of beta crystallin A3 and A4. The most abundant light-induced up-regulated phosphoproteins were crystallins of 15–25-kDa, including beta crystallin S and zeta crystallin. Phosphorylation of beta crystallin suggests an anti-apoptotic chaperone function in the RPE. Other chaperones, cytoskeletal proteins, and proteins involved in energy balance were expressed at higher levels in the dark. A detailed analysis of RPE phosphoproteins provides a molecular basis for understanding light-induced signal transduction and anti-apoptosis mechanisms. Our data indicates that phosphorylation of crystallins likely represents an important mechanism for RPE shielding from physiological and pathophysiological light-induced oxidative injury. PMID:21094180

  15. Influence of ultraviolet A radiation on osmolytes transport in human retinal pigment epithelial cells

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    Da-Yang Wu

    2014-04-01

    Full Text Available AIM: To demonstrate that ultraviolet A(UVAinduces osmolytes accumulation in retinal pigment epithelial(RPEcells.METHODS: Under different experimental conditions such as UVA exposure, hyperosmotic stress condition and hypoosmotic stress condition, RPE cells were cultured for different time periods. The betaine /γ-amino- n-butyric acid(GABAtransporter, the sodium-dependent myoinositol transporter and the taurine transporter(TAUTmRNA were measured by quantitative PCR. The radioactive labeled osmolytes were measured to evaluate the level of osmolytes transportation. RESULTS: This study demonstrated that RPE expressed mRNA specific for the betaine/GABA transporter, for the sodium-dependent myoinositol transporter and for the TAUT. In comparison to norm osmotic(300mosmol/Lcontrols, a 3-5-fold induction of mRNA expression for the betaine/GABA transporter, the sodium-dependent myoinositol transporter and the TAUT was observed within 6-24h after hyperosmotic exposure(400mosmol/L. Expression of osmolyte transporters was associated with an increased uptake of radioactive labeled osmolytes. Conversely, hypoosmotic(200mosmol/Lstimulation induced significant efflux of these osmolytes. UVA significantly stimulated osmolyte uptake. Increased osmolyte uptake was associated with upregulation of mRNA steady-state levels for osmolyte transporters in irradiated cells.CONCLUSION: UVA induces osmolyte uptake in RPE. It is similar reaction to hyperosmotic stress. This suggests that osmolyte uptake response by UVA may be important to maintain homeostasis.

  16. Regulation of Autophagy by High Glucose in Human Retinal Pigment Epithelium

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    Jin Yao

    2014-01-01

    Full Text Available Background: Autophagy is a self-degradative process that is important for balancing sources of energy at critical times in development and in response to nutrient stress. Retinal pigment epithelium (RPE works as the outer blood retina barrier and is vulnerable to energy stress-induced injury. However, the effect of high glucose treatment on autophagy is still unclear in RPE. Methods: Transmission electron microscopy was used to detect the generation of autophagosome. Small interfering RNA (siRNA and MTT was used to determine the effect of autophagy on cell viability. Western blots and immunohistochemistry were used to detect the expression pattern of autophagic markers, including LC3 and p62. Results: High glucose treatment results in a significant increase in the generation of autophagosome and altered expression of LC3 and p62. High glucose-induced autophagy is independent of mTOR signaling, but is mainly regulated via ROS-mediated ER stress signaling. Conclusion: In the scenario of high glucose-induced oxidative stress, autophagy may be required for the removal of damaged proteins, and provide a default mechanism to prevent high glucose-induced injury in RPE.

  17. Retinal pigment epithelial fine structure in the red-backed salamander (Plethodon cinereus).

    Science.gov (United States)

    Braekevelt, C R

    1992-07-01

    The retinal pigment epithelium (RPE) of the red-backed salamander (Plethodon cinerus) consists of a single layer of large squamous shaped cells. The RPE cells are but minimally infolded basally (sclerally) but show many large apical (vitreal) processes interdigitating with the rod outer segments. These epithelial cells are joined laterally by prominent tight junctions located in the mid region of the cells. Internally smooth endoplasmic reticulum is very plentiful while rough endoplasmic reticulum is not. Polysomes, small dense mitochondria and small round to oval melanosomes are plentiful. Golgi zones and lysosome-like bodies are also present as are phagosomes of outer segment material and myeloid bodies. The RPE cell nucleus is large and vesicular. It is felt that the melanosomes undergo retinomotor movements but as only light-adapted specimens were examined it is not known how extensive are these movements. Bruch's membrane or complexus basalis shows the typical pentalaminate structure noted for most vertebrates. The choriocapillaris is a single layer of large anastomosing capillaries which are minimally fenestrated facing Bruch's membrane.

  18. Defining the proteome of human iris, ciliary body, retinal pigment epithelium, and choroid.

    Science.gov (United States)

    Zhang, Pingbo; Kirby, David; Dufresne, Craig; Chen, Yan; Turner, Randi; Ferri, Sara; Edward, Deepak P; Van Eyk, Jennifer E; Semba, Richard D

    2016-04-01

    The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false-positive rates of body, and RPE/choroid. Four "missing proteins" were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.

  19. The Anti-Proliferative Effect of Inhibitor of Telomerase on Cultured Retinal Pigment Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    In order to provide a new method for treating proliferative vitreoretinopathy (PVR), the effects of anti-proliferation and apoptosis induction of inhibitors of telomerase and heat shock protein 90 (Hsp90) on the cultured retinal pigment epithelial (RPE) cells were investigated. The rate of apoptosis cells was measured by using TUNEL on the cultured RPE cells, the co-cultured RPE cells with inhibitor of telomerase (camptothecin) or the co-cultured RPE cells with inhibitor of Hsp90 (geldanamycin). The cell proliferation status was measured in the above three groups by using MTT method. The rate of apoptosis in the RPE cells co-cultured with camptothecin or geldanamycin was increased remarkably (P<0.05). MTT showed the rate of growth inhibition was 8.4 %, 32.3 % and 72.3 % at the concentrations of camptothecin 1 μmol/L, 5 μmol/L, 10 μmol/L, respectively, and 6.5 %, 30.9 %, 71.9 % at the concentrations of geldanamycin 1 μmol/L, 5 μmol/L, 10 μmol/L, respectively. It was concluded that telomerase and Hsp90 can promote the proliferation of the cultured RPE cells, while the inhibitor of them can induce apoptosis and inhibit the growth of the RPE cells.

  20. Caspase-14 Expression Impairs Retinal Pigment Epithelium Barrier Function: Potential Role in Diabetic Macular Edema

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    Selina Beasley

    2014-01-01

    Full Text Available We recently showed that caspase-14 is a novel molecule in retina with potential role in accelerated vascular cell death during diabetic retinopathy (DR. Here, we evaluated whether caspase-14 is implicated in retinal pigment epithelial cells (RPE dysfunction under hyperglycemia. The impact of high glucose (HG, 30 mM D-glucose on caspase-14 expression in human RPE (ARPE-19 cells was tested, which showed significant increase in caspase-14 expression compared with normal glucose (5 mM D-glucose + 25 mM L-glucose. We also evaluated the impact of modulating caspase-14 expression on RPE cells barrier function, phagocytosis, and activation of other caspases using ARPE-19 cells transfected with caspase-14 plasmid or caspase-14 siRNA. We used FITC-dextran flux assay and electric cell substrate impedance sensing (ECIS to test the changes in RPE cell barrier function. Similar to HG, caspase-14 expression in ARPE-19 cells increased FITC-dextran leakage through the confluent monolayer and decreased the transcellular electrical resistance (TER. These effects of HG were prevented by caspase-14 knockdown. Furthermore, caspase-14 knockdown prevented the HG-induced activation of caspase-1 and caspase-9, the only activated caspases by HG. Phagocytic activity was unaffected by caspase-14 expression. Our results suggest that caspase-14 contributes to RPE cell barrier disruption under hyperglycemic conditions and thus plays a role in the development of diabetic macular edema.

  1. Biological effects of cigarette smoke in cultured human retinal pigment epithelial cells.

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    Alice L Yu

    Full Text Available The goal of the present study was to determine whether treatment with cigarette smoke extract (CSE induces cell loss, cellular senescence, and extracellular matrix (ECM synthesis in primary human retinal pigment epithelial (RPE cells. Primary cultured human RPE cells were exposed to 2, 4, 8, and 12% of CSE concentration for 24 hours. Cell loss was detected by cell viability assay. Lipid peroxidation was assessed by loss of cis-parinaric acid (PNA fluorescence. Senescence-associated ß-galactosidase (SA-ß-Gal activity was detected by histochemical staining. Expression of apolipoprotein J (Apo J, connective tissue growth factor (CTGF, fibronectin, and laminin were examined by real-time PCR, western blot, or ELISA experiments. The results showed that exposure of cells to 12% of CSE concentration induced cell death, while treatment of cells with 2, 4, and 8% CSE increased lipid peroxidation. Exposure to 8% of CSE markedly increased the number of SA-ß-Gal positive cells to up to 82%, and the mRNA expression of Apo J, CTGF, and fibronectin by approximately 3-4 fold. Treatment with 8% of CSE also increased the protein expression of Apo J and CTGF and the secretion of fibronectin and laminin. Thus, treatment with CSE can induce cell loss, senescent changes, and ECM synthesis in primary human RPE cells. It may be speculated that cigarette smoke could be involved in cellular events in RPE cells as seen in age-related macular degeneration.

  2. Effect of retinoic acid on proliferation and polyamine metabolism in cultured bovine retinal pigment epithelial cells.

    Science.gov (United States)

    Yasunari, T; Yanagihara, N; Komatsu, T; Moriwaki, M; Shiraki, K; Miki, T; Yano, Y; Otani, S

    1999-01-01

    Reports regarding the effect of all-trans-retinoic acid (RA) on the cell growth of retinal pigment epithelial cells (RPE) have been contradictory. The aims of this study are to clarify the in vitro effect of RA on RPE cells and to examine polyamine metabolism after RA stimulation. A 4-day incubation of fetal-calf-serum (FCS)-stimulated RPE cells with 10 or 25 microM RA significantly increased both cell number and [3H]thymidine incorporation. RPE cells grown over an extended period for 8 days also increased in number and reached full confluency. However, if the incubation was further extended to 12 days, no further increase in cell number was detected. RA treatment of FCS-stimulated RPE cells shifted the peak of ornithine decarboxylase (ODC) activity from 16 to 4 h. S-adenosylmethionine decarboxylase (SAMDC) activity and spermidine/spermine N1-acetyltransferase (SAT) activity of RA-treated RPE cells were significantly greater until 8 and 16 h after incubation, respectively. The putrescine content was significantly increased in RA-treated RPE cells up until 24 h, while spermidine, spermine and N1-acetylspermidine contents were significantly increased until 16 h. Our findings suggest that RA treatment increases the intracellular polyamine concentration of RPE cells via activation of ODC, SAMDC and SAT and that this results in the promotion of RPE cell growth until the cells reach full confluency.

  3. Honeycomb porous films as permeable scaffold materials for human embryonic stem cell-derived retinal pigment epithelium.

    Science.gov (United States)

    Calejo, Maria Teresa; Ilmarinen, Tanja; Jongprasitkul, Hatai; Skottman, Heli; Kellomäki, Minna

    2016-07-01

    Age-related macular degeneration (AMD) is a leading cause of blindness in developed countries, characterised by the degeneration of the retinal pigment epithelium (RPE), a pigmented cell monolayer that closely interacts with the photoreceptors. RPE transplantation is thus considered a very promising therapeutic option to treat this disease. In this work, porous honeycomb-like films are for the first time investigated as scaffold materials for human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE). By changing the conditions during film preparation, it was possible to produce films with homogeneous pore distribution and adequate pore size (∼3-5 µm), that is large enough to ensure high permeability but small enough to enable cell adherence and spreading. A brief dip-coating procedure with collagen type IV enabled the homogeneous adsorption of the protein to the walls and bottom of pores, increasing the hydrophilicity of the surface. hESC-RPE adhered and proliferated on all the collagen-coated materials, regardless of small differences in pore size. The differentiation of hESC-RPE was confirmed by the detection of specific RPE protein markers. These results suggest that the porous honeycomb films can be promising candidates for hESC-RPE tissue engineering, importantly enabling the free flow of ions and molecules across the material. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1646-1656, 2016.

  4. Detection of oxidative stress biomarker-induced assembly of gold nanoparticles in retinal pigment epithelial cells

    Science.gov (United States)

    Yasmin, Z.; Lee, Y.; Maswadi, S.; Glickman, R.; Nash, K. L.

    2013-02-01

    Oxidative stress (OS) is increasingly implicated as an underlying pathogenic mechanism in a wide range of diseases, resulting from an imbalance between the production of reactive oxygen species (ROS) and the system's ability to detoxify the reactive intermediates or repair the resulting damage. ROS can be difficult to detect directly; however, they can be detected indirectly from the effects on oxidative stress biomarkers (OSB), such as glutathione (GSH), 3-nitrotyrosine, homocysteine, and cysteine. Moreover the reaction of transition metals with thiol-containing amino acids (for example GSH) oxidized by ROS can yield reactive products that accumulate with time and contribute to aging and diseases. The study of the interaction between OSB using functionalized nanoparticles (fNPs) has attracted interest because of potential applications in bio-sensors and biomedical diagnostics. A goal of the present work is to use fNPs to detect and ultimately quantitate OS in retinal pigment epithelial (RPE) cells subjected to external stressors, e.g. nonionizing (light) and ionizing (gamma) radiation. Specifically, we are investigating the assembly of gold fNPs mediated by the oxidation of GSH in irradiated RPE cells. The dynamic interparticle interactions had been characterized in previously reported work by monitoring the evolution of the surface plasmon resonance band using spectroscopic analysis (UV-VIS absorption). Here we are comparing the dynamic evolution of fNP assembly using photoacoustic spectroscopy (PAS). We expect that PAS will provide a more sensitive measure allowing these fNP sensors to measure OS in cell-based models without the artifacts limiting the use of current methods, such as fluorescent indicators.

  5. The oxysterol 27-hydroxycholesterol increases β-amyloid and oxidative stress in retinal pigment epithelial cells

    Directory of Open Access Journals (Sweden)

    Dasari Bhanu

    2010-09-01

    Full Text Available Abstract Background Alzheimer's disease (AD and age-related macular degeneration (AMD share several pathological features including β-amyloid (Aβ peptide accumulation, oxidative damage, and cell death. The causes of AD and AMD are not known but several studies suggest disturbances in cholesterol metabolism as a culprit of these diseases. We have recently shown that the cholesterol oxidation metabolite 27-hydroxycholesterol (27-OHC causes AD-like pathology in human neuroblastoma SH-SY5Y cells and in organotypic hippocampal slices. However, the extent to which and the mechanisms by which 27-OHC may also cause pathological hallmarks related to AMD are ill-defined. In this study, the effects of 27-OHC on AMD-related pathology were determined in ARPE-19 cells. These cells have structural and functional properties relevant to retinal pigmented epithelial cells, a target in the course of AMD. Methods ARPE-19 cells were treated with 0, 10 or 25 μM 27-OHC for 24 hours. Levels of Aβ peptide, mitochondrial and endoplasmic reticulum (ER stress markers, Ca2+ homeostasis, glutathione depletion, reactive oxygen species (ROS generation, inflammation and cell death were assessed using ELISA, Western blot, immunocytochemistry, and specific assays. Results 27-OHC dose-dependently increased Aβ peptide production, increased levels of ER stress specific markers caspase 12 and gadd153 (also called CHOP, reduced mitochondrial membrane potential, triggered Ca2+ dyshomeostasis, increased levels of the nuclear factor κB (NFκB and heme-oxygenase 1 (HO-1, two proteins activated by oxidative stress. Additionally, 27-OHC caused glutathione depletion, ROS generation, inflammation and apoptotic-mediated cell death. Conclusions The cholesterol metabolite 27-OHC is toxic to RPE cells. The deleterious effects of this oxysterol ranged from Aβ accumulation to oxidative cell damage. Our results suggest that high levels of 27-OHC may represent a common pathogenic factor for

  6. Metabolism of 4-Hydroxy-7-oxo-5-heptenoic Acid (HOHA) Lactone by Retinal Pigmented Epithelial Cells.

    Science.gov (United States)

    Wang, Hua; Linetsky, Mikhail; Guo, Junhong; Yu, Annabelle O; Salomon, Robert G

    2016-07-18

    4-Hydroxy-7-oxo-5-heptenic acid (HOHA)-lactone is a biologically active oxidative truncation product released (t1/2 = 30 min at 37 °C) by nonenzymatic transesterification/deacylation from docosahexaenoate lipids. We now report that HOHA-lactone readily diffuses into retinal pigmented epithelial (RPE) cells where it is metabolized. A reduced glutathione (GSH) Michael adduct of HOHA-lactone is the most prominent metabolite detected by LC-MS in both the extracellular medium and cell lysates. This molecule appeared inside of ARPE-19 cells within seconds after exposure to HOHA-lactone. The intracellular level reached a maximum concentration at 30 min and then decreased with concomitant increases in its level in the extracellular medium, thus revealing a unidirectional export of the reduced GSH-HOHA-lactone adduct from the cytosol to extracellular medium. This metabolism is likely to modulate the involvement of HOHA-lactone in the pathogenesis of human diseases. HOHA-lactone is biologically active, e.g., low concentrations (0.1-1 μM) induce secretion of vascular endothelial growth factor (VEGF) from ARPE-19 cells. HOHA-lactone is also a precursor of 2-(ω-carboxyethyl)pyrrole (CEP) derivatives of primary amino groups in proteins and ethanolamine phospholipids that have significant pathological and physiological relevance to age-related macular degeneration (AMD), cancer, and wound healing. Both HOHA-lactone and the derived CEP can contribute to the angiogenesis that defines the neovascular "wet" form of AMD and that promotes the growth of tumors. While GSH depletion can increase the lethality of radiotherapy, because it will impair the metabolism of HOHA-lactone, the present study suggests that GSH depletion will also increase levels of HOHA-lactone and CEP that may promote recurrence of tumor growth.

  7. Retinal Pigment Epithelial Features in Central Serous Chorioretinopathy Identified by Polarization-Sensitive Optical Coherence Tomography.

    Science.gov (United States)

    Roberts, Philipp; Baumann, Bernhard; Lammer, Jan; Gerendas, Bianca; Kroisamer, Julia; Bühl, Wolf; Pircher, Michael; Hitzenberger, Christoph K; Schmidt-Erfurth, Ursula; Sacu, Stefan

    2016-04-01

    To determine the subclinical RPE lesions detected by tissue selective polarization-sensitive optical coherence tomography (PS-OCT) in eyes with central serous chorioretinopathy (CSC) and to compare PS-OCT findings to current imaging standards. In this prospective observational case series, individuals with unilateral or bilateral active CSC were imaged using PS-OCT at baseline and after resolution of serous retinal detachment. Features seen on PS-OCT were compared with corresponding lesions as seen on conventional, intensity-based spectral-domain OCT (SD-OCT), fluorescein angiography, and indocyanine green angiography (ICGA). Features of RPE evaluated by PS-OCT were as follows: area and volume of pigment epithelium detachment (PED), presence of RPE aggregations, RPE skip lesions, RPE thickening, and RPE atrophy. Twenty-five study eyes and 23 fellow eyes of 25 participants (2 women, 23 men; mean age ± standard deviation = 40.5 ± 7.4 years) were included and followed for 6.1 ± 3 months. Study eyes and fellow eyes with recurrent CSC showed more RPE abnormalities in PS-OCT than eyes with acute CSC, which correlated well with lesions in ICGA. Closure of the leakage site was observed only in eight (32%) eyes after resolution of subretinal fluid (SRF). All study eyes showed widespread RPE aggregates and 23 (92%) eyes showed RPE skip lesions after resolution of SRF. Features of RPE indicative of previous episodes of CSC detected by PS-OCT correspond well to choroidal lesions in ICGA. In addition, noninvasive PS-OCT imaging enables detection of RPE microrips and aggregations invisible to clinical examination or SD-OCT, thus providing valuable information about disease processes in vivo.

  8. Escin activates AKT-Nrf2 signaling to protect retinal pigment epithelium cells from oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Kaijun [Eye Center, The 2nd Affiliated Hospital, Medical College of Zhejiang University, Hangzhou (China); Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou (China); Jiang, Yiqian [The First People Hospital of Xiaoshan, Hangzhou (China); Wang, Wei; Ma, Jian [Eye Center, The 2nd Affiliated Hospital, Medical College of Zhejiang University, Hangzhou (China); Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou (China); Chen, Min, E-mail: eyedrchenminzj@163.com [Eye Center, The 2nd Affiliated Hospital, Medical College of Zhejiang University, Hangzhou (China); Zhejiang Provincial Key Lab of Ophthalmology, Hangzhou (China)

    2015-12-25

    Here we explored the anti-oxidative and cytoprotective potentials of escin, a natural triterpene-saponin, against hydrogen peroxide (H{sub 2}O{sub 2}) in retinal pigment epithelium (RPE) cells. We showed that escin remarkably attenuated H{sub 2}O{sub 2}-induced death and apoptosis of established (ARPE-19) and primary murine RPE cells. Meanwhile, ROS production and lipid peroxidation by H{sub 2}O{sub 2} were remarkably inhibited by escin. Escin treatment in RPE cells resulted in NF-E2-related factor 2 (Nrf2) signaling activation, evidenced by transcription of anti-oxidant-responsive element (ARE)-regulated genes, including HO-1, NQO-1 and SRXN-1. Knockdown of Nrf2 through targeted shRNAs/siRNAs alleviated escin-mediated ARE gene transcription, and almost abolished escin-mediated anti-oxidant activity and RPE cytoprotection against H{sub 2}O{sub 2}. Reversely, escin was more potent against H{sub 2}O{sub 2} damages in Nrf2-over-expressed ARPE-19 cells. Further studies showed that escin-induced Nrf2 activation in RPE cells required AKT signaling. AKT inhibitors (LY294002 and perifosine) blocked escin-induced AKT activation, and dramatically inhibited Nrf2 phosphorylation, its cytosol accumulation and nuclear translocation in RPE cells. Escin-induced RPE cytoprotection against H{sub 2}O{sub 2} was also alleviated by the AKT inhibitors. Together, these results demonstrate that escin protects RPE cells from oxidative stress possibly through activating AKT-Nrf2 signaling.

  9. Cytotoxic effect of ZnS nanoparticles on primary mouse retinal pigment epithelial cells.

    Science.gov (United States)

    Bose, Karthikeyan; Lakshminarasimhan, Harini; Sundar, Krishnan; Kathiresan, Thandavarayan

    2016-11-01

    The multiple properties of zinc sulphide nanoparticles (ZnS-NPs) are attracting great attention in the field of chemical and biological research. ZnS-NPs also find their application in biosensor and photocatalysis. Zinc is an important metal ion in retina and its deficiency leads to age-related macular degeneration. As of now, not much research is available on bio-interaction of ZnS as nanoform with retinal pigment epithelial (RPE) cells. RPE cells in the retina help in maintaining normal photoreceptor function and vision. To begin with, ZnS-NPs were synthesized and characterized using UV-visible spectra, X-ray diffraction, Fourier transform infrared spectrum, transmission electron microscopy and dynamic light scattering. Followed by the confirmation of nanoparticles, our study extended to investigate the impact of ZnS-NPs in primary mouse RPE (MRPE) cells at different concentrations. ZnS-NPs showed dose-dependent cytotoxicity in MRPE cells and no changes were observed in cells' tight intactness at minimal concentration. In addition, exposure to ZnS-NPs increased cellular permeability in dose- and time-dependent manner in MRPE cells. The findings from DCFH-DA analysis revealed that ZnS-NPs-treated cells had elevated level of reactive oxygen species and partial activation of cell apoptosis was identified after exposure to ZnS-NPs at higher concentration. Furthermore, pre-treatment of the primary MRPE cells with ZnS-NPs led to phosphorylation of Akt (Ser 473), which indicates the crucial role of ZnS-NPs in regulating cell survival at minimal concentration. Altogether, this study enumerates requisite dose of using ZnS-NPs to maintain healthy RPE cells and contributes to future studies in development of therapeutic drug and drug carrier for ocular-related disorders.

  10. Photodisruption increases the free-radical reactivity of melanosomes isolated from retinal pigment epithelium

    Science.gov (United States)

    Glickman, Randolph D.; Jacques, Steven L.; Schwartz, Jon A.; Rodriguez, Tom; Lam, Kwok-Wai; Buhr, Gwen

    1996-05-01

    Melanin in vivo is usually packaged in melanosomes with protein coats that restrict direct interaction of the melanin with the surrounding medium. We found that disruption of the melanosomes by exposure to a pulsed laser increased the ability of the melanin radicals to oxidize NADPH in a photochemical reaction. Retinal pigment epithelial (RPE) melanosomes were prepared from fresh bovine eyes in 0.25 M sucrose. A reaction mixture of 7 mM NADPH, approximately 7500 RPE melanosomes, and 80 mM Tris buffer, pH 7.2, was prepared in a volume of 60 (mu) l. Of the two 25-(mu) l aliquots taken from this mixture, one was pre-exposed to the 2nd-harmonic output of a Q-switched Nd:YAG laser (532 nm, 1800 10-nsec pulses at 10 Hz), and then was exposed to an Argon ion continuous wave (CW) laser (488.1 and 514.5 nm) for five minutes. The other aliquot was exposed only to the Argon laser. The CW exposure excited the melanin radicals to a reactive state that oxidized NADPH, as assayed by the loss of absorbance at 340 nm. Native melanosomes oxidized less NADPH during Ar+ laser pumping than did melanosomes pre-exposed to the YAG laser. The YAG laser's stimulatory effect on melanosomes reactivity increased as the total energy it delivered rose above 3.5 J (0.14 J/cm2/pulse X 1800 pulses), up to a maximum NADPH oxidation at about 20 J (0.2 J/cm2/pulse X 1800 pulses, beam broadened at higher pulse energy). Electron microscopic analysis of the melanosomes confirmed the progressive physical disruption of melanosomes as the YAG pulse energy increased.

  11. Induction of necrotic cell death by oxidative stress in retinal pigment epithelial cells.

    Science.gov (United States)

    Hanus, J; Zhang, H; Wang, Z; Liu, Q; Zhou, Q; Wang, S

    2013-12-12

    Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNFα in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry

  12. Notch signaling modulates proliferative vitreoretinopathy via regulating retinal pigment epithelial-to-mesenchymal transition.

    Science.gov (United States)

    Zhang, Jingjing; Yuan, Gongqiang; Dong, Muchen; Zhang, Ting; Hua, Gao; Zhou, Qingjun; Shi, Weiyun

    2016-09-07

    Elevated Notch signaling has been verified in a large range of fibrotic diseases developed in the kidney, liver, and lung, inducing the development of the epithelial-mesenchymal transition (EMT). The aim of this study was to observe the involvement of Notch signaling in the EMT of retinal pigment epithelial (RPE) cells and the pathogenesis of proliferative vitreoretinopathy (PVR). In vitro cultivated human RPE cells (ARPE-19) were treated with 10 ng/mL transforming growth factor (TGF)-β1 for 24, 48, and 72 h. The expression levels of ZO-1, α-SMA, vimentin, Notch1 intracellular domain (NICD1), and Hes-1 were evaluated with quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence staining or Western blot. TGF-β1 induced EMT and the activation of Notch signaling in ARPE-19 cells. To examine the effect of Notch inhibition on TGF-β1-induced EMT and PVR formation, ARPE-19 cells were preincubated with γ-secretase inhibitor LY411575 before TGF-β1 treatment. Mouse PVR model was used for in vivo study. ARPE-19 cells were injected intravitreously with or without the LY411575 to examine the effect of Notch inhibition on PVR formation. LY411575 significantly attenuated EMT by inhibiting the Notch signaling activation in vitro. PVR was induced by intravitreal injections of ARPE-19 cells, while LY411575 inhibited mouse PVR formation in vivo. Notch signaling plays a critical role in TGF-β1-induced EMT in vitro and mice PVR model, which provides a novel insight into the pathogenesis of PVR. The specific inhibition of Notch signaling by γ-secretase inhibitor may provide a new approach for the prevention of PVR.

  13. Bevacizumab modulates retinal pigment epithelial-tomesenchymal transition via regulating Notch signaling

    Institute of Scientific and Technical Information of China (English)

    Jing-Jing; Zhang; San-Jun; Chu; Xiao-Lei; Sun; Ting; Zhang; Wei-Yun; Shi

    2015-01-01

    AIM: To investigate the effect of bevacizumab treatment on Notch signaling and the induction of epithelial-of-mesenchymal transition(EMT) in human retinal pigment epithelial cells(ARPE-19) in vitro.METHODS: In vitro cultivated ARPE-19 cells were treated with 0.25 mg/m L bevacizumab for 12, 24, and 48 h.Cell morphology changes were observed under an inverted microscope. The expression of zonula occludens-1(ZO-1), vimentin and Notch-1 intracellular domain(NICD) was examined by immunofluorescence.The m RNA levels of ZO-1, α-SMA, Notch-1, Notch-2,Notch-4, Dll4, Jagged-1, RBP-Jk and Hes-1 expression were evaluated with quantitative real-time polymerase chain reaction(q RT-PCR). The protein levels of α-SMA,NICD, Hes-1 and Dll-4 expression were examined with Western blot.RESULTS: Bevacizumab stimulation increased the expression of α-SMA and vimentin in ARPE-19 cells which changed into spindle-shaped fibroblast-like cells.Meanwhile, the m RNA expression of Hes-1 increased and the protein expression of Hes-1 and NICD also increased, which Notch signaling was activated. The m RNA expression of Notch-1, Jagged-1 and RBP-Jk increased at 48 h, and while Dll4 m RNA and protein expression did not change after bevacizumab treatment.CONCLUSION: Jagged-1/Notch-1 signaling may play a critical role in bevacizumab-induced EMT in ARPE-19 cells, which provides a novel insight into the pathogenesis of intravitreal bevacizumab-associated complication.

  14. Osmotic Induction of Angiogenic Growth Factor Expression in Human Retinal Pigment Epithelial Cells

    Science.gov (United States)

    Reichenbach, Andreas; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2016-01-01

    Background Although systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE) cells. Methodology/Principal Findings Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5) expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-β1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-β1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38α/β MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38α/β MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-β1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-β1

  15. Osmotic Induction of Angiogenic Growth Factor Expression in Human Retinal Pigment Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Moritz Veltmann

    Full Text Available Although systemic hypertension is a risk factor of age-related macular degeneration, antihypertensive medications do not affect the risk of the disease. One condition that induces hypertension is high intake of dietary salt resulting in increased blood osmolarity. In order to prove the assumption that, in addition to hypertension, high osmolarity may aggravate neovascular retinal diseases, we determined the effect of extracellular hyperosmolarity on the expression of angiogenic cytokines in cultured human retinal pigment epithelial (RPE cells.Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Hypoxia and oxidative stress were induced by the addition of the hypoxia mimetic CoCl2 and H2O2, respectively. Alterations in gene expression were determined with real-time RT-PCR. Secretion of bFGF was evaluated by ELISA. Cell viability was determined by trypan blue exclusion. Nuclear factor of activated T cell 5 (NFAT5 expression was knocked down with siRNA. Hyperosmolarity induced transcriptional activation of bFGF, HB-EGF, and VEGF genes, while the expression of other cytokines such as EGF, PDGF-A, TGF-β1, HGF, and PEDF was not or moderately altered. Hypoxia induced increased expression of the HB-EGF, EGF, PDGF-A, TGF-β1, and VEGF genes, but not of the bFGF gene. Oxidative stress induced gene expression of HB-EGF, but not of bFGF. The hyperosmotic expression of the bFGF gene was dependent on the activation of p38α/β MAPK, JNK, PI3K, and the transcriptional activity of NFAT5. The hyperosmotic expression of the HB-EGF gene was dependent on the activation of p38α/β MAPK, ERK1/2, and JNK. The hyperosmotic expression of bFGF, HB-EGF, and VEGF genes was reduced by inhibitors of TGF-β1 superfamily activin receptor-like kinase receptors and the FGF receptor kinase, respectively. Hyperosmolarity induced secretion of bFGF that was reduced by inhibition of autocrine/paracrine TGF-β1 signaling and by NFAT5 si

  16. Poly(trimethylene carbonate) as an elastic biodegradable film for human embryonic stem cell-derived retinal pigment epithelial cells.

    Science.gov (United States)

    Sorkio, Anni; Haimi, Suvi; Verdoold, Vincent; Juuti-Uusitalo, Kati; Grijpma, Dirk; Skottman, Heli

    2017-01-04

    Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great promise for these RPE cell therapies. The aim of the present study was to assess whether a flexible, elastic and biodegradable poly(trimethylene carbonate) (PTMC) film promotes the formation of functional hESC-RPE and performs better than often used biodegradable poly(d,l-lactide) (PDLLA) film. Human ESC-RPE maturation and functionality on PTMC films was assessed by cell proliferation assays, RPE-specific gene and protein expression, phagocytic activity and growth factor secretion. It is demonstrated that the mechanical properties of PTMC films have close resemblance to those of the native Bruch's membrane and support the formation hESC-RPE monolayer in serum-free culture conditions with high degree of functionality. In contrast, use of PDLLA films did not lead to the formation of confluent monolayers of hESC-RPE cells and had unsuitable mechanical properties for retinal application. In conclusion, the present study indicates that flexible and elastic biodegradable PTMC films show potential for retinal tissue engineering applications. Copyright © 2017 John Wiley & Sons, Ltd.

  17. Photocoagulation of human retinal pigment epithelium in vitro: unravelling the effects on ARPE-19 by transcriptomics and proteomics.

    Science.gov (United States)

    Tababat-Khani, Poya; de la Torre, Carolina; Canals, Francesc; Bennet, Hedvig; Simo, Rafael; Hernandez, Cristina; Fex, Malin; Agardh, Carl-David; Hansson, Ola; Agardh, Elisabet

    2015-06-01

    Despite the extensive use of retinal photocoagulation for ischaemia and vascular leakage in retinal vascular disease, the molecular mechanisms behind its clinical beneficial effects are still poorly understood. One important target of laser irradiation is the retinal pigment epithelium (RPE). In this study, we aimed at identifying the isolated effects of photocoagulation of RPE at both the mRNA and protein expression levels. Human ARPE-19 cells were exposed to photocoagulation. Gene expression and protein expression were compared to untreated cells using microarray and liquid chromatography-mass spectrometry analysis. Genes and proteins queried by microarray and mass spectrometry were subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database pathway analyses. Laser irradiation resulted in an induction of the cytoprotective heat-shock protein subfamily Hsp70 as well as in a suppression of the vascular permeability factor carbonic anhydrase 9 (CA9). These expression patterns were evident at both the mRNA and protein levels. KEGG pathway analyses revealed genes and proteins involved in cellular turnover, repair and inflammation. By characterizing the transcriptional and translational effects of laser coagulation on the RPE cells in culture, we have revealed responses, which might contribute to some of the beneficial effects obtained by photocoagulation for ischaemia and vascular leakage in retinal vascular disease. © 2015 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  18. Early LPS-induced ERK activation in retinal pigment epithelium cells is dependent on PIP 2 -PLC.

    Science.gov (United States)

    Mateos, Melina V; Kamerbeek, Constanza B; Giusto, Norma M; Salvador, Gabriela A

    2016-06-01

    This article presents additional data regarding the study "The phospholipase D pathway mediates the inflammatory response of the retinal pigment epithelium" [1]. The new data presented here show that short exposure of RPE cells to lipopolysaccharide (LPS) induces an early and transient activation of the extracellular signal-regulated kinase (ERK1/2). This early ERK1/2 activation is dependent on phosphatidylinositol bisphosphate-phospholipase C (PIP2-PLC). On the contrary, neither the phospholipase D 1 (PLD1) nor the PLD2 inhibition is able to modulate the early ERK1/2 activation induced by LPS in RPE cells.

  19. Histochemical study of retinal photoreceptors development during pre- and postnatal period and their association with retinal pigment epithelium

    Directory of Open Access Journals (Sweden)

    Vahid Ebrahimi

    2014-07-01

    Conclusion: According to our findings, we suggest that the generation of the eye photoreceptors begins from pre- natal period and their final differentiations will continue to postnatal period. Glycoconjugates including (β-D-Gal [1–3]-D-GalNac and (β-D-Gal terminal sugars play a critical role in the pre- and postnatal development and differentiation of retinal photoreceptors.

  20. Chronic cigarette smoke causes oxidative damage and apoptosis to retinal pigmented epithelial cells in mice.

    Directory of Open Access Journals (Sweden)

    Masashi Fujihara

    Full Text Available The purpose of this study was to determine whether mice exposed to chronic cigarette smoke develop features of early age-related macular degeneration (AMD. Two month old C57Bl6 mice were exposed to either filtered air or cigarette smoke in a smoking chamber for 5 h/day, 5 days/week for 6 months. Eyes were fixed in 2.5% glutaraldehyde/2% paraformaldehyde and examined for ultrastructural changes by transmission electron microscopy. The contralateral eye was fixed in 2% paraformaldehyde and examined for oxidative injury to the retinal pigmented epithelium (RPE by 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG immunolabeling and apoptosis by TUNEL labeling. Mice exposed to cigarette smoke had immunolabeling for 8-OHdG in 85+/-3.7% of RPE cells counted compared to 9.5+/-3.9% in controls (p<0.00001. Bruch membrane was thicker in mice exposed to smoke (1086+/-332 nm than those raised in air (543+/-132 nm; p = 0.0069. The two most pronounced ultrastructural changes (severity grading scale from 0-3 seen were a loss of basal infoldings (mean difference in grade = 1.98; p<0.0001, and an increase in intracellular vacuoles (mean difference in grade = 1.7; p<0.0001. Ultrastructural changes to Bruch membrane in cigarette-smoke exposed mice were smaller in magnitude but consistently demonstrated significantly higher grade injury in cigarette-exposed mice, including basal laminar deposits (mean difference in grade = 0.54; p<0.0001, increased outer collagenous layer deposits (mean difference in grade = 0.59; p = 0.002, and increased basal laminar deposit continuity (mean difference in grade = 0.4; p<0.0001. TUNEL assay showed a higher percentage of apoptotic RPE from mice exposed to cigarette smoke (average 8.0+/-1.1% than room air (average 0+/-0%; p = 0.043. Mice exposed to chronic cigarette smoke develop evidence of oxidative damage with ultrastructural degeneration to the RPE and Bruch membrane, and RPE cell apoptosis. This model could be useful for studying the

  1. DJ-1-dependent regulation of oxidative stress in the retinal pigment epithelium (RPE.

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    Karen G Shadrach

    Full Text Available BACKGROUND: DJ-1 is found in many tissues, including the brain, where it has been extensively studied due to its association with Parkinson's disease. DJ-1 functions as a redox-sensitive molecular chaperone and transcription regulator that robustly protects cells from oxidative stress. METHODOLOGY: Retinal pigment epithelial (RPE cultures were treated with H2O2 for various times followed by biochemical and immunohistological analysis. Cells were transfected with adenoviruses carrying the full-length human DJ-1 cDNA and a mutant construct, which has the cysteine residues at amino acid 46, 53 and 106 mutated to serine (C to S prior to stress experiments. DJ-1 localization, levels of expression and reactive oxygen species (ROS generation were also analyzed in cells expressing exogenous DJ-1 under baseline and oxidative stress conditions. The presence of DJ-1 and oxidized DJ-1 was evaluated in human RPE total lysates. The distribution of DJ-1 was assessed in AMD and non-AMD cryosectionss and in isolated human Bruch's membrane (BM/choroid from AMD eyes. PRINCIPAL FINDINGS: DJ-1 in RPE cells under baseline conditions, displays a diffuse cytoplasmic and nuclear staining. After oxidative challenge, more DJ-1 was associated with mitochondria. Increasing concentrations of H2O2 resulted in a dose-dependent increase in DJ-1. Overexpression of DJ-1 but not the C to S mutant prior to exposure to oxidative stress led to significant decrease in the generation of ROS. DJ-1 and oxDJ-1 intensity of immunoreactivity was significantly higher in the RPE lysates from AMD eyes. More DJ-1 was localized to RPE cells from AMD donors with geographic atrophy and DJ-1 was also present in isolated human BM/choroid from AMD eyes. CONCLUSIONS/SIGNIFICANCE: DJ-1 regulates RPE responses to oxidative stress. Most importantly, increased DJ-1 expression prior to oxidative stress leads to decreased generation of ROS, which will be relevant for future studies of AMD since oxidative

  2. Regulation of estrogen receptors and MMP-2 expression by estrogens in human retinal pigment epithelium.

    Science.gov (United States)

    Marin-Castaño, Maria E; Elliot, Sharon J; Potier, Mylen; Karl, Michael; Striker, Liliane J; Striker, Gary E; Csaky, Karl G; Cousins, Scott W

    2003-01-01

    Age-related macular degeneration (ARMD) is characterized by progressive thickening and accumulation of various lipid-rich extracellular matrix (ECM) deposits under the retinal pigment epithelium (RPE). ECM dysregulation probably contributes to the pathologic course of ARMD. By activating estrogen receptors (ERs), estrogens regulate the expression of genes relevant in the turnover of ECM, among them matrix metalloproteinase (MMP)-2. Estrogen deficiency may predispose to dysregulated synthesis and degradation of ECM, leading to accumulation of collagens and other proteins between the RPE and its basement membrane. The purposes in the current study were to confirm the expression of ERs in human RPE, to elucidate whether these ERs are functional, and to test whether 17beta-estradiol (E(2)) regulates expression of ERs and MMP-2. Expression of ERs was examined in freshly isolated human RPE monolayer and in cultured human RPE cells, by using total RNA for RT-PCR and protein extracts for Western blot analysis. Supernatants were collected from freshly isolated human RPE and from cultured human RPE to assess MMP-2 activity by zymography and protein expression by Western blot. The transcriptional activity of ERs was studied in transfection experiments with an estrogen-responsive reporter construct. All these studies were preformed in the presence or absence of E(2) (10(-11) and 10(-7) M). Human RPE isolated from female and male individuals expressed both ER subtypes alpha and beta at the mRNA and protein levels. Treatment of cultured RPE cells with 10(-10) M E(2) increased expression of mRNA and protein of both receptor subtypes. E(2) (10(-10) M) also increased MMP-2 activity (approximately 2.2-fold) and protein expression (approximately 2.5-fold). In contrast, there was no change in ER levels and MMP-2 activity at higher E(2) concentrations (10(-8) M), compared with baseline. Preincubation of cells with 10(-7) M pyrrolidinedithiocarbamate (PDTC), an inhibitor of nuclear

  3. Pigment Epithelium-Derived Factor Inhibits Retinal Microvascular Dysfunction Induced By 12/15-Lipoxygenase-Derived Eicosanoids

    Science.gov (United States)

    Ibrahim, Ahmed S.; Tawfik, Amany M.; Hussein, Khaled A; Elshafey, Sally; Markand, Shanu; Rizk, Nasser; Duh, Elia J.; Smith, Sylvia B.; Al-Shabrawey, Mohamed

    2015-01-01

    We recently demonstrated that 12/15-lipoxygenase (LOX) derived metabolites, hydroxyeicosatetraenoic acids (HETEs), contribute to diabetic retinopathy (DR) via NADPH oxidase (NOX) and disruption of the balance in retinal levels of the vascular endothelial growth factor (VEGF) and Pigment Epithelium-Derived Factor (PEDF). Here, we test whether PEDF ameliorates retinal vascular injury induced by HETEs and the underlying mechanisms. Furthermore, we pursue the causal relationship between LOX-NOX system and regulation of PEDF expression during DR. For these purposes, we used an experimental eye model in which normal mice were injected intravitreally with 12/15HETE with/without PEDF. Thereafter, Fluorescein Angiography (FA) was used to evaluate the vascular leakage, followed by Optical coherence tomography (OCT) to assess the presence of angiogenesis. FA and OCT reported an increased vascular leakage and pre-retinal neovascularization, respectively, in response to 12-HETE that were not observed in PEDF-treated group. Moreover, PEDF significantly attenuated the increased levels of vascular cell and intercellular adhesion molecules, VCAM-1 and ICAM-1, elicited by 12-HETE injection. Accordingly, the direct relationship between HETE and PEDF has been explored through in-vitro studies using Müller cells (rMCs) and human retinal endothelial cells (HRECs). The results showed that HETEs triggered the secretion of TNF-α and IL-6, as well as activation of NFκB in rMCs and significantly increased permeability and reduced zonula occludens protein-1 (ZO-1) immunoreactivity in HRECs. All these effects were prevented in PEDF-treated cells. Furthermore, interest in PEDF regulation during DR has been expanded to include NOX system. Retinal PEDF was significantly restored in diabetic mice treated with NOX inhibitor, apocynin, or lacking NOX2 up to 80% of the control level. Collectively, our findings suggest that interfering with LOX-NOX signaling opens up a new direction for treating DR

  4. Photoreceptors repair by autologous transplantation of retinal pigment epithelium and partial-thickness choroid graft in rabbits.

    Science.gov (United States)

    Zhang, Taoran; Hu, Yuntao; Li, Ying; Wu, Jianguo; Zhao, Lin; Wang, Changguan; Liu, Yuling; Yin, Zhengqin; Ma, Zhizhong

    2009-06-01

    To investigate whether autologous retinal pigment epithelium (RPE) and a partial-thickness graft can repair degenerated photoreceptors overlying a mechanically damaged Bruch's membrane. Twenty-one pigmented rabbits were used in the study. Abrasive debridement of the RPE was performed with a metal cannula after superior retinal bleb detachment in 20 rabbits. The graft was prepared beneath the inferior retina and was transplanted to the debridement area 14 days later. Debridement-only sites served as the control. Tissue sections were evaluated by light microscopy and transmission electron microscopy at 7 days, 1 month, and 3 months after transplantation, corresponding to 21 days, 45 days, and 3 months after debridement, respectively. When analyzed at 7 days after transplantation, short buds of inner segment with regularly organized outer nuclear layer were observed. The outer segments (OS) were of insufficient length to be observed, but by 1 and 3 months, a significant elongation of the OS was detected. In control retinas from 21 days (corresponding to 7 days after transplantation) to 3 months after RPE debridement, the outer nuclear layer cells were disorganized and diminished. This study showed that autologous RPE and partial-thickness choroid graft have the capacity not only to support photoreceptor cell survival, but also to initiate early repair mechanisms, as exhibited by outer segment regeneration.

  5. A2E induces IL-1ß production in retinal pigment epithelial cells via the NLRP3 inflammasome.

    Directory of Open Access Journals (Sweden)

    Owen A Anderson

    Full Text Available AIMS: With ageing extracellular material is deposited in Bruch's membrane, as drusen. Lipofuscin is deposited in retinal pigment epithelial cells. Both of these changes are associated with age related macular degeneration, a disease now believed to involve chronic inflammation at the retinal-choroidal interface. We hypothesise that these molecules may act as danger signals, causing the production of inflammatory chemokines and cytokines by the retinal pigment epithelium, via activation of pattern recognition receptors. METHODS: ARPE-19 cells were stimulated in vitro with the following reported components of drusen: amyloid-ß (1-42, Carboxyethylpyrrole (CEP modified proteins (CEP-HSA, Nε-(Carboxymethyllysine (CML modified proteins and aggregated vitronectin. The cells were also stimulated with the major fluorophore of lipofuscin: N-retinylidene-N-retinylethanolamine (A2E. Inflammatory chemokine and cytokine production was assessed using Multiplex assays and ELISA. The mechanistic evaluation of the NLRP3 inflammasome pathway was assessed in a stepwise fashion. RESULTS: Of all the molecules tested only A2E induced inflammatory chemokine and cytokine production. 25 µM A2E induced the production of significantly increased levels of the chemokines IL-8, MCP-1, MCG and MIP-1α, the cytokines IL-1ß, IL-2, IL-6, and TNF-α, and the protein VEGF-A. The release of IL-1ß was studied further, and was determined to be due to NLRP3 inflammasome activation. The pathway of activation involved endocytosis of A2E, and the three inflammasome components NLRP3, ASC and activated caspase-1. Immunohistochemical staining of ABCA4 knockout mice, which show progressive accumulation of A2E levels with age, showed increased amounts of IL-1ß proximal to the retinal pigment epithelium. CONCLUSIONS: A2E has the ability to stimulate inflammatory chemokine and cytokine production by RPE cells. The pattern recognition receptor NLRP3 is involved in this process. This

  6. Retinal pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization

    Directory of Open Access Journals (Sweden)

    Haibo Wang

    2016-01-01

    Full Text Available To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV, self-complementary adeno-associated virus 2 (scAAV2 with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con or constitutively active Rap1a (scAAV2-CARap1a showed strong GFP at the 5 × 108 viral particle/μl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/β-catenin complexes, increased transepithelial electrical resistance through an interaction of β-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes.

  7. Retinal pigment epithelial cell expression of active Rap 1a by scAAV2 inhibits choroidal neovascularization.

    Science.gov (United States)

    Wang, Haibo; Han, Xiaokun; Bretz, Colin A; Becker, Silke; Gambhir, Deeksha; Smith, George W; Samulski, R Jude; Wittchen, Erika S; Quilliam, Lawrence A; Chrzanowska-Wodnicka, Magdalena; Hartnett, M Elizabeth

    2016-01-01

    To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV), self-complementary adeno-associated virus 2 (scAAV2) with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con) or constitutively active Rap1a (scAAV2-CARap1a) showed strong GFP at the 5 × 10(8) viral particle/µl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS)-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE)/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/β-catenin complexes, increased transepithelial electrical resistance through an interaction of β-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC) transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes.

  8. Regulation of molecular clock oscillations and phagocytic activity via muscarinic Ca2+ signaling in human retinal pigment epithelial cells

    Science.gov (United States)

    Ikarashi, Rina; Akechi, Honami; Kanda, Yuzuki; Ahmad, Alsawaf; Takeuchi, Kouhei; Morioka, Eri; Sugiyama, Takashi; Ebisawa, Takashi; Ikeda, Masaaki; Ikeda, Masayuki

    2017-01-01

    Vertebrate eyes are known to contain circadian clocks, however, the intracellular mechanisms regulating the retinal clockwork remain largely unknown. To address this, we generated a cell line (hRPE-YC) from human retinal pigmental epithelium, which stably co-expressed reporters for molecular clock oscillations (Bmal1-luciferase) and intracellular Ca2+ concentrations (YC3.6). The hRPE-YC cells demonstrated circadian rhythms in Bmal1 transcription. Also, these cells represented circadian rhythms in Ca2+-spiking frequencies, which were canceled by dominant-negative Bmal1 transfections. The muscarinic agonist carbachol, but not photic stimulation, phase-shifted Bmal1 transcriptional rhythms with a type-1 phase response curve. This is consistent with significant M3 muscarinic receptor expression and little photo-sensor (Cry2 and Opn4) expression in these cells. Moreover, forskolin phase-shifted Bmal1 transcriptional rhythm with a type-0 phase response curve, in accordance with long-lasting CREB phosphorylation levels after forskolin exposure. Interestingly, the hRPE-YC cells demonstrated apparent circadian rhythms in phagocytic activities, which were abolished by carbachol or dominant-negative Bmal1 transfection. Because phagocytosis in RPE cells determines photoreceptor disc shedding, molecular clock oscillations and cytosolic Ca2+ signaling may be the driving forces for disc-shedding rhythms known in various vertebrates. In conclusion, the present study provides a cellular model to understand molecular and intracellular signaling mechanisms underlying human retinal circadian clocks. PMID:28276525

  9. G-quartet oligonucleotide mediated delivery of proteins into photoreceptors and retinal pigment epithelium via intravitreal injection.

    Science.gov (United States)

    Leaderer, Derek; Cashman, Siobhan M; Kumar-Singh, Rajendra

    2016-04-01

    There is currently no available method to efficiently deliver proteins across the plasma membrane of photoreceptor or retinal pigment epithelium (RPE) cells in vivo. Thus, current clinical application of recombinant proteins in ophthalmology is limited to the use of proteins that perform their biological function extracellularly. The ability to traverse biological membranes would enable the mobilization of a significantly larger number of proteins with previously well characterized properties. Nucleolin is abundantly present on the surface of rapidly dividing cells including cancer cells. Surprisingly, nucleolin is also present on the surface of photoreceptor cell bodies. Here we investigated whether nucleolin can be utilized as a gateway for the delivery of proteins into retinal cells following intravitreal injection. AS1411 is a G-quartet aptamer capable of targeting nucleolin. Subsequent to intravitreal injection, fluorescently labeled AS1411 localized to various retinal cell types including the photoreceptors and RPE. AS1411 linked to streptavidin (a ∼50 kDa protein) via a biotin bridge enabled the uptake of Streptavidin into photoreceptors and RPE. AS1411-Streptavidin conjugate applied topically to the cornea allowed for uptake of the conjugate into the nucleus and cytoplasm of corneal endothelial cells. Clinical relevance of AS1411 as a delivery vehicle was strongly indicated by demonstration of the presence of cell surface nucleolin on the photoreceptors, inner neurons and ganglion cells of human retina. These data support exploration of AS1411 as a means of delivering therapeutic proteins to diseased retina.

  10. Identification of an Alternative Splicing Product of the Otx2 Gene Expressed in the Neural Retina and Retinal Pigmented Epithelial Cells.

    Science.gov (United States)

    Kole, Christo; Berdugo, Naomi; Da Silva, Corinne; Aït-Ali, Najate; Millet-Puel, Géraldine; Pagan, Delphine; Blond, Frédéric; Poidevin, Laetitia; Ripp, Raymond; Fontaine, Valérie; Wincker, Patrick; Zack, Donald J; Sahel, José-Alain; Poch, Olivier; Léveillard, Thierry

    2016-01-01

    To investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene.

  11. Identification of an Alternative Splicing Product of the Otx2 Gene Expressed in the Neural Retina and Retinal Pigmented Epithelial Cells

    Science.gov (United States)

    Kole, Christo; Berdugo, Naomi; Da Silva, Corinne; Aït-Ali, Najate; Millet-Puel, Géraldine; Pagan, Delphine; Blond, Frédéric; Poidevin, Laetitia; Ripp, Raymond; Fontaine, Valérie; Wincker, Patrick; Zack, Donald J.; Sahel, José-Alain; Poch, Olivier; Léveillard, Thierry

    2016-01-01

    To investigate the complexity of alternative splicing in the retina, we sequenced and analyzed a total of 115,706 clones from normalized cDNA libraries from mouse neural retina (66,217) and rat retinal pigmented epithelium (49,489). Based upon clustering the cDNAs and mapping them with their respective genomes, the estimated numbers of genes were 9,134 for the mouse neural retina and 12,050 for the rat retinal pigmented epithelium libraries. This unique collection of retinal of messenger RNAs is maintained and accessible through a web-base server to the whole community of retinal biologists for further functional characterization. The analysis revealed 3,248 and 3,202 alternative splice events for mouse neural retina and rat retinal pigmented epithelium, respectively. We focused on transcription factors involved in vision. Among the six candidates suitable for functional analysis, we selected Otx2S, a novel variant of the Otx2 gene with a deletion within the homeodomain sequence. Otx2S is expressed in both the neural retina and retinal pigmented epithelium, and encodes a protein that is targeted to the nucleus. OTX2S exerts transdominant activity on the tyrosinase promoter when tested in the physiological environment of primary RPE cells. By overexpressing OTX2S in primary RPE cells using an adeno associated viral vector, we identified 10 genes whose expression is positively regulated by OTX2S. We find that OTX2S is able to bind to the chromatin at the promoter of the retinal dehydrogenase 10 (RDH10) gene. PMID:26985665

  12. Threshold determinations for selective retinal pigment epithelium damage with repetitive pulsed microsecond laser systems in rabbits.

    Science.gov (United States)

    Framme, Carsten; Schuele, Georg; Roider, Johann; Kracht, Dietmar; Birngruber, Reginald; Brinkmann, Ralf

    2002-01-01

    In both clinical and animal studies, it has been shown that repetitive short laser pulses can cause selective retinal pigment epithelium damage (RPE) with sparing of photoreceptors. Our purpose was to determine the ophthalmoscopic and angiographic damage thresholds as a function of pulse durations by using different pulsed laser systems to optimize treatment modalities. Chinchilla-breed rabbits were narcotized and placed in a special holding system. Laser lesions were applied using a commercial laser slit lamp, contact lens, and irradiation with a frequency-doubled Nd:YLF laser (wave-length: 527 nm; repetition rate: 500 Hz; number of pulses: 100; pulse duration: 5 micros, 1.7 micros, 200 ns) and an argon-ion laser (514 nm, 500 Hz, 100 pulses, 5 micros and 200 ms). In all eyes, spots with different energies were placed into the regio macularis with a diameter of 102 microm (tophat profile). After treatment, fundus photography and fluorescein angiography were performed and radiant exposure for ED50 damage determined. Speckle measurements at the fiber tips were performed to determine intensity peaks in the beam profile. Using the Nd:YLF laser system, the ophthalmoscopic ED50 threshold energies were 25.4 microJ (5 micros), 32 microJ (1.7 micros), and 30 microJ (200 ns). The angiographic ED50 thresholds were 13.4 microJ (5 micros), 9.2 microJ (1.7 micros), and 6.7 microJ (200 ns). With the argon laser, the angiographic threshold for 5 micros pulses was 5.5 microJ. The ophthalmoscopic threshold could not be determined because of a lack of power; however, it was > 12 microJ. For 200 ms, the ED50 radiant exposures were 20.4 mW ophthalmoscopically and 19.2 mW angiographically. Speckle factors were found to be 1.225 for the Nd:YLF and 3.180 for the argon laser. Thus, the maximal ED50 -threshold radiant exposures for the Nd:YLF were calculated to be 362 mJ/cM2 (5 micros), 478 mJ/cm2 (1.7 micros), and 438 mJ/cm2 (200 ns) ophthalmoscopically. Angiographically, the thresholds

  13. Neonatal human retinal pigment epithelial cells secrete limited trophic factors in vitro and in vivo following striatal implantation in parkinsonian rats

    DEFF Research Database (Denmark)

    Russ, Kaspar; Flores, Joseph; Brudek, Tomasz

    2015-01-01

    Human retinal pigment epithelial (hRPE) cell implants into the striatum have been investigated as a potential cell-based treatment for Parkinson's disease in a Phase II clinical trial that recently failed. We hypothesize that the trophic factor potential of the hRPE cells could potentially influe...

  14. Superantigen presentation by human retinal pigment epithelial cells to T cells is dependent on CD2-CD58 and CD18-CD54 molecule interactions

    DEFF Research Database (Denmark)

    Jørgensen, A; Junker, N; Kaestel, C G

    2001-01-01

    Human retinal pigment epithelial (RPE) cells are capable of presenting bacterial superantigens (SAg) to T cells in vitro by ligation of MHC class II molecules on RPE cells with the T cell receptor. The purpose of this study was to evaluate the involvement of adhesion molecules in presentation...

  15. Lutein Inhibits the Migration of Retinal Pigment Epithelial Cells via Cytosolic and Mitochondrial Akt Pathways (Lutein Inhibits RPE Cells Migration

    Directory of Open Access Journals (Sweden)

    Ching-Chieh Su

    2014-08-01

    Full Text Available During the course of proliferative vitreoretinopathy (PVR, the retinal pigment epithelium (RPE cells will de-differentiate, proliferate, and migrate onto the surfaces of the sensory retina. Several studies have shown that platelet-derived growth factor (PDGF can induce migration of RPE cells via an Akt-related pathway. In this study, the effect of lutein on PDGF-BB-induced RPE cells migration was examined using transwell migration assays and Western blot analyses. We found that both phosphorylation of Akt and mitochondrial translocation of Akt in RPE cells induced by PDGF-BB stimulation were suppressed by lutein. Furthermore, the increased migration observed in RPE cells with overexpressed mitochondrial Akt could also be suppressed by lutein. Our results demonstrate that lutein can inhibit PDGF-BB induced RPE cells migration through the inhibition of both cytoplasmic and mitochondrial Akt activation.

  16. The immune privilege of the eye: human retinal pigment epithelial cells selectively modulate T-cell activation in vitro

    DEFF Research Database (Denmark)

    Kaestel, Charlotte G; Lovato, Paola; Ødum, Niels

    2005-01-01

    PURPOSE: To examine the effect of human retinal pigment epithelial (RPE) cells on phytohemagglutinin (PHA) activation of T cells. METHODS: Resting peripheral blood lymphocytes (PBLs) were stimulated with PHA with or without the presence of gamma-irradiated RPE cells. Proliferation and the cell...... cycle profile were thereafter investigated by 3H-thymidine incorporation and flow cytometric analysis. In addition, the PBLs expression of CD69, major histocompatibility complex (MHC) class I and II, CD3, as well as the IL-2 receptor chains were evaluated by flow cytometry, and the content of IL-2...... in cell culture supernatant was measured by ELISA. RESULTS: Human RPE cells were found to suppress PHA-induced proliferation, cyclin A, IL-2R-alpha and -gamma, and CD71 expression and decrease the production of IL-2; but RPE cells do not inhibit the PHA-induced expression of early activation markers CD69...

  17. Automatic detection of subretinal fluid and sub-retinal pigment epithelium fluid in optical coherence tomography images.

    Science.gov (United States)

    Ding, Weiguang; Young, Mei; Bourgault, Serge; Lee, Sieun; Albiani, David A; Kirker, Andrew W; Forooghian, Farzin; Sarunic, Marinko V; Merkur, Andrew B; Beg, Mirza Faisal

    2013-01-01

    Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. Subretinal fluid (SRF) and sub-retinal pigment epithelium (sub-RPE) fluid are signs of AMD and can be detected in optical coherence tomography images. However, manual detection and segmentation of SRFs and sub-RPE fluids are laborious and time consuming. In this paper, a novel pipeline is proposed for automatic detection of SRFs and sub-RPE fluids. First, top and bottom layers of retina are segmented using a graph cut method. Then, a Split Bregman-based segmentation method is used to segment dark regions between layers. These segmented regions are considered as potential fluid candidates, on which a set of features are generated. After that, a random forest classifier is trained to distinguish between the true fluid regions from the falsely detected fluid regions. This method shows reasonable performance in a leave-one-out evaluation using a dataset from 21 patients.

  18. [Combined hamartoma of the retina and retinal pigment epithelium. Anti-VEGF treatment of the associated choroidal neovascular membranes].

    Science.gov (United States)

    Echevarría, L; Villena, O; Nievas, T; Bellido, R

    2015-02-01

    A 58 year-old female was diagnosed with a juxtapapillary combined hamartoma of the retina and retinal pigment epithelium (CHR-RPE) in her left eye 14 years ago. Her visual acuity in that eye was 20/20. Recently, she came to our department with a sudden visual loss and metamorphopsis in her left eye. After performing funduscopy, angiography and OCT, she was diagnosed with choroidal neovascular membrane (CNVM) at lesion border, and started on antiangiogenic therapy. CHR-RPE, despite being a benign condition, may become complicated with severe visual impairment. Antiangiogenic therapy provides a good alternative to photodynamic therapy or laser photocoagulation for treatment of CNVM, avoiding adding iatrogenesis from these treatment to the complications associated with this pathology. Copyright © 2014 Sociedad Española de Oftalmología. Published by Elsevier España, S.L.U. All rights reserved.

  19. Semi-automated discrimination of retinal pigmented epithelial cells in two-photon fluorescence images of mouse retinas

    Science.gov (United States)

    Alexander, Nathan S.; Palczewska, Grazyna; Palczewski, Krzysztof

    2015-01-01

    Automated image segmentation is a critical step toward achieving a quantitative evaluation of disease states with imaging techniques. Two-photon fluorescence microscopy (TPM) has been employed to visualize the retinal pigmented epithelium (RPE) and provide images indicating the health of the retina. However, segmentation of RPE cells within TPM images is difficult due to small differences in fluorescence intensity between cell borders and cell bodies. Here we present a semi-automated method for segmenting RPE cells that relies upon multiple weak features that differentiate cell borders from the remaining image. These features were scored by a search optimization procedure that built up the cell border in segments around a nucleus of interest. With six images used as a test, our method correctly identified cell borders for 69% of nuclei on average. Performance was strongly dependent upon increasing retinosome content in the RPE. TPM image analysis has the potential of providing improved early quantitative assessments of diseases affecting the RPE. PMID:26309765

  20. Early changes in gene expression induced by blue light irradiation of A2E-laden retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    van der Burght, Barbro W; Hansen, Morten; Olsen, Jørgen;

    2013-01-01

    Purpose:  Accumulation of bisretinoids as lipofuscin in retinal pigment epithelial (RPE) cells is implicated in the pathogenesis of some blinding diseases including age-related macular degeneration (AMD). To identify genes whose expression may change under conditions of bisretinoid accumulation, we...... investigated the differential gene expression in RPE cells that had accumulated the lipofuscin fluorophore A2E and were exposed to blue light (430 nm). Methods:  A2E-laden RPE cells were exposed to blue light (A2E/430 nm) at various time intervals. Cell death was quantified using Dead Red staining, and RNA...... levels for the entire genome was determined using DNA microarrays (Affymetrix GeneChip Human Genome 2.0 Plus). Array results for selected genes were confirmed by real-time reverse-transcriptase polymerase chain reaction. Results:  Principal component analysis revealed that the A2E-laden RPE cells...

  1. Retinal pigment epithelium protein of 65 kDA gene-linked retinal degeneration is not modulated by chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C/ chondroitin sulfate proteoglycan 5.

    Science.gov (United States)

    Cottet, Sandra; Jüttner, René; Voirol, Nathalie; Chambon, Pierre; Rathjen, Fritz G; Schorderet, Daniel F; Escher, Pascal

    2013-01-01

    To analyze in vivo the function of chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C (gene symbol: Cspg5) during retinal degeneration in the Rpe65⁻/⁻ mouse model of Leber congenital amaurosis. We resorted to mice with targeted deletions in the Cspg5 and retinal pigment epithelium protein of 65 kDa (Rpe65) genes (Cspg5⁻/⁻/Rpe65⁻/⁻). Cone degeneration was assessed with cone-specific peanut agglutinin staining. Transcriptional expression of rhodopsin (Rho), S-opsin (Opn1sw), M-opsin (Opn1mw), rod transducin α subunit (Gnat1), and cone transducin α subunit (Gnat2) genes was assessed with quantitative PCR from 2 weeks to 12 months. The retinal pigment epithelium (RPE) was analyzed at P14 with immunodetection of the retinol-binding protein membrane receptor Stra6. No differences in the progression of retinal degeneration were observed between the Rpe65⁻/⁻ and Cspg5⁻/⁻/Rpe65⁻/⁻ mice. No retinal phenotype was detected in the late postnatal and adult Cspg5⁻/⁻ mice, when compared to the wild-type mice. Despite the previously reported upregulation of Cspg5 during retinal degeneration in Rpe65⁻/⁻ mice, no protective effect or any involvement of Cspg5 in disease progression was identified.

  2. Cytoplasmic and nuclear anti-apoptotic roles of αB-crystallin in retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Woo Jin Jeong

    Full Text Available In addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO can alter the function of the basement membrane of retinal pigment epithelial (RPE cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In this study, we examined the mechanisms underlying the anti-apoptotic activity of αB-crystallin in the human retinal pigment epithelial cell line ARPE-19 following MGO treatment using various assays, including nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, western blot analysis, confocal microscopy and co-immunoprecipitation assays. To directly assess the role of phosphorylation of αB-crystallin, we used site-directed mutagenesis to convert relevant serine residues to alanine residues. Using these techniques, we demonstrated that MGO induces apoptosis in ARPE-19 cells. Silencing αB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that αB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in untreated control ARPE-19 cells and that MGO treatment caused the dissociation of these caspase subtypes from αB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of αB-crystallin from the nuclei of untreated control RPE cells leading to the release of caspase subtypes. Additionally, transfection of these mutants enhanced MGO-induced apoptosis in ARPE-19 cells, indicating that phosphorylation of nuclear αB-crystallin on serine residues 19, 45 and 59 plays a pivotal role in preventing apoptosis in ARPE-19 cells. Taken together, these results suggest that αB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis.

  3. Lecithin-Bound Iodine Prevents Disruption of Tight Junctions of Retinal Pigment Epithelial Cells under Hypoxic Stress

    Directory of Open Access Journals (Sweden)

    Masahiko Sugimoto

    2016-01-01

    Full Text Available Aim. We investigated whether lecithin-bound iodine (LBI can protect the integrity of tight junctions of retinal pigment epithelial cells from hypoxia. Method. Cultured human retinal pigment epithelial (ARPE-19 cells were pretreated with LBI. To mimic hypoxic conditions, cells were incubated with CoCl2. We compared the integrity of the tight junctions (TJs of control to cells with either LBI alone, CoCl2 alone, or LBI + CoCl2. The levels of cytokines in the conditioned media were also determined. Results. Significant decrease in the zonula occludens-1 (ZO-1 intensity in the CoCl2 group compared to the control (5787.7 ± 4126.4 in CoCl2 group versus 29244.6 ± 2981.2 in control; average ± standard deviation. But the decrease was not significant in the LBI + CoCl2 (27189.0 ± 11231.1. The levels of monocyte chemoattractant protein-1 (MCP-1 and Chemokine (C-C Motif Ligand 11 (CCL-11 were significantly higher in the CoCl2 than in the control (340.8 ± 43.3 versus 279.7 ± 68.3 pg/mL for MCP-1, and 15.2 ± 12.9 versus 12.5 ± 6.1 pg/mL for CCL-11. With LBI pretreatment, the levels of both cytokines were decreased to 182.6 ± 23.8 (MCP-1 and 5.46 ± 1.9 pg/mL for CCL-11. Blockade of MCP-1 or CCL-11 also shows similar result representing TJ protection from hypoxic stress. Conclusions. LBI results in a protective action from hypoxia.

  4. The impact of cHS4 insulators on DNA transposon vector mobilization and silencing in retinal pigment epithelium cells.

    Directory of Open Access Journals (Sweden)

    Nynne Sharma

    Full Text Available DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB, piggyBac (PB, and Tol2 transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5'-HS4 chicken β-globin (cHS4 insulator element in the context of SB, PB, and Tol2 transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells.

  5. Fenretinide induces ubiquitin-dependent proteasomal degradation of stearoyl-CoA desaturase in human retinal pigment epithelial cells.

    Science.gov (United States)

    Samuel, William; Kutty, R Krishnan; Duncan, Todd; Vijayasarathy, Camasamudram; Kuo, Bryan C; Chapa, Krysten M; Redmond, T Michael

    2014-08-01

    Stearoyl-CoA desaturase (SCD, SCD1), an endoplasmic reticulum (ER) resident protein and a rate-limiting enzyme in monounsaturated fatty acid biosynthesis, regulates cellular functions by controlling the ratio of saturated to monounsaturated fatty acids. Increase in SCD expression is strongly implicated in the proliferation and survival of cancer cells, whereas its decrease is known to impair proliferation, induce apoptosis, and restore insulin sensitivity. We examined whether fenretinide, (N-(4-hydroxyphenyl)retinamide, 4HPR), which induces apoptosis in cancer cells and recently shown to improve insulin sensitivity, can modulate the expression of SCD. We observed that fenretinide decreased SCD protein and enzymatic activity in the ARPE-19 human retinal pigment epithelial cell line. Increased expression of BiP/GRP78, ATF4, and GADD153 implicated ER stress. Tunicamycin and thapsigargin, compounds known to induce ER stress, also decreased the SCD protein. This decrease was completely blocked by the proteasome inhibitor MG132. In addition, PYR41, an inhibitor of ubiquitin activating enzyme E1, blocked the fenretinide-mediated decrease in SCD. Immunoprecipitation analysis using anti-ubiquitin and anti-SCD antibodies and the blocking of SCD loss by PYR41 inhibition of ubiquitination further corroborate that fenretinide mediates the degradation of SCD in human RPE cells via the ubiquitin-proteasome dependent pathway. Therefore, the effect of fenretinide on SCD should be considered in its potential therapeutic role against cancer, type-2 diabetes, and retinal diseases.

  6. Lectin from Agaricus Bisporus Suppresses Akt Phosphorylation and Arrests Cell Cycle Progression in Primary Human Retinal Pigment Epithelial Cells

    Directory of Open Access Journals (Sweden)

    Y. H. Cheung

    2011-05-01

    Full Text Available Anomalous retinal pigment epithelial (RPE cells have been implicated in the development of retinal diseases. Lectin from the edible mushroom Agaricus bisporus (ABL was found to inhibit growth of RPE cells. To elucidate the mechanism through which ABL inhibits RPE cell proliferation, we investigated the changes in cell proliferation-related signaling pathways and cell cycle distribution patterns. Primary human RPE cells were grown with or without the lectin (ABL supplement (20ug or 90ug/ml for three days. Phosphorylation statuses of Akt, Jnk and p38 as well as p53 expression level were investigated by Western blotting. Cellular distributions in various cell cycle phases were investigated using flow cytometry. After ABL treatment (90ug/ml, Akt was found to be hypo-phosphorylated while the expression levels of p53, phosphorylated-Jnk and phosphorylated-p38 were not altered. The amount of cells present at S phase was reduced. Our results showed that ABL hypo-phosphorylated Akt and this observation is in line with the finding that ABL could attenuate cell proliferation. As the level of p53 was not significantly altered by ABL, this suggested that the mechanism in which ABL arrested cell proliferation was independent of Akt-mediated MDM2 activation but was possibly mediated by altering G1 to S phase transition.

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

    Science.gov (United States)

    Leung, Kar Wah; Barnstable, Colin J; Tombran-Tink, Joyce

    2009-04-01

    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-gamma, 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-gamma and MCP-1, and is toxic to RPE cells at LC(50)=17.7 microg/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.

  8. Cyclin-dependent kinase inhibitor roscovitine induces cell cycle arrest and apoptosis in rabbit retinal pigment epithelial cells.

    Science.gov (United States)

    Wu, Pei-Chang; Tai, Ming-Hong; Hu, Dan-Ning; Lai, Chien-Hsiung; Chen, Yi-Hao; Wu, Yi-Chen; Tsai, Chia-Ling; Shin, Shyi-Jang; Kuo, Hsi-Kung

    2008-02-01

    Cyclin-dependent kinases (CDKs) play essential roles in the intracellular control of the cell cycle. It has been postulated that roscovitine, a potent CDK2, CDK5, and CDC2 inhibitor, might inhibit cellular proliferation by arresting the cell cycle. This in vitro study investigated the antiproliferative and apoptotic effects of roscovitine in cultured rabbit retinal pigment epithelial (RPE) cells. Experiments using rabbit RPE from young pigmented rabbits were carried out using roscovitine dissolved in dimethylsulfoxide at concentrations ranging from 1 to 100 micromol. Cell proliferation was measured by an MTT assay. The cell cycle response of RPE cells to roscovitine was analyzed by flow cytometry of propidium iodide-stained nuclei. Proteins related to DNA damage in the RPE cells were then assayed by Western blot. Roscovitine inhibited proliferation of RPE cells in a dose-dependent manner. Cell cycle analysis after treatment demonstrated an accumulation of cells arrested in the S- and G2/M phases. Flow cytometry showed that 40 microM of roscovitine increased the cell population in the sub-G1 peak, which is considered a marker of cell death by apoptosis. Western blot analysis revealed Bcl-2 decreased and Bax increased after treatment of RPE cells with roscovitine. This study of the response of RPE cells to roscovitine demonstrated a bidirectional relationship between cell cycle control and apoptosis.

  9. Bystander effects elicited by single-cell photo-oxidative blue-light stimulation in retinal pigment epithelium cell networks

    Science.gov (United States)

    Ishii, Masaaki; Rohrer, Bärbel

    2017-01-01

    ‘Bystander effect’ refers to the induction of biological effects in cells not directly targeted. The retinal pigment epithelium consists of hexagonal cells, forming a monolayer interconnected by gap junctions (GJs). Oxidative stress initiated in an individual cell by photostimulation (488 nm) triggered changes in reactive oxygen species (ROS), Ca2+ and mitochondrial membrane potential (ψm). The Ca2+ signal was transmitted to neighboring cells slowly and non-uniformly; the ROS signal spread fast and radially. Increased Ca2+ levels were associated with a loss in ψm. GJ blockers prevented the spreading of the Ca2+, but not the ROS-related signal. The GJ-mediated Ca2+ wave was associated with cell death by 24 h, requiring endoplasmic reticulum–mitochondria Ca2+ transfer. Ensuing cell death was correlated with baseline Ca2+ levels, and baseline Ca2+ levels were correlated with pigmentation. Hence, local oxidative stress in a donor cell can trigger changes in certain connected recipient cells, a signal that required GJ communication and an ROS-Ca2+ dual-hit. Finally, damage apparently occurred in susceptible cells, which correlated with baseline Ca2+ levels. PMID:28179989

  10. Epiretinal membrane surgery for combined hamartoma of the retina and retinal pigment epithelium: role of multimodal analysis

    Directory of Open Access Journals (Sweden)

    Bruè C

    2013-01-01

    Full Text Available Claudia Bruè, Andrea Saitta, Michele Nicolai, Cesare Mariotti, Alfonso GiovanniniOphthalmology, Department of Neuroscience, Marche Polytechnic University, Ancona, ItalyBackground: The purpose of this study was to evaluate the role of spectral domain optical coherence tomography (SD-OCT, MP-1 microperimetry, and fundus autofluorescence imaging for planning surgical procedures in combined hamartomas of the retina and retinal pigment epithelium (CHR-RPE and following epiretinal membrane removal.Methods: In an interventional retrospective case series, six consecutive subjects with CHR-RPE underwent vitrectomy and epiretinal membrane peeling, with 4 years of follow-up. Each underwent complete ophthalmic examination, including best corrected visual acuity, fundus examination, fundus fluorescein angiography, SD-OCT, MP-1, and fundus autofluorescence at one, 6, 12, and 48 months.Results: Six eyes from six subjects with CHR-RPE were studied (mean age 31 ± 14 years. All patients were phakic and five were male (83.3%. Lesions were unilateral, ie, three macular, two juxtapapillary and macular, and one pericentral. Preoperative best corrected visual acuity was 0.3 ± 0.08 Snellen, with significant improvement to 0.9 ± 0.17 Snellen (P = 0.001 at 4 years of follow-up. Mean retinal sensitivity within the central 20° field improved from 16.6 ± 1.84 dB to 18.8 ± 0.96 dB (P = 0.07. There was also a statistically significant reduction in the visual defect (P = 0.04. SD-OCT demonstrated that the epiretinal membranes were completely removed in all but one patient, with significantly decreased macular edema on follow-up at one, 6, 12, and 48 months (P = 0.001. A positive correlation was shown between preoperative macular sensitivity and postoperative best corrected visual acuity. Fundus autofluorescence demonstrated a block in background autofluorescence at the site of the lesion, and hyperautofluorescsence at the edematous retina overlain by the epiretinal

  11. Review of spectral domain-enhanced depth imaging optical coherence tomography of tumors of the retina and retinal pigment epithelium in children and adults

    Directory of Open Access Journals (Sweden)

    Carol L Shields

    2015-01-01

    Full Text Available Background: Spectral domain (SD enhanced depth imaging optical coherence tomography (EDI-OCT is a useful tool for anatomic, cross-sectional imaging of retinal conditions. Aims: The aim was to identify characteristic patterns of retinal and retinal pigment epithelial tumors on EDI-OCT in children and adults. Settings and Design: Retrospective review. Materials and Methods: Analysis of published reports and personal observations using office-based EDI-OCT for adults and portable hand-held SD OCT for infants and children. Results: Using EDI-OCT, retinal tumors such as small retinoblastoma, astrocytic hamartoma, and hemangioblastoma arose abruptly from the retina, immediately adjacent to normal retina. Small exophytic retinoblastoma and retinal hemangioblastoma showed the full-thickness, homogeneous retinal disorganization with surrounding normal retina "draping" over the margins. Retinoblastoma occasionally had intralesional cavities and surrounding subretinal fluid. Hemangioblastoma often had adjacent intraretinal edema and subretinal fluid. Astrocytic hamartoma arose within the nerve fiber layer and sometimes with a "moth-eaten" or cavitary appearance. Retinal pigment epithelial (RPE lesions such as congenital hypertrophy of RPE appeared flat with shadowing, occasional subretinal cleft, and abrupt photoreceptor loss. Congenital simple hamartoma showed an abrupt elevation from the inner retina with crisp, dark posterior shadowing. Combined hamartoma of the retina/RPE showed vitreoretinal traction causing "sawtooth mini-peak" or gently "maxi-peak" folding of the retina. RPE adenoma often produces remote macular edema or epiretinal membrane and the tumor has an irregular, "rugged" surface with deep shadowing. Conclusions: Enhanced depth imaging optical coherence tomography shows characteristic patterns that are suggestive of certain retinal and RPE tumors.

  12. Cotransport of H+, lactate, and H2O in porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Hamann, Steffen; Kiilgaard, Jens Folke; la Cour, Morten;

    2003-01-01

    and placed in a perfusion chamber in which the solution facing the retinal membrane could be changed rapidly. Two types of experiments were performed: Changes in cell water volume were measured by self-quenching of the fluorescent dye Calcein, and changes in intracellular pH were measured ratiometrically......) for the H(+) and lactate fluxes. The data suggest that H(2)O is cotransported along with H(+) and lactate ions in MCT1 localized to the retinal membrane. The study emphasizes the importance of this cotransporter in the maintenance of water homeostasis and pH in the subretinal space of a mammalian tissue...... and supports our previous study performed by an invasive technique in an amphibian tissue....

  13. Cotransport of H+, lactate, and H2O in porcine retinal pigment epithelial cells

    DEFF Research Database (Denmark)

    Hamann, Steffen; Kiilgaard, Jens Folke; la Cour, Morten

    2003-01-01

    ) for the H(+) and lactate fluxes. The data suggest that H(2)O is cotransported along with H(+) and lactate ions in MCT1 localized to the retinal membrane. The study emphasizes the importance of this cotransporter in the maintenance of water homeostasis and pH in the subretinal space of a mammalian tissue...... and supports our previous study performed by an invasive technique in an amphibian tissue....

  14. The generation of induced pluripotent stem cells for macular degeneration as a drug screening platform: identification of curcumin as a protective agent for retinal pigment epithelial cells against oxidative stress

    National Research Council Canada - National Science Library

    Chang, Yun-Ching; Chang, Wei-Chao; Hung, Kuo-Hsuan; Yang, Der-Ming; Cheng, Yung-Hsin; Liao, Yi-Wen; Woung, Lin-Chung; Tsai, Ching-Yao; Hsu, Chih-Chien; Lin, Tai-Chi; Liu, Jorn-Hon; Chiou, Shih-Hwa; Peng, Chi-Hsien; Chen, Shih-Jen

    2014-01-01

    .... Its pathogenesis remains unclear, but oxidative stress inducing retinal pigment epithelial (RPE) cells damage is perhaps responsible for the aging sequence of retina and may play an important role in macular degeneration...

  15. Action spectrum for photochemical retinal pigment epithelium (RPE) disruption in an in vivo monkey model

    Science.gov (United States)

    Zhang, Jie; Sabarinathan, Ranjani; Bubel, Tracy; Williams, David R.; Hunter, Jennifer J.

    2016-03-01

    Observations of RPE disruption and autofluorescence (AF) photobleaching at light levels below the ANSI photochemical maximum permissible exposure (MPE) (Morgan et al., 2008) indicates a demand to modify future light safety standards to protect the retina from harm. To establish safe light exposures, we measured the visible light action spectrum for RPE disruption in an in vivo monkey model with fluorescence adaptive optics retinal imaging. Using this high resolution imaging modality can provide insight into the consequences of light on a cellular level and allow for longitudinal monitoring of retinal changes. The threshold retinal radiant exposures (RRE) for RPE disruption were determined for 4 wavelengths (460, 488, 544, and 594 nm). The anaesthetized macaque retina was exposed to a uniform 0.5° × 0.5° field of view (FOV). Imaging within a 2° × 2° FOV was performed before, immediately after and at 2 week intervals for 10 weeks. At each wavelength, multiple RREs were tested with 4 repetitions each to determine the threshold for RPE disruption. For qualitative analysis, RPE disruption is defined as any detectable change from the pre exposure condition in the cell mosaic in the exposed region relative to the corresponding mosaic in the immediately surrounding area. We have tested several metrics to evaluate the RPE images obtained before and after exposure. The measured action spectrum for photochemical RPE disruption has a shallower slope than the current ANSI photochemical MPE for the same conditions and suggests that longer wavelength light is more hazardous than other measurements would suggest.

  16. Research resource: nuclear receptor atlas of human retinal pigment epithelial cells: potential relevance to age-related macular degeneration.

    Science.gov (United States)

    Dwyer, Mary A; Kazmin, Dmitri; Hu, Peng; McDonnell, Donald P; Malek, Goldis

    2011-02-01

    Retinal pigment epithelial (RPE) cells play a vital role in retinal physiology by forming the outer blood-retina barrier and supporting photoreceptor function. Retinopathies including age-related macular degeneration (AMD) involve physiological and pathological changes in the epithelium, severely impairing the retina and effecting vision. Nuclear receptors (NRs), including peroxisome proliferator-activated receptor and liver X receptor, have been identified as key regulators of physiological pathways such as lipid metabolic dysregulation and inflammation, pathways that may also be involved in development of AMD. However, the expression levels of NRs in RPE cells have yet to be systematically surveyed. Furthermore, cell culture lines are widely used to study the biology of RPE cells, without knowledge of the differences or similarities in NR expression and activity between these in vitro models and in vivo RPE. Using quantitative real-time PCR, we assessed the expression patterns of all 48 members of the NR family plus aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator in human RPE cells. We profiled freshly isolated cells from donor eyes (in vivo), a spontaneously arising human cell line (in vitro), and primary cell culture lines (in vitro) to determine the extent to which NR expression in the cultured cell lines reflects that of in vivo. To evaluate the validity of using cell culture models for investigating NR receptor biology, we determined transcriptional activity and target gene expression of several moderately and highly expressed NRs in vitro. Finally, we identified a subset of NRs that may play an important role in pathobiology of AMD.

  17. Snail involves in the transforming growth factor β1-mediated epithelial-mesenchymal transition of retinal pigment epithelial cells.

    Directory of Open Access Journals (Sweden)

    Hui Li

    Full Text Available BACKGROUND: The proliferation of retinal pigment epithelium (RPE cells resulting from an epithelial-mesenchymal transition (EMT plays a key role in proliferative vitreoretinopathy (PVR, which leads to complex retinal detachment and the loss of vision. Genes of Snail family encode the zinc finger transcription factors that have been reported to be essential in EMT during embryonic development and cancer metastasis. However, the function of Snail in RPE cells undergoing EMT is largely unknown. PRINCIPAL FINDINGS: Transforming growth factor beta(TGF-β-1 resulted in EMT in human RPE cells (ARPE-19, which was characterized by the expected decrease in E-cadherin and Zona occludin-1(ZO-1 expression, and the increase in fibronectin and α-smooth muscle actin (α-SMA expression, as well as the associated increase of Snail expression at both mRNA and protein levels. Furthermore, TGF-β1 treatment caused a significant change in ARPE-19 cells morphology, with transition from a typical epithelial morphology to mesenchymal spindle-shaped. More interestingly, Snail silencing significantly attenuated TGF-β1-induced EMT in ARPE-19 cells by decreasing the mesenchymal markers fibronectin and a-SMA and increasing the epithelial marker E-cadherin and ZO-1. Snail knockdown could effectively suppress ARPE-19 cell migration. Finally, Snail was activated in epiretinal membranes from PVR patients. Taken together, Snail plays very important roles in TGF-β-1-induced EMT in human RPE cells and may contribute to the development of PVR. SIGNIFICANCE: Snail transcription factor plays a critical role in TGF-β1-induced EMT in human RPE cells, which provides deep insight into the pathogenesis of human PVR disease. The specific inhibition of Snail may provide a new approach to treat and prevent PVR.

  18. Large-scale purification of porcine or bovine photoreceptor outer segments for phagocytosis assays on retinal pigment epithelial cells.

    Science.gov (United States)

    Parinot, Célia; Rieu, Quentin; Chatagnon, Jonathan; Finnemann, Silvia C; Nandrot, Emeline F

    2014-12-12

    Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

  19. Effects of Nerve Growth Factor on Proliferation and DNA Synthesis of Cultured Human Fetal Retinal Pigment Epithelium Cells

    Institute of Scientific and Technical Information of China (English)

    Wensheng Li; Jun Wen; Deyong Jiang; Jianguang Ding

    2002-01-01

    Objective: To investigate the effects of nerve growth factor(NGF)on proliferation and DNAthesis of cultured human fetal retinal pigment epithelium (RPE)cells in vitro.Methods: Primary culture and subculture of human fetal retinal pigment epithelium cellswere established in vitro first. Cultured RPE cells were treated with NGF by variousconcentrations 0μg/L, 50μg/L, 100μg/L, 200μg/L and 300μg/L(final concentration)for 48 hs.After 48 hs, cells proliferation was measured with methyl thiazolyl tetrazolium(MTT)assay method and the amount of DNA was determined by the absorbance at 280nm of nucleic acid & protein analysis.Results: The A values of 100 μg/L, 200 μg/L, 300 μg/L NGF was(0. 213 7 ± 0. 23 3),(0. 218 8 ±0. 018 1), (0. 232 2 ±0. 016 4) as compared with(0. 189 7 ±0. 015 2) of Avalue of 0 μg/L NGF respectively, q value was 3.63,4.40, 6. 42 and P value was0. 015, 0. 000, 0. 000(q-test). The DNA concentrations of 100 μg/L, 200 μg/L, 300μg/L and 400 μg/L NGF was (981. 220 4 ± 123.535 7), (1 375. 848 4 ±244. 471 8),(1 658.707 1 ± 176. 938 1), (2 353.086 3 ±609. 906 4) μg/ml as compared with(666. 818 8 ± 141. 330 2) μg/ml of DNA concentration of 0 μg/L NGF respectively, qvalue was 3.63,8.20,11.47,19.46, P value was 0. 024,0. 000,0. 000,0. 000 (q-test).Conclusion: The data suggested that NGF could stimulate the proliferation and DNAsynthesis of cultured of hRPE cells in vitro in a dose-dependent manner.

  20. C3a Increases VEGF and Decreases PEDF mRNA Levels in Human Retinal Pigment Epithelial Cells

    Science.gov (United States)

    Long, Qin; Cao, Xiaoguang; Bian, Ailing

    2016-01-01

    Complement activation, specifically complement 3 (C3) activation and C3a generation, contributes to an imbalance between angiogenic stimulation by vascular endothelial growth factor (VEGF) and angiogenic inhibition by pigment epithelial derived factor (PEDF), leading to pathological angiogenesis. This study aimed to investigate the effects of C3a and small interfering RNA (siRNA) targeting C3 on the levels of VEGF and PEDF mRNAs in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were cultured in the presence of exogenous C3a at 0.1 μM and 0.3 μM C3a for 24, 48, and 72 hours. 0.1 pmol/μL duplexes of siRNA targeting C3 were applied for C3a inhibition by transfecting ARPE-19 cells for 48 hours. RT-PCR was performed to examine the level of VEGF and PEDF mRNA. A random siRNA duplex was set for control siRNA. Results demonstrated that exogenous C3a significantly upregulated VEGF and downregulated PEDF mRNA levels in cultured ARPE-19 cells, and siRNA targeting C3 transfection reversed the above changes, significantly reducing VEGF and enhancing PEDF mRNAs level in ARPE-19 cells compared to the control. The present data provided evidence that reducing C3 activation can decreases VEGF and increase PEDF mRNA level in RPE and may serve as a potential therapy in pathological angiogenesis.

  1. Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization

    Science.gov (United States)

    Wang, Haibo; Jiang, Yanchao; Shi, Dallas; Quilliam, Lawrence A.; Chrzanowska-Wodnicka, Magdalena; Wittchen, Erika S.; Li, Dean Y.; Hartnett, M. Elizabeth

    2014-01-01

    Activation of Rap1 GTPase can improve the integrity of the barrier of the retina pigment epithelium (RPE) and reduce choroidal neovascularization (CNV). Inhibition of NADPH oxidase activation also reduces CNV. We hypothesize that Rap1 inhibits NADPH oxidase-generated ROS and thereby reduces CNV formation. Using a murine model of laser-induced CNV, we determined that reduced Rap1 activity in RPE/choroid occurred with CNV formation and that activation of Rap1 by 2′-O-Me-cAMP (8CPT)-reduced laser-induced CNV via inhibiting NADPH oxidase-generated ROS. In RPE, inhibition of Rap1 by Rap1 GTPase-activating protein (Rap1GAP) increased ROS generation, whereas activation of Rap1 by 8CPT reduced ROS by interfering with the assembly of NADPH oxidase membrane subunit p22phox with NOX4 or cytoplasmic subunit p47phox. Activation of NADPH oxidase with Rap1GAP reduced RPE barrier integrity via cadherin phosphorylation and facilitated choroidal EC migration across the RPE monolayer. Rap1GAP-induced ROS generation was inhibited by active Rap1a, but not Rap1b, and activation of Rap1a by 8CPT in Rap1b−/− mice reduced laser-induced CNV, in correlation with decreased ROS generation in RPE/choroid. These findings provide evidence that active Rap1 reduces CNV by interfering with the assembly of NADPH oxidase subunits and increasing the integrity of the RPE barrier.—Wang, H., Jiang, Y., Shi, D., Quilliam, L. A., Chrzanowska-Wodnicka, M., Wittchen, E. S., Li, D. Y., Hartnett, M. E. Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization. PMID:24043260

  2. Regulation of the hyperosmotic induction of aquaporin 5 and VEGF in retinal pigment epithelial cells: Involvement of NFAT5

    Science.gov (United States)

    Vogler, Stefanie; Reichenbach, Andreas; Wiedemann, Peter; Bringmann, Andreas; Kohen, Leon

    2015-01-01

    Purpose High intake of dietary salt increases extracellular osmolarity, which results in hypertension, a risk factor of neovascular age-related macular degeneration. Neovascular retinal diseases are associated with edema. Various factors and channels, including vascular endothelial growth factor (VEGF) and aquaporins (AQPs), influence neovascularization and the development of edema. Therefore, we determined whether extracellular hyperosmolarity alters the expression of VEGF and AQPs in cultured human retinal pigment epithelial (RPE) cells. Methods Human RPE cells obtained within 48 h of donor death were prepared and cultured. Hyperosmolarity was induced by the addition of 100 mM NaCl or sucrose to the culture medium. Alterations in gene expression and protein secretion were determined with real-time RT–PCR and ELISA, respectively. The levels of signaling proteins and nuclear factor of activated T cell 5 (NFAT5) were determined by western blotting. DNA binding of NFAT5 was determined with EMSA. NFAT5 was knocked down with siRNA. Results Extracellular hyperosmolarity stimulated VEGF gene transcription and the secretion of VEGF protein. Hyperosmolarity also increased the gene expression of AQP5 and AQP8, induced the phosphorylation of p38 MAPK and ERK1/2, increased the expression of HIF-1α and NFAT5, and induced the DNA binding of NFAT5. The hyperosmotic expression of VEGF was dependent on the activation of p38 MAPK, ERK1/2, JNK, PI3K, HIF-1, and NFAT5. The hyperosmotic induction of AQP5 was in part dependent on the activation of p38 MAPK, ERK1/2, NF-κB, and NFAT5. Triamcinolone acetonide inhibited the hyperosmotic expression of VEGF but not AQP5. The expression of AQP5 was decreased by hypoosmolarity, serum, and hypoxia. Conclusions Hyperosmolarity induces the gene transcription of AQP5, AQP8, and VEGF, as well as the secretion of VEGF from RPE cells. The data suggest that high salt intake resulting in osmotic stress may aggravate neovascular retinal diseases and

  3. Gene expression regulation in retinal pigment epithelial cells induced by viral RNA and viral/bacterial DNA

    Science.gov (United States)

    Brosig, Anton; Kuhrt, Heidrun; Wiedemann, Peter; Kohen, Leon; Bringmann, Andreas

    2015-01-01

    Purpose The pathogenesis of age-related macular degeneration (AMD) is associated with systemic and local inflammation. Various studies suggested that viral or bacterial infection may aggravate retinal inflammation in the aged retina. We compared the effects of synthetic viral RNA (poly(I:C)) and viral/bacterial DNA (CpG-ODN) on the expression of genes known to be involved in the development of AMD in retinal pigment epithelial (RPE) cells. Methods Cultured human RPE cells were stimulated with poly(I:C; 500 µg/ml) or CpG-ODN (500 nM). Alterations in gene expression and protein secretion were determined with real-time RT–PCR and ELISA, respectively. Phosphorylation of signal transduction molecules was revealed by western blotting. Results Poly(I:C) induced gene expression of the pattern recognition receptor TLR3, transcription factors (HIF-1α, p65/NF-κB), the angiogenic factor bFGF, inflammatory factors (IL-1β, IL-6, TNFα, MCP-1, MIP-2), and complement factors (C5, C9, CFB). Poly(I:C) also induced phosphorylation of ERK1/2 and p38 MAPK proteins, and the secretion of bFGF and TNFα from the cells. CpG-ODN induced moderate gene expression of transcription factors (p65/NF-κB, NFAT5) and complement factors (C5, C9), while it had no effect on the expression of various TLR, angiogenic factor, and inflammatory factor genes. The activities of various signal transduction pathways and transcription factors were differentially involved in mediating the poly(I:C)-induced transcriptional activation of distinct genes. Conclusions The widespread effects of viral RNA, and the restricted effects of viral/bacterial DNA, on the gene expression pattern of RPE cells may suggest that viral RNA rather than viral/bacterial DNA induces physiologic alterations of RPE cells, which may aggravate inflammation in the aged retina. The data also suggest that selective inhibition of distinct signal transduction pathways or individual transcription factors may not be effective to inhibit

  4. Fine structure of the retinal pigment epithelium in the Port Jackson shark (Heterodontus phillipi).

    Science.gov (United States)

    Braekevelt, C R

    1994-11-01

    The structure of the retinal epithelium (RPE), choriocapillaris and Bruch's membrane (complexus basalis) has been studied by light and electron microscopy in the Port Jackson shark (Heterodontus phillipi). In this elasmobranch the RPE consists of a single layer of low cuboidal cells which show basal (scleral) infolding and apical (vitreal) processes that enclose photoreceptor outer segments. Laterally these epithelial cells are joined by a series of apically located tight junctions. The RPE cells display a large vesicular nucleus, abundant smooth endoplasmic reticulum as well as numerous polysomes and mitochondria. Phagosomes are present, rough endoplasmic reticulum is scarce and myeloid bodies were not observed. Melanosomes are absent over the choroidally located tapetum lucidum, but are not abundant even in extratapetal areas. This paucity of melanosomes probably makes retinomotor movements unimportant. Bruch's membrane or complexus basalis is a pentalaminate structure. The endothelium of the choriocapillaris is thin but minimally fenestrated.

  5. Proof of concept for AAV2/5-mediated gene therapy in iPSC-derived retinal pigment epithelium of a choroideremia patient

    OpenAIRE

    2014-01-01

    Inherited retinal dystrophies (IRDs) comprise a large group of genetically and clinically heterogeneous diseases that lead to progressive vision loss, for which a paucity of disease-mimicking animal models renders preclinical studies difficult. We sought to develop pertinent human cellular IRD models, beginning with choroideremia, caused by mutations in the CHM gene encoding Rab escort protein 1 (REP1). We reprogrammed REP1-deficient fibroblasts from a CHM -/y patient into induced pluripotent...

  6. Epigallocatechin-gallate (EGCG) regulates autophagy in human retinal pigment epithelial cells: A potential role for reducing UVB light-induced retinal damage

    Energy Technology Data Exchange (ETDEWEB)

    Li, Chao-Peng; Yao, Jin; Tao, Zhi-Fu; Li, Xiu-Miao; Jiang, Qin, E-mail: jqin710@vip.sina.com; Yan, Biao, E-mail: yanbiao1982@hotmail.com

    2013-09-06

    Highlights: •UVB irradiation induces RPE autophagy. •EGCG treatment represses UVB-mediated autophagy. •EGCG regulates UVB-mediated autophagy through mTOR signaling pathway. •EGCG sensitizes RPE cells to UVB-induced damage in an autophagy-dependent manner. -- Abstract: Autophagy is an intracellular catabolic process involved in protein and organelle degradation via the lysosomal pathway that has been linked in the pathogenesis of age-related macular degeneration (AMD). UVB irradiation-mediated degeneration of the macular retinal pigment epithelial (RPE) cells is an important hallmark of AMD, which is along with the change in RPE autophagy. Thus, pharmacological manipulation of RPE autophagy may offer an alternative therapeutic target in AMD. Here, we found that epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, plays a regulatory role in UVB irradiation-induced autophagy in RPE cells. UVB irradiation results in a marked increase in the amount of LC3-II protein in a dose-dependent manner. EGCG administration leads to a significant reduction in the formation of LC3-II and autophagosomes. mTOR signaling activation is required for EGCG-induced LC3-II formation, as evidenced by the fact that EGCG-induced LC3-II formation is significantly impaired by rapamycin administration. Moreover, EGCG significantly alleviates the toxic effects of UVB irradiation on RPE cells in an autophagy-dependent manner. Collectively, our study reveals a novel role of EGCG in RPE autophagy. EGCG may be exploited as a potential therapeutic reagent for the treatment of pathological conditions associated with abnormal autophagy.

  7. iPSC-Derived Retinal Pigment Epithelium Allografts Do Not Elicit Detrimental Effects in Rats: A Follow-Up Study

    Directory of Open Access Journals (Sweden)

    Peter D. Westenskow

    2016-01-01

    Full Text Available Phototransduction is accomplished in the retina by photoreceptor neurons and retinal pigment epithelium (RPE cells. Photoreceptors rely heavily on the RPE, and death or dysfunction of RPE is characteristic of age-related macular degeneration (AMD, a very common neurodegenerative disease for which no cure exists. RPE replacement is a promising therapeutic intervention for AMD, and large numbers of RPE cells can be generated from pluripotent stem cells. However, questions persist regarding iPSC-derived RPE (iPS-RPE viability, immunogenicity, and tumorigenesis potential. We showed previously that iPS-RPE prevent photoreceptor atrophy in dystrophic rats up until 24 weeks after implantation. In this follow-up study, we longitudinally monitored the same implanted iPS-RPE, in the same animals. We observed no gross abnormalities in the eyes, livers, spleens, brains, and blood in aging rats with iPSC-RPE grafts. iPS-RPE cells that integrated into the subretinal space outlived the photoreceptors and survived for as long as 2 1/2 years while nonintegrating RPE cells were ingested by host macrophages. Both populations could be distinguished using immunohistochemistry and electron microscopy. iPSC-RPE could be isolated from the grafts and maintained in culture; these cells also phagocytosed isolated photoreceptor outer segments. We conclude that iPS-RPE grafts remain viable and do not induce any obvious associated pathological changes.

  8. Progressive atrophy of retinal pigment epithelium after trypan-blue-assisted ILM peeling for macular hole surgery

    Directory of Open Access Journals (Sweden)

    Sachin Jain

    2013-01-01

    Full Text Available We report a case of progressive atrophy of the retinal pigment epithelium (RPE after trypan-blue-assisted peeling of internal limiting membrane (ILM for macular hole surgery. A 68-year-old Caucasian female underwent a 20-g pars plana vitrectomy for a chronic stage-3 macular hole. The ILM was stained with 0.06% trypan blue (VisionBlue™, DORC Netherlands for 2 min after fluid air exchange. Dye was reapplied for another 2 min due to poor staining. The ILM was completely removed around the macular hole with forceps. RPE atrophy was noticed at the edge of the hole 1 month after surgery. It progressively increased in intensity and enlarged over 2 years. Her final visual acuity was counting fingers, significantly worse compared to her presenting visual acuity of 20/200. Progressive atrophy of RPE in our patient was most likely due to the toxicity of trypan blue. Reapplication of the dye may increase the likelihood of toxicity.

  9. Peroxidation stimulated by lipid hydroperoxides on bovine retinal pigment epithelium mitochondria: effect of cellular retinol-binding protein.

    Science.gov (United States)

    Terrasa, Ana M; Guajardo, Margarita H; Catalá, Angel

    2003-07-01

    This study analyzes the effect of cellular retinol-binding protein (CRBP), partially purified from retinal pigment epithelium (RPE) cytosol, on the non-enzymatic lipid peroxidation induced by fatty acid hydroperoxides of mitochondrial membranes isolated from bovine RPE. The effect of different amounts (50, 75 and 100 nmol) of linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) on the lipid peroxidation of RPE mitochondria was studied; RPE mitochondria deprived of exogenously added hydroperoxide was utilized as control. The process was measured simultaneously by determining chemiluminescence as well as polyunsaturated fatty acid (PUFA) degradation of total lipids isolated from RPE mitochondria. The addition of hydroperoxides to RPE mitochondria produces a marked increase in light emission that was hydroperoxide concentration dependent. The highest value of activation was produced by LHP. The major difference in the fatty acid composition of total lipids isolated from native and peroxidized RPE mitochondria incubated with and without hydroperoxides was found in the docosahexaenoic acid content, this decreased 40.90+/-3.01% in the peroxidized group compared to native RPE mitochondria. The decrease was significantly high: 86.32+/-2.57% when the lipid peroxidation was stimulated by 100 nmol of LHP. Inhibition of lipid peroxidation (decrease of chemiluminescence) was observed with the addition of increasing amounts (100-600 microg) of CRBP to RPE mitochondria. The inhibitory effect reaches the highest values in the presence of LHP.

  10. Retinal pigment epithelium cell-derived exosomes: Possible relevance to CNV in wet-age related macular degeneration.

    Science.gov (United States)

    Tong, Yao; Zhou, Ya-Li; Wang, Yi-Xiao; Zhao, Pei-Quan; Wang, Zhao-Yang

    2016-12-01

    Exosomes are small vesicles that are released by almost every cell type and play a crucial role in many physiological and pathological processes associated with different diseases. Specifically, they promote angiogenesis in the pathogenesis of some diseases. According to previous research, the proteins of exosomes taken from the aqueous humor (AH) of patients with wet-age related macular degeneration (AMD) may function as a new diagnostic biomarker of AMD, suggesting that exosomes may play an important role in the occurrence and development of choroidal neovascularization (CNV). Moreover, additional research has revealed that the levels of some protein makers of exosomes are up-regulated in aged retinal pigment epithelium (RPE) and that drusen and oxidative stress may promote the secretion of exosomes derived from RPE cells. Consequently, we hypothesize that RPE cell-derived exosomes may be relevant to CNV in wet AMD. If this hypothesis is proven correct, future studies based on this link may also help to elucidate the molecular mechanisms of wet AMD and to find new therapeutic targets for the treatment of AMD.

  11. Possible role of HIWI2 in modulating tight junction proteins in retinal pigment epithelial cells through Akt signaling pathway.

    Science.gov (United States)

    Sivagurunathan, Suganya; Palanisamy, Karthikka; Arunachalam, Jayamuruga Pandian; Chidambaram, Subbulakshmi

    2017-03-01

    PIWI subfamily of proteins is shown to be primarily expressed in germline cells. They maintain the genomic integrity by silencing the transposable elements. Although the role of PIWI proteins in germ cells has been documented, their presence and function in somatic cells remains unclear. Intriguingly, we detected all four members of PIWI-like proteins in human ocular tissues and somatic cell lines. When HIWI2 was knocked down in retinal pigment epithelial cells, the typical honeycomb morphology was affected. Further analysis showed that the expression of tight junction (TJ) proteins, CLDN1, and TJP1 were altered in HIWI2 knockdown. Moreover, confocal imaging revealed disrupted TJP1 assembly at the TJ. Previous studies report the role of GSK3β in regulating TJ proteins. Accordingly, phospho-kinase proteome profiler array indicated increased phosphorylation of Akt and GSK3α/β in HIWI2 knockdown, suggesting that HIWI2 might affect TJ proteins through Akt-GSK3α/β signaling axis. Moreover, treating the HIWI2 knockdown cells with wortmannin increased the levels of TJP1 and CLDN1. Taken together, our study demonstrates the presence of PIWI-like proteins in somatic cells and the possible role of HIWI2 in preserving the functional integrity of epithelial cells probably by modulating the phosphorylation status of Akt.

  12. Fucoidan reduces secretion and expression of vascular endothelial growth factor in the retinal pigment epithelium and reduces angiogenesis in vitro.

    Directory of Open Access Journals (Sweden)

    Michaela Dithmer

    Full Text Available Fucoidan is a polysaccharide isolated from brown algae which is of current interest for anti-tumor therapy. In this study, we investigated the effect of fucoidan on the retinal pigment epithelium (RPE, looking at physiology, vascular endothelial growth factor (VEGF secretion, and angiogenesis, thus investigating a potential use of fucoidan for the treatment of exudative age-related macular degeneration. For this study, human RPE cell line ARPE-19 and primary porcine RPE cells were used, as well as RPE/choroid perfusion organ cultures. The effect of fucoidan on RPE cells was investigated with methyl thiazolyl tetrazolium--assay, trypan blue exclusion assay, phagocytosis assay and a wound healing assay. VEGF expression was evaluated in immunocytochemistry and Western blot, VEGF secretion was evaluated in ELISA. The effect of fucoidan on angiogenesis was tested in a Matrigel assay using calcein-AM vital staining, evaluated by confocal laser scanning microcopy and quantitative image analysis. Fucoidan displays no toxicity and does not diminish proliferation or phagocytosis, but reduces wound healing in RPE cells. Fucoidan decreases VEGF secretion in RPE/choroid explants and RPE cells. Furthermore, it diminishes VEGF expression in RPE cells even when co-applied with bevacizumab. Furthermore, fucoidan reduces RPE-supernatant- and VEGF-induced angiogenesis of peripheral endothelial cells. In conclusion, fucoidan is a non-toxic agent that reduces VEGF expression and angiogenesis in vitro and may be of interest for further studies as a potential therapy against exudative age-related macular degeneration.

  13. Expression of a novel alternative transcript of the novel retinal pigment epithelial cell gene NORPEG in human testes

    Institute of Scientific and Technical Information of China (English)

    Wa Yuan; Ying Zheng; Ran Huo; Li Lu; Xiao-Yan Huang; Lan-Lan Yin; Jian-Min Li; Zuo-Min Zhou; Jia-Hao Sha

    2005-01-01

    Aim: To identify a novel alternative transcript of the novel retinal pigment epithelial cell gene (NORPEG) expressed in the human testis. Methods: A human testis cDNA microarray was established and hybridized with cDNA probes from human fetal testes, adult testes and human spermatozoa. Differentially expressed clones were sequenced and analyzed. One of these clones was a short transcript of NORPEG which we proceeded to analyze by RT-PCR.Results: The novel short alternative transcript of NORPEG was isolated and named sNORPEG. It was 3486 bp in length and contained a 2952-bp open reading frame, encoding a 110.4-kDa protein of 983 amino acids. Amino acid sequence analysis showed that the sNORPEG protein contains six ankyrin repeats and two coiled-coil domains. It shares a high homology with the NORPEG and ankycorbin proteins in both its sequence and motifs. Blasting the human genome database localized sNORPEG to human chromosome 5p13.2-13.3. Expression profiles showed that sNORPEG was expressed in human fetal testes, adult testes and spermatozoa. Moreover, sNORPEG was found to be ubiquitously expressed in human tissues. Conclusion: sNORPEG is expressed in different developmental stages of the testis and encodes a protein that may have roles in human testis development and spermatogenesis.

  14. Plasma polymer coatings to aid retinal pigment epithelial growth for transplantation in the treatment of age related macular degeneration.

    Science.gov (United States)

    Kearns, Victoria; Mistry, Anita; Mason, Sharon; Krishna, Yamini; Sheridan, Carl; Short, Robert; Williams, Rachel L

    2012-08-01

    Subretinal transplantation of functioning retinal pigment epithelial (RPE) cells grown on a synthetic substrate is a potential treatment for age-related macular degeneration (AMD), a common cause of irreversible vision loss in developed countries. Plasma polymers give the opportunity to tailor the surface chemistry of the artificial substrate whilst maintaining the bulk properties. In this study, plasma polymers with different functionalities were investigated in terms of their effect on RPE attachment and growth. Plasma polymers of acrylic acid (AC), allyl amine (AM) and allyl alcohol (AL) were fabricated and characterised using X-ray photoelectron spectroscopy (XPS) and water contact angle measurements. Octadiene (OD) hydrocarbon films and tissue culture polystyrene were used as controls. Wettability varied from hydrophobic OD to relatively hydrophilic AC. XPS demonstrated four very different surfaces with the expected functionalities. Attachment, proliferation and morphological examination of an RPE cell line and primary RPE cells were investigated. Both cell types grew on all surfaces, with the exception of OD, although the proliferation rate of primary cells was low. Good epithelial morphology was also demonstrated. Plasma polymerised films show potential as cell carrier surfaces for RPE cells in the treatment of AMD.

  15. Deriving retinal pigment epithelium (RPE) from induced pluripotent stem (iPS) cells by different sizes of embryoid bodies.

    Science.gov (United States)

    Muñiz, Alberto; Ramesh, Kaini R; Greene, Whitney A; Choi, Jae-Hyek; Wang, Heuy-Ching

    2015-02-04

    Pluripotent stem cells possess the ability to proliferate indefinitely and to differentiate into almost any cell type. Additionally, the development of techniques to reprogram somatic cells into induced pluripotent stem (iPS) cells has generated interest and excitement towards the possibility of customized personal regenerative medicine. However, the efficiency of stem cell differentiation towards a desired lineage remains low. The purpose of this study is to describe a protocol to derive retinal pigment epithelium (RPE) from iPS cells (iPS-RPE) by applying a tissue engineering approach to generate homogenous populations of embryoid bodies (EBs), a common intermediate during in vitro differentiation. The protocol applies the formation of specific size of EBs using microwell plate technology. The methods for identifying protein and gene markers of RPE by immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR) are also explained. Finally, the efficiency of differentiation in different sizes of EBs monitored by fluorescence-activated cell sorting (FACS) analysis of RPE markers is described. These techniques will facilitate the differentiation of iPS cells into RPE for future applications.

  16. Lack of T Cell Response to iPSC-Derived Retinal Pigment Epithelial Cells from HLA Homozygous Donors

    Directory of Open Access Journals (Sweden)

    Sunao Sugita

    2016-10-01

    Full Text Available Allografts of retinal pigment epithelial (RPE cells have been considered for the treatment of ocular diseases. We recently started the transplantation of induced pluripotent stem cell (iPSC-derived RPE cells for patients with age-related macular degeneration (autogenic grafts. However, there are at least two problems with this approach: (1 high cost, and (2 uselessness for acute patients. To resolve these issues, we established RPE cells from induced iPSCs in HLA homozygote donors. In vitro, human T cells directly recognized allogeneic iPSC-derived RPE cells that expressed HLA class I/II antigens. However, these T cells failed to respond to HLA-A, -B, and -DRB1-matched iPSC-derived RPE cells from HLA homozygous donors. Because of the lack of T cell response to iPSC-derived RPE cells from HLA homozygous donors, we can use these allogeneic iPSC-derived RPE cells in future clinical trials if the recipient and donor are HLA matched.

  17. The Developmental Stage of Adult Human Stem Cell-Derived Retinal Pigment Epithelium Cells Influences Transplant Efficacy for Vision Rescue

    Directory of Open Access Journals (Sweden)

    Richard J. Davis

    2017-07-01

    Full Text Available Age-related macular degeneration (AMD is a common cause of central visual loss in the elderly. Retinal pigment epithelial (RPE cell loss occurs early in the course of AMD and RPE cell transplantation holds promise to slow disease progression. We report that subretinal transplantation of RPE stem cell (RPESC-derived RPE cells (RPESC-RPE preserved vision in a rat model of RPE cell dysfunction. Importantly, the stage of differentiation that RPESC-RPE acquired prior to transplantation influenced the efficacy of vision rescue. Whereas cells at all stages of differentiation tested rescued photoreceptor layer morphology, an intermediate stage of RPESC-RPE differentiation obtained after 4 weeks of culture was more consistent at vision rescue than progeny that were differentiated for 2 weeks or 8 weeks of culture. Our results indicate that the developmental stage of RPESC-RPE significantly influences the efficacy of RPE cell replacement, which affects the therapeutic application of these cells for AMD.

  18. Light-induced damage and its diagnosis in two-photon excited autofluorescence imaging of retinal pigment epithelium cells

    Science.gov (United States)

    Chen, Danni; Qu, Junle; Xu, Gaixia; Zhao, Lingling; Niu, Hanben

    2007-05-01

    In this paper, a novel method for the differentiation of the retinal pigment epithelium (RPE) cells after light-induced damage by two-photon excitation is presented. Fresh samples of RPE cells of pig eyes are obtained from local slaughterhouse. Light-induced damage is produced by the output from Ti: sapphire laser which is focused onto the RPE layer. We study the change of the autofluorescence properties of RPE after two-photon excitation with the same wavelength. Preliminary results show that after two-photon excitation, there are two clear changes in the emission spectrum. The first change is the blue-shift of the emission peak. The emission peak of the intact RPE is located at 592nm, and after excitation, it shifts to 540nm. It is supposed that the excitation has led to the increased autofluorescence of flavin whose emission peak is located at 540nm. The second change is the increased intensity of the emission peak, which might be caused by the accelerated aging because the autofluorescence of RPE would increase during aging process. Experimental results indicate that two-photon excitation could not only lead to the damage of the RPE cells in multiphoton RPE imaging, but also provide an evaluation of the light-induced damage.

  19. Memory in induced pluripotent stem cells: reprogrammed human retinal-pigmented epithelial cells show tendency for spontaneous redifferentiation.

    Science.gov (United States)

    Hu, Qirui; Friedrich, Amy M; Johnson, Lincoln V; Clegg, Dennis O

    2010-11-01

    Induced pluripotent stem (iPS) cells have been generated from a variety of somatic cell types via introduction of transcription factors that mediate pluripotency. However, it is unknown that all cell types can be reprogrammed and whether the origin of the parental cell ultimately determines the behavior of the resultant iPS cell line. We sought to determine whether human retinal-pigmented epithelial (RPE) cells could be reprogrammed, and to test the hypothesis that reprogrammed cells retain a "memory" of their origin in terms of propensity for differentiation. We reprogrammed primary fetal RPE cells via lentiviral expression of OCT4, SOX2, LIN28, and Nanog. The iPS cell lines derived from RPE exhibited morphologies similar to human embryonic stem cells and other iPS cell lines, expressed stem cell markers, and formed teratomas-containing derivatives of all three germ layers. To test whether these iPS cells retained epigenetic imprints from the parental RPE cells, we analyzed their propensity for spontaneous differentiation back into RPE after removal of FGF2. We found that some, but not all, iPS lines exhibited a marked preference for redifferentiation into RPE. Our results show that RPE cells can be reprogrammed to pluripotency, and suggest that they often retain a memory of their previous state of differentiation.

  20. Fisetin and luteolin protect human retinal pigment epithelial cells from oxidative stress-induced cell death and regulate inflammation

    Science.gov (United States)

    Hytti, Maria; Piippo, Niina; Korhonen, Eveliina; Honkakoski, Paavo; Kaarniranta, Kai; Kauppinen, Anu

    2015-01-01

    Degeneration of retinal pigment epithelial (RPE) cells is a clinical hallmark of age-related macular degeneration (AMD), the leading cause of blindness among aged people in the Western world. Both inflammation and oxidative stress are known to play vital roles in the development of this disease. Here, we assess the ability of fisetin and luteolin, to protect ARPE-19 cells from oxidative stress-induced cell death and to decrease intracellular inflammation. We also compare the growth and reactivity of human ARPE-19 cells in serum-free and serum-containing conditions. The absence of serum in the culture medium did not prevent ARPE-19 cells from reaching full confluency but caused an increased sensitivity to oxidative stress-induced cell death. Both fisetin and luteolin protected ARPE-19 cells from oxidative stress-induced cell death. They also significantly decreased the release of pro-inflammatory cytokines into the culture medium. The decrease in inflammation was associated with reduced activation of MAPKs and CREB, but was not linked to NF- κB or SIRT1. The ability of fisetin and luteolin to protect and repair stressed RPE cells even after the oxidative insult make them attractive in the search for treatments for AMD. PMID:26619957

  1. JAM-C maintains VEGR2 expression to promote retinal pigment epithelium cell survival under oxidative stress.

    Science.gov (United States)

    Jia, Xin; Zhao, Chen; Chen, Qishan; Du, Yuxiang; Huang, Lijuan; Ye, Zhimin; Ren, Xiangrong; Wang, Shasha; Lee, Chunsik; Tang, Zhongshu; Li, Xuri; Ju, Rong

    2017-04-03

    Junctional adhesion molecule-C (JAM-C) has been shown to play critical roles during development and in immune responses. However, its role in adult eyes under oxidative stress remains poorly understood. Here, we report that JAM-C is abundantly expressed in adult mouse retinae and choroids in vivo and in cultured retinal pigment epithelium (RPE) and photoreceptor cells in vitro. Importantly, both JAM-C expression and its membrane localisation are downregulated by H2O2-induced oxidative stress. Under H2O2-induced oxidative stress, JAM-C is critically required for the survival of human RPE cells. Indeed, loss of JAM-C by siRNA knockdown decreased RPE cell survival. Mechanistically, we show that JAM-C is required to maintain VEGFR2 expression in RPE cells, and VEGFR2 plays an important role in keeping the RPE cells viable since overexpression of VEGFR2 partially restored impaired RPE survival caused by JAM-C knockdown and increased RPE survival. We further show that JAM-C regulates VEGFR2 expression and, in turn, modulates p38 phosphorylation. Together, our data demonstrate that JAM-C plays an important role in maintaining VEGR2 expression to promote RPE cell survival under oxidative stress. Given the vital importance of RPE in the eye, approaches that can modulate JAM-C expression may have therapeutic values in treating diseases with impaired RPE survival.

  2. Downregulation of VEGF mRNA expression by triamcinolone acetonide acetate-loaded chitosan derivative nanoparticles in human retinal pigment epithelial cells

    Directory of Open Access Journals (Sweden)

    Zhou H

    2012-08-01

    Full Text Available Huaisheng Zhou,1 Liqun Yang,2,* Huajie Li,2 Haijun Gong,1 Liangzheng Cheng,2 Haisheng Zheng,1 Li-Ming Zhang,2 Yuqing Lan1,*1Department of Ophthalmology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, 2Institute of Polymer Science, School of Chemistry and Chemical Engineering, Key Laboratory of Designed Synthesis and Application of Polymer Material, Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, Sun Yat-sen University, Guangzhou, China*Both corresponding authors contributed equally to this workBackground: The purpose of this study was to investigate the downregulation of mRNA expression of vascular endothelial growth factor (VEGF by triamcinolone acetonide acetate (TAA-loaded chitosan nanoparticles in human retinal pigment epithelial cells.Methods: TAA-loaded deoxycholic acid-modified chitosan (TAA/DA-Chit nanoparticles were prepared via a self-assembly mechanism, and their morphology and zeta potential were examined by transmission electron microscopy and zeta potential analysis, respectively. DA-Chit and TAA/DA-Chit nanoparticle toxicity was evaluated using a Cell Counting Kit-8 assay. The efficiency of cellular uptake was determined using fluorescein isothiocyanate-labeled DA-Chit nanoparticles, in place of TAA/DA-Chit nanoparticles, assessed by both inverted fluorescence microscopy and flow cytometry. Downregulation of VEGF mRNA expression by TAA/DA-Chit nanoparticles was further investigated by real-time reverse transcription polymerase chain reaction (RT-PCR assay of the treated human retinal pigment epithelial cells.Results: TAA/DA-Chit nanoparticles were prepared with a TAA-loading capacity in the range of 12%–82%, which increased the water solubility of TAA from 0.3 mg/mL to 2.1 mg/mL. These nanoparticles showed oblate shapes 100–550 nm in size in transmission electron microscopic images and had positive zeta potentials. The Cell Counting Kit-8 assay indicated that the DA-Chit and

  3. In vivo imaging of retinal pigment epithelium cells in age related macular degeneration.

    Science.gov (United States)

    Rossi, Ethan A; Rangel-Fonseca, Piero; Parkins, Keith; Fischer, William; Latchney, Lisa R; Folwell, Margaret A; Williams, David R; Dubra, Alfredo; Chung, Mina M

    2013-01-01

    Morgan and colleagues demonstrated that the RPE cell mosaic can be resolved in the living human eye non-invasively by imaging the short-wavelength autofluorescence using an adaptive optics (AO) ophthalmoscope. This method, based on the assumption that all subjects have the same longitudinal chromatic aberration (LCA) correction, has proved difficult to use in diseased eyes, and in particular those affected by age-related macular degeneration (AMD). In this work, we improve Morgan's method by accounting for chromatic aberration variations by optimizing the confocal aperture axial and transverse placement through an automated iterative maximization of image intensity. The increase in image intensity after algorithmic aperture placement varied depending upon patient and aperture position prior to optimization but increases as large as a factor of 10 were observed. When using a confocal aperture of 3.4 Airy disks in diameter, images were obtained using retinal radiant exposures of less than 2.44 J/cm(2), which is ~22 times below the current ANSI maximum permissible exposure. RPE cell morphologies that were strikingly similar to those seen in postmortem histological studies were observed in AMD eyes, even in areas where the pattern of fluorescence appeared normal in commercial fundus autofluorescence (FAF) images. This new method can be used to study RPE morphology in AMD and other diseases, providing a powerful tool for understanding disease pathogenesis and progression, and offering a new means to assess the efficacy of treatments designed to restore RPE health.

  4. Effects of mechanical stress and vitreous samples in retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Eri, E-mail: eritakahashi@fc.kuh.kumamoto-u.ac.jp; Fukushima, Ayako; Haga, Akira; Inomata, Yasuya; Ito, Yasuhiro; Fukushima, Mikiko; Tanihara, Hidenobu

    2016-02-12

    In rhegmatogenous retinal detachment (RRD), scattered RPE cells from the basement membrane into the vitreous cavity undergo an epithelial mesenchymal transition (EMT) and form the intraocular fibrous membrane in response to vitreous fluid. We investigated whether exposure to vitreous samples was associated with EMT-associated signals and mesenchymal characters. Human vitreous samples were collected from patients with RRD, epiretinal membrane (ERM), or macular hole (MH). We evaluated the effects of vitreous on ARPE-19 cells in suspension cultures using poly 2-hydroxyethyl methacrylate-coated dishes and three-dimensional (3D) Matrigel cultures. We found that exposure to vitreous samples did not induce morphological changes or accelerate wound closure in monolayers. Several samples showed increased phosphorylation of Smad2 and nuclear translocation of nuclear factor-κB. Mechanical stress triggered an elevation of phosphorylation levels in Smad2. In addition, exposure to vitreous fluid increased the phosphorylation of p38 mitogen-activated protein kinase in cell suspension cultures after mechanical stress. Moreover, ARPE-19 cells showed a stellate invasive phenotype in 3D Matrigel cultures with vitreous samples. In this study, we demonstrated that mechanical stress and vitreous were associated with EMT-associated signals and invasive phenotypes in 3D cultures but not in monolayers. These results have important implications for the role of vitreous humor in the induction of EMT and intraocular fibrosis.

  5. Retinal pigment epithelial fine structure in the red-tailed hawk (Buto jamaicensis).

    Science.gov (United States)

    Braekevelt, C R

    1992-03-01

    As part of a comparative morphological study, the fine structure of the retinal epithelium (RPE), choriocapillaris and Bruch's membrane (complexus basalis) has been studied by electron microscopy in the red-tailed hawk (Buteo jamaicensis). In this species the RPE consists of a single layer of low cuboidal cells which display numerous basal (scleral) infoldings and extensive apical (vitreal) processes which interdigitate with photoreceptor outer segments. These epithelial cells are joined laterally by a series of basally located tight junctions. Internally SER is the most abundant cell organelle while only small amounts of RER are present. Polysomes are however abundant as are mitochondria. The RPE cell nucleus is large and vesicular. Melanosomes are mainly located in the apical processes of the RPE cells in light-adaptation. Myeloid bodies are large and numerous in light-adaptation and often show ribosomes on their outer border. Bruch's membrane (complexus basalis) shows the typical pentalaminate structure noted in most vertebrates but with only a poorly defined central elastic layer. The endothelium of the choriocapillaris is very thin facing the RPE but is only moderately fenestrated. The choriocapillaris in this species is unusual however in that many of the fenestrae show a double-layered diaphragm.

  6. Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy

    Science.gov (United States)

    Qi, Xiaoping; Beli, Eleni; Rao, Haripriya V.; Ding, Jindong; Ip, Colin S.; Gu, Hongmei; Akin, Debra; Dunn, William A.; Bowes Rickman, Catherine; Lewin, Alfred S.; Grant, Maria B.; Boulton, Michael E.

    2017-01-01

    p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative

  7. Oxidative stress-mediated NFκB phosphorylation upregulates p62/SQSTM1 and promotes retinal pigmented epithelial cell survival through increased autophagy.

    Science.gov (United States)

    Song, Chunjuan; Mitter, Sayak K; Qi, Xiaoping; Beli, Eleni; Rao, Haripriya V; Ding, Jindong; Ip, Colin S; Gu, Hongmei; Akin, Debra; Dunn, William A; Bowes Rickman, Catherine; Lewin, Alfred S; Grant, Maria B; Boulton, Michael E

    2017-01-01

    p62 is a scaffolding adaptor implicated in the clearance of protein aggregates by autophagy. Reactive oxygen species (ROS) can either stimulate or inhibit NFκB-mediated gene expression influencing cellular fate. We studied the effect of hydrogen peroxide (H2O2)-mediated oxidative stress and NFκB signaling on p62 expression in the retinal pigment epithelium (RPE) and investigated its role in regulation of autophagy and RPE survival against oxidative damage. Cultured human RPE cell line ARPE-19 and primary human adult and fetal RPE cells were exposed to H2O2-induced oxidative stress. The human apolipoprotein E4 targeted-replacement (APOE4) mouse model of AMD was used to study expression of p62 and other autophagy proteins in the retina. p62, NFκB p65 (total, phosphorylated, nuclear and cytoplasmic) and ATG10 expression was assessed by mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was determined using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFκB p65 were evaluated after cellular fractionation by Western blotting. We report that p62 is up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFκB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFκB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in APOE4-HFC AMD mouse model, suggesting reduction of autophagic flux in disease conditions. Our findings suggest that p62 is necessary for RPE cytoprotection under oxidative

  8. High speed optical holography of retinal blood flow

    CERN Document Server

    Pellizzari, Mathilde; Degardin, Julie; Sahel, Jose-Alain; Fink, Mathias; Paques, Michel; Atlan, Michael

    2016-01-01

    We performed non-invasive video imaging of retinal blood flow in a pigmented rat by holographic interferometry of near-infrared laser light backscattered by retinal tissue, beating against an off-axis reference beam sampled at a frame rate of 39 kHz with a high throughput camera. Local Doppler contrasts emerged from the envelopes of short-time Fourier transforms and the phase of autocorrelation functions of holograms rendered by Fresnel transformation. This approach permitted imaging of blood flow in large retinal vessels (30 microns diameter) over 400 by 400 pixels with a spatial resolution of 8 microns and a temporal resolution of 6.5 ms.

  9. Microglia in the mouse retina alter the structure and function of retinal pigmented epithelial cells: a potential cellular interaction relevant to AMD.

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    Wenxin Ma

    Full Text Available BACKGROUND: Age-related macular degeneration (AMD is a leading cause of legal blindness in the elderly in the industrialized word. While the immune system in the retina is likely to be important in AMD pathogenesis, the cell biology underlying the disease is incompletely understood. Clinical and basic science studies have implicated alterations in the retinal pigment epithelium (RPE layer as a locus of early change. Also, retinal microglia, the resident immune cells of the retina, have been observed to translocate from their normal position in the inner retina to accumulate in the subretinal space close to the RPE layer in AMD eyes and in animal models of AMD. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we examined the effects of retinal microglia on RPE cells using 1 an in vitro model where activated retinal microglia are co-cultured with primary RPE cells, and 2 an in vivo mouse model where retinal microglia are transplanted into the subretinal space. We found that retinal microglia induced in RPE cells 1 changes in RPE structure and distribution, 2 increased expression and secretion of pro-inflammatory, chemotactic, and pro-angiogenic molecules, and 3 increased extent of in vivo choroidal neovascularization in the subretinal space. CONCLUSIONS/SIGNIFICANCE: These findings share similarities with important pathological features found in AMD and suggest the relevance of microglia-RPE interactions in AMD pathogenesis. We speculate that the migration of retinal microglia into the subretinal space in early stages of the disease induces significant changes in RPE cells that perpetuate further microglial accumulation, increase inflammation in the outer retina, and fosters an environment conducive for the formation of neovascular changes responsible for much of vision loss in advanced AMD.

  10. Optimizing visualization in enhanced depth imaging OCT in healthy subjects and patients with retinal pigment epithelial detachment

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    Kampik A

    2012-11-01

    Full Text Available Lukas Reznicek, Efstathios Vounotrypidis, Florian Seidensticker, Karsten Kortuem, Anselm Kampik, Aljoscha S Neubauer, Armin WolfDepartment of Ophthalmology, Ludwig Maximilians University Muenchen, Munich, GermanyBackground: This study’s objective was to optimize the visualization of three different spectral-domain optical coherence tomography (SD-OCT display modalities and evaluate enhanced depth imaging (EDI by comparing the maximum depth of assessment in conventional versus inverted cross-sectional OCT images in healthy subjects and in patients with retinal pigment epithelial detachment (PED.Methods: Cross-sectional SD-OCT conventional and inverted images were obtained with the HRA2 (Heidelberg Retina Angiograph II, Heidelberg Engineering, Heidelberg, Germany. Horizontal as well as vertical sections in three different display modes were blinded for evaluation by three independent, experienced graders for maximal imaging depth of the deep ocular fundus layers.Results: The mean imaging depth as measured from the inner segment/outer segment (IS/OS to the outer choroid of all 14 healthy subjects was 197 ± 44 µm vs 263 ± 56 µm for conventional vs EDI scans: in black/white mode, it was significantly lower (P < 0.001 than in white/black mode (249 ± 42 µm vs 337 ± 71 µm and color/heat mode (254 ± 48 µm vs 354 ± 73 µm. The mean imaging depth of all 14 study eyes with PED was 240 ± 78 µm vs 345 ± 100 µm for conventional vs EDI scans in black/white mode, and was significantly lower (P < 0.001 than in white/black mode (393 ± 104 µm vs 464 ± 126 µm and in color/heat mode (373 ± 106 µm vs 453 ± 114 µm. In each display modality of healthy subjects and of patients with PED, EDI scans showed a significantly higher imaging depth than the corresponding conventional scans.Conclusion: White/black and color/heat modes allow increased imaging depth, compared to black/white mode using both conventional or EDI OCT scans in healthy subjects or

  11. Morphological changes in the retinal pigment epithelium on spectral-domain OCT in the unaffected eyes with idiopathic central serous chorioretinopathy.

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    Gupta, Pawan; Gupta, Vishali; Dogra, M R; Singh, Ramandeep; Gupta, Amod

    2010-04-01

    To report the changes seen in the retinal pigment epithelium (RPE) morphology in the asymptomatic eyes of patients with idiopathic central serous chorioretinopathy (ICSC) using spectral-domain Cirrus (TM) high-definition optical coherence tomography (HD-OCT). In a prospective case series, 17 consecutive patients with unilateral ICSC underwent spectral-domain Cirrus (TM) HD-OCT scans for both affected and opposite asymptomatic eye. Three-dimensional single-layer RPE map was studied in both eyes for morphological alterations, and findings were correlated with clinical presentation, fluorescein angiogram, and 5 Line raster scan. Additionally, three-dimensional (3D) single-layer RPE maps done in 111 healthy volunteers served as control. In patients with ICSC, 3D single-layer RPE analysis of asymptomatic eyes showed presence of RPE bumps in 16 (94%) eyes and pigment epithelium detachment (PED) in 2 (11.8%) eyes. The 5 Line raster scan was normal in all eyes. Of 222 normal (control) scans, 18 showed RPE bumps and none showed PED. Conclusions 3D single-layer RPE map showed abnormal pattern in the asymptomatic eyes of patients with unilateral ICSC. Spectral-domain optical coherence tomography showed morphologic alterations in retinal pigment epithelium in both eyes of patients with idiopathic central serous chorioretinopathy.

  12. Osmotic induction of placental growth factor in retinal pigment epithelial cells in vitro: contribution of NFAT5 activity.

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    Hollborn, Margrit; Reichmuth, Konrad; Prager, Philipp; Wiedemann, Peter; Bringmann, Andreas; Kohen, Leon

    2016-08-01

    One risk factor of neovascular age-related macular degeneration is systemic hypertension; hypertension is mainly caused by extracellular hyperosmolarity after consumption of dietary salt. In retinal pigment epithelial (RPE) cells, high extracellular osmolarity induces vascular endothelial growth factor (VEGF)-A (Hollborn et al. in Mol Vis 21:360-377, 2015). The aim of the present study was to determine whether extracellular hyperosmolarity and chemical hypoxia trigger the expression of further VEGF family members including placental growth factor (PlGF) in human RPE cells. Hyperosmotic media were made up by addition of 100 mM NaCl or sucrose. Chemical hypoxia was induced by CoCl2. Gene expression was quantified by real-time RT-PCR, and secretion of PlGF-2 was investigated with ELISA. Nuclear factor of activated T cell 5 (NFAT5) was depleted using siRNA. Extracellular hyperosmolarity triggered expression of VEGF-A, VEGF-D, and PlGF genes, and secretion of PlGF-2. Hypoosmolarity decreased PlGF gene expression. Hypoxia induced expression of VEGF-A, VEGF-B, VEGF-D, and PlGF genes. Extracellular hyperosmolarity and hypoxia produced additive PlGF gene expression. Both hyperosmolarity and hypoxia induced expression of KDR and FLT-4 receptor genes, while hyperosmolarity caused neuropilin-2 and hypoxia neuropilin-1 gene expression. The hyperosmotic, but not the hypoxic, PlGF gene expression was in part mediated by NFAT5. The expression of PlGF in RPE cells depends on the extracellular osmolarity. The data suggest that high consumption of dietary salt may exacerbate the angiogenic response of RPE cells in the hypoxic retina via transcriptional activation of various VEGF family member genes.

  13. The influences of purple sweet potato anthocyanin on the growth characteristics of human retinal pigment epithelial cells

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    Min Sun

    2015-06-01

    Full Text Available Background: Anthocyanins have been proven to be beneficial to the eyes. However, information is scarce about the effects of purple sweet potato (Ipomoea batatas, L. anthocyanin (PSPA, a class of anthocyanins derived from purple sweet potato roots, on visual health. Objective: The aim of this study was to investigate whether PSPA could have influences on the growth characteristics (cellular morphology, survival, and proliferation of human retinal pigment epithelial (RPE cells, which perform essential functions for the visual process. Methods: The RPE cell line D407 was used in the present study. The cytotoxicity of PSPA was assessed by MTT assay. Then, cellular morphology, viability, cell cycle, Ki67expression, and PI3K/MAPK activation of RPE cells treated with PSPA were determined. Results: PSPA exhibited dose-dependent promotion of RPE cell proliferation at concentrations ranging from 10 to 1,000 µg/ml. RPE cells treated with PSPA demonstrated a predominantly polygonal morphology in a mosaic arrangement, and colony-like cells displayed numerous short apical microvilli and typical ultrastructure. PSPA treatment also resulted in a better platform growing status, statistically higher viability, an increase in the S-phase, and more Ki67+ cells. However, neither pAkt nor pERK were detected in either group. Conclusions: We found that PSPA maintained high cell viability, boosted DNA synthesis, and preserved a high percentage of continuously cycling cells to promote cell survival and division without changing cell morphology. This paper lays the foundation for further research about the damage-protective activities of PSPA on RPE cells or human vision.

  14. ROCK Inhibition Promotes Attachment, Proliferation, and Wound Closure in Human Embryonic Stem Cell-Derived Retinal Pigmented Epithelium.

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    Croze, Roxanne H; Thi, William J; Clegg, Dennis O

    2016-11-01

    Nonexudative (dry) age-related macular degeneration (AMD), a leading cause of blindness in the elderly, is associated with the loss of retinal pigmented epithelium (RPE) cells and the development of geographic atrophy, which are areas devoid of RPE cells and photoreceptors. One possible treatment option would be to stimulate RPE attachment and proliferation to replace dying/dysfunctional RPE and bring about wound repair. Clinical trials are underway testing injections of RPE cells derived from pluripotent stem cells to determine their safety and efficacy in treating AMD. However, the factors regulating RPE responses to AMD-associated lesions are not well understood. Here, we use cell culture to investigate the role of RhoA coiled coil kinases (ROCKs) in human embryonic stem cell-derived RPE (hESC-RPE) attachment, proliferation, and wound closure. H9 hESC were spontaneously differentiated into RPE cells. hESC-RPE cells were treated with a pan ROCK1/2 or a ROCK2 only inhibitor; attachment, and proliferation and cell size within an in vitro scratch assay were examined. Pharmacological inhibition of ROCKs promoted hESC-RPE attachment and proliferation, and increased the rate of closure of in vitro wounds. ROCK inhibition decreased phosphorylation of cofilin and myosin light chain, suggesting that regulation of the cytoskeleton underlies the mechanism of action of ROCK inhibition. ROCK inhibition promotes attachment, proliferation, and wound closure in H9 hESC-RPE cells. ROCK isoforms may have different roles in wound healing. Modulation of the ROCK-cytoskeletal axis has potential in stimulating wound repair in transplanted RPE cells and attachment in cellular therapies.

  15. αvβ5 Integrin/FAK/PGC-1α Pathway Confers Protective Effects on Retinal Pigment Epithelium.

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    Murilo F Roggia

    Full Text Available To elucidate the mechanism of the induction of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α by photoreceptor outer segments (POS and its effects on retinal pigment epithelium (RPE.PGC-1α upregulation by POS was confirmed in ARPE-19 cells and in RPE ex vivo. To elucidate the mechanism, siRNAs against β5 integrin, CD36, Mer tyrosine kinase (MerTK, and Atg5, blocking antibodies against CD36 and MerTK, and a specific inhibitor for focal adhesion kinase (FAK were used. We examined the effect of POS-induced PGC-1α upregulation on the levels of reactive oxygen species (ROS, mitochondrial biogenesis, senescence-associated β-galactosidase (SA-β-gal after H2O2 treatment, and lysosomal activity. Lysosomal activity was evaluated through transcriptional factor EB and its target genes, and the activity of cathepsin D. Lipid metabolism after POS treatment was assessed using Oil Red O and BODIPY C11. RPE phenotypes of PGC-1α-deficient mice were examined.POS-induced PGC-1α upregulation was suppressed by siRNA against β5 integrin and a FAK inhibitor. siRNAs and blocking antibodies against CD36 and MerTK enhanced the effect of POS on PGC-1α. The upregulation of PGC-1α increased the levels of mRNA for antioxidant enzymes and stimulated mitochondrial biogenesis, decreased ROS levels, and reduced SA-β-gal staining in H2O2-treated ARPE-19 cells. PGC-1α was critical for lysosomal activity and lipid metabolism after POS treatment. PGC-1α-deficient mice demonstrated an accumulation of type 2 lysosomes in RPE, thickening of Bruch's membrane, and poor choriocapillaris vasculature.The binding, but not the internalization of POS confers protective effects on RPE cells through the αvβ5 integrin/FAK/PGC-1α pathway.

  16. Polarisation-sensitive OCT is useful for evaluating retinal pigment epithelial lesions in patients with neovascular AMD

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    Schütze, Christopher; Teleky, Katharina; Baumann, Bernhard; Pircher, Michael; Götzinger, Erich; Hitzenberger, Christoph K; Schmidt-Erfurth, Ursula

    2016-01-01

    Background/aims To examine the reproducibility of lesion dimensions of the retinal pigment epithelium (RPE) in neovascular age-related macular degeneration (AMD) with polarisation-sensitive optical coherence tomography (PS-OCT), specifically imaging the RPE. Methods Twenty-six patients (28 eyes) with neovascular AMD were included in this study, and examined by a PS-OCT prototype. Each patient was scanned five times at a 1-day visit. The PS-OCT B-scan located closest to the macular centre presenting with RPE atrophy was identified, and the longitudinal diameter of the lesion was quantified manually using AutoCAD 2008. This procedure was followed for the identical B-scan position in all five scans per eye and patient. Reproducibility of qualitative changes in PS-OCT was evaluated. Interobserver variability was assessed. Results were compared with intensity-based spectral-domain OCT (SD-OCT) imaging. Results Mean variability of all atrophy lesion dimensions was 0.10 mm (SD±=0.06 mm). Coefficient of variation (SD±/mean) was 0.06 on average (SD±=0.03). Interobserver variability assessment showed a mean difference of 0.02 mm across all patients regarding RPE lesion size evaluation (paired t test: p=0.38). Spearman correlation coefficient was r=0.98, p<0.001. Results revealed a good overall reproducibility of ∼90%. PS-OCT specifically detected the RPE in all eyes compared with conventional intensity-based SD-OCT that was not capable to clearly identify RPE atrophy in 25 eyes (89.3%, p<0.01). Conclusions PS-OCT offers good reproducibility of RPE atrophy assessment in neovascular AMD, and may be suitable for precise RPE evaluation in clinical practice. PS-OCT unambiguously identifies RPE changes in choroidal neovascularisation compared with intensity-based SD-OCT that does not identify the RPE status reliably. PMID:26183936

  17. ROCK Inhibition Promotes Attachment, Proliferation, and Wound Closure in Human Embryonic Stem Cell–Derived Retinal Pigmented Epithelium

    Science.gov (United States)

    Croze, Roxanne H.; Thi, William J.; Clegg, Dennis O.

    2016-01-01

    Purpose Nonexudative (dry) age-related macular degeneration (AMD), a leading cause of blindness in the elderly, is associated with the loss of retinal pigmented epithelium (RPE) cells and the development of geographic atrophy, which are areas devoid of RPE cells and photoreceptors. One possible treatment option would be to stimulate RPE attachment and proliferation to replace dying/dysfunctional RPE and bring about wound repair. Clinical trials are underway testing injections of RPE cells derived from pluripotent stem cells to determine their safety and efficacy in treating AMD. However, the factors regulating RPE responses to AMD-associated lesions are not well understood. Here, we use cell culture to investigate the role of RhoA coiled coil kinases (ROCKs) in human embryonic stem cell–derived RPE (hESC-RPE) attachment, proliferation, and wound closure. Methods H9 hESC were spontaneously differentiated into RPE cells. hESC-RPE cells were treated with a pan ROCK1/2 or a ROCK2 only inhibitor; attachment, and proliferation and cell size within an in vitro scratch assay were examined. Results Pharmacological inhibition of ROCKs promoted hESC-RPE attachment and proliferation, and increased the rate of closure of in vitro wounds. ROCK inhibition decreased phosphorylation of cofilin and myosin light chain, suggesting that regulation of the cytoskeleton underlies the mechanism of action of ROCK inhibition. Conclusions ROCK inhibition promotes attachment, proliferation, and wound closure in H9 hESC-RPE cells. ROCK isoforms may have different roles in wound healing. Translational Relevance Modulation of the ROCK-cytoskeletal axis has potential in stimulating wound repair in transplanted RPE cells and attachment in cellular therapies. PMID:27917311

  18. In Vitro Response of Retinal Pigment Epithelial Cells Exposed to Chitosan Materials Prepared with Different Cross-Linkers

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    Jui-Yang Lai

    2010-12-01

    Full Text Available The interaction between cells and biopolymers is the evaluation indicator of the biocompatibility of materials. The purpose of this work was to examine the responses of retinal pigment epithelial (RPE cells to genipin (GP or glutaraldehyde (GTA cross-linked chitosan by means of cell viability assays, cytokine expression analyses, and apoptosis assays. Evaluations of non-cross-linked chitosan were conducted simultaneously for comparison. Both GP and GTA treated samples with the same extent of cross-linking (around 80% were prepared by varying cross-linking time. Our results showed that GP cross-linking was carried out by either radical polymerization of the monomers or SN2 nucleophilic substitution reaction involving the replacement of the ester group on the monomer with a secondary amide linkage. On the other hand, GTA could react with free amino groups of chitosan, leading to the formation of either the Schiff bases or the Michael-type adducts with terminal aldehydes. The biocompatibility of non-cross-linked chitosan membranes was demonstrated by the absence of any signs of toxicity or inflammation reaction. The present study showed that the ARPE-19 cells exposed to GTA cross-linked chitosan membranes had significantly higher cytotoxicity, interleukin-6 levels, and number of TUNEL-positive nuclei than did those exposed to GP treated samples. In addition, the materials modified with GTA trigger apoptosis at an early stage and may induce toxicity in the RPE cells later. The findings suggest that while the chitosan molecules bridged by GP are satisfactorily cytocompatible, the counterparts treated by GTA do not seem to be tolerated. In terms of material safety, the GP cross-linked chitosan may be compatible with human RPE cells and may have a potential application as delivery carriers in the treatment of posterior segment diseases.

  19. Smoke Exposure Causes Endoplasmic Reticulum Stress and Lipid Accumulation in Retinal Pigment Epithelium through Oxidative Stress and Complement Activation*

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    Kunchithapautham, Kannan; Atkinson, Carl; Rohrer, Bärbel

    2014-01-01

    Age-related macular degeneration (AMD) is a complex disease caused by genetic and environmental factors, including genetic variants in complement components and smoking. Smoke exposure leads to oxidative stress, complement activation, endoplasmic reticulum (ER) stress, and lipid dysregulation, which have all been proposed to be associated with AMD pathogenesis. Here we examine the effects of smoke exposure on the retinal pigment epithelium (RPE). Mice were exposed to cigarette smoke or filtered air for 6 months. RPE cells grown as stable monolayers were exposed to 5% cigarette smoke extract (CSE). Effects of smoke were determined by biochemical, molecular, and histological measures. Effects of the alternative pathway (AP) of complement and complement C3a anaphylatoxin receptor signaling were analyzed using knock-out mice or specific inhibitors. ER stress markers were elevated after smoke exposure in RPE of intact mice, which was eliminated in AP-deficient mice. To examine this relationship further, RPE monolayers were exposed to CSE. Short term smoke exposure resulted in production and release of complement C3, the generation of C3a, oxidative stress, complement activation on the cell membrane, and ER stress. Long term exposure to CSE resulted in lipid accumulation, and secretion. All measures were reversed by blocking C3a complement receptor (C3aR), alternative complement pathway signaling, and antioxidant therapy. Taken together, our results provide clear evidence that smoke exposure results in oxidative stress and complement activation via the AP, resulting in ER stress-mediated lipid accumulation, and further suggesting that oxidative stress and complement act synergistically in the pathogenesis of AMD. PMID:24711457

  20. Ultraviolet vision in lacertid lizards: evidence from retinal structure, eye transmittance, SWS1 visual pigment genes and behaviour.

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    Pérez i de Lanuza, Guillem; Font, Enrique

    2014-08-15

    Ultraviolet (UV) vision and UV colour patches have been reported in a wide range of taxa and are increasingly appreciated as an integral part of vertebrate visual perception and communication systems. Previous studies with Lacertidae, a lizard family with diverse and complex coloration, have revealed the existence of UV-reflecting patches that may function as social signals. However, confirmation of the signalling role of UV coloration requires demonstrating that the lizards are capable of vision in the UV waveband. Here we use a multidisciplinary approach to characterize the visual sensitivity of a diverse sample of lacertid species. Spectral transmission measurements of the ocular media show that wavelengths down to 300 nm are transmitted in all the species sampled. Four retinal oil droplet types can be identified in the lacertid retina. Two types are pigmented and two are colourless. Fluorescence microscopy reveals that a type of colourless droplet is UV-transmitting and may thus be associated with UV-sensitive cones. DNA sequencing shows that lacertids have a functional SWS1 opsin, very similar at 13 critical sites to that in the presumed ancestral vertebrate (which was UV sensitive) and other UV-sensitive lizards. Finally, males of Podarcis muralis are capable of discriminating between two views of the same stimulus that differ only in the presence/absence of UV radiance. Taken together, these results provide convergent evidence of UV vision in lacertids, very likely by means of an independent photopigment. Moreover, the presence of four oil droplet types suggests that lacertids have a four-cone colour vision system.

  1. Complement system dysregulation and inflammation in the retinal pigment epithelium of a mouse model for Stargardt macular degeneration.

    Science.gov (United States)

    Radu, Roxana A; Hu, Jane; Yuan, Quan; Welch, Darcy L; Makshanoff, Jacob; Lloyd, Marcia; McMullen, Stephen; Travis, Gabriel H; Bok, Dean

    2011-05-27

    Accumulation of vitamin A-derived lipofuscin fluorophores in the retinal pigment epithelium (RPE) is a pathologic feature of recessive Stargardt macular dystrophy, a blinding disease caused by dysfunction or loss of the ABCA4 transporter in rods and cones. Age-related macular degeneration, a prevalent blinding disease of the elderly, is strongly associated with mutations in the genes for complement regulatory proteins (CRP), causing chronic inflammation of the RPE. Here we explore the possible relationship between lipofuscin accumulation and complement activation in vivo. Using the abca4(-/-) mouse model for recessive Stargardt, we investigated the role of lipofuscin fluorophores (A2E-lipofuscin) on oxidative stress and complement activation. We observed higher expression of oxidative-stress genes and elevated products of lipid peroxidation in eyes from abca4(-/-) versus wild-type mice. We also observed higher levels of complement-activation products in abca4(-/-) RPE cells. Unexpectedly, expression of multiple CRPs, which protect cells from attack by the complement system, were lower in abca4(-/-) versus wild-type RPE. To test whether acute exposure of healthy RPE cells to A2E-lipofuscin affects oxidative stress and expression of CRPs, we fed cultured fetal-derived human RPE cells with rod outer segments from wild-type or abca4(-/-) retinas. In contrast to RPE cells in abca4(-/-) mice, human RPE cells exposed to abca4(-/-) rod outer segments adaptively increased expression of both oxidative-stress and CRP genes. These results suggest that A2E accumulation causes oxidative stress, complement activation, and down-regulation of protective CRP in the Stargardt mouse model. Thus, Stargardt disease and age-related macular degeneration may both be caused by chronic inflammation of the RPE.

  2. Locational heterogeneity of maturation by changes in migratory behaviors of human retinal pigment epithelial cells in culture.

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    Sonoi, Rie; Kim, Mee-Hae; Kino-oka, Masahiro

    2015-01-01

    To better characterize human retinal pigment epithelial (RPE) cells, their maturation was studied by time-lapse observation and immunostaining of the tight junction protein ZO-1. During subconfluency with active migration, the cells had an elongated shape. During cell division to reach confluency, RPE cells became small and tight, exhibiting cobblestone-like morphology. In addition, RPE maturation at the peripheral region of the culture vessel was delayed when compared with the central region, demonstrating local heterogeneity during maturation. To correlate cellular migration and maturation, we compared frequencies of migration rate and number of ZO-1-positive cells at the central and peripheral regions. Cells having migration rates less than 5.0 μm/h in the central region were 1.4-fold higher than in the peripheral region at day 5. Regardless of locational differences in the culture vessel, the frequency of cells having migration rates less than 5.0 μm/h showed 90% agreement with the frequency of ZO-1-positive cells. To inhibit cell migration, RPE cells were exposed to medium containing 50 μg/ml Rac1 inhibitor at day 5. Frequencies of ZO-1-positive cells and cells having migration rates less than 5.0 μm/h at the peripheral region were similar to those at the central region. The results show that migration is an important factor affecting maturation, and demonstrate that location heterogeneity during maturation is caused by different migratory behaviors in the culture vessel. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  3. Reversal of the Caspase-Dependent Apoptotic Cytotoxicity Pathway by Taurine from Lycium barbarum (Goji Berry in Human Retinal Pigment Epithelial Cells: Potential Benefit in Diabetic Retinopathy

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    M. K. Song

    2012-01-01

    Full Text Available Diabetic retinopathy is a preventable microvascular diabetic complication and a leading cause of vision loss. Retinal pigment epithelial cell apoptosis is an early event in diabetic retinopathy. Taurine is reportedly beneficial for diabetic retinopathy and is abundant in the fruit of Lycium barbarum (LB. We have investigated the effect of pure taurine and an extract of LB rich in taurine on a model of diabetic retinopathy, the retinal ARPE-19 cell line exposed to high glucose. We demonstrate for the first time that LB extract and the active ligand, taurine, dose dependently enhance cell viability following high glucose treatment in the ARPE-19 retinal epithelial cell line. This cytoprotective effect was associated with the attenuation of high glucose-induced apoptosis, which was shown by characteristic morphological staining and the dose-dependent decrease in the number of apoptotic cells, determined by flow cytometry. Moreover, we have shown that LB extract and taurine dose dependently downregulate caspase-3 protein expression and the enzymatic activity of caspase-3. We conclude that taurine, a major component of LB, and the LB extract, have a cytoprotective effect against glucose exposure in a human retinal epithelial cell line and may provide useful approaches to delaying diabetic retinopathy progression.

  4. Mechanism of riboflavin uptake by cultured human retinal pigment epithelial ARPE-19 cells: possible regulation by an intracellular Ca2+-calmodulin-mediated pathway

    OpenAIRE

    Said, Hamid M.; Wang, S.L.; Ma, T Y

    2005-01-01

    In mammalian cells (including those of the ocular system), the water-soluble vitamin B-2 (riboflavin, RF) assumes an essential role in a variety of metabolic reactions and is critical for normal cellular functions, growth and development. Cells of the human retinal pigment epithelium (hRPE) play an important role in providing a sufficient supply of RF to the retina, but nothing is known about the mechanism of the vitamin uptake by these cells and its regulation. Our aim in the present study w...

  5. The generation of induced pluripotent stem cells for macular degeneration as a drug screening platform: identification of curcumin as a protective agent for retinal pigment epithelial cells against oxidative stress

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    Yun-Ching eChang

    2014-08-01

    Full Text Available Age-related macular degeneration (AMD is one retinal aging process that may lead to irreversible vision loss in the elderly. Its pathogenesis remains unclear, but oxidative stress inducing retinal pigment epithelial (RPE cells damage is perhaps responsible for the aging sequence of retina and may play an important role in macular degeneration. In this study, we have reprogrammed T cells from patients with dry type AMD into induced pluripotent stem cells (iPSCs via integration-free episomal vectors and differentiated them into RPE cells that were used as an expandable platform for investigating pathogenesis of the AMD and in-vitro drug screening. These patient-derived RPEs with the AMD-associated background (AMD-RPEs exhibited reduced antioxidant ability, compared with normal RPE cells. Among several screened candidate drugs, curcumin caused most significant reduction of ROS in AMD-RPEs. Pre-treatment of curcumin protected these AMD-RPEs from H2O2-induced cell death and also increased the cytoprotective effect against the oxidative stress of H2O2 through the reduction of ROS levels. In addition, curcumin with its versatile activities modulated the expression of many oxidative stress-regulating genes such as PDGF, VEGF, IGFBP-2, HO1, SOD2 and GPX1. Our findings indicated that the RPE cells derived from AMD patients have decreased antioxidative defense, making RPE cells more susceptible to oxidative damage and thereby leading to AMD formation. Curcumin represented an ideal drug that can effectively restore the neuronal functions in AMD patient-derived RPE cells, rendering this drug an effective option for macular degeneration therapy and an agent against aging-associated oxidative stress.

  6. Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

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    Huang, Xionggao [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China); Department of Ophthalmology, Hainan Medical College, Haikou (China); Wei, Yantao; Ma, Haizhi [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China); Zhang, Shaochong, E-mail: zhshaochong@163.com [State Key Ophthalmic Laboratory, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou (China)

    2012-03-09

    Highlights: Black-Right-Pointing-Pointer Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. Black-Right-Pointing-Pointer Rac1 is activated in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells induced by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that vitreous

  7. A constitutively active Gαi3 protein corrects the abnormal retinal pigment epithelium phenotype of Oa1-/- mice.

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    Alejandra Young

    Full Text Available PURPOSE: Ocular Albinism type 1 (OA1 is a disease caused by mutations in the OA1 gene and characterized by the presence of macromelanosomes in the retinal pigment epithelium (RPE as well as abnormal crossing of the optic axons at the optic chiasm. We showed in our previous studies in mice that Oa1 activates specifically Gαi3 in its signaling pathway and thus, hypothesized that a constitutively active Gαi3 in the RPE of Oa1-/- mice might keep on the Oa1 signaling cascade and prevent the formation of macromelanosomes. To test this hypothesis, we have generated transgenic mice that carry the constitutively active Gαi3 (Q204L protein in the RPE of Oa1-/- mice and are now reporting the effects that the transgene produced on the Oa1-/- RPE phenotype. METHODS: Transgenic mice carrying RPE-specific expression of the constitutively active Gαi3 (Q204L were generated by injecting fertilized eggs of Oa1-/- females with a lentivirus containing the Gαi3 (Q204L cDNA. PCR, Southern blots, Western blots and confocal microscopy were used to confirm the presence of the transgene in the RPE of positive transgenic mice. Morphometrical analyses were performed using electron microscopy to compare the size and number of melanosomes per RPE area in putative Oa1-/-, Gαi3 (Q204L transgenic mice with those of wild-type NCrl and Oa1-/- mice. RESULTS: We found a correlation between the presence of the constitutively active Gαi3 (Q204L transgene and the rescue of the normal phenotype of RPE melanosomes in Oa1-/-, Gαi3 (Q204L mice. These mice have higher density of melanosomes per RPE area and a larger number of small melanosomes than Oa1-/- mice, and their RPE phenotype is similar to that of wild-type mice. CONCLUSIONS: Our results show that a constitutively active Gαi3 protein can by-pass the lack of Oa1 protein in Oa1-/- mice and consequently rescue the RPE melanosomal phenotype.

  8. Combined Hamartoma of the Retina and Retinal Pigment Epithelium in a Patient with Gorlin Syndrome: Spontaneous Partial Resolution of Traction Caused by Epiretinal Membrane

    Science.gov (United States)

    Sánchez-Vicente, José L.; Rueda, Trinidad; Rodríguez de la Rúa-Franch, Enrique; Molina-Socola, Fredy E.; Vital-Berral, Cristina; Alfaro-Juárez, Asunción; López-Herrero, Fernando; Muñoz-Morales, Ana

    2016-01-01

    Purpose. To describe the case of spontaneous resolution of epiretinal membrane in a patient with Combined Hamartoma of the Retina and Retinal Pigment Epithelium (CHR-RPE), in the clinical context of Gorlin Syndrome (GS). Methods. Observational case report of a 12-year-old female patient is presented. The diagnosis of CHRRPE was made by OCT and fundus examination, which showed a mound of disorganized tissue originating from retina and retinal pigment epithelium. Epiretinal membrane (EM) was also detected. Genetic study was performed to confirm the diagnosis of GS. Results. The patient was observed for 39 months, showing spontaneous resolution of the traction caused by the EM and improvement in visual acuity (VA), which was 20/80 at initial presentation, rising to 20/40 after follow-up period. Conclusions. The presence of EM in CHR-REP is a cause of reduction of visual acuity. Management of this condition is controversial; however, we would like to highlight that spontaneous resolution of the traction caused by EM is possible, resulting in recovery of VA. PMID:27595027

  9. Pigment Epithelium-Derived Factor Reduces Apoptosis and Pro-Inflammatory Cytokine Gene Expression in a Murine Model of Focal Retinal Degeneration

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    Yujuan Wang

    2013-10-01

    Full Text Available AMD (age-related macular degeneration is a neurodegenerative disease causing irreversible central blindness in the elderly. Apoptosis and inflammation play important roles in AMD pathogenesis. PEDF (pigment epithelium-derived factor is a potent neurotrophic and anti-inflammatory glycoprotein that protects the retinal neurons and photoreceptors against cell death caused by pathological insults. We studied the effects of PEDF on focal retinal lesions in DKO rd8 (Ccl2 −/− /Cx3cr1 −/− on C57BL/6N [Crb1rd8 ] mice, a model for progressive, focal rd (retinal degeneration. First, we found a significant decrease in PEDF transcript expression in DKO rd8 mouse retina and RPE (retinal pigment epithelium than WT (wild-type, C57BL/6N. Next, cultured DKO rd8 RPE cells secreted lower levels of PEDF protein in the media than WT. Then the right eyes of DKO rd8 mice were injected intravitreously with recombinant human PEDF protein (1 μg, followed by a subconjunctival injection of PEDF (3 μg 4 weeks later. The untreated left eyes served as controls. The effect of PEDF was assessed by fundoscopy, ocular histopathology and A2E {[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl-1E,3E,5E,7E-octatetra-enyl]-1-(2-hydroxyethyl-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl 1E,3E,5E,7E-hexatrienyl]-pyridinium} levels, as well as apoptotic and inflammatory molecules. The PEDF-treated eyes showed slower progression or attenuation of the focal retinal lesions, fewer and/or smaller photoreceptor and RPE degeneration, and significantly lower A2E, relative to the untreated eyes. In addition, lower expression of apoptotic and inflammatory molecules were detected in the PEDF-treated than untreated eyes. Our results establish that PEDF potently stabilizes photoreceptor degeneration via suppression of both apoptotic and inflammatory pathways. The multiple beneficial effects of PEDF represent a novel approach for potential AMD treatment.

  10. Foxg1-Cre Mediated Lrp2 Inactivation in the Developing Mouse Neural Retina, Ciliary and Retinal Pigment Epithelia Models Congenital High Myopia.

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    Olivier Cases

    Full Text Available Myopia is a common ocular disorder generally due to increased axial length of the eye-globe. Its extreme form high myopia (HM is a multifactorial disease leading to retinal and scleral damage, visual impairment or loss and is an important health issue. Mutations in the endocytic receptor LRP2 gene result in Donnai-Barrow (DBS and Stickler syndromes, both characterized by HM. To clearly establish the link between Lrp2 and congenital HM we inactivated Lrp2 in the mouse forebrain including the neural retina and the retinal and ciliary pigment epithelia. High resolution in vivo MRI imaging and ophthalmological analyses showed that the adult Lrp2-deficient eyes were 40% longer than the control ones mainly due to an excessive elongation of the vitreal chamber. They had an apparently normal intraocular pressure and developed chorioretinal atrophy and posterior scleral staphyloma features reminiscent of human myopic retinopathy. Immunomorphological and ultrastructural analyses showed that increased eye lengthening was first observed by post-natal day 5 (P5 and that it was accompanied by a rapid decrease of the bipolar, photoreceptor and retinal ganglion cells, and eventually the optic nerve axons. It was followed by scleral thinning and collagen fiber disorganization, essentially in the posterior pole. We conclude that the function of LRP2 in the ocular tissues is necessary for normal eye growth and that the Lrp2-deficient eyes provide a unique tool to further study human HM.

  11. Foxg1-Cre Mediated Lrp2 Inactivation in the Developing Mouse Neural Retina, Ciliary and Retinal Pigment Epithelia Models Congenital High Myopia

    Science.gov (United States)

    Obry, Antoine; Santin, Mathieu D.; Ben-Yacoub, Sirine; Pâques, Michel; Amsellem-Levera, Sabine; Bribian, Ana; Simonutti, Manuel; Augustin, Sébastien; Debeir, Thomas; Sahel, José Alain; Christ, Annabel; de Castro, Fernando; Lehéricy, Stéphane; Cosette, Pascal; Kozyraki, Renata

    2015-01-01

    Myopia is a common ocular disorder generally due to increased axial length of the eye-globe. Its extreme form high myopia (HM) is a multifactorial disease leading to retinal and scleral damage, visual impairment or loss and is an important health issue. Mutations in the endocytic receptor LRP2 gene result in Donnai-Barrow (DBS) and Stickler syndromes, both characterized by HM. To clearly establish the link between Lrp2 and congenital HM we inactivated Lrp2 in the mouse forebrain including the neural retina and the retinal and ciliary pigment epithelia. High resolution in vivo MRI imaging and ophthalmological analyses showed that the adult Lrp2-deficient eyes were 40% longer than the control ones mainly due to an excessive elongation of the vitreal chamber. They had an apparently normal intraocular pressure and developed chorioretinal atrophy and posterior scleral staphyloma features reminiscent of human myopic retinopathy. Immunomorphological and ultrastructural analyses showed that increased eye lengthening was first observed by post-natal day 5 (P5) and that it was accompanied by a rapid decrease of the bipolar, photoreceptor and retinal ganglion cells, and eventually the optic nerve axons. It was followed by scleral thinning and collagen fiber disorganization, essentially in the posterior pole. We conclude that the function of LRP2 in the ocular tissues is necessary for normal eye growth and that the Lrp2-deficient eyes provide a unique tool to further study human HM. PMID:26107939

  12. X-box binding protein 1 is essential for the anti-oxidant defense and cell survival in the retinal pigment epithelium.

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    Yimin Zhong

    Full Text Available Damage to the retinal pigment epithelium (RPE is an early event in the pathogenesis of age-related macular degeneration (AMD. X-box binding protein 1 (XBP1 is a key transcription factor that regulates endoplasmic reticulum (ER homeostasis and cell survival. This study aimed to delineate the role of endogenous XBP1 in the RPE. Our results show that in a rat model of light-induced retinal degeneration, XBP1 activation was suppressed in the RPE/choroid complex, accompanied by decreased anti-oxidant genes and increased oxidative stress. Knockdown of XBP1 by siRNA resulted in reduced expression of SOD1, SOD2, catalase, and glutathione synthase and sensitized RPE cells to oxidative damage. Using Cre/LoxP system, we generated a mouse line that lacks XBP1 only in RPE cells. Compared to wildtype littermates, RPE-XBP1 KO mice expressed less SOD1, SOD2, and catalase in the RPE, and had increased oxidative stress. At age 3 months and older, these mice exhibited apoptosis of RPE cells, decreased number of cone photoreceptors, shortened photoreceptor outer segment, reduced ONL thickness, and deficit in retinal function. Electron microscopy showed abnormal ultrastructure, Bruch's membrane thickening, and disrupted basal membrane infolding in XBP1-deficient RPE. These results indicate that XBP1 is an important gene involved in regulation of the anti-oxidant defense in the RPE, and that impaired activation of XBP1 may contribute to RPE dysfunction and cell death during retinal degeneration and AMD.

  13. Retina-specific nuclear receptor: A potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Müller glial cells.

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    Chen, F; Figueroa, D J; Marmorstein, A D; Zhang, Q; Petrukhin, K; Caskey, C T; Austin, C P

    1999-12-21

    In an effort to identify nuclear receptors important in retinal disease, we screened a retina cDNA library for nuclear receptors. Here we describe the identification of a retina-specific nuclear receptor (RNR) from both human and mouse. Human RNR is a splice variant of the recently published photoreceptor cell-specific nuclear receptor [Kobayashi, M., Takezawa, S., Hara, K., Yu, R. T., Umesono, Y., Agata, K., Taniwaki, M., Yasuda, K. & Umesono, K. (1999) Proc. Natl. Acad. Sci. USA 96, 4814-4819] whereas the mouse RNR is a mouse ortholog. Northern blot and reverse transcription-PCR analyses of human mRNA samples demonstrate that RNR is expressed exclusively in the retina, with transcripts of approximately 7.5 kb, approximately 3.0 kb, and approximately 2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrates that RNR is expressed in the retinal pigment epithelium and in Müller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR). These data raise the possibility that RNR acts to regulate the visual cycle through its interaction with cellular retinaldehyde binding protein and therefore may be a target for retinal diseases such as retinitis pigmentosa and age-related macular degeneration.

  14. Structure and barrier properties of human embryonic stem cell-derived retinal pigment epithelial cells are affected by extracellular matrix protein coating.

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    Sorkio, Anni; Hongisto, Heidi; Kaarniranta, Kai; Uusitalo, Hannu; Juuti-Uusitalo, Kati; Skottman, Heli

    2014-02-01

    Extracellular matrix (ECM) interactions play a vital role in cell morphology, migration, proliferation, and differentiation of cells. We investigated the role of ECM proteins on the structure and function of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells during their differentiation and maturation from hESCs into RPE cells in adherent differentiation cultures on several human ECM proteins found in native human Bruch's membrane, namely, collagen I, collagen IV, laminin, fibronectin, and vitronectin, as well as on commercial substrates of xeno-free CELLstart™ and Matrigel™. Cell pigmentation, expression of RPE-specific proteins, fine structure, as well as the production of basal lamina by hESC-RPE on different protein coatings were evaluated after 140 days of differentiation. The integrity of hESC-RPE epithelium and barrier properties on different coatings were investigated by measuring transepithelial resistance. All coatings supported the differentiation of hESC-RPE cells as demonstrated by early onset of cell pigmentation and further maturation to RPE monolayers after enrichment. Mature RPE phenotype was verified by RPE-specific gene and protein expression, correct epithelial polarization, and phagocytic activity. Significant differences were found in the degree of RPE cell pigmentation and tightness of epithelial barrier between different coatings. Further, the thickness of self-assembled basal lamina and secretion of the key ECM proteins found in the basement membrane of the native RPE varied between hESC-RPE cultured on compared protein coatings. In conclusion, this study shows that the cell culture substrate has a major effect on the structure and basal lamina production during the differentiation and maturation of hESC-RPE potentially influencing the success of cell integrations and survival after cell transplantation.

  15. Quantum rendering

    Science.gov (United States)

    Lanzagorta, Marco O.; Gomez, Richard B.; Uhlmann, Jeffrey K.

    2003-08-01

    In recent years, computer graphics has emerged as a critical component of the scientific and engineering process, and it is recognized as an important computer science research area. Computer graphics are extensively used for a variety of aerospace and defense training systems and by Hollywood's special effects companies. All these applications require the computer graphics systems to produce high quality renderings of extremely large data sets in short periods of time. Much research has been done in "classical computing" toward the development of efficient methods and techniques to reduce the rendering time required for large datasets. Quantum Computing's unique algorithmic features offer the possibility of speeding up some of the known rendering algorithms currently used in computer graphics. In this paper we discuss possible implementations of quantum rendering algorithms. In particular, we concentrate on the implementation of Grover's quantum search algorithm for Z-buffering, ray-tracing, radiosity, and scene management techniques. We also compare the theoretical performance between the classical and quantum versions of the algorithms.

  16. Cisto vítreo e retinose pigmentária: relato de caso Vitreous cyst and retinitis pigmentosa: case report

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    Maria Frasson

    2010-04-01

    Full Text Available Cistos vítreos são achados raros do segmento posterior ocular. Podem ocorrer em olhos com doenças oculares preexistentes ou em olhos aparentemente normais. Este estudo é um relato de caso de um paciente com retinose pigmentária e cisto vítreo, e descreve sua apresentação clínica e ultrassonográfica.Vitreous cyst is a rare condition of the posterior segment of the eye. It can occur in eyes with coexistent ocular diseases or in eyes that are otherwise normal. This study reports a case of vitreous cyst in a patient with retinitis pigmentosa and presents its clinical and ultrasonographic features.

  17. Mechanism of retinal pigment epithelium tear formation following intravitreal anti-vascular endothelial growth factor therapy revealed by spectral-domain optical coherence tomography

    DEFF Research Database (Denmark)

    Nagiel, Aaron; Freund, K Bailey; Spaide, Richard F;

    2013-01-01

    PURPOSE: To demonstrate the mechanism by which retinal pigment epithelium (RPE) tears occur in eyes with neovascular age-related macular degeneration (AMD) treated with intravitreal anti-vascular endothelial growth factor (VEGF) agents using spectral-domain optical coherence tomography (OCT......). DESIGN: Retrospective observational case series. METHODS: OCT images of 8 eyes that developed RPE tears following the administration of intravitreal anti-VEGF agents for neovascular AMD were evaluated. Pretear and posttear images were compared in order to elucidate the mechanism by which RPE tears occur...... to the retracted RPE. In all eyes, the RPE ruptured along a segment of bare RPE not in contact with the CNV or Bruch membrane. CONCLUSIONS: Eyes with vascularized PEDs secondary to AMD may show specific OCT findings that increase the risk for RPE tear following intravitreal anti-VEGF injection. Rapid involution...

  18. 3,3'-Diindolylmethane inhibits VEGF expression through the HIF-1α and NF-κB pathways in human retinal pigment epithelial cells under chemical hypoxic conditions.

    Science.gov (United States)

    Park, Hongzoo; Lee, Dae-Sung; Yim, Mi-Jin; Choi, Yung Hyun; Park, Saegwang; Seo, Su-Kil; Choi, Jung Sik; Jang, Won Hee; Yea, Sung Su; Park, Won Sun; Lee, Chang-Min; Jung, Won-Kyo; Choi, Il-Whan

    2015-07-01

    Oxidative stress in the retinal pigment epithelium (RPE) can lead to the pathological causes of age-related macular degeneration (AMD). Hypoxia induces oxidative damage in retinal pigment epithelial cells (RPE cells). In this study, we investigated the capacity of 3,3'-diindolylmethane (DIM) to reduce the expression of vascular endothelial growth factor (VEGF) under hypoxic conditions, as well as the molecular mechanisms involved. Human RPE cells (ARPE-19 cells) were treated with cobalt chloride (CoCl2, 200 µM) and/or DIM (10 and 20 µM). The production of VEGF was measured by enzyme-linked immunosorbent assay. The translocation of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB (NF-κB) was determined by western blot analysis. The binding activity of HIF-1α and NF-κB was analyzed by electrophoretic mobility shift assay. The phosphorylation levels of mitogen-activated protein kinases (MAPKs) were measured by western blot analysis. The levels of mitochondrial reactive oxygen species (ROS) were detected by fluorescence microplate assay. The results revealed that DIM significantly attenuated the CoCl2-induced expression of VEGF in the ARPE-19 cells. The CoCl2-induced translocation and activation of HIF-1α and NF-κB were also attenuated by treatment with DIM. In addition, DIM inhibited the CoCl2-induced activation of p38 MAPK in the ARPE-19 cells. Pre-treatment with YCG063, a mitochondrial ROS inhibitor, led to the downregulation of the CoCl2-induced production of VEGF by suppressing HIF-1α and NF-κB activity. Taken together, the findings of our study demonstrate that DIM inhibits the CoCl2-induced production of VEGF by suppressing mitochondrial ROS production, thus attenuating the activation of HIF-1α and p38 MAPK/NF-κB.

  19. B-Scan and “En-Face” Spectral-Domain Optical Coherence Tomography Imaging for the Diagnosis and Followup of Acute Retinal Pigment Epitheliitis

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    Flore De Bats

    2013-01-01

    Full Text Available Purpose. To report B-scan and “En-face” spectral-domain optical coherence tomography (SD-OCT findings in acute retinal pigment epitheliitis (ARPE. Methods. Two patients (3 eyes with ARPE were examined. Fluorescein and indocyanine green (ICGA angiography, B-scan, and “En-face” SD-OCT were performed in each patient at initial and follow-up visits. Results. Both patients presented with acute onset of blurred vision, and one with bilateral involvement. B-can OCT revealed disruption of the macular retinal pigment epithelial (RPE inner band layer and photoreceptors’ inner and outer segment (IS-OS junction. Hyperreflective dots were observed in the outer nuclear layer (ONL above the RPE/IS-OS disruption. Just around these hyperreflective dots, slight thickening of the hyperreflective IS/OS junction was observed. During the late phase, indocyanine green angiography (ICGA showed a macular cockade-like hyperfluorescent halo. “En-face” OCT showed the same cockade-like appearance with a hyporeflective center and a hyperreflective border matching the pattern observed on ICGA. At followup, as vision improved without treatment, B-scan OCT demonstrated progressive resolution of the hyperreflective and disrupted lesions; “en-face” OCT also showed disappearance of the macular cockade-like halo with a transient discrete hyperreflective macular star at the RPE level in one eye. Conclusion. “En-face” OCT associated with B-scan SD-OCT analysis appears to be very helpful in the diagnosis and followup of ARPE. The pathophysiology of ARPE remains complex and still poorly understood. These techniques help define the location and extent of structural damage occurring in this disease.

  20. Effects of the vegetable polyphenols epigallocatechin-3-gallate, luteolin, apigenin, myricetin, quercetin, and cyanidin in primary cultures of human retinal pigment epithelial cells

    Science.gov (United States)

    Chen, Rui; Grosche, Antje; Reichenbach, Andreas; Wiedemann, Peter; Bringmann, Andreas; Kohen, Leon

    2014-01-01

    Purpose Vegetable polyphenols (bioflavonoids) have been suggested to represent promising drugs for treating cancer and retinal diseases. We compared the effects of various bioflavonoids (epigallocatechin-3-gallate [EGCG], luteolin, apigenin, myricetin, quercetin, and cyanidin) on the physiological properties and viability of cultured human retinal pigment epithelial (RPE) cells. Methods Human RPE cells were obtained from several donors within 48 h of death. Secretion of vascular endothelial growth factor (VEGF) was determined with enzyme-linked immunosorbent assay. Messenger ribonucleic acid levels were determined with real-time reverse transcription polymerase chain reaction. Cellular proliferation was investigated with a bromodeoxyuridine immunoassay, and chemotaxis was examined with a Boyden chamber assay. The number of viable cells was determined by Trypan Blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation enzyme-linked immunosorbent assay. The phosphorylation level of signaling proteins was revealed by western blotting. Results With the exception of EGCG, all flavonoids tested decreased dose-dependently the RPE cell proliferation, migration, and secretion of VEGF. EGCG inhibited the secretion of VEGF evoked by CoCl2-induced hypoxia. The gene expression of VEGF was reduced by myricetin at low concentrations and elevated at higher concentrations. Luteolin, apigenin, myricetin, and quercetin induced significant decreases in the cell viability at higher concentration, by triggering cellular necrosis. Cyanidin reduced the rate of RPE cell necrosis. Myricetin caused caspase-3 independent RPE cell necrosis mediated by free radical generation and activation of calpain and phospholipase A2. The myricetin- and quercetin-induced RPE cell necrosis was partially inhibited by necrostatin-1, a blocker of programmed necrosis. Most flavonoids tested diminished the phosphorylation levels of extracellular signal-regulated kinases 1/2 and Akt

  1. αB crystallin is apically secreted within exosomes by polarized human retinal pigment epithelium and provides neuroprotection to adjacent cells.

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    Parameswaran G Sreekumar

    Full Text Available 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

  2. The role of helper lipids in the intracellular disposition and transfection efficiency of niosome formulations for gene delivery to retinal pigment epithelial cells.

    Science.gov (United States)

    Ojeda, Edilberto; Puras, Gustavo; Agirre, Mireia; Zarate, Jon; Grijalvo, Santiago; Eritja, Ramon; DiGiacomo, Luca; Caracciolo, Giulio; Pedraz, Jose-Luis

    2016-04-30

    In this work, we carried out a comparative study of four different niosome formulations based on the same cationic lipid and non-ionic tensoactive. The niosomes prepared by oil-in-water emulsion technique (o/w) only differed in the helper lipid composition: squalene, cholesterol, squalane or no helper lipid. Niosomes and nioplexes elaborated upon the addition of pCMS-EGFP reporter plasmid were characterized in terms of size, zeta potential and polydispersity index. The capacity of the niosomes to condense, release and protect the DNA against enzymatic degradation was evaluated by agarose gel electrophoresis. In vitro experiments were carried out to evaluate transfection efficiency and cell viability in retinal pigment epithelial cells. Moreover, uptake and intracellular trafficking studies were performed to further understand the role of the helper lipids in the transfection process. Interestingly, among all tested formulations, niosomes elaborated with squalene as helper lipid were the most efficient transfecting cells. Such transfection efficiency could be attributed to their higher cellular uptake and the particular entry pathways used, where macropinocytosis pathway and lysosomal release played an important role. Therefore, these results suggest that helper lipid composition is a crucial step to be considered in the design of niosome formulation for retinal gene delivery applications since clearly modulates the cellular uptake, internalization mechanism and consequently, the final transfection efficiency.

  3. A pilot study on expression of toll like receptors (TLRs in response to herpes simplex virus (HSV infection in acute retinal pigment epithelial cells (ARPE cells

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    S Moses

    2014-01-01

    Full Text Available Introduction: Toll like receptors (TLRs have been proven to play an important role in mounting the innate immune response in an infected host. The expression of TLRs against herpes simplex virus (HSV have not been studied in retinitis. Therefore, the current study was undertaken to determine the same using the retinal pigment epithelial (ARPE-19 cell line. Materials and Methods: APRE cells cultured in vitro were challenged with HSV 1 and 2 standard strains and 20 other clinical isolates. The cells were observed for cytopathic changes. The cell culture harvest was subjected to RNA extraction using a Total RNA mini kit. The RNA was subjected to reverse transcriptase polymerase chain reaction (PCR for the amplification of TLRs 3, 4 and 9 and GAPDH housekeeping gene. The amplified products were subjected to electrophoresis on a 2% agarose gel and viewed under a transilluminator. Results: TLR 3 and 4 were expressed by ARPE treated with all the 22 isolates. TLR 9 expression was seen in 16 of the 22 isolates. Bacterial contamination was ruled out by subjecting the harvests to PCR amplification of 16sRNA gene amplification of the eubacterial genome. Conclusions: The expression of TLR 4 has been reported for the first time in HSV infection. TLR 4 along with TLR 3 and 9 is responsible for the antiviral response in HSV infections.

  4. Lycopene inhibits PDGF-BB-induced retinal pigment epithelial cell migration by suppression of PI3K/Akt and MAPK pathways

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Chi-Ming [School of Medicine, Fu Jen Catholic University, Taipei Hsien, Taiwan, ROC (China); Department of Ophthalmology, Cardinal Tien Hospital, Taipei Hsien, Taiwan, ROC (China); Fang, Jia-You [Pharmaceutics Laboratory, Graduate Institute of Natural Products, Chang Gung University, Kweishan, Taoyuan, Taiwan, ROC (China); Lin, Hsin-Huang [School of Medicine, Fu Jen Catholic University, Taipei Hsien, Taiwan, ROC (China); Yang, Chi-Yea [Department of Biotechnology, Vanung University, Taoyuan, Taiwan, ROC (China); Hung, Chi-Feng, E-mail: 054317@mail.fju.edu.tw [School of Medicine, Fu Jen Catholic University, Taipei Hsien, Taiwan, ROC (China)

    2009-10-09

    Retinal pigment epithelial (RPE) cells play a dominant role in the development of proliferative vitreoretinopathy (PVR), which is the leading cause of failure in retinal reattachment surgery. Several studies have shown that platelet-derived growth factor (PDGF) exhibits chemotaxis and proliferation effects on RPE cells in PVR. In this study, the inhibitory effect of lycopene on PDGF-BB-induced ARPE19 cell migration is examined. In electric cell-substrate impedance sensing (ECIS) and Transwell migration assays, significant suppression of PDGF-BB-induced ARPE19 cell migration by lycopene is observed. Cell viability assays show no cytotoxicity of lycopene on RPE cells. Lycopene shows no effect on ARPE19 cell adhesion and is found to inhibit PDGF-BB-induced tyrosine phosphorylation and the underlying signaling pathways of PI3K, Akt, ERK and p38 activation. However, PDGF-BB and lycopene show no effects on JNK activation. Taken together, our results demonstrate that lycopene inhibits PDGF-BB-induced ARPE19 cell migration through inhibition of PI3K/Akt, ERK and p38 activation.

  5. Down-regulation of pigment epithelium-derived factor in uveitic lesion associates with focal vascular endothelial growth factor expression and breakdown of the blood-retinal barrier.

    Science.gov (United States)

    Deeg, Cornelia A; Altmann, Frank; Hauck, Stefanie M; Schoeffmann, Stephanie; Amann, Barbara; Stangassinger, Manfred; Ueffing, Marius

    2007-05-01

    Spontaneous equine recurrent uveitis (ERU) is an incurable autoimmune disease affecting the eye. Identifying biological markers or pathways associated with this disease may allow the understanding of its pathogenesis at a molecular level. The vitreous is the body fluid closest to the disease-affected tissue and possibly also an effector of pathological processes relevant for ERU. Surgical removal of vitreous leads to cessation of relapses in spontaneous uveitis of both man and horse, therefore vitreous composites are likely to contribute to disease progression. Uveitic vitreous is likely to contain potential biomarkers in relatively undiluted quantities. With the goal to identify these markers, we systematically compared vitreous from healthy and disease-affected eyes by proteomic profiling. Nine differentially expressed proteins were identified, that are functionally related to immune response, inflammation, and maintenance of the blood-retinal barrier. One of these, pigment epithelium-derived factor, a protein involved in maintaining a proper blood-retina barrier as well as protecting from neoangiogenesis was additionally found to be down-regulated within uveitic retinal lesions whereas, conversely, vascular endothelial growth factor was found to be up-regulated at these sites. Together, these changes point to as of yet undiscovered biological pathways involved in the pathogenesis of this autoimmune disease.

  6. Mislocalisation of BEST1 in iPSC-derived retinal pigment epithelial cells from a family with autosomal dominant vitreoretinochoroidopathy (ADVIRC)

    Science.gov (United States)

    Carter, David A.; Smart, Matthew J. K.; Letton, William V. G.; Ramsden, Conor M.; Nommiste, Britta; Chen, Li Li; Fynes, Kate; Muthiah, Manickam N.; Goh, Pollyanna; Lane, Amelia; Powner, Michael B.; Webster, Andrew R.; da Cruz, Lyndon; Moore, Anthony T.; Coffey, Peter J.; Carr, Amanda-Jayne F.

    2016-01-01

    Autosomal dominant vitreoretinochoroidopathy (ADVIRC) is a rare, early-onset retinal dystrophy characterised by distinct bands of circumferential pigmentary degeneration in the peripheral retina and developmental eye defects. ADVIRC is caused by mutations in the Bestrophin1 (BEST1) gene, which encodes a transmembrane protein thought to function as an ion channel in the basolateral membrane of retinal pigment epithelial (RPE) cells. Previous studies suggest that the distinct ADVIRC phenotype results from alternative splicing of BEST1 pre-mRNA. Here, we have used induced pluripotent stem cell (iPSC) technology to investigate the effects of an ADVIRC associated BEST1 mutation (c.704T > C, p.V235A) in patient-derived iPSC-RPE. We found no evidence of alternate splicing of the BEST1 transcript in ADVIRC iPSC-RPE, however in patient-derived iPSC-RPE, BEST1 was expressed at the basolateral membrane and the apical membrane. During human eye development we show that BEST1 is expressed more abundantly in peripheral RPE compared to central RPE and is also expressed in cells of the developing retina. These results suggest that higher levels of mislocalised BEST1 expression in the periphery, from an early developmental stage, could provide a mechanism that leads to the distinct clinical phenotype observed in ADVIRC patients. PMID:27653836

  7. Reduced Expression of Cytoskeletal and Extracellular Matrix Genes in Human Adult Retinal Pigment Epithelium Cells Exposed to Simulated Microgravity

    DEFF Research Database (Denmark)

    Corydon, Thomas J; Mann, Vivek; Slumstrup, Lasse;

    2016-01-01

    BACKGROUND/AIMS: Microgravity (µg) has adverse effects on the eye of humans in space. The risk of visual impairment is therefore one of the leading health concerns for NASA. The impact of µg on human adult retinal epithelium (ARPE-19) cells is unknown. METHODS: In this study we investigated the i...

  8. Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model

    Directory of Open Access Journals (Sweden)

    Alvaro Plaza Reyes

    2016-01-01

    Full Text Available Human embryonic stem cell (hESC-derived retinal pigment epithelial (RPE cells could replace lost tissue in geographic atrophy (GA but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model.

  9. Benzo(a)pyrene and X-rays induce reversions of the pink-eyed unstable mutation in the retinal pigment epithelium of mice.

    Science.gov (United States)

    Bishop, A J; Kosaras, B; Sidman, R L; Schiestl, R H

    2000-12-20

    The pink-eyed unstable (p(un)) mutation is the result of a 70kb tandem duplication within the murine p gene. Homologous deletion/recombination of the locus to wild-type occurs spontaneously in embryos and results in pigmented spots in the fur and eye that persist for life. Such deletion events are also inducible by a variety of DNA damaging agents, as we have observed previously with the fur spot assay. Here, we describe the use of the retinal pigment epithelium (RPE) of the eye to detect reversion events induced with two differently acting agents. Benzo(a)pyrene (B(a)P) induces a high frequency, and X-ray exposure a more modest increase, of p(un) reversion in both the fur and the eye. The eye-spot assay requires fewer mice for significant results than the fur spot assay. Previous work had elucidated the cell proliferation pattern in the RPE and a position effect variegation phenotype in the pattern of p(un) reversions, which we have confirmed. Acute exposure to B(a)P or X-rays resulted in an increased frequency of reversion events. The majority of the spontaneous reversions lie toward the periphery of the RPE whereas induced events are found more centrally, closer to the optic nerve head. The induced distribution corresponds to the major sites of cell proliferation in the RPE at the time of exposure, and further advocates the proposal that dividing cells are at highest risk to develop deletions.

  10. The 5HT1a receptor agonist 8-Oh DPAT induces protection from lipofuscin accumulation and oxidative stress in the retinal pigment epithelium.

    Directory of Open Access Journals (Sweden)

    Prajitha Thampi

    Full Text Available Age-related macular degeneration (AMD, a major cause of blindness in the elderly, is associated with oxidative stress, lipofuscin accumulation and retinal degeneration. The aim of this study was to determine if a 5-HT(1A receptor agonist can reduce lipofuscin accumulation, reduce oxidative damage and prevent retinal cell loss both in vitro and in vivo. Autophagy-derived and photoreceptor outer segment (POS-derived lipofuscin formation was assessed using FACS analysis and confocal microscopy in cultured retinal pigment epithelial (RPE cells in the presence or absence of the 5-HT(1A receptor agonist, 8-OH DPAT. 8-OH DPAT treatment resulted in a dose-dependent reduction in both autophagy- and POS-derived lipofuscin compared to control. Reduction in autophagy-induced lipofuscin was sustained for 4 weeks following removal of the drug. The ability of 8-OH DPAT to reduce oxidative damage following exposure to 200 µM H(2O(2 was assessed. 8-OH DPAT reduced superoxide generation and increased mitochondrial superoxide dismutase (MnSOD levels and the ratio of reduced glutathione to the oxidized form of glutathione in H(2O(2-treated cells compared to controls and protected against H(2O(2-initiated lipid peroxidation, nitrotyrosine levels and mitochondrial damage. SOD2 knockdown mice, which have an AMD-like phenotype, received daily subcutaneous injections of either saline, 0.5 or 5.0 mg/kg 8-OH DPAT and were evaluated at monthly intervals. Systemic administration of 8-OH DPAT improved the electroretinogram response in SOD2 knockdown eyes of mice compared to knockdown eyes receiving vehicle control. There was a significant increase in the ONL thickness in mice treated with 8-OH DPAT at 4 months past the time of MnSOD knockdown compared to untreated controls together with a 60% reduction in RPE lipofuscin. The data indicate that 5-HT(1A agonists can reduce lipofuscin accumulation and protect the retina from oxidative damage and mitochondrial dysfunction. 5-HT

  11. Transplante autólogo do epitélio pigmentado da retina na degeneração macular relacionada com a idade Autologous transplantation of retinal pigment epithelium in age related macular degeneration

    Directory of Open Access Journals (Sweden)

    Rubens Camargo Siqueira

    2009-02-01

    Full Text Available Acredita-se que a disfunção do epitélio pigmentado da retina é a principal causa de muitas doenças debilitantes da retina das quais a degeneração macular relacionada com a idade é a mais comum. Nesta doença a disfunção das células do epitélio pigmentado da retina leva a um comprometimento secundário dos fotorreceptores com perda visual grave. O epitélio pigmentado da retina e membrana de Bruch sofrem dano acumulativo com o tempo o qual induz à degeneração macular relacionada com a idade em indivíduos susceptíveis. Nos últimos 20 anos, uma grande quantidade de pesquisas tem sido conduzida na área de transplante do epitélio pigmentado da retina. A técnica tem como objetivo, restaurar a anatomia sub-retiniana e restabelecer a interação crítica entre o epitélio pigmentado da retina e o fotorreceptor, o qual é fundamental para a visão. O transplante autólogo do epitélio pigmentado da retina tem sido usado em alguns casos de degeneração macular relacionada com a idade através de duas técnicas: epitélio pigmentado da retina em suspensão e transplante de espessura total do epitélio pigmentado da retina-coróide. Apesar da viabilidade desta técnica, pesquisas buscando uma fonte de células para repor epitélio pigmentado da retina autólogo como células-tronco embrionárias, células-tronco derivadas da medula óssea e derivadas do cordão umbilical continuam em andamento. A combinação do transplante de células com outras modalidades de tratamento como transferência de genes permanecem como excitante perspectiva futura.Retinal pigment epithelial dysfunction is believed to be the main cause of many debilitating retinal diseases of which age-related macular degeneration is the most common. In this disease, the retinal pigment epithelial dysfunction leads to photoreceptors damage causing severe vision loss. The retinal pigment epithelium and Bruch's membrane suffer cumulative damage over lifetime, which is

  12. Cotransport of H+, lactate and H2O by membrane proteins in retinal pigment epithelium of bullfrog

    DEFF Research Database (Denmark)

    Zeuthen, T; Hamann, S; la Cour, M

    1996-01-01

    solution concentration and/or osmolarity. 3. Two parallel pathways for water transport were identified across the retinal membrane, an osmotic one with a hydraulic water permeability of 3.2 x 10(-4) cm s-1 (osmol l-1)-1 and one which depended on the presence of lactate. 4. Addition of sodium lactate...... the effect. The influx of water could proceed against osmotic gradients elicited by mannitol. 6. The interdependence of the fluxes of H+, lactate and H2O can be described as cotransport: the fluxes had a fixed ratio of about 109 mmol of lactic acid per litre of water, the flux of one species was able...

  13. Understanding age-related macular degeneration (AMD): relationships between the photoreceptor/retinal pigment epithelium/Bruch's membrane/choriocapillaris complex.

    Science.gov (United States)

    Bhutto, Imran; Lutty, Gerard

    2012-08-01

    There is a mutualistic symbiotic relationship between the components of the photoreceptor/retinal pigment epithelium (RPE)/Bruch's membrane (BrMb)/choriocapillaris (CC) complex that is lost in AMD. Which component in the photoreceptor/RPE/BrMb/CC complex is affected first appears to depend on the type of AMD. In atrophic AMD (~85-90% of cases), it appears that large confluent drusen formation and hyperpigmentation (presumably dysfunction in RPE) are the initial insult and the resorption of these drusen and loss of RPE (hypopigmentation) can be predictive for progression of geographic atrophy (GA). The death and dysfunction of photoreceptors and CC appear to be secondary events to loss in RPE. In neovascular AMD (~10-15% of cases), the loss of choroidal vasculature may be the initial insult to the complex. Loss of CC with an intact RPE monolayer in wet AMD has been observed. This may be due to reduction in blood supply because of large vessel stenosis. Furthermore, the environment of the CC, basement membrane and intercapillary septa, is a proinflammatory milieu with accumulation of complement components as well as proinflammatory molecules like CRP during AMD. In this toxic milieu, CC die or become dysfunction making adjacent RPE hypoxic. These hypoxic cells then produce angiogenic substances like VEGF that stimulate growth of new vessels from CC, resulting in choroidal neovascularization (CNV). The loss of CC might also be a stimulus for drusen formation since the disposal system for retinal debris and exocytosed material from RPE would be limited. Ultimately, the photoreceptors die of lack of nutrients, leakage of serum components from the neovascularization, and scar formation. Therefore, the mutualistic symbiotic relationship within the photoreceptor/RPE/BrMb/CC complex is lost in both forms of AMD. Loss of this functionally integrated relationship results in death and dysfunction of all of the components in the complex.

  14. Translational read-through of the RP2 Arg120stop mutation in patient iPSC-derived retinal pigment epithelium cells

    Science.gov (United States)

    Schwarz, Nele; Carr, Amanda-Jayne; Lane, Amelia; Moeller, Fabian; Chen, Li Li; Aguilà, Mònica; Nommiste, Britta; Muthiah, Manickam N.; Kanuga, Naheed; Wolfrum, Uwe; Nagel-Wolfrum, Kerstin; da Cruz, Lyndon; Coffey, Peter J.; Cheetham, Michael E.; Hardcastle, Alison J.

    2015-01-01

    Mutations in the RP2 gene lead to a severe form of X-linked retinitis pigmentosa. RP2 patients frequently present with nonsense mutations and no treatments are currently available to restore RP2 function. In this study, we reprogrammed fibroblasts from an RP2 patient carrying the nonsense mutation c.519C>T (p.R120X) into induced pluripotent stem cells (iPSC), and differentiated these cells into retinal pigment epithelial cells (RPE) to study the mechanisms of disease and test potential therapies. RP2 protein was undetectable in the RP2 R120X patient cells, suggesting a disease mechanism caused by complete lack of RP2 protein. The RP2 patient fibroblasts and iPSC-derived RPE cells showed phenotypic defects in IFT20 localization, Golgi cohesion and Gβ1 trafficking. These phenotypes were corrected by over-expressing GFP-tagged RP2. Using the translational read-through inducing drugs (TRIDs) G418 and PTC124 (Ataluren), we were able to restore up to 20% of endogenous, full-length RP2 protein in R120X cells. This level of restored RP2 was sufficient to reverse the cellular phenotypic defects observed in both the R120X patient fibroblasts and iPSC-RPE cells. This is the first proof-of-concept study to demonstrate successful read-through and restoration of RP2 function for the R120X nonsense mutation. The ability of the restored RP2 protein level to reverse the observed cellular phenotypes in cells lacking RP2 indicates that translational read-through could be clinically beneficial for patients. PMID:25292197

  15. Enhanced Ca(2+) response and stimulation of prostaglandin release by the bradykinin B2 receptor in human retinal pigment epithelial cells primed with proinflammatory cytokines.

    Science.gov (United States)

    Catalioto, Rose-Marie; Valenti, Claudio; Maggi, Carlo Alberto; Giuliani, Sandro

    2015-09-15

    Kallikrein, kininogen and kinin receptors are present in human ocular tissues including the retinal pigment epithelium (RPE), suggesting a possible role of bradykinin (BK) in physiological and/or pathological conditions. To test this hypothesis, kinin receptors expression and function was investigated for the first time in human fetal RPE cells, a model close to native RPE, in both control conditions and after treatment with proinflammatory cytokines. Results showed that BK evoked intracellular Ca(2+) transients in human RPE cells by activating the kinin B2 receptor. Pretreatment of the cells with TNF-α and/or IL-1β enhanced Ca(2+) response in a time- and concentration-dependent additive manner, whereas the potency of BK and that of the selective B2 receptor antagonist, fasitibant chloride, both in the nanomolar range, remained unaffected. Cytokines have no significant effect on cell number and viability and on the activity of other GPCRs such as the kinin B1, acetylcholine, ATP and thrombin receptors. Immunoblot analysis and immunofluorescence studies revealed that cytokines treatment was associated with an increase in both kinin B2 receptor and COX-2 expression and with the secretion of prostaglandin E1 and E2 into the extracellular medium. BK, through activation of the kinin B2 receptor, potentiated the COX-2 mediated prostaglandin release in cytokines-primed RPE cells while new protein synthesis and prostaglandin production contribute to the potentiating effect of cytokines on BK-induced Ca(2+) response. In conclusion, overall data revealed a cross-talk between the kinin B2 receptor and cytokines in human RPE in promoting inflammation, a key feature in retinal pathologies including diabetic retinopathy and macular edema.

  16. Illumination from light-emitting diodes (LEDs) disrupts pathological cytokines expression and activates relevant signal pathways in primary human retinal pigment epithelial cells.

    Science.gov (United States)

    Shen, Ye; Xie, Chen; Gu, Yangshun; Li, Xiuyi; Tong, Jianping

    2016-04-01

    Age-related macular degeneration (AMD) is the leading cause of blindness in the aged people. The latest systemic review of epidemiological investigations revealed that excessive light exposure increases the risk of AMD. With the drastically increasing use of high-energy light-emitting diodes (LEDs) light in our domestic environment nowadays, it is supposed to pose a potential oxidative threat to ocular health. Retinal pigment epithelium (RPE) is the major ocular source of pathological cytokines, which regulate local inflammation and angiogenesis. We hypothesized that high-energy LED light might disrupt the pathological cytokine expression of retinal pigment epithelium (RPE), contributing to the pathogenesis of AMD. Primary human RPE cells were isolated from eyecups of normal eye donors and seeded into plate wells for growing to confluence. Two widely used multichromatic white light-emitting diodes (LEDs) with correlated color temperatures (CCTs) of 2954 and 7378 K were used in this experiment. The confluent primary RPE cells were under white LEDs light exposure until 24 h. VEGF-A, IL-6, IL-8 and MCP-1 proteins and mRNAs were measured using an ELISA kit and RT-PCR, respectively. Activation of mitogen-activated protein kinases (MAPKs), Akt, Janus kinase (JAK)2 and Nuclear factor (NF)-κB signal pathways after LEDs illumination were evaluated by western blotting analysis. The level of reactive oxygen species (ROS) using chloromethyl- 2',7'-dichlorodihydrofluorescein diacetate. Inhibitors of relevant signal pathways and anti-oxidants were added to the primary RPE cells before LEDs illumination to evaluate their biological functions. We found that 7378 K light, but not 2954 K upregulated the VEGF-A, IL-6, IL-8 and downregulated MCP-1 proteins and mRNAs levels in a time-dependent manner. In parallel, initial activation of MAPKs and NF-κB signal pathways were also observed after 7378 K light exposure. Mechanistically, antioxidants for eliminating reactive oxygen

  17. The molecular basis for UV vision in birds: spectral characteristics, cDNA sequence and retinal localization of the UV-sensitive visual pigment of the budgerigar (Melopsittacus undulatus).

    Science.gov (United States)

    Wilkie, S E; Vissers, P M; Das, D; Degrip, W J; Bowmaker, J K; Hunt, D M

    1998-02-15

    Microspectrophotometric (msp) studies have shown that the colour-vision system of many bird species is based on four pigments with absorption peaks in the red, green, blue and UV regions of the spectrum. The existence of a fourth pigment (UV) is the major difference between the trichromacy of humans and the tetrachromacy of such birds, and recent studies have shown that it may play a determining role in such diverse aspects of behaviour as mate selection and detection of food. Avian visual pigments are composed of an opsin protein covalently bound via a Schiff-base linkage to the chromophore 11-cis-retinal. Here we report the cDNA sequence of a UV opsin isolated from an avian species, Melopsittacus undulatus (budgerigar or small parakeet). This sequence has been expressed using the recombinant baculovirus system; the pigment generated from the expressed protein on addition of 11-cis-retinal yielded an absorption spectrum typical of a UV photopigment, with lambdamax 365+/-3 nm. This is the first UV opsin from an avian species to be sequenced and expressed in a heterologous system. In situ hybridization of this sequence to budgerigar retinas selectively labelled a sub-set of UV cones, representing approx. 9% of the total cone population, that are distributed in a semi-regular pattern across the entire retina.

  18. Sulforaphane Enhances the Ability of Human Retinal Pigment Epithelial Cell against Oxidative Stress, and Its Effect on Gene Expression Profile Evaluated by Microarray Analysis

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    Liang Ye

    2013-01-01

    Full Text Available To gain further insights into the molecular basis of Sulforaphane (SF mediated retinal pigment epithelial (RPE 19 cell against oxidative stress, we investigated the effects of SF on the regulation of gene expression on a global scale and tested whether SF can endow RPE cells with the ability to resist apoptosis. The data revealed that after exposure to H2O2, RPE 19 cell viability was increased in the cells pretreated with SF compared to the cell not treated with SF. Microarray analysis revealed significant changes in the expression of 69 genes in RPE 19 cells after 6 hours of SF treatment. Based on the functional relevance, eight of the SF-responsive genes, that belong to antioxidant redox system, and inflammatory responsive factors were validated. The up-regulating translation of thioredoxin-1 (Trx1 and the nuclear translocation of Nuclear factor-like2 (Nrf2 were demonstrated by immunoblot analysis in SF treated RPE cells. Our data indicate that SF increases the ability of RPE 19 cell against oxidative stress through up-regulating antioxidative enzymes and down-regulating inflammatory mediators and chemokines. The results suggest that the antioxidant, SF, may be a valuable supplement for preventing and retarding the development of Age Related Macular Degeneration.

  19. Suppression of the proliferation of hypoxia-Induced retinal pigment epithelial cell by rapamycin through the /mTOR/HIF-1α/VEGF/ signaling.

    Science.gov (United States)

    Liu, Ning-Ning; Zhao, Ning; Cai, Na

    2015-06-01

    Rapamycin, a highly specific inhibitor of mammalian target of rapamycin (mTOR), exhibits significant antitumor/antiangiogenic activity in human cancer cells. Its effect on the retinal pigment epithelial (RPE) cells was rarely investigated. This study assessed the proliferation of hypoxia-induced RPE and the inhibitory effects of rapamycin using 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and examined the expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in RPE cells with or without rapamycin under normoxic and hypoxic conditions using real-time PCR and Western blot. We found that hypoxia increased the levels of mTOR, HIF-1α, and VEGF. The suppression of HIF-1α and VEGF by rapamycin was associated with dephosphorylation of mTOR and the downstream effector ribosomal protein S6 kinase (P70S6K) and 4E-binding protein-1 (4E-BP1) of mTORC1. Rapamycin only inhibited the protein levels and did not change the mRNA expression of HIF-1α. No cytotoxicity to the RPE cells by rapamycin was caused under either normoxia or hypoxia. Our data suggest that rapamycin suppresses hypoxia-induced RPE cell proliferation through a mechanism related to the targeting of mTOR/HIF-1α/VEGF signaling. Rapamycin may potentially provide a safe and effective novel treatment for choroidal vascular disease.

  20. Inhibition of DNA methyltransferase or histone deacetylase protects retinal pigment epithelial cells from DNA damage induced by oxidative stress by the stimulation of antioxidant enzymes.

    Science.gov (United States)

    Tokarz, Paulina; Kaarniranta, Kai; Blasiak, Janusz

    2016-04-05

    Epigenetic modifications influence DNA damage response (DDR). In this study we explored the role of DNA methylation and histone acetylation in DDR in cells challenged with acute or chronic oxidative stress. We used retinal pigment epithelial cells (ARPE-19), which natively are exposed to oxidative stress due to permanent exposure to light and high blood flow. We employed a DNA methyltransferase inhibitor - RG108 (RG), or a histone deacetylase inhibitor - valproic acid (VA). ARPE-19 cells were exposed to tert-butyl hydroperoxide, an acute oxidative stress inducer, or glucose oxidase, which slowly liberates low-doses of hydrogen peroxide in the presence of glucose, creating chronic conditions. VA and RG reduced level of intracellular reactive oxygen species and DNA damage in ARPE-19 cells in normal condition and in oxidative stress. This protective effect of VA and RG was associated with the up-regulated expression of antioxidant enzyme genes: CAT, GPx1, GPx4, SOD1 and SOD2. RG decreased the number of cells in G2/M checkpoint in response to chronic oxidative stress. Neither RG nor VA changed the DNA repair or apoptosis induced by oxidative stress. Therefore, certain epigenetic manipulations may protect ARPE-19 cells from detrimental effects of oxidative stress by modulation of antioxidative enzyme gene expression, which may be further explored in pharmacological studies on oxidative stress-related eye diseases.

  1. Inhibition by miR-410 facilitates direct retinal pigment epithelium differentiation of umbilical cord blood-derived mesenchymal stem cells

    Science.gov (United States)

    Choi, Soon Won; Kim, Jae-Jun; Seo, Min-Soo; Park, Sang-Bum; Shin, Tae-Hoon; Shin, Ji-Hee; Seo, Yoojin; Kim, Hyung-Sik

    2017-01-01

    Retinal pigment epithelium (RPE) is a major component of the eye. This highly specialized cell type facilitates maintenance of the visual system. Because RPE loss induces an irreversible visual impairment, RPE generation techniques have recently been investigated as a potential therapeutic approach to RPE degeneration. The microRNA-based technique is a new strategy for producing RPE cells from adult stem cell sources. Previously, we identified that antisense microRNA-410 (anti-miR-410) induces RPE differentiation from amniotic epithelial stem cells. In this study, we investigated RPE differentiation from umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) via anti-miR-410 treatment. We identified miR-410 as a RPE-relevant microRNA in UCB-MSCs from among 21 putative human RPE-depleted microRNAs. Inhibition of miR-410 induces overexpression of immature and mature RPE-specific factors, including MITF, LRAT, RPE65, Bestrophin, and EMMPRIN. The RPE-induced cells were able to phagocytize microbeads. Results of our microRNA-based strategy demonstrated proof-of-principle for RPE differentiation in UCB-MSCs by using anti-miR-410 treatment without the use of additional factors or exogenous transduction. PMID:27297412

  2. Salvianolic Acid B (Sal B Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Cell Death by Activating Glutaredoxin 1 (Grx1

    Directory of Open Access Journals (Sweden)

    Xiaobin Liu

    2016-11-01

    Full Text Available Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG between cysteine residues and glutathione (GSH, can lead to cell death. Glutaredoxin 1 (Grx1 is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B, a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H2O2-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2, the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H2O2-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage.

  3. Salvianolic Acid B (Sal B) Protects Retinal Pigment Epithelial Cells from Oxidative Stress-Induced Cell Death by Activating Glutaredoxin 1 (Grx1).

    Science.gov (United States)

    Liu, Xiaobin; Xavier, Christy; Jann, Jamieson; Wu, Hongli

    2016-11-03

    Protein glutathionylation, defined as the formation of protein mixed disulfides (PSSG) between cysteine residues and glutathione (GSH), can lead to cell death. Glutaredoxin 1 (Grx1) is a thiol repair enzyme which catalyzes the reduction of PSSG. Therefore, Grx1 exerts strong anti-apoptotic effects by improving the redox state, especially in times of oxidative stress. However, there is currently no compound that is identified as a Grx1 activator. In this study, we identified and characterized Salvianolic acid B (Sal B), a natural compound, as a Grx1 inducer, which potently protected retinal pigment epithelial (RPE) cells from oxidative injury. Our results showed that treatment with Sal B protected primary human RPE cells from H₂O₂-induced cell damage. Interestingly, we found Sal B pretreatment upregulated Grx1 expression in RPE cells in a time- and dose-dependent manner. Furthermore, NF-E2-related factor 2 (Nrf2), the key transcription factor that regulates the expression of Grx1, was activated in Sal B treated RPE cells. Further investigation showed that knockdown of Grx1 by small interfering RNA (siRNA) significantly reduced the protective effects of Sal B. We conclude that Sal B protects RPE cells against H₂O₂-induced cell injury through Grx1 induction by activating Nrf2 pathway, thus preventing lethal accumulation of PSSG and reversing oxidative damage.

  4. Comparison of Progression Rate of Retinal Pigment Epithelium Loss in Patients with Neovascular Age-Related Macular Degeneration Treated with Ranibizumab and Aflibercept

    Directory of Open Access Journals (Sweden)

    Juliana Wons

    2017-01-01

    Full Text Available Purpose. Retinal pigment epithelium (RPE loss in neovascular age-related macular degeneration (nAMD seem to have a linear progression but might be influenced by the treatment. The purpose of the study is the comparison of RPE loss over three years in patients treated with intravitreal ranibizumab to patients who were switched to aflibercept. Methods. A retrospective analysis with 96 eyes switched to aflibercept was conducted. The progression rate of RPE loss was evaluated in patients who showed atrophy one year prior to switch (n=17 or on switch date (n=19. The RPE loss was evaluated by spectral domain optical coherence tomography (SD-OCT. Further, 22 eyes from patients treated with ranibizumab were compared. Results. The median yearly progression of RPE loss after square root transformation showed no significant difference in the year prior to switch compared to the year after switch (p=0.854. In patients who received only ranibizumab, the median yearly progression of RPE loss was 0.15 mm/y, for aflibercept patients, 0.13 mm/y. This difference was not statistically significant (p=0.172. Conclusions. There seems to be a linear progression rate of RPE loss in patients treated with ranibizumab as well as in patients with aflibercept. No significant increase of progression rate was found after switch to aflibercept.

  5. Oxalomalate reduces expression and secretion of vascular endothelial growth factor in the retinal pigment epithelium and inhibits angiogenesis: Implications for age-related macular degeneration

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    Sung Hwan Kim

    2016-12-01

    Full Text Available Clinical and experimental observations indicate a critical role for vascular endothelial growth factor (VEGF, secreted by the retinal pigment epithelium (RPE, in pathological angiogenesis and the development of choroidal neovascularization (CNV in age-related macular degeneration (AMD. RPE-mediated VEGF expression, leading to angiogenesis, is a major signaling mechanism underlying ocular neovascular disease. Inhibiting this signaling pathway with a therapeutic molecule is a promising anti-angiogenic strategy to treat this disease with potentially fewer side effects. Oxalomalate (OMA is a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase (IDH, which plays an important role in cellular signaling pathways regulated by reactive oxygen species (ROS. Here, we have investigated the inhibitory effect of OMA on the expression of VEGF, and the associated underlying mechanism of action, using in vitro and in vivo RPE cell models of AMD. We found that OMA reduced the expression and secretion of VEGF in RPE cells, and consequently inhibited CNV formation. This function of OMA was linked to its capacity to activate the pVHL-mediated HIF-1α degradation in these cells, partly via a ROS-dependent ATM signaling axis, through inhibition of IDH enzymes. These findings reveal a novel role for OMA in inhibiting RPE-derived VEGF expression and angiogenesis, and suggest unique therapeutic strategies for treating pathological angiogenesis and AMD development.

  6. PRMT1 and PRMT4 Regulate Oxidative Stress-Induced Retinal Pigment Epithelial Cell Damage in SIRT1-Dependent and SIRT1-Independent Manners

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    Dong-Il Kim

    2015-01-01

    Full Text Available Oxidative stress-induced retinal pigment epithelial (RPE cell damage is involved in the progression of diabetic retinopathy. Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs has emerged as an important histone modification involved in diverse diseases. Sirtuin (SIRT1 is a protein deacetylase implicated in the onset of metabolic diseases. Therefore, we examined the roles of type I PRMTs and their relationship with SIRT1 in human RPE cells under H2O2-induced oxidative stress. H2O2 treatment increased PRMT1 and PRMT4 expression but decreased SIRT1 expression. Similar to H2O2 treatment, PRMT1 or PRMT4 overexpression increased RPE cell damage. Moreover, the H2O2-induced RPE cell damage was attenuated by PRMT1 or PRMT4 knockdown and SIRT1 overexpression. In this study, we revealed that SIRT1 expression was regulated by PRMT1 but not by PRMT4. Finally, we found that PRMT1 and PRMT4 expression is increased in the RPE layer of streptozotocin-treated rats. Taken together, we demonstrated that oxidative stress induces apoptosis both via PRMT1 in a SIRT1-dependent manner and via PRMT4 in a SIRT1-independent manner. The inhibition of the expression of type I PRMTs, especially PRMT1 and PRMT4, and increased SIRT1 could be therapeutic approaches for diabetic retinopathy.

  7. MERTK signaling in the retinal pigment epithelium regulates the tyrosine phosphorylation of GDP dissociation inhibitor alpha from the GDI/CHM family of RAB GTPase effectors.

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    Shelby, Shameka J; Feathers, Kecia L; Ganios, Anna M; Jia, Lin; Miller, Jason M; Thompson, Debra A

    2015-11-01

    Photoreceptor outer segments (OS) in the vertebrate retina undergo a process of continual renewal involving shedding of disc membranes that are cleared by phagocytic uptake into the retinal pigment epithelium (RPE). In dystrophic Royal College of Surgeons (RCS) rats, OS phagocytosis is blocked by a mutation in the gene encoding the receptor tyrosine kinase MERTK. To identify proteins tyrosine-phosphorylated downstream of MERTK in the RPE, MALDI-mass spectrometry with peptide-mass fingerprinting was used in comparative studies of RCS congenic and dystrophic rats. At times corresponding to peak phagocytic activity, the RAB GTPase effector GDP dissociation inhibitor alpha (GDI1) was found to undergo tyrosine phosphorylation only in congenic rats. In cryosections of native RPE/choroid, GDI1 colocalized with MERTK and the intracellular tyrosine-kinase SRC. In cultured RPE-J cells, and in transfected heterologous cells, MERTK stimulated SRC-mediated tyrosine phosphorylation of GDI1. In OS-fed RPE-J cells, GDI1 colocalized with MERTK and SRC on apparent phagosomes located near the apical membrane. In addition, both GDI1 and RAB5, a regulator of vesicular transport, colocalized with ingested OS. Taken together, these findings identify a novel role of MERTK signaling in membrane trafficking in the RPE that is likely to subserve mechanisms of phagosome formation.

  8. Changes in retinal pigment epithelium related to cigarette smoke: possible relevance to smoking as a risk factor for age-related macular degeneration.

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    Ai Ling Wang

    Full Text Available Age-related Macular Degeneration (AMD is a major cause of central vision loss in the elderly and smoking is a primary risk factor associated with the prevalence and incidence of AMD. To better understand the cellular and molecular bases for the association between smoking and AMD, we determined the effects of Benzo(aPyrene (B(aP, a toxic element in cigarette smoke, on cultured retinal pigment epithelia (RPE and we examined the RPE/choroid from mice exposed to chronic cigarette smoke. We measured: mitochondrial DNA (mtDNA damage, phagocytic activity, lysosomal enzymes, exosome markers and selected complement pathway components. In the presence of a non-cytotoxic dose of B(aP, there was extensive mtDNA damage but no nuclear DNA damage. RPE phagocytic activity was not altered but there were increased lysosomal activity, exocytotic activity and complement pathway components. Retinas from mice exposed to cigarette smoke contained markers for mtDNA damage, exosomes and complement pathway components surrounding Bruch's membrane. Markers for these processes are found in drusen from AMD patients. Thus, smoking may cause damage to mtDNA and increased degradative processes in the RPE. These altered cell biological processes in the RPE may contribute to the formation of drusen in individuals who are cigarette smokers and underlie susceptibility to genetic mutations associated with AMD.

  9. Silencing heme oxygenase-1 gene expression in retinal pigment epithelial cells inhibits proliferation, migration and tube formation of cocultured endothelial cells

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    Zhang, Wenjie [Ophthalmology Hospital, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Harbin 150001 (China); Zhang, Xiaomei, E-mail: zhangxm667@163.com [Ophthalmology Hospital, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Harbin 150001 (China); Lu, Hong [Ophthalmology Hospital, The First Affiliated Hospital of Harbin Medical University, 23 Youzheng Street, Harbin 150001 (China); Matsukura, Makoto [Laboratory of Clinical Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082 (Japan); Zhao, Jien; Shinohara, Makoto [Ashikita Institution for Developmental Disabilities, 2813 Oaza Ashikita, Ashikita-machi, Ashikita, Kumamoto 869-5461 (Japan)

    2013-05-10

    Highlights: •HO-1 is highly induced in RPE cells by hypoxia. •Inhibition of HO-1 activity and knockdown of HO-1 expression inhibit VEGF expression in RPE cells under hypoxia. •Knockdown of HO-1 in RPE cells inhibits angiogenesis of endothelial cells in vitro. -- Abstract: Heme oxygenase-1 (HO-1) plays an important role in the vasculature and in the angiogenesis of tumors, wounds and other environments. Retinal pigment epithelial (RPE) cells and choroidal endothelial cells (CECs) are the main cells involved in choroidal neovascularization (CNV), a process in which hypoxia plays an important role. Our aim was to evaluate the role of human RPE-cell HO-1 in the angiogenic activities of cocultured endothelial cells under hypoxia. Small interfering RNA (siRNA) for HO-1 was transfected into human RPE cell line ARPE-19, and zinc protoporphyrin (ZnPP) was used to inhibit HO-1 activity. Knockdown of HO-1 expression and inhibition of HO-1 activity resulted in potent reduction of the expression of vascular endothelial growth factor (VEGF) under hypoxia. Furthermore, knockdown of HO-1 suppressed the proliferation, migration and tube formation of cocultured endothelial cells. These findings indicated that HO-1 might have an angiogenic effect in CNV through modulation of VEGF expression and might be a potential target for treating CNV.

  10. Long Non-Coding RNA MALAT1 Mediates Transforming Growth Factor Beta1-Induced Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells.

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    Shuai Yang

    Full Text Available To study the role of long non-coding RNA (lncRNA MALAT1 in transforming growth factor beta 1 (TGF-β1-induced epithelial-mesenchymal transition (EMT of retinal pigment epithelial (RPE cells.ARPE-19 cells were cultured and exposed to TGF-β1. The EMT of APRE-19 cells is confirmed by morphological change, as well as the increased expression of alpha-smooth muscle actin (αSMA and fibronectin, and the down-regulation of E-cadherin and Zona occludin-1(ZO-1 at both mRNA and protein levels. The expression of lncRNA MALAT1 in RPE cells were detected by quantitative real-time PCR. Knockdown of MALAT1 was achieved by transfecting a small interfering RNA (SiRNA. The effect of inhibition of MALAT1 on EMT, migration, proliferation, and TGFβ signalings were observed. MALAT1 expression was also detected in primary RPE cells incubated with proliferative vitreoretinopathy (PVR vitreous samples.The expression of MALAT1 is significantly increased in RPE cells incubated with TGFβ1. MALAT1 silencing attenuates TGFβ1-induced EMT, migration, and proliferation of RPE cells, at least partially through activating Smad2/3 signaling. MALAT1 is also significantly increased in primary RPE cells incubated with PVR vitreous samples.LncRNA MALAT1 is involved in TGFβ1-induced EMT of human RPE cells and provides new understandings for the pathogenesis of PVR.

  11. 视网膜脱离外路显微手术中色素颗粒进入玻璃体临床观察%Clinical observation of intravitreal dispersion of retinal pigment epithelium cells during external approach microsurgery of retinal detachment

    Institute of Scientific and Technical Information of China (English)

    许大玲; 刘文; 霍鸣; 罗彤

    2008-01-01

    Objective It is a common phenomenon that a lot of pigmented debris disperses into vitreous cavity through retinal breaks during the cryotherapy or location of retinal breaks while the external approach microsurgery for rhegmatogenous retinal detachment (RRD) is performed.The study was designed for observing the surgical outcomes of this complication.Methods A consecutive thirty-eight patients (39eyes) with RRD,which were found intravitreal dispersion of retinal pigment epithelium cells during external approach mierosurgery of retinal detachment,were retrospectively analyzed.All of them were primary RRD and proliferative vitreoretinopathy (PVR) was classified equal to or under B.The numbers of average retinal breaks were 2.67.The size of the retinal break ranged from 1/7DD to 1.5 clock hour.The scleral buckling was performed in 11 eyes,and the circling and buckling in 28 eyes.Results The following-up times were 6 months at least.The retinal pigment epithelium cells of intravitreal dispersion gradually decreased and faded away.Retinal reattachment was achieved in 38 eyes (96.7%) after the primary surgery.A new retinal break was formed in one case and caused retinal redetachment.After vitreous surgery,this case got retinal reattachment.The final rate of reattachment was 100%.The excessive cryotherapy and severe PVR were not occurred.Conclusion As long as the holes or tears are closed effectively,the intravitreal dispersion of retinal pigment epithelium cells during external approach microsurgery of retinal detachment doesn't cause PVR develop or aggravate.The surgical outcomes are excellent.%目的 观察在视网膜脱离外路显微手术中做视网膜裂孔冷凝或定位时,色素颗粒从裂孔播散入玻璃体腔,对手术效果的影响.方法 回顾性统计分析视网膜脱离外路显微手术中有色素颗粒从裂孔涌人玻璃体腔的连续38例39只眼,均是初发裂孔性视网膜脱离患者,PVR分级为B级以下.平均每只眼裂孔数2.67

  12. High speed optical holography of retinal blood flow.

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    Pellizzari, M; Simonutti, M; Degardin, J; Sahel, J-A; Fink, M; Paques, M; Atlan, M

    2016-08-01

    We performed noninvasive video imaging of retinal blood flow in a pigmented rat by holographic interferometry of near-infrared laser light backscattered by retinal tissue, beating against an off-axis reference beam sampled at a frame rate of 39 kHz with a high throughput camera. Local Doppler contrasts emerged from the envelopes of short-time Fourier transforms and the phase of autocorrelation functions of holograms rendered by Fresnel transformation. This approach permitted imaging of blood flow in large retinal vessels (∼30 microns diameter) over 400×400  pixels with a spatial resolution of ∼8 microns and a temporal resolution of ∼6.5  ms.

  13. Reduced Expression of Cytoskeletal and Extracellular Matrix Genes in Human Adult Retinal Pigment Epithelium Cells Exposed to Simulated Microgravity

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    Thomas J. Corydon

    2016-11-01

    Full Text Available Background/Aims: Microgravity (µg has adverse effects on the eye of humans in space. The risk of visual impairment is therefore one of the leading health concerns for NASA. The impact of µg on human adult retinal epithelium (ARPE-19 cells is unknown. Methods: In this study we investigated the influence of simulated µg (s-µg; 5 and 10 days (d, using a Random Positioning Machine (RPM, on ARPE-19 cells. We performed phase-contrast/fluorescent microscopy, qRT-PCR, Western blotting and pathway analysis. Results: Following RPM-exposure a subset of ARPE-19 cells formed multicellular spheroids (MCS, whereas the majority of the cells remained adherent (AD. After 5d, alterations of F-actin and fibronectin were observed which reverted after 10d-exposure, suggesting a time-dependent adaptation to s-µg. Gene expression analysis of 12 genes involved in cell structure, shape, adhesion, migration, and angiogenesis suggested significant changes after a 10d-RPM-exposure. 11 genes were down-regulated in AD and MCS 10d-RPM-samples compared to 1g, whereas FLK1 was up-regulated in 5d- and 10d-RPM-MCS-samples. Similarly, TIMP1 was up-regulated in 5d-RPM-samples, whereas the remaining genes were down-regulated in 5d-RPM-samples. Western blotting revealed similar changes in VEGF, β-actin, laminin and fibronectin of 5d-RPM-samples compared to 10d, whereas different alterations of β-tubulin and vimentin were observed. The pathway analysis showed complementing effects of VEGF and integrin β-1. Conclusions: These findings clearly show that s-µg induces significant alterations in the F-actin-cytoskeleton and cytoskeleton-related proteins of ARPE-19, in addition to changes in cell growth behavior and gene expression patterns involved in cell structure, growth, shape, migration, adhesion and angiogenesis.

  14. Regulation of Na,K-ATPase β1-subunit in TGF-β2-mediated epithelial-to-mesenchymal transition in human retinal pigmented epithelial cells.

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    Mony, Sridevi; Lee, Seung Joon; Harper, Jeffrey F; Barwe, Sonali P; Langhans, Sigrid A

    2013-10-01

    Proliferative vitreo retinopathy (PVR) is associated with extracellular matrix membrane (ECM) formation on the neural retina and disruption of the multilayered retinal architecture leading to distorted vision and blindness. During disease progression in PVR, retinal pigmented epithelial cells (RPE) lose cell-cell adhesion, undergo epithelial-to-mesenchymal transition (EMT), and deposit ECM leading to tissue fibrosis. The EMT process is mediated via exposure to vitreous cytokines and growth factors such as TGF-β2. Previous studies have shown that Na,K-ATPase is required for maintaining a normal polarized epithelial phenotype and that decreased Na,K-ATPase function and subunit levels are associated with TGF-β1-mediated EMT in kidney cells. In contrast to the basolateral localization of Na,K-ATPase in most epithelia, including kidney, Na,K-ATPase is found on the apical membrane in RPE cells. We now show that EMT is also associated with altered Na,K-ATPase expression in RPE cells. TGF-β2 treatment of ARPE-19 cells resulted in a time-dependent decrease in Na,K-ATPase β1 mRNA and protein levels while Na,K-ATPase α1 levels, Na,K-ATPase activity, and intracellular sodium levels remained largely unchanged. In TGF-β2-treated cells reduced Na,K-ATPase β1 mRNA inversely correlated with HIF-1α levels and analysis of the Na,K-ATPase β1 promoter revealed a putative hypoxia response element (HRE). HIF-1α bound to the Na,K-ATPase β1 promoter and inhibiting the activity of HIF-1α blocked the TGF-β2 mediated Na,K-ATPase β1 decrease suggesting that HIF-1α plays a potential role in Na,K-ATPase β1 regulation during EMT in RPE cells. Furthermore, knockdown of Na,K-ATPase β1 in ARPE-19 cells was associated with a change in cell morphology from epithelial to mesenchymal and induction of EMT markers such as α-smooth muscle actin and fibronectin, suggesting that loss of Na,K-ATPase β1 is a potential contributor to TGF-β2-mediated EMT in RPE cells.

  15. Mechanism of riboflavin uptake by cultured human retinal pigment epithelial ARPE-19 cells: possible regulation by an intracellular Ca2+-calmodulin-mediated pathway.

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    Said, Hamid M; Wang, Shuling; Ma, Thomas Y

    2005-07-15

    In mammalian cells (including those of the ocular system), the water-soluble vitamin B2 (riboflavin, RF) assumes an essential role in a variety of metabolic reactions and is critical for normal cellular functions, growth and development. Cells of the human retinal pigment epithelium (hRPE) play an important role in providing a sufficient supply of RF to the retina, but nothing is known about the mechanism of the vitamin uptake by these cells and its regulation. Our aim in the present study was to address this issue using the hRPE ARPE-19 cells as the retinal epithelial model. Our results show RF uptake in the hRPE to be: (1) energy and temperature dependent and occurring without metabolic alteration in the transported substrate, (2) pH but not Na+ dependent, (3) saturable as a function of concentration with an apparent Km of 80 +/- 14 nM, (4) trans-stimulated by unlabelled RF and its structural analogue lumiflavine, (5) cis-inhibited by the RF structural analogues lumiflavine and lumichrome but not by unrelated compounds, and (6) inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) as well as by the Na+ -H+ exchange inhibitor amiloride and the sulfhydryl group inhibitor p-chloromercuriphenylsulphonate (p-CMPS). Maintaining the hRPE cells in a RF-deficient medium led to a specific and significant up-regulation in RF uptake which was mediated via changes in the number and affinity of the RF uptake carriers. While modulating the activities of intracellular protein kinase A (PKA)-, protein kinase C (PKC)-, protein tyrosine kinase (PTK)-, and nitric oxide (NO)-mediated pathways were found to have no role in regulating RF uptake, a role for the Ca2+ -calmodulin-mediated pathway was observed. These studies demonstrate for the first time the involvement of a specialized carrier-mediated mechanism for RF uptake by hRPE cells and show that the process is

  16. Multiphoton absorption is probably not the primary threshold damage mechanism for femtosecond laser pulse exposures in the retinal pigment epithelium

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    Glickman, Randolph D.; Johnson, Thomas E.

    2004-07-01

    Laser induced breakdown has the lowest energy threshold in the femtosecond domain, and is responsible for production of threshold ocular lesions. It has been proposed that multiphoton absorption may also contribute to ultrashort-pulse tissue damage, based on the observation that 33 fs, 810 nm pulse laser exposures caused more DNA breakage in cultured, primary RPE cells, compared to CW laser exposures delivering the same average power. Subsequent studies, demonstrating two-photon excitation of fluorescence in isolated RPE melanosomes, appeared to support the role of multiphoton absorption, but mainly at suprathreshold irradiance. Additional experiments have not found a consistent difference in the DNA strand breakage produced by ultrashort and CW threshold exposures. DNA damage appears to be dependent on the amount of melanin pigmentation in the cells, rather than the pulsewidth of the laser; current studies have found that, at threshold, CW and ultrashort pulse laser exposures produce almost identical amounts of DNA breakage. A theoretical analysis suggest that the number of photons delivered to the RPE melanosome during a single 33-fsec pulse at the ED50 irradiance is insufficient to produce multiphoton excitation. This result appears to exclude the melanosome as a locus for two- or three-photon excitation; however, a structure with a larger effective absorption cross-section than the melanosome may interact with the laser pulses. One possibility is that the nuclear chromatin acts as a unit absorber of photons resulting in DNA damage, but this does not explain the near equivalence of ultrashort and CW exposures in the comet assay model. This equivalence indicated that multiphoton absorption is not a major contributor to the ultrashort pulse laser damage threshold in the near infrared.

  17. Cigarette smoke-related hydroquinone dysregulates MCP-1, VEGF and PEDF expression in retinal pigment epithelium in vitro and in vivo.

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    Marianne Pons

    Full Text Available BACKGROUND: Age-related macular degeneration (AMD is the leading cause of legal blindness in the elderly population. Debris (termed drusen below the retinal pigment epithelium (RPE have been recognized as a risk factor for dry AMD and its progression to wet AMD, which is characterized by choroidal neovascularization (CNV. The underlying mechanism of how drusen might elicit CNV remains undefined. Cigarette smoking, oxidative damage to the RPE and inflammation are postulated to be involved in the pathophysiology of the disease. To better understand the cellular mechanism(s linking oxidative stress and inflammation to AMD, we examined the expression of pro-inflammatory monocyte chemoattractant protein-1 (MCP-1, pro-angiogenic vascular endothelial growth factor (VEGF and anti-angiogenic pigment epithelial derived factor (PEDF in RPE from smoker patients with AMD. We also evaluated the effects of hydroquinone (HQ, a major pro-oxidant in cigarette smoke on MCP-1, VEGF and PEDF expression in cultured ARPE-19 cells and RPE/choroids from C57BL/6 mice. PRINCIPAL FINDINGS: MCP-1, VEGF and PEDF expression was examined by real-time PCR, Western blot, and ELISA. Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls. Both MCP-1 mRNA and protein were downregulated in ARPE-19 cells and RPE/choroids from C57BL/6 mice after 5 days and 3 weeks of exposure to HQ-induced oxidative injury. VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio. Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo. CONCLUSION: We propose that impaired RPE-derived MCP-1-mediated scavenging macrophages recruitment and phagocytosis might lead to incomplete clearance of proinflammatory debris and infiltration of proangiogenic macrophages which along with increased VEGF/PEDF ratio favoring

  18. Protective Effects of Human iPS-Derived Retinal Pigmented Epithelial Cells in Comparison with Human Mesenchymal Stromal Cells and Human Neural Stem Cells on the Degenerating Retina in rd1 mice.

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    Sun, Jianan; Mandai, Michiko; Kamao, Hiroyuki; Hashiguchi, Tomoyo; Shikamura, Masayuki; Kawamata, Shin; Sugita, Sunao; Takahashi, Masayo

    2015-05-01

    Retinitis pigmentosa (RP) is a group of visual impairments characterized by progressive rod photoreceptor cell loss due to a genetic background. Pigment epithelium-derived factor (PEDF) predominantly secreted by the retinal pigmented epithelium (RPE) has been reported to protect photoreceptors in retinal degeneration models, including rd1. In addition, clinical trials are currently underway outside Japan using human mesenchymal stromal cells and human neural stem cells to protect photoreceptors in RP and dry age-related macular degeneration, respectively. Thus, this study aimed to investigate the rescue effects of induced pluripotent stem (iPS)-RPE cells in comparison with those types of cells used in clinical trials on photoreceptor degeneration in rd1 mice. Cells were injected into the subretinal space of immune-suppressed 2-week-old rd1 mice. The results demonstrated that human iPS-RPE cells significantly attenuated photoreceptor degeneration on postoperative days (PODs) 14 and 21 and survived longer up to at least 12 weeks after operation than the other two types of graft cells with less immune responses and apoptosis. The mean PEDF concentration in the intraocular fluid in RPE-transplanted eyes was more than 1 µg/ml at PODs 14 and 21, and this may have contributed to the protective effect of RPE transplantation. Our findings suggest that iPS-RPE cells serve as a competent source to delay photoreceptor degeneration through stable survival in degenerating ocular environment and by releasing neuroprotective factors such as PEDF.

  19. Mitochondrial "movement" and lens optics following oxidative stress from UV-B irradiation: cultured bovine lenses and human retinal pigment epithelial cells (ARPE-19) as examples.

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    Bantseev, Vladimir; Youn, Hyun-Yi

    2006-12-01

    Mitochondria provide energy generated by oxidative phosphorylation and at the same time play a central role in apoptosis and aging. As a byproduct of respiration, the electron transport chain is known to be the major intracellular site for the generation of reactive oxygen species (ROS). Exposure to solar and occupational ultraviolet (UV) radiation, and thus production of ROS and subsequent cell death, has been implicated in a large spectrum of skin and ocular pathologies, including cataract. Retinal pigment epithelial cell apoptosis generates photoreceptor dysfunction and ultimately visual impairment. The purpose of this article was to characterize in vitro changes following oxidative stress with UV-B radiation in (a) ocular lens optics and cellular function in terms of mitochondrial dynamics of bovine lens epithelium and superficial cortical fiber cells and (b) human retinal pigment epithelial (ARPE-19) cells. Cultured bovine lenses and confluent cultures of ARPE-19 cells were irradiated with broadband UV-B radiation at energy levels of 0.5 and 1.0 J/cm(2). Lens optical function (spherical aberration) was monitored daily up to 14 days using an automated laser scanning system that was developed at the University of Waterloo. This system consists of a single collimated scanning helium-neon laser source that projects a thin (0.05 mm) laser beam onto a plain mirror mounted at 45 degrees on a carriage assembly. This mirror reflects the laser beam directly up through the scanner table surface and through the lens under examination. A digital camera captures the actual position and slope of the laser beam at each step. When all steps have been made, the captured data for each step position is used to calculate the back vertex distance for each position and the difference in that measurement between beams. To investigate mitochondrial movement, the mitochondria-specific fluorescent dye Rhodamine 123 was used. Time series were acquired with a Zeiss 510 (configuration Meta

  20. Retinal pigmented epithelial cells cytotoxicity and apoptosis through activation of the mitochondrial intrinsic pathway: role of indocyanine green, brilliant blue and implications for chromovitrectomy.

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    Fernando M Penha

    Full Text Available PURPOSE: To investigate the in vitro effect of four vital dyes on toxicity and apoptosis in a human retinal pigment epithelial (RPE cell line. METHODS: ARPE-19 cells were exposed to brilliant blue (BriB, methyl blue (MetB, acid violet (AcV and indocyanine green (ICG. Balanced salt solution was used as control. Five different concentrations of each dye (1, 0.5, 0.25, 0.05 and 0.005 mg/mL and two exposure times (3 and 30 min were tested. Cell viability was determined by cell count and MTS assay and cell toxicity by LDH assay. Real-time PCR and Western blotting were used to access the apoptosis process. RESULTS: ICG significantly reduced cell viability after 3 minutes of exposure at all concentrations (p<0.01. BriB was safe at concentrations up to 0.25 mg/mL and MetB at concentrations up to 0.5 mg/mL, while AcV was safe up to 0.05 mg/ml, after 3 minutes of exposure. Toxicity was higher, when the cells were treated for 30 minutes. Expression of Bax, cytochrome c and caspase-9 was upregulated at the mRNA and protein level after ICG exposure, while Bcl-2 was downregulated. AcV and MetB were similar to control. However, BriB resulted in upregulation of Bcl-2, an antiapoptotic protein. CONCLUSIONS: The safest dye used on RPE cells was MetB followed by BriB and AcV. ICG was toxic at all concentrations and exposure times tested. Moreover, ICG was the only dye that induced apoptosis in ARPE-19 cells. BriB significantly increased Bcl-2 protein levels, which might protect against the apoptosis process.

  1. A Possible Role of Acrolein in Diabetic Retinopathy: Involvement of a VEGF/TGFβ Signaling Pathway of the Retinal Pigment Epithelium in Hyperglycemia

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    Grigsby, Jeffery; Betts, Brandi; Vidro-Kotchan, Eileen; Culbert, Richard; Tsin, Andrew

    2015-01-01

    Purpose Acrolein has been implicated in retinal pigment epithelium (RPE) cell death, and has been associated with diabetic retinopathy. Our purpose was to investigate the potential effect of high glucose in influencing acrolein-mediated RPE cytokine production and cell death. We investigated the influence of the acrolein effect on ARPE-19 cells in high glucose conditions and quantified the release of transforming growth factor β (TGFβ1 and 2) and vascular endothelial growth factor (VEGF). We assessed the ability of N-benzylhydroxylamine(NBHA) as well as TGFβ pathway inhibitors SIS3 and SB431542 to prevent this effect of acrolein on ARPE-19 cells. Materials and methods Confluent ARPE-19 cells were treated with acrolein and/or NBHA in both 5.5 and 18.8 mM glucose conditions. Cells were also pretreated with SIS3, a specific inhibitor of the SMAD3 pathway, and SB431542, a specific inhibitor of TGFβ signaling pathway, before treating them with acrolein. Viable cells were counted and ELISAs were performed to measure the cytokines TGFβ1 and 2, and VEGF released into the conditioned media. Results In ARPE-19 cells exposed to acrolein and hyperglycemia there was reduced cell viability and an increase in the cell media of VEGF, TGFβ1, and TGFβ2, which was reversed by NBHA. Acrolein/hyperglycemia-induced cell viability reduction and cytokine overproduction was also reduced by TGFβ pathway blockade. Conclusions We conclude that the effect of acrolein on the reduction of viability and VEGF increase by ARPE-19 cells in hyperglycemic media is conducted through the TGFβ signaling pathway. Our results suggest that benefits of sequestering acrolein by NBHA and the blockage of the TGFβ pathway by SB431542 and SIS3 offer suggestions as to potential useful pharmacological drug candidates for the prevention of diabetes-induced complications in the eye. PMID:22906079

  2. Complement Factor H Expressed by Retinal Pigment Epithelium Cells Can Suppress Neovascularization of Human Umbilical Vein Endothelial Cells: An in vitro Study.

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    Yi Zhang

    Full Text Available Complement factor H (CFH is one of the most important soluble complement regulatory proteins and is closely associated with age-related macular degeneration (AMD, the leading cause of irreversible central vision loss in the elderly population in developed countries. Our study searches to investigate whether CFH expression is changed in oxidative damaged retinal pigment epithelium (RPE cells and the role of CFH in the in vitro neovascularization. First, it was confirmed by immunofluorescence staining that CFH was expressed by ARPE-19 cells. CFH mRNA and protein in oxidative (H2O2 damaged ARPE-19 cells were both reduced, as determined by Real-time PCR and Western blotting analysis. Enzyme-linked immunosorbent assay (ELISA also showed that ARPE-19 cells treated with H2O2 caused an increase in C3a content, which indicates complement activation. Then, wound assays were performed to show that CFH expression suppression promoted human umbilical vein endothelial cell (HUVECs migration. Thereafter, ARPE-19 cells were transfected with CFH-specific siRNA and CFH knockdown was confirmed with the aid of Real-time PCR, immunofluorescence staining and Western blotting. The ELISA results showed that specific CFH knockdown in ARPE-19 cells activated the complement system. Finally, in vitro matrigel tube formation assay was performed to determine whether change of CFH expression in RPE would affect tube formation by HUVECs. More tubes were formed by HUVECs co-cultured with ARPE-19 cells transfected with CFH specific-siRNA when compared with controls. Our results suggested that RPE cells might be the local CFH source, and RPE cell injuries (such as oxidative stress may cause CFH expression suppression, which in turn may lead to complement activation and promotion of tube formation by HUVECs. This finding is of importance in elucidating the role of complement in the pathogenesis of ocular neovascularization including choroidal neovascularization.

  3. Ormocomp-modified glass increases collagen binding and promotes the adherence and maturation of human embryonic stem cell-derived retinal pigment epithelial cells.

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    Käpylä, Elli; Sorkio, Anni; Teymouri, Shokoufeh; Lahtonen, Kimmo; Vuori, Leena; Valden, Mika; Skottman, Heli; Kellomäki, Minna; Juuti-Uusitalo, Kati

    2014-12-09

    In in vitro live-cell imaging, it would be beneficial to grow and assess human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells on thin, transparent, rigid surfaces such as cover glasses. In this study, we assessed how the silanization of glass with 3-aminopropyltriethoxysilane (APTES), 3-(trimethoxysilyl)propyl methacrylate (MAPTMS), or polymer-ceramic material Ormocomp affects the surface properties, protein binding, and maturation of hESC-RPE cells. The surface properties were studied by contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and a protein binding assay. The cell adherence and proliferation were evaluated by culturing hESCRPE cells on collagen IV-coated untreated or silanized surfaces for 42 days. The Ormocomp treatment significantly increased the hydrophobicity and roughness of glass surfaces compared to the APTES and MAPTMS treatments. The XPS results indicated that the Ormocomp treatment changes the chemical composition of the glass surface by increasing the carbon content and the number of C-O/═O bonds. The protein-binding test confirmed that the Ormocomp-treated surfaces bound more collagen IV than did APTES- or MAPTMS-treated surfaces. All of the silane treatments increased the number of cells: after 42 days of culture, Ormocomp had 0.38, APTES had 0.16, MAPTMS had 0.19, and untreated glass had only 0.062, all presented as million cells cm(-2). There were no differences in cell numbers compared to smoother to rougher Ormocomp surfaces, suggesting that the surface chemistry and, more specifically, the collagen binding in combination with Ormocomp are beneficial to hESC-RPE cell culture. This study clearly demonstrates that Ormocomp treatment combined with collagen coating significantly increases hESC-RPE cell attachment compared to commonly used silanizing agents APTES and MAPTMS. Ormocomp silanization could thus enable the use of microscopic live cell imaging methods for h

  4. TNF-{alpha} promotes human retinal pigment epithelial (RPE) cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression through activation of Akt/mTORC1 signaling

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    Wang, Cheng-hu; Cao, Guo-Fan [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Jiang, Qin, E-mail: Jqin710@vip.sina.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China); Yao, Jin, E-mail: dryaojin@yahoo.com [The Affiliated Eye Hospital of Nanjing Medical University, Nanjing 210029 (China)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-added active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.

  5. TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

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    Chung, Eun Jee [Department of Ophthalmology, National Health Insurance Corporation Ilsan Hospital, Gyeonggi-do (Korea, Republic of); Chun, Ji Na; Jung, Sun-Ah [Konyang University Myunggok Medical Research Institute, Kim' s Eye Hospital, Konyang University College of Medicine, Seoul (Korea, Republic of); Cho, Jin Won [Department of Biology, Yonsei University, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Lee, Joon H., E-mail: joonhlee@konyang.ac.kr [Konyang University Myunggok Medical Research Institute, Kim' s Eye Hospital, Konyang University College of Medicine, Seoul (Korea, Republic of)

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology in pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful information

  6. Retinal pigment epithelial cells secrete neurotrophic factors and synthesize dopamine: possible contribution to therapeutic effects of RPE cell transplantation in Parkinson's disease

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    Gu Qing

    2009-06-01

    Full Text Available Abstract Background New strategies for the treatment of Parkinson's disease (PD are shifted from dopamine (DA replacement to regeneration or restoration of the nigro-striatal system. A cell therapy using human retinal pigment epithelial (RPE cells as substitution for degenerated dopaminergic (DAergic neurons has been developed and showed promising prospect in clinical treatment of PD, but the exact mechanism underlying this therapy is not fully elucidated. In the present study, we investigated whether the beneficial effects of this therapy are related to the trophic properties of RPE cells and their ability to synthesize DA. Methods We evaluated the protective effects of conditioned medium (CM from cultured RPE cells on the DAergic cells against 6-hydroxydopamine (6-OHDA- and rotenone-induced neurotoxicity and determined the levels of glial cell derived neurotrophic factor (GDNF and brain derived neurotrophic factor (BDNF released by RPE cells. We also measured the DA synthesis and release. Finally we transplanted microcarriers-RPE cells into 6-OHDA lesioned rats and observed the improvement in apomorphine-induced rotations (AIR. Results We report here: (1 CM from RPE cells can secret trophic factors GDNF and BDNF, and protect DAergic neurons against the 6-OHDA- and rotenone-induced cell injury; (2 cultured RPE cells express L-dopa decarboxylase (DDC and synthesize DA; (3 RPE cells attached to microcarriers can survive in the host striatum and improve the AIR in 6-OHDA-lesioned animal model of PD; (4 GDNF and BDNF levels are found significantly higher in the RPE cell-grafted tissues. Conclusion These findings indicate the RPE cells have the ability to secret GDNF and BDNF, and synthesize DA, which probably contribute to the therapeutic effects of RPE cell transplantation in PD.

  7. Blockade of Jagged/Notch pathway abrogates transforming growth factor β2-induced epithelial-mesenchymal transition in human retinal pigment epithelium cells.

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    Chen, X; Xiao, W; Liu, X; Zeng, M; Luo, L; Wu, M; Ye, S; Liu, Y

    2014-05-01

    The epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells plays a key role in proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), which lead to the loss of vision. The Jagged/Notch pathway has been reported to be essential in EMT during embryonic development, fibrotic diseases and cancer metastasis. However, the function of Jagged/Notch signaling in EMT of RPE cells is unknown. Thus, we hypothesized that a crosstalk between Notch and transforming growth factor β2 (TGF-β2) signaling could induce EMT in RPE cells, which subsequently contributes to PVR and PDR. Here, we demonstrate that Jagged-1/Notch pathway is involved in the TGF-β2-mediated EMT of human RPE cells. Blockade of Notch pathway with DAPT (a specific inhibitor of Notch receptor cleavage) and knockdown of Jagged-1 expression inhibited TGF-β2-induced EMT through regulating the expression of Snail, Slug and ZEB1. Besides the canonical Smad signaling pathway, the noncanonical PI3K/Akt and MAPK pathway also contributed to TGF-β2-induced up-regulation of Jagged-1 in RPE cells. Overexpression of Jagged-1 could mimic TGF-β2 induce EMT. Our data suggest that the Jagged-1/Notch signaling pathway plays a critical role in TGF-β2-induced EMT in human RPE cells, and may contribute to the development of PVR and PDR. Inhibition of the Jagged/Notch signaling pathway, therefore, may have therapeutic value in the prevention and treatment of PVR and PDR.

  8. Change of retinal pigment epithelial atrophy after anti-vascular endothelial growth factor treatment in exudative age-related macular degeneration

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    Moosang Kim

    2016-01-01

    Full Text Available Purpose: The study aimed to investigate the quantitative changes of retinal pigment epithelial (RPE atrophy during a 24-month follow-up period of anti-vascular endothelial growth factor (VEGF for exudative age-related macular degeneration (AMD. Materials and Methods: This is a retrospective study. Sixty-five eyes of 62 consecutive patients with naοve exudative AMD who had received treatment with anti-VEGF therapy and followed for more 24 months were enrolled. All patients received three initial monthly injections of anti-VEGF (ranibizumab or bevacizumab, followed by pro re nata or treat-and-extend protocol. Color fundus image, optical coherence tomography, and fundus autofluorescence were evaluated for RPE atrophy. Multiple regression analysis was performed to investigate the predictive factors found during univariate analysis to identify an association with increased RPE atrophic areas. Results: The mean number of anti-VEGF treatments was 9.18. RPE atrophic area was 1.293 ± 1.298 mm 2 at baseline and enlarged to 2.394 ± 1.940 mm 2 after 24 months, which differed significantly (P = 0.001. Multiple regression analysis revealed that larger areas of RPE atrophy at month 4 and larger numbers of anti-VEGF treatments were associated with increased RPE atrophic areas. Conclusions: RPE atrophy progresses in eyes with exudative AMD during anti-VEGF treatment. Larger areas of RPE atrophy at month 4 and larger numbers of anti-VEGF injections were associated with an increased risk of progression of RPE atrophy the following treatment. These findings may be useful to clinicians using intravitreal anti-VEGF for the treatment of exudative AMD, both for selecting an appropriate treatment plan and for predicting the progression of RPE atrophy.

  9. The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit

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    Lobato-Álvarez, Jorge A.; Roldán, María L.; López-Murillo, Teresa del Carmen; González-Ramírez, Ricardo; Bonilla-Delgado, José; Shoshani, Liora

    2016-01-01

    Na+, K+-ATPase, or the Na+ pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β1 subunit of Na+, K+-ATPase plays an important role in this mechanism because homotypic β1-β1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na+ pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na+, K+-ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na+ pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β2 subunit. qPCR results showed a time-dependent increase in the level of β2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α2 subunit in that domain. Our results demonstrate that the apical polarization of Na+, K+-ATPase in RPE cells depends on the expression of the β2 subunit. PMID:27774068

  10. The Apical Localization of Na+, K+-ATPase in Cultured Human Retinal Pigment Epithelial Cells Depends on Expression of the β2 Subunit

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    Jorge Lobato Álvarez

    2016-10-01

    Full Text Available Na+, K+-ATPase, or the Na+ pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the β1 subunit of Na+, K+-ATPase plays an important role in this mechanism because homotypic β1-β1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE, the Na+ pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its β2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na+, K+-ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS, ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na+ pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the β2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β2 subunit. qPCR results showed a time-dependent increase in the level of β2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α2 subunit in that domain. Our results demonstrate that the apical polarization of Na+, K+-ATPase in RPE cells depends on the expression of the β2 subunit.

  11. The effect of 17beta-estradiol on IL-6 secretion and NF-kappaB DNA-binding activity in human retinal pigment epithelial cells.

    Science.gov (United States)

    Paimela, Tuomas; Ryhänen, Tuomas; Mannermaa, Eliisa; Ojala, Johanna; Kalesnykas, Giedrius; Salminen, Antero; Kaarniranta, Kai

    2007-06-15

    Toll-like receptors (TLRs) and inflammatory cascades participate in the pathology of age-related macular degeneration (AMD). The effect of estrogens on the development of AMD is poorly understood, although many studies indicate that these compounds can modulate inflammatory responses. In this study, we investigated the regulatory role of TLR agonists and 17beta-estradiol (E(2)) on IL-6 expression and NF-kappaB DNA-binding activity in human retinal pigment epithelial cells (ARPE-19). The inflammatory response of ARPE-19 cells to various TLR agonists, e.g. Pam, zymosan, flagellin, SLTA and lipopolysaccharide (LPS) exposures were examined via the secretion of IL-6 cytokine as analyzed by ELISA. In addition, the IL-6 responses to the estrogen-receptor agonist, E(2), and to the estrogen-receptor antagonist ICI 182.780 as well as to the NF-kappaB inhibitor helenalin were compared. The DNA-binding activity of NF-kappaB transcription factor of nuclear cell extracts was analyzed by the gel mobility shift assay (EMSA). TLR4 gene expression was studied by quantitave PCR. The TLR4 agonist, LPS, caused a clear IL-6 response that was attenuated by E(2) in ARPE-19-cells. The anti-inflammatory properties of E(2) were mediated through estrogen receptors and were associated with decreased NF-kappaB DNA-binding activity. The level of TLR4 gene expression was not affected by LPS exposure. Our results indicate that IL-6 expression is regulated through NF-kappaB transcription factor and stereoid-receptor signalling pathways in ARPE-19 cells.

  12. Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture.

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    Gangalum, Rajendra K; Bhat, Ankur M; Kohan, Sirus A; Bhat, Suraj P

    2016-06-17

    Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein αB-crystallin (αB) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of αB via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where αB expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and αB-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of αB results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for αB in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes.

  13. Transforming growth factor-β2 increases the capacity of retinal pigment epithelial cells to induce the generation of regulatory T cells.

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    Yan, Feng; He, Jin; Tang, Li; Kong, Yi; Shi, Yuhua; Chen, Suihua; Huang, Zhenping

    2016-02-01

    The present study investigated the underlying mechanism of the induction of regulatory T cells (Tregs) by retinal pigment epithelial (RPE) cells and the characteristics of these Tregs. Human RPE cells were cultured in the presence or absence of transforming growth factor-β 2 (TGF-β2), and reverse-transcription quantitative PCR was performed to determine the mRNA expression of indoleamine 2,3-dioxygenase (IDO) and nuclear factor erythroid 2-related factor (Nrf2). Supernatants of RPE cell cultures were added to CD4+ T cells to induce Tregs. The RPE-induced Tregs were purified by two-step magnetic cell sorting. The natural Tregs were isolated from the peripheral blood mononuclear cells of healthy volunteers. Purified CD4+ CD25- T cells (2 x 10(5)/well) were cultured alone or with Tregs (various densities, natural or RPE-induced). The proliferation of CD4+ CD25- T cells was determined by 3H-thymidine incorporation. After 24 h of stimulation with TGF-β2, the mRNA expression of IDO in RPE cells was upregulated. The highest level of IDO mRNA expression was reached after 72 h of stimulation with TGF-β2. However, the Nrf2 mRNA expression was slightly decreased after 24 h of stimulation with TGF-β2 and significantly increased after 48-72 h of TGF-β2 stimulation. Increased levels of CD25 expression were observed on CD4+ T cells exposed to supernatants of RPE cell cultures treated with TGF-β2 and recombinant interleukin-2. The RPE-induced Tregs were more effective at suppressing the proliferation of CD4+ CD25- T cells compared with native Tregs. These findings suggested that IDO may be a signaling protein in RPE cells which is implicated in the induction of Tregs. RPE-induced Tregs have the potential to be applied for immunotherapy for ocular inflammatory diseases.

  14. Myeloid cells expressing VEGF and arginase-1 following uptake of damaged retinal pigment epithelium suggests potential mechanism that drives the onset of choroidal angiogenesis in mice.

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    Jian Liu

    Full Text Available Whilst data recognise both myeloid cell accumulation during choroidal neovascularisation (CNV as well as complement activation, none of the data has presented a clear explanation for the angiogenic drive that promotes pathological angiogenesis. One possibility that is a pre-eminent drive is a specific and early conditioning and activation of the myeloid cell infiltrate. Using a laser-induced CNV murine model, we have identified that disruption of retinal pigment epithelium (RPE and Bruch's membrane resulted in an early recruitment of macrophages derived from monocytes and microglia, prior to angiogenesis and contemporaneous with lesional complement activation. Early recruited CD11b(+ cells expressed a definitive gene signature of selective inflammatory mediators particularly a pronounced Arg-1 expression. Accumulating macrophages from retina and peripheral blood were activated at the site of injury, displaying enhanced VEGF expression, and notably prior to exaggerated VEGF expression from RPE, or earliest stages of angiogenesis. All of these initial events, including distinct VEGF (+ Arg-1(+ myeloid cells, subsided when CNV was established and at the time RPE-VEGF expression was maximal. Depletion of inflammatory CCR2-positive monocytes confirmed origin of infiltrating monocyte Arg-1 expression, as following depletion Arg-1 signal was lost and CNV suppressed. Furthermore, our in vitro data supported a myeloid cell uptake of damaged RPE or its derivatives as a mechanism generating VEGF (+ Arg-1(+ phenotype in vivo. Our results reveal a potential early driver initiating angiogenesis via myeloid-derived VEGF drive following uptake of damaged RPE and deliver an explanation of why CNV develops during any of the stages of macular degeneration and can be explored further for therapeutic gain.

  15. Highly sensitive in vitro methods for detection of residual undifferentiated cells in retinal pigment epithelial cells derived from human iPS cells.

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    Takuya Kuroda

    Full Text Available Human induced pluripotent stem cells (hiPSCs possess the capabilities of self-renewal and differentiation into multiple cell types, and they are free of the ethical problems associated with human embryonic stem cells (hESCs. These characteristics make hiPSCs a promising choice for future regenerative medicine research. There are significant obstacles, however, preventing the clinical use of hiPSCs. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. To locate residual undifferentiated cells, in vivo teratoma formation assays have been performed with immunodeficient animals, which is both costly and time-consuming. Here, we examined three in vitro assay methods to detect undifferentiated cells (designated an in vitro tumorigenicity assay: soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR. Although the soft agar colony formation assay was unable to detect hiPSCs even in the presence of a ROCK inhibitor that permits survival of dissociated hiPSCs/hESCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of Lin28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×10⁴ RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived products for future regenerative medicine research.

  16. Angiotensin-2-mediated Ca2+ signaling in the retinal pigment epithelium: role of angiotensin-receptor-associated-protein and TRPV2 channel.

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    Rene Barro-Soria

    Full Text Available Angiotensin II (AngII receptor (ATR is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap, and transient-receptor-potential channel-V2 (TRPV2. AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE cells by AngII results in biphasic increases in intracellular free Ca(2+inhibited by losartan. Xestospongin C (xest C and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca(2+response. RPE cells from Atrap(-/- mice showed smaller AngII-evoked Ca(2+peak (by 22% and loss of sustained Ca(2+elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD at 15 µM stimulates intracellular Ca(2+-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM SKF96365 (a TRPV channel inhibitor reduced the cannabidiol-induced Ca(2+-rise. Application of SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca(2+transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca(2+transients in the RPE by releasing Ca(2+from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca(2+elevation.

  17. Tumorigenicity studies of induced pluripotent stem cell (iPSC-derived retinal pigment epithelium (RPE for the treatment of age-related macular degeneration.

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    Hoshimi Kanemura

    Full Text Available Basic studies of human pluripotential stem cells have advanced rapidly and stem cell products are now seeing therapeutic applications. However, questions remain regarding the tumorigenic potential of such cells. Here, we report the tumorigenic potential of induced pluripotent stem cell (iPSC-derived retinal pigment epithelium (RPE for the treatment of wet-type, age-related macular degeneration (AMD. First, immunodeficient mouse strains (nude, SCID, NOD-SCID and NOG were tested for HeLa cells' tumor-forming capacity by transplanting various cell doses subcutaneously with or without Matrigel. The 50% Tumor Producing Dose (TPD50 value is the minimal dose of transplanted cells that generated tumors in 50% of animals. For HeLa cells, the TPD50 was the lowest when cells were embedded in Matrigel and transplanted into NOG mice (TPD50 = 10(1.1, n = 75. The TPD50 for undifferentiated iPSCs transplanted subcutaneously to NOG mice in Matrigel was 10(2.12; (n = 30. Based on these experiments, 1×10(6 iPSC-derived RPE were transplanted subcutaneously with Matrigel, and no tumor was found during 15 months of monitoring (n = 65. Next, to model clinical application, we assessed the tumor-forming potential of HeLa cells and iPSC 201B7 cells following subretinal transplantation of nude rats. The TPD50 for iPSCs was 10(4.73 (n = 20 and for HeLa cells 10(1.32 (n = 37 respectively. Next, the tumorigenicity of iPSC-derived RPE was tested in the subretinal space of nude rats by transplanting 0.8-1.5×10(4 iPSC-derived RPE in a collagen-lined (1 mm×1 mm sheet. No tumor was found with iPSC-derived RPE sheets during 6-12 months of monitoring (n = 26. Considering the number of rodents used, the monitoring period, the sensitivity of detecting tumors via subcutaneous and subretinal administration routes and the incidence of tumor formation from the iPSC-derived RPE, we conclude that the tumorigenic potential of the iPSC-derived RPE was

  18. Inhibition of vascular endothelial growth factor gene expression by T7-siRNAs in cultured human retinal pigment epithelial cells

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    LI Guang-yu; FAN Bin; WU Ya-zhen; WANG Xin-rui; WANG Yao-hui; WU Jia-xiang

    2005-01-01

    Background Retinal pigment epithelial (RPE) cells play an important role in the occurrence of choroidal neovascularization (CNV). Vascular endothelial growth factor (VEGF) as a positive regulatory growth factor is produced by the RPE in an autocrine or paracrine manner, promoting CNV development. Duplexes of 21 nt RNAs, known as short interfering RNAs (siRNAs), efficiently inhibit gene expression by RNA interference when introduced into mammalian cells. We searched for an efficient siRNA to interfere with VEGF expression in RPE cells and shed light on the treatment of CNV.Methods Human primary RPE (hRPE) cells were cultured and identified. Three pairs of siRNAs were designed according to the sequence of VEGF 1-5 extrons and synthesized by T7 RNA polymerase transcription in vitro. To evaluate the inhibitory activity of T7-siRNAs, hRPE cells were transfected via siPORT Amine. The interfering effect of T7-siRNAs in hRPE cells was examined by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence. Results Three pairs of T7-siRNAs synthesized by in vitro transcription with T7 RNA polymerase suppressed VEGF gene expression with efficiency from 65% to 90%. T7-siRNA (B), targeted region at 207 nt to 228 nt and double stranded for 21 nt with 2 nt UU 3' overhangs, was the most effective sequence tested for inhibition of VEGF expression in hRPE cells. Compared with nontransfected cells, the mean fluorescence in hRPE cells transfected with T7-sRNAs was significantly less (P<0.01). siRNA with a single-base mismatch and ssRNA(+) did not show suppressing effect. Furthermore, it was found that siRNAs had a dose dependent inhibitory effect (5 to 10 pmol).Conclusion T7-siRNA can effectively and specifically suppress VEGF expression in hRPE cells and may be a new way to treat CNV.

  19. Cholesterol enhances amyloid {beta} deposition in mouse retina by modulating the activities of A{beta}-regulating enzymes in retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jiying [Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan); Ohno-Matsui, Kyoko, E-mail: k.ohno.oph@tmd.ac.jp [Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan); Morita, Ikuo [Section of Cellular Physiological Chemistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer Cholesterol-treated RPE produces more A{beta} than non-treated RPE. Black-Right-Pointing-Pointer Neprilysin expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer {alpha}-Secretase expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer Cholesterol-enriched diet induced subRPE deposits in aged mice. Black-Right-Pointing-Pointer A{beta} were present in cholesterol-enriched-diet-induced subRPE deposits in aged mice. -- Abstract: Subretinally-deposited amyloid {beta} (A{beta}) is a main contributor of developing age-related macular degeneration (AMD). However, the mechanism causing A{beta} deposition in AMD eyes is unknown. Hypercholesterolemia is a significant risk for developing AMD. Thus, we investigated the effects of cholesterol on A{beta} production in retinal pigment epithelial (RPE) cells in vitro and in the mouse retina in vivo. RPE cells isolated from senescent (12-month-old) C57BL/6 mice were treated with 10 {mu}g/ml cholesterol for 48 h. A{beta} amounts in culture supernatants were measured by ELISA. Activity and expression of enzymes and proteins that regulate A{beta} production were examined by activity assay and real time PCR. The retina of mice fed cholesterol-enriched diet was examined by transmission electron microscopy. Cholesterol significantly increased A{beta} production in cultured RPE cells. Activities of A{beta} degradation enzyme; neprilysin (NEP) and anti-amyloidogenic secretase; {alpha}-secretase were significantly decreased in cell lysates of cholesterol-treated RPE cells compared to non-treated cells, but there was no change in the activities of {beta}- or {gamma}-secretase. mRNA levels of NEP and {alpha}-secretase (ADAM10 and ADAM17) were significantly lower in cholesterol-treated RPE cells than non-treated cells. Senescent (12-month-old) mice fed cholesterol-enriched chow developed subRPE deposits containing A{beta}, whereas

  20. Specific interaction of Gαi3 with the Oa1 G-protein coupled receptor controls the size and density of melanosomes in retinal pigment epithelium.

    Directory of Open Access Journals (Sweden)

    Alejandra Young

    Full Text Available BACKGROUND: Ocular albinism type 1, an X-linked disease characterized by the presence of enlarged melanosomes in the retinal pigment epithelium (RPE and abnormal crossing of axons at the optic chiasm, is caused by mutations in the OA1 gene. The protein product of this gene is a G-protein-coupled receptor (GPCR localized in RPE melanosomes. The Oa1-/- mouse model of ocular albinism reproduces the human disease. Oa1 has been shown to immunoprecipitate with the Gαi subunit of heterotrimeric G proteins from human skin melanocytes. However, the Gαi subfamily has three highly homologous members, Gαi1, Gαi2 and Gαi3 and it is possible that one or more of them partners with Oa1. We had previously shown by in-vivo studies that Gαi3-/- and Oa1-/- mice have similar RPE phenotype and decussation patterns. In this paper we analyze the specificity of the Oa1-Gαi interaction. METHODOLOGY: By using the genetic mouse models Gαi1-/-, Gαi2-/-, Gαi3-/- and the double knockout Gαi1-/-, Gαi3-/- that lack functional Gαi1, Gαi2, Gαi3, or both Gαi1 and Gαi3 proteins, respectively, we show that Gαi3 is critical for the maintenance of a normal melanosomal phenotype and that its absence is associated with changes in melanosomal size and density. GST-pull-down and immunoprecipitation assays conclusively demonstrate that Gαi3 is the only Gαi that binds to Oa1. Western blots show that Gαi3 expression is barely detectable in the Oa1-/- RPE, strongly supporting a previously unsuspected role for Gαi3 in melanosomal biogenesis. CONCLUSION: Our results identify the Oa1 transducer Gαi3 as the first downstream component in the Oa1 signaling pathway.

  1. Protective effect of autophagy on human retinal pigment epithelial cells against lipofuscin fluorophore A2E: implications for age-related macular degeneration.

    Science.gov (United States)

    Zhang, J; Bai, Y; Huang, L; Qi, Y; Zhang, Q; Li, S; Wu, Y; Li, X

    2015-11-12

    Age-related macular degeneration (AMD) is the leading cause of central vision loss in the elderly. Degeneration of retinal pigment epithelial (RPE) cells is a crucial causative factor responsible for the onset and progression of AMD. A2E, a major component of toxic lipofuscin implicated in AMD, is deposited in RPE cells with age. However, the mechanism whereby A2E may contribute to the pathogenesis of AMD remains unclear. We demonstrated that A2E was a danger signal of RPE cells, which induced autophagy and decreased cell viability in a concentration- and time-dependent manner. Within 15 min after the treatment of RPE with 25 μM A2E, the induction of autophagosome was detected by transmission electron microscopy. After continuous incubating RPE cells with A2E, intense punctate staining of LC3 and increased expression of LC3-II and Beclin-1 were identified. Meanwhile, the levels of intercellular adhesion molecule (ICAM), interleukin (IL)1β, IL2, IL-6, IL-8, IL-17A, IL-22, macrophage cationic peptide (MCP)-1, stromal cell-derived factor (SDF)-1, and vascular endothelial growth factor A (VEGFA) were elevated. The autophagic inhibitor 3-methyladenine (3-MA) and activator rapamycin were also used to verify the effect of autophagy on RPE cells against A2E. Our results revealed that 3-MA decreased the autophagosomes and LC3 puncta induced by A2E, increased inflammation-associated protein expression including ICAM, IL1β, IL2, IL-6, IL-8, IL-17A, IL-22, and SDF-1, and upregulated VEGFA expression. Whereas rapamycin augmented the A2E-mediated autophagy, attenuated protein expression of inflammation-associated and angiogenic factors, and blocked the Akt/mTOR pathway. Taken together, A2E induces autophagy in RPE cells at the early stage of incubation, and this autophagic response can be inhibited by 3-MA or augmented by rapamycin via the mTOR pathway. The enhancement of autophagy has a protective role in RPE cells against the adverse effects of A2E by reducing the

  2. Practical Parallel Rendering

    CERN Document Server

    Chalmers, Alan

    2002-01-01

    Meeting the growing demands for speed and quality in rendering computer graphics images requires new techniques. Practical parallel rendering provides one of the most practical solutions. This book addresses the basic issues of rendering within a parallel or distributed computing environment, and considers the strengths and weaknesses of multiprocessor machines and networked render farms for graphics rendering. Case studies of working applications demonstrate, in detail, practical ways of dealing with complex issues involved in parallel processing.

  3. Sex-specific retinal pigmentation results in sexually dimorphic long-wavelength-sensitive photoreceptors in the eastern pale clouded yellow butterfly, Colias erate

    NARCIS (Netherlands)

    Ogawa, Yuri; Kinoshita, Michiyo; Stavenga, Doekele G.; Arikawa, Kentaro

    The compound eyes of the eastern pale clouded yellow butterfly, Colias erate, contain three types of ommatidia (I, II and III), identifiable by the differing arrangements of pigment clusters around the rhabdoms. The pigment color is red in all ommatidial types except for type II ommatidia of

  4. Sex-specific retinal pigmentation results in sexually dimorphic long-wavelength-sensitive photoreceptors in the eastern pale clouded yellow butterfly, Colias erate

    NARCIS (Netherlands)

    Ogawa, Yuri; Kinoshita, Michiyo; Stavenga, Doekele G.; Arikawa, Kentaro

    2013-01-01

    The compound eyes of the eastern pale clouded yellow butterfly, Colias erate, contain three types of ommatidia (I, II and III), identifiable by the differing arrangements of pigment clusters around the rhabdoms. The pigment color is red in all ommatidial types except for type II ommatidia of females

  5. Tyrosinase-Cre-Mediated Deletion of the Autophagy Gene Atg7 Leads to Accumulation of the RPE65 Variant M450 in the Retinal Pigment Epithelium of C57BL/6 Mice

    Science.gov (United States)

    Sukseree, Supawadee; Chen, Ying-Ting; Laggner, Maria; Gruber, Florian; Petit, Valérie; Nagelreiter, Ionela-Mariana; Mlitz, Veronika; Rossiter, Heidemarie; Pollreisz, Andreas; Schmidt-Erfurth, Ursula; Larue, Lionel; Tschachler, Erwin

    2016-01-01

    Targeted gene knockout mouse models have helped to identify roles of autophagy in many tissues. Here, we investigated the retinal pigment epithelium (RPE) of Atg7f/f Tyr-Cre mice (on a C57BL/6 background), in which Cre recombinase is expressed under the control of the tyrosinase promoter to delete the autophagy gene Atg7. In line with pigment cell-directed blockade of autophagy, the RPE and the melanocytes of the choroid showed strong accumulation of the autophagy adaptor and substrate, sequestosome 1 (Sqstm1)/p62, relative to the levels in control mice. Immunofluorescence and Western blot analysis demonstrated that the RPE, but not the choroid melanocytes, of Atg7f/f Tyr-Cre mice also had strongly increased levels of retinoid isomerohydrolase RPE65, a pivotal enzyme for the maintenance of visual perception. In contrast to Sqstm1, genes involved in retinal regeneration, i.e. Lrat, Rdh5, Rgr, and Rpe65, were expressed at higher mRNA levels. Sequencing of the Rpe65 gene showed that Atg7f/f and Atg7f/f Tyr-Cre mice carry a point mutation (L450M) that is characteristic for the C57BL/6 mouse strain and reportedly causes enhanced degradation of the RPE65 protein by an as-yet unknown mechanism. These results suggest that the increased abundance of RPE65 M450 in the RPE of Atg7f/f Tyr-Cre mice is, at least partly, mediated by upregulation of Rpe65 transcription; however, our data are also compatible with the hypothesis that the RPE65 M450 protein is degraded by Atg7-dependent autophagy in Atg7f/f mice. Further studies in mice of different genetic backgrounds are necessary to determine the relative contributions of these mechanisms. PMID:27537685

  6. Retinal Pigment Epithelium and Müller Progenitor Cell Interaction Increase Müller Progenitor Cell Expression of PDGFR and Ability to Induce Proliferative Vitreoretinopathy in a Rabbit Model

    Directory of Open Access Journals (Sweden)

    Gisela Velez

    2012-01-01

    Full Text Available Purpose. Proliferative vitreoretinopathy (PVR is a complication of retinal detachment characterized by redetachment of the retina as a result of membrane formation and contraction. A variety of retinal cells, including retinal pigment epithelial (RPE and Müller glia, and growth factors may be responsible. Platelet-derived growth factor receptor alpha (PDGFRα is found in large quantities in PVR membranes, and is intrinsic to the development of PVR in rabbit models. This study explores the expression of PDGFR in cocultures of RPE and Müller cells over time to examine how these two cell types may collaborate in the development of PVR. We also examine how changes in PDGFRα expression alter Müller cell pathogenicity. Methods. Human MIO-M1 Müller progenitor (MPC and ARPE19 cells were studied in a transmembrane coculture system. Immunocytochemistry and Western blot were used to look at PDGFRα, PDGFRβ, and GFAP expression. A transfected MPC line cell line expressing the PDGFRα (MIO-M1α was generated, and tested in a rabbit model for its ability to induce PVR. Results. The expression of PDGFRα and PDGFRβ was upregulated in MIO-M1 MPCs cocultured with ARPE19 cells; GFAP was slightly decreased. Increased expression of PDGFRα in the MIO-M1 cell line resulted in increased pathogenicity and enhanced ability to induce PVR in a rabbit model. Conclusions. Müller and RPE cell interaction can lead to upregulation of PDGFRα and increased Müller cell pathogenicity. Müller cells may play a more active role than previously thought in the development of PVR membranes, particularly when stimulated by an RPE-cell-rich environment. Additional studies of human samples and in animal models are warranted.

  7. Duration of rhegmatogenous retinal detachment predicts recovery of retinal sensitivity

    Directory of Open Access Journals (Sweden)

    Rose Rose

    2016-02-01

    Full Text Available The decision to treat a disease is often based on the presence or absence of symptoms, one prototype case being rhegmatogenous retinal detachment. Detachment of the neural retina from the pigment epithelium is a major cause of anatomical and functional dysfunction of the retina, where retinal recovery is inversely related to duration of detachment. The purpose of retinal reattachment is to effect recovery of the photoreceptors and pigment epithelium from degeneration. The aim of this study was to determine the critical duration of rhegmatogenous retinal detachment resulting in optimal retinal recovery after reattachment. A prospective study was conducted at a private hospital in Yogyakarta. Thirty five eyes were involved in this study. Three months after reattachment, central retinal recovery was measured by means of a Goldmann manual kinetic perimeter. The results showed that retinal recovery developed three months after surgery if the onset of rhegmatogenous retinal detachment was less than 28 days before surgery. The results were not significant if the onset of rhegmatogenous retinal detachment was more than 35 days. Although the Goldmann manual kinetic perimeter can efficiently detect central retinal sensitivity, it should be supported by more sensitive tools to evaluate the anatomy and function of the retina.

  8. Retinitis pigmentosa

    Directory of Open Access Journals (Sweden)

    Hamel Christian

    2006-10-01

    Full Text Available Abstract Retinitis pigmentosa (RP is an inherited retinal dystrophy caused by the loss of photoreceptors and characterized by retinal pigment deposits visible on fundus examination. Prevalence of non syndromic RP is approximately 1/4,000. The most common form of RP is a rod-cone dystrophy, in which the first symptom is night blindness, followed by the progressive loss in the peripheral visual field in daylight, and eventually leading to blindness after several decades. Some extreme cases may have a rapid evolution over two decades or a slow progression that never leads to blindness. In some cases, the clinical presentation is a cone-rod dystrophy, in which the decrease in visual acuity predominates over the visual field loss. RP is usually non syndromic but there are also many syndromic forms, the most frequent being Usher syndrome. To date, 45 causative genes/loci have been identified in non syndromic RP (for the autosomal dominant, autosomal recessive, X-linked, and digenic forms. Clinical diagnosis is based on the presence of night blindness and peripheral visual field defects, lesions in the fundus, hypovolted electroretinogram traces, and progressive worsening of these signs. Molecular diagnosis can be made for some genes, but is not usually performed due to the tremendous genetic heterogeneity of the disease. Genetic counseling is always advised. Currently, there is no therapy that stops the evolution of the disease or restores the vision, so the visual prognosis is poor. The therapeutic approach is restricted to slowing down the degenerative process by sunlight protection and vitaminotherapy, treating the complications (cataract and macular edema, and helping patients to cope with the social and psychological impact of blindness. However, new therapeutic strategies are emerging from intensive research (gene therapy, neuroprotection, retinal prosthesis.

  9. 基因转染的虹膜色素上皮细胞移植后RCS鼠视网膜BDNF表达观察%Retinal BDNF expressions in RCS rats after transplantation of gene transfected iris pigment epithelium

    Institute of Scientific and Technical Information of China (English)

    张英瑜; 高朋芬; 杨丽霞

    2011-01-01

    目的 探讨脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)基因转染的虹膜色素上皮细胞(AAV-BDNF-IPE)移植入皇家外科学院(royal college of surgeons,RCS)大鼠视网膜下腔后,不同时期视网膜组织BDNF表达变化.方法 通过外路途径将BDNF基因转染的虹膜色素上皮细胞移植到RCS大鼠视网膜下腔,术后3、5、7、9、11周分别取RCS大鼠手术眼及对照组动物眼视网膜组织,用酶联免疫吸附法(Elisa)检测视网膜组织中BDNF的表达水平,比较分析这些数据.结果 对照组RCS大鼠出生后3周龄时视网膜组织中BDNF仍保持较高水平,其后迅速降低,其中3周龄组与其它周龄组比较,P<0.01;手术组RCS大鼠术时、术后3、5、7、9、11周各组间两两比较,BDNF表达无显著差异(P>0.05);出生后6周龄直到14周龄的不同时期,AAV-BDNF-IPE移植手术组RCS大鼠视网膜BDNF表达水平均明显高于对照组(其中6周龄组P<0.05,其它各周龄组P<0.01).结论 BDNF基因转染的虹膜色素上皮细胞在RCS大鼠视网膜下腔移植后,视网膜组织中BDNF可以持续稳定高水平表达,这为临床开发新的神经营养因子给药方式提供了实验依据.%Objective To investigate the retinal brain derived neurotrophic factor( BDNF) expressions in different phases of royal college of surgeons( RCS) rats after BDNF transfected iris pigment epithelium( AAV-BDNF-IPE) being transplanted into the subretinal space of RCS rat. Methods AAV-BDNF-IPEs were transplanted into the subretinal space of RCS rats. BDNF expressions in retinal tissue of intact RCS rats and surgery RCS rats were detected by enzyme linked immunosorbent assay ( Elisa) at 3 ,5 ,7 ,9and 11 weeks after surgery. Results BDNF expressions in retinal tissue of intact RCS rats were still high at postnatal 3w and were sharply decreased into low level later; retinal BDNF expression of intact RCS rats at postnatal 3w were much higher than those at other

  10. Video-based rendering

    CERN Document Server

    Magnor, Marcus A

    2005-01-01

    Driven by consumer-market applications that enjoy steadily increasing economic importance, graphics hardware and rendering algorithms are a central focus of computer graphics research. Video-based rendering is an approach that aims to overcome the current bottleneck in the time-consuming modeling process and has applications in areas such as computer games, special effects, and interactive TV. This book offers an in-depth introduction to video-based rendering, a rapidly developing new interdisciplinary topic employing techniques from computer graphics, computer vision, and telecommunication en

  11. Safety and Proof-of-Concept Study of Oral QLT091001 in Retinitis Pigmentosa Due to Inherited Deficiencies of Retinal Pigment Epithelial 65 Protein (RPE65 or Lecithin:Retinol Acyltransferase (LRAT.

    Directory of Open Access Journals (Sweden)

    Hendrik P N Scholl

    Full Text Available Restoring vision in inherited retinal degenerations remains an unmet medical need. In mice exhibiting a genetically engineered block of the visual cycle, vision was recently successfully restored by oral administration of 9-cis-retinyl acetate (QLT091001. Safety and visual outcomes of a once-daily oral dose of 40 mg/m2/day QLT091001 for 7 consecutive days was investigated in an international, multi-center, open-label, proof-of-concept study in 18 patients with RPE65- or LRAT-related retinitis pigmentosa. Eight of 18 patients (44% showed a ≥20% increase and 4 of 18 (22% showed a ≥40% increase in functional retinal area determined from Goldmann visual fields; 12 (67% and 5 (28% of 18 patients showed a ≥5 and ≥10 ETDRS letter score increase of visual acuity, respectively, in one or both eyes at two or more visits within 2 months of treatment. In two patients who underwent fMRI, a significant positive response was measured to stimuli of medium contrast, moving, pattern targets in both left and right hemispheres of the occipital cortex. There were no serious adverse events. Treatment-related adverse events were transient and the most common included headache, photophobia, nausea, vomiting, and minor biochemical abnormalities. Measuring the outer segment length of the photoreceptor layer with high-definition optical coherence tomography was highly predictive of treatment responses with responders having a significantly larger baseline outer segment thickness (11.7 ± 4.8 μm, mean ± 95% CI than non-responders (3.5 ± 1.2 μm. This structure-function relationship suggests that treatment with QLT091001 is more likely to be efficacious if there is sufficient photoreceptor integrity.ClinicalTrials.gov NCT01014052.

  12. Safety and Proof-of-Concept Study of Oral QLT091001 in Retinitis Pigmentosa Due to Inherited Deficiencies of Retinal Pigment Epithelial 65 Protein (RPE65) or Lecithin:Retinol Acyltransferase (LRAT).

    Science.gov (United States)

    Scholl, Hendrik P N; Moore, Anthony T; Koenekoop, Robert K; Wen, Yuquan; Fishman, Gerald A; van den Born, L Ingeborgh; Bittner, Ava; Bowles, Kristen; Fletcher, Emily C; Collison, Frederick T; Dagnelie, Gislin; Degli Eposti, Simona; Michaelides, Michel; Saperstein, David A; Schuchard, Ronald A; Barnes, Claire; Zein, Wadih; Zobor, Ditta; Birch, David G; Mendola, Janine D; Zrenner, Eberhart

    2015-01-01

    Restoring vision in inherited retinal degenerations remains an unmet medical need. In mice exhibiting a genetically engineered block of the visual cycle, vision was recently successfully restored by oral administration of 9-cis-retinyl acetate (QLT091001). Safety and visual outcomes of a once-daily oral dose of 40 mg/m2/day QLT091001 for 7 consecutive days was investigated in an international, multi-center, open-label, proof-of-concept study in 18 patients with RPE65- or LRAT-related retinitis pigmentosa. Eight of 18 patients (44%) showed a ≥20% increase and 4 of 18 (22%) showed a ≥40% increase in functional retinal area determined from Goldmann visual fields; 12 (67%) and 5 (28%) of 18 patients showed a ≥5 and ≥10 ETDRS letter score increase of visual acuity, respectively, in one or both eyes at two or more visits within 2 months of treatment. In two patients who underwent fMRI, a significant positive response was measured to stimuli of medium contrast, moving, pattern targets in both left and right hemispheres of the occipital cortex. There were no serious adverse events. Treatment-related adverse events were transient and the most common included headache, photophobia, nausea, vomiting, and minor biochemical abnormalities. Measuring the outer segment length of the photoreceptor layer with high-definition optical coherence tomography was highly predictive of treatment responses with responders having a significantly larger baseline outer segment thickness (11.7 ± 4.8 μm, mean ± 95% CI) than non-responders (3.5 ± 1.2 μm). This structure-function relationship suggests that treatment with QLT091001 is more likely to be efficacious if there is sufficient photoreceptor integrity. ClinicalTrials.gov NCT01014052.

  13. Whole number, distribution and co-expression of brn3 transcription factors in retinal ganglion cells of adult albino and pigmented rats.

    Directory of Open Access Journals (Sweden)

    Francisco M Nadal-Nicolás

    Full Text Available The three members of the Pou4f family of transcription factors: Pou4f1, Pou4f2, Pou4f3 (Brn3a, Brn3b and Brn3c, respectively play, during development, essential roles in the differentiation and survival of sensory neurons. The purpose of this work is to study the expression of the three Brn3 factors in the albino and pigmented adult rat. Animals were divided into these groups: i untouched; ii fluorogold (FG tracing from both superior colliculli; iii FG-tracing from one superior colliculus; iv intraorbital optic nerve transection or crush. All retinas were dissected as flat-mounts and subjected to single, double or triple immunohistofluorescence The total number of FG-traced, Brn3a, Brn3b, Brn3c or Brn3 expressing RGCs was automatically quantified and their spatial distribution assessed using specific routines. Brn3 factors were studied in the general RGC population, and in the intrinsically photosensitive (ip-RGCs and ipsilateral RGC sub-populations. Our results show that: i 70% of RGCs co- express two or three Brn3s and the remaining 30% express only Brn3a (26% or Brn3b; ii the most abundant Brn3 member is Brn3a followed by Brn3b and finally Brn3c; iii Brn3 a-, b- or c- expressing RGCs are similarly distributed in the retina; iv The vast majority of ip-RGCs do not express Brn3; v The main difference between both rat strains was found in the population of ipsilateral-RGCs, which accounts for 4.2% and 2.5% of the total RGC population in the pigmented and albino strain, respectively. However, more ipsilateral-RGCs express Brn3 factors in the albino than in the pigmented rat; vi RGCs that express only Brn3b and RGCs that co-express the three Brn3 members have the biggest nuclei; vii After axonal injury the level of Brn3a expression in the surviving RGCs decreases compared to control retinas. Finally, this work strengthens the validity of Brn3a as a marker to identify and quantify rat RGCs.

  14. Anti-apoptotic effects of Curcuma longa L. extract and its curcuminoids against blue light-induced cytotoxicity in A2E-laden human retinal pigment epithelial cells.

    Science.gov (United States)

    Park, Sang-Il; Lee, Eun Hye; Kim, So Ra; Jang, Young Pyo

    2017-03-01

    The purpose of the study was to investigate the protective effect of the Curcuma longa L. extract (CLE) and its curcuminoids against blue light-induced cytotoxicity in human retinal pigment epithelial (RPE) cells laded with A2E. A2E has been concerned in age-related macular degeneration (AMD). To perform this study, A2E-accumulated ARPE-19 cells were exposed to blue light to induce cytotoxicity. The cytotoxicity and apoptotic gene expression levels were evaluated using a lactate dehydrogenase (LDH) assay and real-time PCR analysis, respectively. Curcuma longa L. extract was found to exert a protective effect in a dose-dependent manner. At a concentration of 15 μm, curcumin, demethoxycurcumin and bisdemethoxycurcumin exerted significant protective effects against blue light-induced cytotoxicity. Treatment with CLE and curcuminoids meaningfully reduced the mRNA levels of c-Abl and p53, which was known to be augmented in apoptotic RPE cells. Demethoxycurcumin and bisdemethoxycurcumin were found to inhibit p38 expression, which is increased in blue light-irradiated A2E-accumulated RPE cells. Curcuma longa L. extract and its curcuminoids provided significant protection against photooxidative damage and apoptosis in the RPE cells. Our results suggest that curcuminoids may show potential in the treatment of AMD. © 2017 Royal Pharmaceutical Society.

  15. Wnt信号通路在视网膜色素上皮发育中的作用%The roles of Wnt signaling pathway in the development of retinal pigment epithelium

    Institute of Scientific and Technical Information of China (English)

    殷秋菊; 赵娉婷; 杨春波; 李筱荣

    2014-01-01

    Wnt signaling plays important roles in embryonic development and cell differentiation, especially during central nervous system (CNS) development. At the early stage of eye development, retinal pigment epithelium (RPE) in the dorsal optic vesicle shows intensive Wnt/β-catenin signaling activity, which is critical for the development of neural retina and RPE. This review focuses on recent advances in the fields of Wnt signaling pathway, Wnt protein family and the relationship between Wnt signaling and RPE development and differentiation.%Wnt(wingless-type MMTV integration site family members)信号通路与细胞的发育分化密切相关,尤其对动物胚胎期中枢神经系统的发育至关重要。在眼的早期发育中,视泡背部视网膜色素上皮细胞(RPE)Wnt/βcatenin信号通路高度活跃,对神经视网膜及RPE的发育调控起重要作用。本文结合目前该领域研究进展,综合评述Wnt信号通路、Wnt蛋白家族以及Wnt信号通路与RPE发育的关系。

  16. Resveratrol inhibits transforming growth factor-β2-induced epithelial-to-mesenchymal transition in human retinal pigment epithelial cells by suppressing the Smad pathway

    Science.gov (United States)

    Chen, Ching-Long; Chen, Yi-Hao; Tai, Ming-Cheng; Liang, Chang-Min; Lu, Da-Wen; Chen, Jiann-Torng

    2017-01-01

    Proliferative vitreoretinopathy (PVR) is the main cause of failure following retinal detachment surgery. Transforming growth factor (TGF)-β2-induced epithelial-to-mesenchymal transition (EMT) plays an important role in the development of PVR, and EMT inhibition decreases collagen gel contraction and fibrotic membrane formation, resulting in prevention of PVR. Resveratrol is naturally found in red wine and has inhibitory effects on EMT. Resveratrol is widely used in cardioprotection, neuroprotection, chemotherapy, and antiaging therapy. The purpose of this study was to investigate the effects of resveratrol on TGF-β2-induced EMT in ARPE-19 cells in vitro. We found that resveratrol suppressed the decrease of zona occludens-1 (ZO-1) and caused an increase of alpha-smooth muscle actin expression in TGF-β2-treated ARPE-19 cells, assessed using Western blots; moreover, it also suppressed the decrease in ZO-1 and the increase of vimentin expression, observed using immunocytochemistry. Resveratrol attenuated TGF-β2-induced wound closure and cell migration in ARPE-19 cells in a scratch wound test and modified Boyden chamber assay, respectively. We also found that resveratrol reduced collagen gel contraction – assessed by collagen matrix contraction assay – and suppressed the phosphorylation of Smad2 and Smad3 in TGF-β2-treated ARPE-19 cells. These results suggest that resveratrol mediates anti-EMT effects, which could be used in the prevention of PVR.

  17. Resveratrol inhibits transforming growth factor-β2-induced epithelial-to-mesenchymal transition in human retinal pigment epithelial cells by suppressing the Smad pathway.

    Science.gov (United States)

    Chen, Ching-Long; Chen, Yi-Hao; Tai, Ming-Cheng; Liang, Chang-Min; Lu, Da-Wen; Chen, Jiann-Torng

    2017-01-01

    Proliferative vitreoretinopathy (PVR) is the main cause of failure following retinal detachment surgery. Transforming growth factor (TGF)-β2-induced epithelial-to-mesenchymal transition (EMT) plays an important role in the development of PVR, and EMT inhibition decreases collagen gel contraction and fibrotic membrane formation, resulting in prevention of PVR. Resveratrol is naturally found in red wine and has inhibitory effects on EMT. Resveratrol is widely used in cardioprotection, neuroprotection, chemotherapy, and antiaging therapy. The purpose of this study was to investigate the effects of resveratrol on TGF-β2-induced EMT in ARPE-19 cells in vitro. We found that resveratrol suppressed the decrease of zona occludens-1 (ZO-1) and caused an increase of alpha-smooth muscle actin expression in TGF-β2-treated ARPE-19 cells, assessed using Western blots; moreover, it also suppressed the decrease in ZO-1 and the increase of vimentin expression, observed using immunocytochemistry. Resveratrol attenuated TGF-β2-induced wound closure and cell migration in ARPE-19 cells in a scratch wound test and modified Boyden chamber assay, respectively. We also found that resveratrol reduced collagen gel contraction - assessed by collagen matrix contraction assay - and suppressed the phosphorylation of Smad2 and Smad3 in TGF-β2-treated ARPE-19 cells. These results suggest that resveratrol mediates anti-EMT effects, which could be used in the prevention of PVR.

  18. Spontaneous or secondary to intravitreal injections of anti-angiogenic agents retinal pigment epithelial tears in age-related macular degeneration

    Institute of Scientific and Technical Information of China (English)

    Pia; E.; Leon; Sandro; Saviano; Andrea; Zanei; Marco; R.; Pastore; Elvira; Guaglione; Alessandro; Mangogna; Daniele; Tognetto

    2014-01-01

    ·AIM:Toevaluatethevisualfunctionevolutionofretinal pigment epithelial(RPE) tears in patients with age-related macular degeneration(AMD) according to type of occurrence [spontaneous or secondary to anti-vascular endothelial growth factor(anti-VEGF) injection] and the topographic location of the tear after a two-year followup period.·METHODS: A total of 15 eyes of 14 patients with RPE tears in exudative AMD were analyzed retrospectively at the University Eye Clinic of Trieste. Inclusion criteria were: patient age of 50 or older with AMD and RPE tears both spontaneous occurring or post anti-VEGF treatment. Screening included: careful medical history,complete ophthalmological examination, fluorescein angiography(FA), indocyanine green angiography(ICG),autofluorescence and infrared imaging and optical coherence tomography(OCT). Patients were evaluated every month for visual acuity(VA), fundus examination and OCT. Other data reported were: presence of PED,number of injections before the tear, location of the lesion.·RESULTS:Meanfollow-up was24wk(SD±4wk). Atotal of 15 eyes were studied for RPE tear. In 6 cases(40%),the RPE tears occurred within two years of anti-VEGF injections the others occurred spontaneously. In 13cases(86.6%), the RPE tear was associated with pigment epithelial detachment(PED). In 7 cases(46.6%), the RPE tear occurred in the central area of the retina and involved the fovea. Two lesions were found in the parafoveal region, six in the extra-macular area. In all cases visual acuity decreased at the end of the follow-up period(P <0.01) independently of the type or the topographical location of the lesion.·CONCLUSION: RPE tear occurs in exudative AMD as a spontaneous complication or in relation to anti-VEGF injections. Visual acuity decreased significantly and gradually in the follow-up period in all cases. No correlation was found between visual loss and the type of onset or the topographic location of the tears.

  19. Rendering the Topological Spines

    Energy Technology Data Exchange (ETDEWEB)

    Nieves-Rivera, D. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-05-05

    Many tools to analyze and represent high dimensional data already exits yet most of them are not flexible, informative and intuitive enough to help the scientists make the corresponding analysis and predictions, understand the structure and complexity of scientific data, get a complete picture of it and explore a greater number of hypotheses. With this in mind, N-Dimensional Data Analysis and Visualization (ND²AV) is being developed to serve as an interactive visual analysis platform with the purpose of coupling together a number of these existing tools that range from statistics, machine learning, and data mining, with new techniques, in particular with new visualization approaches. My task is to create the rendering and implementation of a new concept called topological spines in order to extend ND²AV's scope. Other existing visualization tools create a representation preserving either the topological properties or the structural (geometric) ones because it is challenging to preserve them both simultaneously. Overcoming such challenge by creating a balance in between them, the topological spines are introduced as a new approach that aims to preserve them both. Its render using OpenGL and C++ and is currently being tested to further on be implemented on ND²AV. In this paper I will present what are the Topological Spines and how they are rendered.

  20. In vitro culture and phagocytosis of human embryonic retinal pigment epithelium%人胚胎视网膜色素上皮细胞的体外培养和吞噬作用

    Institute of Scientific and Technical Information of China (English)

    靳晓亮; 徐国彤; 张文芳; 鲁建华

    2011-01-01

    Objective To analyze the culturing characteristics and phagocytosis of the retinal pigment epithelium (RPE) from human embryo, and to provide the cellular basis for the further research of RPE related diseases. Methods Fresh human embryonic eyeballs (13~15 weeks) were collected, and RPE cells were separated by mechanical and trypsin digestion after microdissec-tion. Human embryonic RPE phagocytosis of retinal outer segments was investigated by using immunofluorescence. Results After the human embryo had developed for 13~15 weeks, the RPE cells could be cultured in vitro according to standard culturing methods. Based on the growth curve, the RPE cells in generations 2~6 were collected for essays and showed the ability of phagocytosis afterwards. Conclusion The method of culturing human embryonic RPE cells is improved, and phagocytosis ability of human embryonic RPE cells is also proved in vitro according to this study.%目的 探讨人胚胎视网膜色素上皮(RPE)细胞分离培养的特性及其吞噬作用.方法 取发育至13~15周的新鲜人胚胎眼球,显微解剖后采用机械胰酶消化法分离培养RPE细胞,应用免疫荧光法观察人胚胎RPE细胞吞噬视网膜光感受细胞外节膜盘.结果 人胚胎发育至13~15周后,RPE细胞可应用标准的培养方法进行体外培养和传代.根据测定的人胚胎RPE细胞生长曲线,取2~6代细胞用于实验培养的人胚胎RPE细胞具备吞噬功能.结论 改进了人胚胎RPE细胞的培养方法,证明人胚胎RPE细胞在体外具有吞噬能力.

  1. Up-regulation of HIF-1α and VEGF Expression by Elevated Glucose Concentration and Hypoxia in Cultured Human Retinal Pigment Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    XIAO Qing; ZENG Shuiqing; LING Shiqi; LV Mingliang

    2006-01-01

    In order to explore the effect of high glucose concentration and high glucose concentration with hypoxia on the production of hypoxia-inducible factor-1 α (HIF-1α) and vascular endothelial growth factor (VEGF), human RPE cells were cultured in 5.56 mmol/L glucose (control group), 5.56 mmol/L glucose with 150 μ mol/L CoCl2 (hypoxic group), 25 mmol/L glucose (high glucose group)and 25 mmol/L glucose with 150 μ mol/L CoCl2 (combination group). RT-PCR was used to detect the expression of HIF-1α and VEGF mRNAs. Western blot analysis was used to measure the levels of HIF-1α and VEGF proteins. Although the small amount of HIF-1α protein was able to be detected in high glucose group but not in control group, there was no significant difference between the expression of HIF-1α mRNA of RPE cells in high glucose group and that of RPE cells in control group.As compared with RPE cells in control group, the mRNA expression and the protein synthesis of VEGF in high glucose group were up-regulated. As compared with RPE cells in hypoxic group, the expression of HIF-1α mRNA of RPE cells in combination group was not different, but the protein synthesis of HIF-1 α, the mRNA expression and the protein synthesis of VEGF were more obviously up-regulated. In conclusion, high concentration glucose mainly influence the protein synthesis of HIF-1α of RPE cell, and HIF-1α protein is able to be accumulated in high concentration glucose.Under hypoxia, the HIF-1α protein induced by high concentration glucose is more stable, and the expression of VEGF is obviously increased. It is suggested that high concentration glucose may play a role in retinal neovascularization, especially at ischemia stage of diabetic retinopathy.

  2. High Fidelity Haptic Rendering

    CERN Document Server

    Otaduy, Miguel A

    2006-01-01

    The human haptic system, among all senses, provides unique and bidirectional communication between humans and their physical environment. Yet, to date, most human-computer interactive systems have focused primarily on the graphical rendering of visual information and, to a lesser extent, on the display of auditory information. Extending the frontier of visual computing, haptic interfaces, or force feedback devices, have the potential to increase the quality of human-computer interaction by accommodating the sense of touch. They provide an attractive augmentation to visual display and enhance t

  3. Selective impairment of a subset of Ran-GTP-binding domains of ran-binding protein 2 (Ranbp2) suffices to recapitulate the degeneration of the retinal pigment epithelium (RPE) triggered by Ranbp2 ablation.

    Science.gov (United States)

    Patil, Hemangi; Saha, Arjun; Senda, Eugene; Cho, Kyoung-in; Haque, MdEmdadul; Yu, Minzhong; Qiu, Sunny; Yoon, Dosuk; Hao, Ying; Peachey, Neal S; Ferreira, Paulo A

    2014-10-24

    Retinal pigment epithelium (RPE) degeneration underpins diseases triggered by disparate genetic lesions, noxious insults, or both. The pleiotropic Ranbp2 controls the expression of intrinsic and extrinsic pathological stressors impinging on cellular viability. However, the physiological targets and mechanisms controlled by Ranbp2 in tissue homeostasis, such as RPE, are ill defined. We show that mice, RPE-cre::Ranbp2(-/-), with selective Ranbp2 ablation in RPE develop pigmentary changes, syncytia, hypoplasia, age-dependent centrifugal and non-apoptotic degeneration of the RPE, and secondary leakage of choriocapillaris. These manifestations are accompanied by the development of F-actin clouds, metalloproteinase-11 activation, deregulation of expression or subcellular localization of critical RPE proteins, atrophic cell extrusions into the subretinal space, and compensatory proliferation of peripheral RPE. To gain mechanistic insights into what Ranbp2 activities are vital to the RPE, we performed genetic complementation analyses of transgenic lines of bacterial artificial chromosomes of Ranbp2 harboring loss of function of selective Ranbp2 domains expressed in a Ranbp2(-/-) background. Among the transgenic lines produced, only Tg(RBD2/3*-HA)::RPE-cre::Ranbp2(-/-)-expressing mutations, which selectively impair binding of RBD2/3 (Ran-binding domains 2 and 3) of Ranbp2 to Ran-GTP, recapitulate RPE degeneration, as observed with RPE-cre::Ranbp2(-/-). By contrast, Tg(RBD2/3*-HA) expression rescues the degeneration of cone photoreceptors lacking Ranbp2. The RPE of RPE-cre::Ranbp2(-/-) and Tg(RBD2/3*-HA)::RPE-cre::Ranbp2(-/-) share proteostatic deregulation of Ran GTPase, serotransferrin, and γ-tubulin and suppression of light-evoked electrophysiological responses. These studies unravel selective roles of Ranbp2 and its RBD2 and RBD3 in RPE survival and functions. We posit that the control of Ran GTPase by Ranbp2 emerges as a novel therapeutic target in diseases promoting

  4. 线粒体DNA损伤与视网膜色素上皮细胞关系的研究进展%The relationship between mitochondrial DNA damage and retinal pigment epithelium cells

    Institute of Scientific and Technical Information of China (English)

    俞永珍; 邹秀兰; 邹玉平

    2015-01-01

    Mitochondrial DNA (mtDNA) is a genetic effect DNA molecule of double closed loop, and is crucial for cells and their functions. Mitochondria take an active part in physiological activities of retinal pigment epithelium (RPE) cells. The oxidative stress is usually occurred in RPE for its active metabolism, which can lead to mitochondria and mtDNA dam⁃age. Once mitochondria and mtDNA lesions have not been repaired timely, the lesions can be accumulated, which can cause dysfunctions and damaged-structures of RPE and mitochondria, and can motivate the progression of cell apoptosis. In the end it can result in some ocular related diseases such as aged-related macular degeneration (AMD). This study reviewed the functional relationship between mtDNA and RPE, and repair and detection methods of mtDNA damage.%线粒体DNA(mtDNA)是线粒体内具有遗传效应的双股闭环DNA分子,对细胞及其功能具有重要作用。视网膜色素上皮(RPE)细胞活动亦由大量线粒体参与。因RPE细胞代谢活跃,当发生氧化应激时可引起线粒体及mtDNA损伤;当线粒体及mtDNA损伤无法及时修复而使损伤积累,可引起RPE及线粒体功能障碍,并诱发启动细胞凋亡,进而引发某些眼病,如年龄相关性黄斑变性等。现就mtDNA与RPE细胞的功能关系、mtDNA损伤修复及检测方法作一综述。

  5. The complex interplay between ERK1/2, TGFβ/Smad, and Jagged/Notch signaling pathways in the regulation of epithelial-mesenchymal transition in retinal pigment epithelium cells.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Chen

    Full Text Available Epithelial-mesenchymal transition (EMT of retinal pigment epithelium (RPE cells is a major pathologic change in the development of proliferative vitreoretinopathy (PVR, which leads to severe visual impairment. ERK1/2 pathway has been reported to play a key role in the carcinogenesis, cancer metastasis, and multiple fibrotic diseases. We hypothesized that ERK1/2 signaling could cross-interact with transforming growth factor β2 (TGFβ2/Smad and Notch signaling pathways in the regulation of EMT in RPE cells. Here, we demonstrated that ERK1/2 signaling was activated in TGFβ2-induced EMT in human RPE cells, while blockade of the canonical TGFβ2/Smad2/3 signaling with SB431542 could not inhibit TGFβ2-induced the activation of ERK1/2. Meanwhile, blockade of ERK1/2 signaling with a specific MEK/ERK1/2 inhibitor U0126 strongly prevented TGFβ2-induced the downregulation of P-cadherin, and the upregulation of α-SMA, collagen type IV, N-cadherin and fibronectin in RPE cells. In addition, we also identified that blockade of ERK1/2 signaling could inhibit not only the canonical TGFβ/Smad signaling, but also the Jagged/Notch pathway. Finally, we found that blockade of Notch pathway with a specific inhibitor DAPT could inhibit TGFβ2-induced the activation of ERK1/2 pathway conversely. Therefore, our study provides evidence that ERK1/2 signaling can cross-interact with the canonical TGFβ/Smad and the Jagged/Notch signaling pathways in RPE cells EMT. ERK1/2 inhibitor may have therapeutic value in the prevention and treatment of PVR and other fibrotic diseases.

  6. The novel triterpenoid RTA 408 protects human retinal pigment epithelial cells against H2O2-induced cell injury via NF-E2-related factor 2 (Nrf2 activation

    Directory of Open Access Journals (Sweden)

    Xiaobin Liu

    2016-08-01

    Full Text Available Oxidative stress-induced retinal pigment epithelial (RPE cell damage is an important factor in the pathogenesis of age-related macular degeneration (AMD. Previous studies have shown that RTA 408, a synthetic triterpenoid compound, potently activates Nrf2. This study aimed to investigate the protective effects of RTA 408 in cultured RPE cells during oxidative stress and to determine the effects of RTA 408 on Nrf2 and its downstream target genes. Primary human RPE cells were pretreated with RTA 408 and then incubated in 200 μM H2O2 for 6 h. Cell viability was measured with the WST-8 assay. Apoptosis was quantitatively measured by annexin V/propidium iodide (PI double staining and Hoechst 33342 fluorescent staining. Reduced (GSH and oxidized glutathione (GSSG were measured using colorimetric assays. Nrf2 activation and its downstream effects on phase II enzymes were examined by Western blot. Treatment of RPE cells with nanomolar ranges (10 and 100 nM of RTA 408 markedly attenuated H2O2-induced viability loss and apoptosis. RTA 408 pretreatment significantly protected cells from oxidative stress-induced GSH loss, GSSG formation and decreased ROS production. RTA 408 activated Nrf2 and increased the expression of its downstream genes, such as HO-1, NQO1, SOD2, catalase, Grx1, and Trx1. Consequently, the enzyme activities of NQO1, Grx1, and Trx1 were fully protected by RTA 408 pretreatment under oxidative stress. Moreover, knockdown of Nrf2 by siRNA significantly reduced the cytoprotective effects of RTA 408. In conclusion, our data suggest that RTA 408 protect primary human RPE cells from oxidative stress-induced damage by activating Nrf2 and its downstream genes.

  7. Bis-Retinoid A2E Induces an Increase of Basic Fibroblast Growth Factor via Inhibition of Extracellular Signal-Regulated Kinases 1/2 Pathway in Retinal Pigment Epithelium Cells and Facilitates Phagocytosis

    Science.gov (United States)

    Balmer, Delphine; Bapst-Wicht, Linda; Pyakurel, Aswin; Emery, Martine; Nanchen, Natacha; Bochet, Christian G.; Roduit, Raphael

    2017-01-01

    Age-related macular degeneration (ARMD) is the leading cause of vision loss in developed countries. Hallmarks of the disease are well known; indeed, this pathology is characterized by lipofuscin accumulation, is principally composed of lipid-containing residues of lysosomal digestion. The N-retinyl-N-retinylidene ethanolamine (A2E) retinoid which is thought to be a cytotoxic component for RPE is the best-characterized component of lipofuscin so far. Even if no direct correlation between A2E spatial distribution and lipofuscin fluorescence has been established in aged human RPE, modified forms or metabolites of A2E could be involved in ARMD pathology. Mitogen-activated protein kinase (MAPK) pathways have been involved in many pathologies, but not in ARMD. Therefore, we wanted to analyze the effects of A2E on MAPKs in polarized ARPE19 and isolated mouse RPE cells. We showed that long-term exposure of polarized ARPE19 cells to low A2E dose induces a strong decrease of the extracellular signal-regulated kinases' (ERK1/2) activity. In addition, we showed that A2E, via ERK1/2 decrease, induces a significant decrease of the retinal pigment epithelium-specific protein 65 kDa (RPE65) expression in ARPE19 cells and isolated mouse RPE. In the meantime, we showed that the decrease of ERK1/2 activity mediates an increase of basic fibroblast growth factor (bFGF) mRNA expression and secretion that induces an increase in phagocytosis via a paracrine effect. We suggest that the accumulation of deposits coming from outer segments (OS) could be explained by both an increase of bFGF-induced phagocytosis and by the decrease of clearance by A2E. The bFGF angiogenic protein may therefore be an attractive target to treat ARMD. PMID:28298893

  8. 胎兔视网膜色素上皮移植的初步研究%Primary study of transplanted embryonic retinal pigment epithelial cells in rabbits

    Institute of Scientific and Technical Information of China (English)

    曲毅; 周芳; 李艳; 李剑桥; 冯进波

    2005-01-01

    目的:观察受体Bruch膜和视网膜色素上皮(retrial pigment epithelial,RPE)受损情况下供体RPE细胞的增殖、分化和凋亡状况.方法:制备有色素胎兔RPE细胞悬液,移植至破坏了Bruch膜和RPE细胞的12只成年新西兰大白兔的视网膜下腔.左眼作为实验组,右眼作为对照,于术后3、7和14d,采用Ki-67免疫组化和TUNEL染色,并提取RPE细胞DNA作琼脂糖凝胶电泳,观察Ki-67阳性RPE细胞及移植的RPE细胞和受体ONL细胞的凋亡百分率,行统计学分析.结果:术后随着时间的延长,实验组或对照组Ki-67阳性RPE细胞数显著增加(P<0.05);术后14 d实验组或对照组的TUNEL染色ONL核阳性百分率较术后3 d显著减少(P<0.05);术后实验组TUNEL染色阳性和细胞核深染的RPE细胞无显著增加(P>0.05);细胞核深染的RPE细胞百分率明显高于TUNEL阳性的细胞(P<0.01).对照组未见TUNEL阳性的RPE细胞.术后14d,移植的RPE细胞DNA电泳出现典型的凋亡带.结论:在受体Bruch膜、RPE受损状态下,供体的RPE细胞具有良好的增殖和分化能力.

  9. Retinal vessel tortuosity associated with central retinal vein occlusion: an optical coherence tomography study.

    Science.gov (United States)

    Muraoka, Yuki; Tsujikawa, Akitaka; Kumagai, Kyoko; Akagi-Kurashige, Yumiko; Ogino, Ken; Murakami, Tomoaki; Miyamoto, Kazuaki; Yoshimura, Nagahisa

    2014-01-07

    We studied morphologic changes of the retinal vasculature in eyes with central retinal vein occlusion (CRVO) through the use of optical coherence tomography (OCT). Major retinal vessels in 35 eyes from 35 consecutive patients with acute CRVO were examined prospectively and longitudinally with sequential thin sectioning and circumpapillary scanning. Anteroposterior venous tortuosity associated with CRVO was quantified on longitudinal OCT images of a randomly selected major temporal vein. On OCT sections of a given vein, we identified the innermost and outermost points of the vessel wall. The degree of anteroposterior venous tortuosity was defined as the difference between the vertical distances from the retinal pigment epithelium to the center of the venous lumen at these two points. The OCT images revealed that the major retinal veins traveled tortuously through the swollen neurosensory retina from the inner retinal surface to the retinal pigment epithelium. The degree of anteroposterior venous tortuosity was correlated with poor visual acuity (r = 0.457, P = 0.017), increased mean foveal thickness (r = 0.671, P retinal detachment was detected around the optic disc, which correlated with anteroposterior venous tortuosity. In 14 (40%) eyes, elongated major retinal veins disrupted the boundary between retinal vessels and parenchyma, which resulted in juxtavenous splitting of the neurosensory retina. In eyes with CRVO, OCT can be used to visualize anteroposterior venous tortuosity and associated structural changes to the retinal parenchyma.

  10. The mechanics of retinal detachment

    Science.gov (United States)

    Chou, Tom; Siegel, Michael

    2013-03-01

    We present a model of the mechanical and fluid forces associated with exudative retinal detachments where the retinal photoreceptor cells separate typically from the underlying retinal pigment epithelium (RPE). By computing the total fluid volume flow arising from transretinal, vascular, and retinal pigment epithelium (RPE) pump currents, we determine the conditions under which the subretinal fluid pressure exceeds the maximum yield stress holding the retina and RPE together, giving rise to an irreversible, extended retinal delamination. We also investigate localized, blister-like retinal detachments by balancing mechanical tension in the retina with both the retina-RPE adhesion energy and the hydraulic pressure jump across the retina. For detachments induced by traction forces, we find a critical radius beyond which the blister is unstable to growth. Growth of a detached blister can also be driven by inflamed tissue within which e.g., the hydraulic conductivities of the retina or choroid increase, the RPE pumps fail, or the adhesion properties change. We determine the parameter regimes in which the blister either becomes unstable to growth, remains stable and finite-sized, or shrinks, allowing possible healing. This work supported by the Army Research Office through grant 58386MA

  11. Mitochondrial dysfunction underlying outer retinal diseases

    DEFF Research Database (Denmark)

    Lefevere, Evy; Toft-Kehler, Anne Katrine; Vohra, Rupali

    2017-01-01

    Dysfunction of photoreceptors, retinal pigment epithelium (RPE) or both contribute to the initiation and progression of several outer retinal disorders. Disrupted Müller glia function might additionally subsidize to these diseases. Mitochondrial malfunctioning is importantly associated with outer...... retina pathologies, which can be classified as primary and secondary mitochondrial disorders. This review highlights the importance of oxidative stress and mitochondrial DNA damage, underlying outer retinal disorders. Indeed, the metabolically active photoreceptors/RPE are highly prone to these hallmarks...... of mitochondrial dysfunction, indicating that mitochondria represent a weak link in the antioxidant defenses of outer retinal cells....

  12. ARE: Ada Rendering Engine

    Directory of Open Access Journals (Sweden)

    Stefano Penge

    2009-10-01

    Full Text Available E' ormai pratica diffusa, nello sviluppo di applicazioni web, l'utilizzo di template e di potenti template engine per automatizzare la generazione dei contenuti da presentare all'utente. Tuttavia a volte la potenza di tali engine è€ ottenuta mescolando logica e interfaccia, introducendo linguaggi diversi da quelli di descrizione della pagina, o addirittura inventando nuovi linguaggi dedicati.ARE (ADA Rendering Engine è€ pensato per gestire l'intero flusso di creazione del contenuto HTML/XHTML dinamico, la selezione del corretto template, CSS, JavaScript e la produzione dell'output separando completamente logica e interfaccia. I templates utilizzati sono puro HTML senza parti in altri linguaggi, e possono quindi essere gestiti e visualizzati autonomamente. Il codice HTML generato è€ uniforme e parametrizzato.E' composto da due moduli, CORE (Common Output Rendering Engine e ALE (ADA Layout Engine.Il primo (CORE viene utilizzato per la generazione OO degli elementi del DOM ed è pensato per aiutare lo sviluppatore nella produzione di codice valido rispetto al DTD utilizzato. CORE genera automaticamente gli elementi del DOM in base al DTD impostato nella configurazioneIl secondo (ALE viene utilizzato come template engine per selezionare automaticamente in base ad alcuni parametri (modulo, profilo utente, tipologia del nodo, del corso, preferenze di installazione il template HTML, i CSS e i file JavaScript appropriati. ALE permette di usare templates di default e microtemplates ricorsivi per semplificare il lavoro del grafico.I due moduli possono in ogni caso essere utilizzati indipendentemente l'uno dall'altro. E' possibile generare e renderizzare una pagina HTML utilizzando solo CORE oppure inviare gli oggetti CORE al template engine ALE che provvede a renderizzare la pagina HTML. Viceversa è possibile generare HTML senza utilizzare CORE ed inviarlo al template engine ALECORE è alla prima release ed è€ già utilizzato all

  13. Parallel hierarchical radiosity rendering

    Energy Technology Data Exchange (ETDEWEB)

    Carter, M.

    1993-07-01

    In this dissertation, the step-by-step development of a scalable parallel hierarchical radiosity renderer is documented. First, a new look is taken at the traditional radiosity equation, and a new form is presented in which the matrix of linear system coefficients is transformed into a symmetric matrix, thereby simplifying the problem and enabling a new solution technique to be applied. Next, the state-of-the-art hierarchical radiosity methods are examined for their suitability to parallel implementation, and scalability. Significant enhancements are also discovered which both improve their theoretical foundations and improve the images they generate. The resultant hierarchical radiosity algorithm is then examined for sources of parallelism, and for an architectural mapping. Several architectural mappings are discussed. A few key algorithmic changes are suggested during the process of making the algorithm parallel. Next, the performance, efficiency, and scalability of the algorithm are analyzed. The dissertation closes with a discussion of several ideas which have the potential to further enhance the hierarchical radiosity method, or provide an entirely new forum for the application of hierarchical methods.

  14. Sea modeling and rendering

    Science.gov (United States)

    Cathala, Thierry; Latger, Jean

    2010-10-01

    More and more defence and civil applications require simulation of marine synthetic environment. Currently, the "Future Anti-Surface-Guided-Weapon" (FASGW) or "anti-navire léger" (ANL) missile needs this kind of modelling. This paper presents a set of technical enhancement of the SE-Workbench that aim at better representing the sea profile and the interaction with targets. The operational scenario variability is a key criterion: the generic geographical area (e.g. Persian Gulf, coast of Somalia,...), the type of situation (e.g. peace keeping, peace enforcement, anti-piracy, drug interdiction,...)., the objectives (political, strategic, or military objectives), the description of the mission(s) (e.g. antipiracy) and operation(s) (e.g. surveillance and reconnaissance, escort, convoying) to achieve the objectives, the type of environment (Weather, Time of day, Geography [coastlines, islands, hills/mountains]). The paper insists on several points such as the dual rendering using either ray tracing [and the GP GPU optimization] or rasterization [and GPU shaders optimization], the modelling of sea-surface based on hypertextures and shaders, the wakes modelling, the buoyancy models for targets, the interaction of coast and littoral, the dielectric infrared modelling of water material.

  15. Protective effect of genistein on human retinal pigment epithelium cell cultured in high glucose%金雀异黄素对高糖诱导的人RPE细胞损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    帅捷; 洪瑾; 袁志兰

    2007-01-01

    目的 探讨金雀异黄素(genistein,Gen)对高糖诱导的人视网膜色素上皮(retinal pigment epithelium,RPE)细胞的保护作用.方法 培养人RPE细胞,分别以Gen 0 μmol·L-1、50 μmol·L-1、100 μmol·L-1、200 μmol·L-1的药物浓度作用高糖(33 mmol·L-1)培养的RPE细胞48 h,噻唑蓝比色法检测细胞活性;流式细胞仪检测细胞周期;激光共聚焦显微镜检测细胞内活性氧(reactive oxygen species,ROS)水平;黄嘌呤氧化酶法测定细胞内超氧化物歧化酶(SOD)活性.结果 高糖组细胞活力(0.308±0.022)明显低于正常对照组(0.424±0.014)(P<0.01);高糖+Gen组细胞活力较高糖组升高(P<0.05),且随Gen浓度增大,细胞活力增加更明显.与正常对照组比较,高糖组细胞增殖受到抑制,被阻滞于S期和G2期;与高糖组比较,Gen可减轻高糖对细胞增殖的抑制作用.与正常对照组比较,高糖组细胞内ROS的产生明显增多;Gen以浓度依赖方式抑制高糖组RPE细胞内ROS的产生.与正常对照组(31.04±0.02)比较,高糖组SOD活性(12.32±0.02)显著降低(P<0.01);与高糖组比较,高糖+Gen组SOD活性随Gen浓度的增加而升高(均P<0.05).结论 Gen可保护和修复高糖诱导的人RPE细胞的损伤,其作用可能与抗氧化、增强SOD活性有关.

  16. Linoleic acid-induced expression of inducible nitric oxide synthase and cyclooxygenase II via p42/44 mitogen-activated protein kinase and nuclear factor-kappaB pathway in retinal pigment epithelial cells.

    Science.gov (United States)

    Fang, I-Mo; Yang, Chang-Hao; Yang, Chung-May; Chen, Muh-Shy

    2007-11-01

    High linoleic acid (LA) intake is known to correlate with age-related macular degeneration (AMD), but the molecular mechanisms remain unclear. This study was conducted to investigate the effects of LA on expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase II (COX-2) and their associated signaling pathways in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were treated with different concentrations of LA. Expressions of iNOS and COX-2 were examined using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Concentrations of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in the culture medium were determined by enzyme-link immunosorbent assay (ELISA). Activation of p42/44, p38, JNK mitogen-activated protein kinase (MAPK) and nuclear factors (NF)-kappaB were evaluated by Western blot analysis and electrophoretic mobility shift assay (EMSA). We found that LA induced expression of iNOS and COX-2 in RPE cells at the mRNA and protein levels in a time-and dose-dependent manner. Upregulation of iNOS and COX-2 resulted in increased production of NO and PGE(2). Moreover, LA caused degradation of IkappaB and increased NF-kappaB DNA binding activity. Effects of LA-induced iNOS and COX-2 expression were inhibited by a NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC). LA activated p42/44, but not p38 or JNK MAPK. Inhibition of p42/44 activity by PD98059 significantly reduced LA-induced activation of NF-kappaB. Linoleic acid-induced expression of iNOS and COX-2 as well as PGE(2) and NO release in RPE cells were sequentially mediated through activation of p42/p44, MAPK, then NF-kappaB. These results may provide new insights into both mechanisms of LA action on RPE cells and pathogenesis of age-related macular degeneration.

  17. Spectral tuning of deep red cone pigments.

    Science.gov (United States)

    Amora, Tabitha L; Ramos, Lavoisier S; Galan, Jhenny F; Birge, Robert R

    2008-04-22

    Visual pigments are G-protein-coupled receptors that provide a critical interface between organisms and their external environment. Natural selection has generated vertebrate pigments that absorb light from the far-UV (360 nm) to the deep red (630 nm) while using a single chromophore, in either the A1 (11- cis-retinal) or A2 (11- cis-3,4-dehydroretinal) form. The fact that a single chromophore can be manipulated to have an absorption maximum across such an extended spectral region is remarkable. The mechanisms of wavelength regulation remain to be fully revealed, and one of the least well-understood mechanisms is that associated with the deep red pigments. We investigate theoretically the hypothesis that deep red cone pigments select a 6- s- trans conformation of the retinal chromophore ring geometry. This conformation is in contrast to the 6- s- cis ring geometry observed in rhodopsin and, through model chromophore studies, the vast majority of visual pigments. Nomographic spectral analysis of 294 A1 and A2 cone pigment literature absorption maxima indicates that the selection of a 6- s- trans geometry red shifts M/LWS A1 pigments by approximately 1500 cm (-1) ( approximately 50 nm) and A2 pigments by approximately 2700 cm (-1) ( approximately 100 nm). The homology models of seven cone pigments indicate that the deep red cone pigments select 6- s- trans chromophore conformations primarily via electrostatic steering. Our results reveal that the generation of a 6- s- trans conformation not only achieves a significant red shift but also provides enhanced stability of the chromophore within the deep red cone pigment binding sites.

  18. Natural pigments and sacred art

    Science.gov (United States)

    Kelekian, Lena, ,, Lady

    2010-05-01

    iconographer uses earthly creations to create divine images: "Thine own of Thine own we offer unto Thee." (Byzantine Liturgy). Thus, by combining geology with art and religion, I can render homage to God through His creation by using minerals of the Planet Earth, as natural pigments in painting His image.

  19. Retinal Vasculitis

    Science.gov (United States)

    Rosenbaum, James T.; Sibley, Cailin H.; Lin, Phoebe

    2016-01-01

    Purpose of review Ophthalmologists and rheumatologists frequently miscommunicate in consulting on patients with retinal vasculitis. This report seeks to establish a common understanding of the term, retinal vasculitis, and to review recent papers on this diagnosis. Recent findings 1) The genetic basis of some rare forms of retinal vascular disease have recently been described. Identified genes include CAPN5, TREX1, and TNFAIP3; 2) Behçet’s disease is a systemic illness that is very commonly associated with occlusive retinal vasculitis; 3) retinal imaging including fluorescein angiography and other newer imaging modalities has proven crucial to the identification and characterization of retinal vasculitis and its complications; 4) although monoclonal antibodies to IL-17A or IL-1 beta failed in trials for Behçet’s disease, antibodies to TNF alpha, either infliximab or adalimumab, have demonstrated consistent benefit in managing this disease. Interferon treatment and B cell depletion therapy via rituximab may be beneficial in certain types of retinal vasculitis. Summary Retinal vasculitis is an important entity for rheumatologists to understand. Retinal vasculitis associated with Behçet’s disease responds to monoclonal antibodies that neutralize TNF, but the many other forms of non-infectious retinal vasculitis may require alternate therapeutic management. PMID:26945335

  20. Regeneration of the retina: toward stem cell therapy for degenerative retinal diseases.

    Science.gov (United States)

    Jeon, Sohee; Oh, Il-Hoan

    2015-04-01

    Degenerative retinal diseases affect millions of people worldwide, which can lead to the loss of vision. However, therapeutic approaches that can reverse this process are limited. Recent efforts have allowed the possibility of the stem cell-based regeneration of retinal cells and repair of injured retinal tissues. Although the direct differentiation of pluripotent stem cells into terminally differentiated photoreceptor cells comprises one approach, a series of studies revealed the intrinsic regenerative potential of the retina using endogenous retinal stem cells. Muller glial cells, ciliary pigment epithelial cells, and retinal pigment epithelial cells are candidates for such retinal stem cells that can differentiate into multiple types of retinal cells and be integrated into injured or developing retina. In this review, we explore our current understanding of the cellular identity of these candidate retinal stem cells and their therapeutic potential for cell therapy against degenerative retinal diseases.

  1. Entropy, color, and color rendering.

    Science.gov (United States)

    Price, Luke L A

    2012-12-01

    The Shannon entropy [Bell Syst. Tech J.27, 379 (1948)] of spectral distributions is applied to the problem of color rendering. With this novel approach, calculations for visual white entropy, spectral entropy, and color rendering are proposed, indices that are unreliant on the subjectivity inherent in reference spectra and color samples. The indices are tested against real lamp spectra, showing a simple and robust system for color rendering assessment. The discussion considers potential roles for white entropy in several areas of color theory and psychophysics and nonextensive entropy generalizations of the entropy indices in mathematical color spaces.

  2. Retinal Biochemistry, Physiology and Cell Biology.

    Science.gov (United States)

    Smith, Ricardo Luiz; Sivaprasad, Sobha; Chong, Victor

    2016-01-01

    The vitreous, the vasculature of the retina, macular pigments, phototransduction, retinal pigment epithelium, Bruch's membrane and the extracellular matrix, all play an important role in the normal function of the retina as well as in diseases. Understanding the pathophysiology allows us to target treatment. As ocular angiogenesis, immunity and inflammation are covered elsewhere, those subjects will not be discussed in this chapter. © 2016 S. Karger AG, Basel.

  3. Translocation of the retinal pigment epithelium and choroidal neovascularization induced by injecting Matrigel to subretinal space of rats%大鼠视网膜下注射Matrigel诱导视网膜色素上皮易位和脉络膜新生血管形成

    Institute of Scientific and Technical Information of China (English)

    汪振芳; 万鹏霞; 何丽文; 袁钊辉; 温容

    2007-01-01

    Objective To establish an animal model of sub-retinal pigment epithelium (sub-RPE)deposits,and to explore the pathological process of the transloeation of retinal pigment epithelium, choroidal neovascularization and the pathogenesis of age-related macular degeneration (AMD). Methods Matrigel was injected to the subretinal space of rats to create an amorphous deposit. Translocation of retinal pigment epithelium and choroidal neovascularization were observed. Results The subretinal deposit of Matrigel induced RPE translocation and therefore it became a sub-RPE deposit. The RPE mobilization required the presence of photoreceptors. Bruch's membrane devoid of RPE attachment became vulnerable to the invasion of new blood vessels from the choroid. Conclusions A novel model of sub-RPE deposit formation is established successfully. The presence of sub-RPE deposit is sufficient to induce choroidal neovascularization to penetrate Bruch's membrane and may cause the exudative form of AMD.%目的 制备视网膜色素上皮(RPE)下沉积物形成动物模型,并探讨其诱导视网膜色素上皮易位和脉络膜新牛血管(CNV)形成的病理过程以及年龄相关性黄斑变性(AMD)的发病机制.方法 将Matrigel基质胶注射于大鼠视网膜下形成视网膜下沉积物,观察视网膜色素上皮的迁移和脉络膜新生血管的形成.结果 Matrigel形成视网膜下沉积物后诱导RPE易位,而沉积物换位成为视网膜色素上皮层下沉积物.REP细胞的动员需要光感受器细胞的参与.缺乏色素上皮细胞贴附的Bruch膜易受脉络膜新生血管的侵袭.结论 建立了一个新的视网膜色素上皮层下沉积物形成的AMD动物模型.视网膜色素上皮层下沉积物足以导致CNV穿透Bruch膜,可能导致湿性AMD发生.

  4. The molecular basis for UV vision in birds: spectral characteristics, cDNA sequence and retinal localization of the UV-sensitive visual pigment of the budgerigar (Melopsittacus undulatus)

    National Research Council Canada - National Science Library

    Wilkie, S E; Vissers, P M; Das, D; Degrip, W J; Bowmaker, J K; Hunt, D M

    1998-01-01

    .... The existence of a fourth pigment (UV) is the major difference between the trichromacy of humans and the tetrachromacy of such birds, and recent studies have shown that it may play a determining role in such diverse aspects of behaviour...

  5. Exposure render: an interactive photo-realistic volume rendering framework.

    Directory of Open Access Journals (Sweden)

    Thomas Kroes

    Full Text Available The field of volume visualization has undergone rapid development during the past years, both due to advances in suitable computing hardware and due to the increasing availability of large volume datasets. Recent work has focused on increasing the visual realism in Direct Volume Rendering (DVR by integrating a number of visually plausible but often effect-specific rendering techniques, for instance modeling of light occlusion and depth of field. Besides yielding more attractive renderings, especially the more realistic lighting has a positive effect on perceptual tasks. Although these new rendering techniques yield impressive results, they exhibit limitations in terms of their exibility and their performance. Monte Carlo ray tracing (MCRT, coupled with physically based light transport, is the de-facto standard for synthesizing highly realistic images in the graphics domain, although usually not from volumetric data. Due to the stochastic sampling of MCRT algorithms, numerous effects can be achieved in a relatively straight-forward fashion. For this reason, we have developed a practical framework that applies MCRT techniques also to direct volume rendering (DVR. With this work, we demonstrate that a host of realistic effects, including physically based lighting, can be simulated in a generic and flexible fashion, leading to interactive DVR with improved realism. In the hope that this improved approach to DVR will see more use in practice, we have made available our framework under a permissive open source license.

  6. Cytomegalovirus retinitis associated with acquired immunodeficiency syndrome

    Institute of Scientific and Technical Information of China (English)

    GENG Shuang; YE Jun-jie; ZHAO Jia-liang; LI Tai-sheng; HAN Yang

    2011-01-01

    Background Cytomegalovirus (CMV) retinitis is the most severe intraocular complication that results in total retinal destruction and loss of visual acuity in patients with acquired immunodeficiency syndrome (AIDS). This study aimed to investigate the fundus characteristics, systemic manifestations and therapeutic outcomes of CMV retinitis associated with AIDS.Methods It was a retrospective case series. CMV retinitis was present in 39 eyes (25 patients). Best corrected visual acuities, anterior segment, fundus features, fundus fluorescence angiography (FFA) and CD4+ T-lymphocyte counts of the patients with CMV retinitis associated with AIDS were analyzed. Intravitreal injections of ganciclovir (400 μg) were performed in 4 eyes (2 patients).Results Retinal vasculitis, dense, full-thickness, yellow-white lesions along vascular distribution with irregular granules at the border, and hemorrhage on the retinal surface were present in 28 eyes. The vitreous was clear or mildly opaque.Late stage of the retinopathy was demonstrated in 8 eyes characterized as atrophic retina, sclerotic and attenuated vessels, retinal pigment epithelium (RPE) atrophy, and optic nerve atrophy. Retinal detachment was found in 3 eyes. The average CD4+ T-lymphocyte count in peripheral blood of the patients with CMV retinitis was (30.6±25.3) ×106/L (range,(0-85) × 106/L). After intravitreal injections of ganciclovir, visual acuity was improved and fundus lesions regressed.Conclusions CMV retinitis is the most severe and the most common intraocular complication in patients with AIDS. For the patients with yellow-white retinal lesions, hemorrhage and retinal vasculitis without clear cause, human immunodeficiency virus (HIV) serology should be performed. Routine eye examination is also indicated in HIV positive patients.

  7. Bucky Paper as a Support Membrane in Retinal Cell Transplantation

    Science.gov (United States)

    Loftus, David J. (Inventor); Leng, Theodore (Inventor); Huie, Philip (Inventor); Fishman, Harvey (Inventor)

    2006-01-01

    A method for repairing a retinal system of an eye, using bucky paper on which a plurality of retina pigment epithelial cells and/or iris pigment epithelial cells and/or stem cells is deposited, either randomly or in a selected cell pattern. The cell-covered bucky paper is positioned in a sub-retinal space to transfer cells to this space and thereby restore the retina to its normal functioning, where retinal damage or degeneration, such as macular degeneration, has occurred.

  8. Dermatoscopic findings of pigmented purpuric dermatosis*

    Science.gov (United States)

    Ozkaya, Dilek Biyik; Emiroglu, Nazan; Su, Ozlem; Cengiz, Fatma Pelin; Bahali, Anil Gulsel; Yildiz, Pelin; Demirkesen, Cuyan; Onsun, Nahide

    2016-01-01

    Background Pigmented purpuric dermatosis is a chronic skin disorder of unknown aetiology characterised by symmetrical petechial and pigmented macules, often confined to the lower limbs. The aetiology of pigmented purpuric dermatosis is unknown. Dermatoscopy is a non-invasive diagnostic technique that allows the visualisation of morphological features invisible to the naked eye; it combines a method that renders the corneal layer of the skin translucent with an optical system that magnifies the image projected onto the retina. Objectives The aim of this study is to investigate the dermatoscopic findings of pigmented purpuric dermatosis. Methods This study enrolled patients diagnosed histopathologically with pigmented purpuric dermatosis who had dermatoscopic records. We reviewed the dermatoscopic images of PPD patients who attended the outpatient clinic in the Istanbul Dermatovenereology Department at the Bezmialem Vakıf University Medical Faculty. Results Dermatoscopy showed: coppery-red pigmentation (97%, n = 31) in the background, a brown network (34%, n = 11), linear vessels (22%, n = 7), round to oval red dots, globules, and patches (69%, n = 22; 75%, n = 24; 34%, n = 11; respectively), brown globules (26%, n = 8) and dots (53%, n = 17), linear brown lines (22%, n = 7), and follicular openings (13%, n = 4). Conclusion To our knowledge, this is the first study to report the dermatoscopy of pigmented purpuric dermatosis. In our opinion, dermatoscopy can be useful in the diagnosis of pigmented purpuric dermatosis. PMID:27828629

  9. Mechanism of the aging phenomenon with passage of human retinal pigment epithelial cell%人胚胎视网膜色素上皮细胞在传代中的衰老及其机制

    Institute of Scientific and Technical Information of China (English)

    赵颖; 牛膺筠

    2012-01-01

    7.620、11.207,P<0.01). 结论 成功制作了RPE细胞随年龄而逐渐衰老的模型,提示衰老细胞线粒体膜电位降低,线粒体功能受损可能是RPE细胞衰老的机制之一.%Background Retinal pigment epithelial (RPE) cell senescence damage the metabolism of photoreceptor,leading to retinal dysfunction and loss of vision.To understand RPE cell senescence mechanism will contribute to the study of age-related macular degeneration ( AMD). Objective The present study was to prepare the ageing RPE cell model with passage and explore its potential mechanisms. Methods This study was approved by the Ethic Committee of Qingdao University Medical College,and the informed consent was obtained from each gravida.Six human eyeballs were obtained from artificial labor fetusl with the gestational age 16-28 weeks.RPE cells were isolated,cultured and passaged in vitro to establish the cell replicative aging model.The third to twelfth cells were collected to be used to this experiment.Human keratin was used to identify the cells by immunochemistry,and MTT method was utilized to assess the proliferation and viability of different generations of cells as the A490 value.The cellular cycles and transmembrane potential (△ψm) of mitochondrion (△ψm) with passage were detected and compared using Flow Cytometry. Results Cultured and passaged cells showed the hexagon in shape with the melanin in 1-2 generations of cells and presented with the brown staining in cytoplasm for human keratin.The melanin was absent in the third generation cells.Vibrant growth statues were seen from the 3-6 generations cells and thereafter the proliferation ability reduced.The cells of G0/G1 phase were gradually elevated with the passage from 3 - 12 generations with a percentage of 68.40% in the third generation of cells to 87.33% in the twelfth generation of cells,showing a significant difference among various generations ( F =180.43,P =0.00),and that of the sixth,ninth and twelfth

  10. RenderMan design principles

    Science.gov (United States)

    Apodaca, Tony; Porter, Tom

    1989-01-01

    The two worlds of interactive graphics and realistic graphics have remained separate. Fast graphics hardware runs simple algorithms and generates simple looking images. Photorealistic image synthesis software runs slowly on large expensive computers. The time has come for these two branches of computer graphics to merge. The speed and expense of graphics hardware is no longer the barrier to the wide acceptance of photorealism. There is every reason to believe that high quality image synthesis will become a standard capability of every graphics machine, from superworkstation to personal computer. The significant barrier has been the lack of a common language, an agreed-upon set of terms and conditions, for 3-D modeling systems to talk to 3-D rendering systems for computing an accurate rendition of that scene. Pixar has introduced RenderMan to serve as that common language. RenderMan, specifically the extensibility it offers in shading calculations, is discussed.

  11. Exposure Render: An Interactive Photo-Realistic Volume Rendering Framework

    NARCIS (Netherlands)

    Kroes, T.; Post, F.H.; Botha, C.P.

    2012-01-01

    The field of volume visualization has undergone rapid development during the past years, both due to advances in suitable computing hardware and due to the increasing availability of large volume datasets. Recent work has focused on increasing the visual realism in Direct Volume Rendering (DVR) by i

  12. Current Concepts in the Treatment of Retinitis Pigmentosa

    Directory of Open Access Journals (Sweden)

    Maria A. Musarella

    2011-01-01

    Full Text Available Inherited retinal degenerations, including retinitis pigmentosa (RP and Leber congenital amaurosis (LCA, affect 1 in 4000 individuals in the general population. A majority of the genes which are mutated in these conditions are expressed in either photoreceptors or the retinal pigment epithelium (RPE. There is considerable variation in the clinical severity of these conditions; the most severe being autosomal recessive LCA, a heterogeneous retinal degenerative disease and the commonest cause of congenital blindness in children. Here, we discuss all the potential treatments that are now available for retinal degeneration. A number of therapeutic avenues are being explored based on our knowledge of the pathophysiology of retinal degeneration derived from research on animal models, including: gene therapy, antiapoptosis agents, neurotrophic factors, and dietary supplementation. Technological advances in retinal implant devices continue to provide the promise of vision for patients with end-stage disease.

  13. Effects of extremely low frequency electromagnetic fields on human retinal pigment epithelium cells%极低频电磁场对体外培养人视网膜色素上皮细胞的影响

    Institute of Scientific and Technical Information of China (English)

    王洁; 朱煌; 杜尔罡

    2015-01-01

    Objective To investigate pathological changes in the molecules of human retinal pigment epithelium (hRPE) exposed to an extremely low frequency electromagnetic field (ELF-EMF),and study its possible significance in the occurrence and development of myopia.Methods In this experimental study,the experimental group was composed of hRPE exposed to 50 Hz electromagnetic fields; the untreated group was composed of hRPE without exposure.The changes in hRPE cell morphology were observed and cell proliferation in hRPE was measured by a CCK8 assay.The expressions of matrix metallo-proteinase-2 (MMP-2),tissue inhibitor of metalloproteinase 2 (TIMP-2),transforming growth factor-β2 (TGF-β2) and fibroblast growth factor 2 (FGF-2) mRNA in hRPE were detected by reverse transcription-polymerase chain reaction (RT-PCR).The concentrations of TGF-β2 and FGF-2 in the culture medium of hRPE were assayed by enzyme-linked immunosorbent assay (ELISA).The difference between the two group using independent t test.Results hRPE cell morphology changed,and the proliferation of hRPE was inhibited (t=-7.069,-3.652,-6.974,P< 0.05).The mRNA expressions of MMP-2,TIMP-2 and TGF-β2 were up-regulated significantly (t=10.562,6.277,4.940,P<0.01).However,the mRNA expression of FGF-2 was down-regulated significantly (t=-6.905,P<0.01).The results of ELISA indicated that after exposure to ELF-EMF,the expression of TGF-β2 in hRPE supernatant medium was up-regulated significantly (t=-4.079,P< 0.05).However,the expression of FGF-2 in hRPE supernatant medium was down-regulated (t=4.441,P<0.05).Conclusion ELF-EMF changed the proliferation of hRPE,and also changed the expressions of MMP-2,TIMP-2,TGF-β2 and FGF-2 mRNA in hRPE,which might induce the occurrence of myopia through pathological changes in the molecules of hRPE.%目的 研究极低频电磁场(ELF-EMF)作用下体外培养的人视网膜色素上皮细胞(hRPE)的分子病理改变,探讨其在近视发生发展

  14. 高级糖基化终末产物对人视网膜色素上皮细胞的作用机制研究%Advanced glycosylation end products and human retinal pigment epithelium cells

    Institute of Scientific and Technical Information of China (English)

    黄焱; 周敏; 郑卫东; 徐国兴

    2013-01-01

    目的 探讨高级糖基化终末产物(AGEs)对人视网膜色素上皮(RPE)细胞的作用机制.方法 原代培养人RPE细胞,在传代细胞生长状态良好的情况下,无血清的Dulbecco改良Eagle培养基同步化24 h,进行分组:(1)C组:BSA浓度为0.1 g/L,各作用24、48 h,分别为C1、C2组;(2)NC组:葡萄糖浓度为5.6 mmol/L,各作用24、48 h,分别为NC1、NC2组;(3)A组:AGEs浓度分别为0.1、0.2、0.4 g/L,各作用24、48 h,作用24 h依次为A1组、A2组、A3组,作用48 h依次为A4组、A5组、A6组.免疫组织化学检测AGEs受体(RAGE)、过氧化物酶体增生物激活受体γ辅助激活因子-1α(PGC-1α)和血管内皮生长因子(VEGF)蛋白的表达;激光共聚焦显微镜检测核因子-κB (NF-κB)的活化.通过图像分析软件IPP6.0和统计软件SPSS 17.0进行定量分析.结果 A组RPE细胞较C组和NC组RAGE、PGC-1α和VEGF蛋白的表达明显增加,且随着AGEs浓度的升高和刺激时间的延长而增加(F=294.5、228.3、241.5,P<0.05);随着AGEs浓度的升高和刺激时间的延长,NF-κB的活化增加.结论 AGEs的堆积可导致其受体RAGE在RPE细胞表达增加,并促进核转录共同激活因子PGC-1α蛋白的表达和NF-cB的活化,促进RPE细胞VEGF的分泌.%Objective To study the effect of advanced glycosylation end products (AGEs) on human retinal pigment epithelium (RPE) cells.Methods Human primary RPE cells were cultured in basal and different concentrations of AGEs with different times.The cells were divided into several groups as follows:group C (control):bovine serum albumin 0.1 g/L,24 hours (C1) and 48 hours (C2); group NC (normal control):basal culture medium with 5.6 mmol/L of glucose,24 hours (NC1) and 48 hours (NC2) ; group A (AGEs):0.1 g/L,24 hours and 48 hours,A1 and A4 ; 0.2 g/L,24 hours and 48 hours,A2 and A5 ; 0.4 g/L,24 hours and 48 hours,A3 and A6.Immunohistochemistry analysis was used to study the protein expression of receptor for AGEs (RAGE

  15. ski基因干扰对人视网膜色素上皮细胞增生和迁移的影响%Effects of ski-siRNA on proliferation and migration of human retinal pigment cells

    Institute of Scientific and Technical Information of China (English)

    郭斌; 刘晓娟; 王莉; 范钦华

    2013-01-01

    目的:设计并化学合成针对ski的siRNA分子片段,转染人视网膜色素上皮(hRPE)细胞抑制ski表达,观察其对hRPE细胞增生和迁移等生物学功能方面的影响。方法设计并化学合成3对针对人ski mRNA(NM_003036)中910、1912和389靶位的siRNAs,以脂质体方法转染hRPE细胞,运用RT-PCR法和Western印迹法检测细胞中ski基因在mRNA水平和蛋白水平的变化。然后利用MTT法和Transwell模型检测转染ski-siRNA的hRPE细胞增生活力和迁移能力等生物学功能方面指标的变化。结果 RT-PCR和Western印迹检测结果证实3条ski-siRNA转染48 h后,均不同程度降低了hRPE细胞中ski基因的mRNA和蛋白表达水平(P<0.05),其中siRNA-C的干扰效果最强,与阴性对照相比抑制率达到45%左右,被用于后续生物学功能实验的转染。转染ski-siRNA后2~6 d hRPE细胞的增生能力明显受到抑制(P<0.05)。与空白对照组和NC组相比,在培养12 h和24 h时,siRNA-C转染hRPE细胞迁移数量明显升高(P<0.01)。结论 siRNA-C (389)是可以高效、特异地沉默hRPE细胞中ski基因。靶向ski基因的siRNA转染可以有效地抑制hRPE细胞增生,但可能促进细胞迁移。ski对hRPE增生和迁移调控可能是PVR治疗的一个新的方向。%Objective To design and prepare siRNAs targeting ski gene and to observe its effects on the biological characteristics of human retinal pigment (hRPE) cells, such as proliferation and migration. Methods Three pairs of siRNAs targeting ski mRNA 910, 1912 and 389 targets were designed and synthesized by utilizing RNA design software. The most effective siRNA was chosen to transfect into hRPE cells with cathodolyte liposome transfection method. Real-time PCR and Western blotting were used to measure ski expression at mRNA and protein levels. The cell proliferation was assessed by MTT assay and recorded by growth curve. The number of cells which migrate through micropores and

  16. Pigments of fly agaric (Amanita muscaria).

    Science.gov (United States)

    Stintzing, Florian; Schliemann, Willibald

    2007-01-01

    The complex pigment pattern of fly agaric (Amanita muscaria) cap skins has been studied by LC-DAD and mass spectrometry. Among the betaxanthins the corresponding derivatives of serine, threonine, ethanolamine, alanine, Dopa, phenylalanine and tryptophan are reported for the first time to contribute to the pigment pattern of fly agarics. Betalamic acid, the chromophoric precursor of betaxanthins and betacyanins, muscaflavin and seco-dopas were also detected. Furthermore, the red-purple muscapurpurin and the red muscarubrin were tentatively assigned while further six betacyanin-like components could not be structurally allocated. Stability studies indicated a high susceptibility of pigment extracts to degradation which led to rapid colour loss thus rendering a complete characterization of betacyanin-like compounds impossible at present. Taking into account these difficulties the presented results may be a starting point for a comprehensive characterization of the pigment composition of fly agarics.

  17. Abnormal pigmentation within cutaneous scars: A complication of wound healing

    Directory of Open Access Journals (Sweden)

    Sarah Chadwick

    2012-01-01

    Full Text Available Abnormally pigmented scars are an undesirable consequence of cutaneous wound healing and are a complication every single individual worldwide is at risk of. They present a challenge for clinicians, as there are currently no definitive treatment options available, and render scars much more noticeable making them highly distressing for patients. Despite extensive research into both wound healing and the pigment cell, there remains a scarcity of knowledge surrounding the repigmentation of cutaneous scars. Pigment production is complex and under the control of many extrinsic and intrinsic factors and patterns of scar repigmentation are unpredictable. This article gives an overview of human skin pigmentation, repigmentation following wounding and current treatment options.

  18. Skin Pigmentation Disorders

    Science.gov (United States)

    Pigmentation means coloring. Skin pigmentation disorders affect the color of your skin. Your skin gets its color from a pigment called melanin. Special cells in the skin make melanin. When these cells become damaged or ...

  19. Retinal iron homeostasis in health and disease

    Directory of Open Access Journals (Sweden)

    Delu eSong

    2013-06-01

    Full Text Available Iron is essential for life, but excess iron can be toxic. As a potent free radical creator, iron generates hydroxyl radicals leading to significant oxidative stress. Since iron is not excreted from the body, it accumulates with age in tissues, including the retina, predisposing to age-related oxidative insult. Both hereditary and acquired retinal diseases are associated with increased iron levels. For example, retinal degenerations have been found in hereditary iron overload disorders, like aceruloplasminemia, Friedreich’s ataxia, and pantothenate kinase-associated neurodegeneration. Similarly, mice with targeted mutation of the iron exporter ceruloplasmin and its homolog hephaestin showed age-related retinal iron accumulation and retinal degeneration with features resembling human age-related macular degeneration (AMD. Post mortem AMD eyes have increased levels of iron in retina compared to age-matched healthy donors. Iron accumulation in AMD is likely to result, in part, from inflammation, hypoxia, and oxidative stress, all of which can cause iron dysregulation. Fortunately, it has been demonstrated by in vitro and in vivo studies that iron in the retinal pigment epithelium and retina is chelatable. Iron chelation protects photoreceptors and retinal pigment epithelial cells (RPE in a variety of mouse models. This has therapeutic potential for diminishing iron-induced oxidative damage to prevent or treat AMD.

  20. Monascus pigments.

    Science.gov (United States)

    Feng, Yanli; Shao, Yanchun; Chen, Fusheng

    2012-12-01

    Monascus pigments (MPs) as natural food colorants have been widely utilized in food industries in the world, especially in China and Japan. Moreover, MPs possess a range of biological activities, such as anti-mutagenic and anticancer properties, antimicrobial activities, potential anti-obesity activities, and so on. So, in the past two decades, more and more attention has been paid to MPs. Up to now, more than 50 MPs have been identified and studied. However, there have been some reviews about red fermented rice and the secondary metabolites produced by Monascus, but no monograph or review of MPs has been published. This review covers the categories and structures, biosynthetic pathway, production, properties, detection methods, functions, and molecular biology of MPs.

  1. Imaging retinal mosaics in the living eye.

    Science.gov (United States)

    Rossi, E A; Chung, M; Dubra, A; Hunter, J J; Merigan, W H; Williams, D R

    2011-03-01

    Adaptive optics imaging of cone photoreceptors has provided unique insight into the structure and function of the human visual system and has become an important tool for both basic scientists and clinicians. Recent advances in adaptive optics retinal imaging instrumentation and methodology have allowed us to expand beyond cone imaging. Multi-wavelength and fluorescence imaging methods with adaptive optics have allowed multiple retinal cell types to be imaged simultaneously. These new methods have recently revealed rod photoreceptors, retinal pigment epithelium (RPE) cells, and the smallest retinal blood vessels. Fluorescence imaging coupled with adaptive optics has been used to examine ganglion cells in living primates. Two-photon imaging combined with adaptive optics can evaluate photoreceptor function non-invasively in the living primate retina.

  2. Vitamin A derivatives as treatment options for retinal degenerative diseases.

    Science.gov (United States)

    Perusek, Lindsay; Maeda, Tadao

    2013-07-12

    The visual cycle is a sequential enzymatic reaction for vitamin A, all-trans-retinol, occurring in the outer layer of the human retina and is essential for the maintenance of vision. The central source of retinol is derived from dietary intake of both retinol and pro-vitamin A carotenoids. A series of enzymatic reactions, located in both the photoreceptor outer segment and the retinal pigment epithelium, transform retinol into the visual chromophore 11-cis-retinal, regenerating visual pigments. Retina specific proteins carry out the majority of the visual cycle, and any significant interruption in this sequence of reactions is capable of causing varying degrees of blindness. Among these important proteins are Lecithin:retinol acyltransferase (LRAT) and retinal pigment epithelium-specific 65-kDa protein (RPE65) known to be responsible for esterification of retinol to all-trans-retinyl esters and isomerization of these esters to 11-cis-retinal, respectively. Deleterious mutations in these genes are identified in human retinal diseases that cause blindness, such as Leber congenital amaurosis (LCA) and retinitis pigmentosa (RP). Herein, we discuss the pathology of 11-cis-retinal deficiency caused by these mutations in both animal disease models and human patients. We also review novel therapeutic strategies employing artificial visual chromophore 9-cis-retinoids which have been employed in clinical trials involving LCA patients.

  3. Vitamin A Derivatives as Treatment Options for Retinal Degenerative Diseases

    Directory of Open Access Journals (Sweden)

    Tadao Maeda

    2013-07-01

    Full Text Available The visual cycle is a sequential enzymatic reaction for vitamin A, all-trans-retinol, occurring in the outer layer of the human retina and is essential for the maintenance of vision. The central source of retinol is derived from dietary intake of both retinol and pro-vitamin A carotenoids. A series of enzymatic reactions, located in both the photoreceptor outer segment and the retinal pigment epithelium, transform retinol into the visual chromophore 11-cis-retinal, regenerating visual pigments. Retina specific proteins carry out the majority of the visual cycle, and any significant interruption in this sequence of reactions is capable of causing varying degrees of blindness. Among these important proteins are Lecithin:retinol acyltransferase (LRAT and retinal pigment epithelium-specific 65-kDa protein (RPE65 known to be responsible for esterification of retinol to all-trans-retinyl esters and isomerization of these esters to 11-cis-retinal, respectively. Deleterious mutations in these genes are identified in human retinal diseases that cause blindness, such as Leber congenital amaurosis (LCA and retinitis pigmentosa (RP. Herein, we discuss the pathology of 11-cis-retinal deficiency caused by these mutations in both animal disease models and human patients. We also review novel therapeutic strategies employing artificial visual chromophore 9-cis-retinoids which have been employed in clinical trials involving LCA patients.

  4. [New drug therapy for retinal degeneration].

    Science.gov (United States)

    Ohguro, Hiroshi

    2008-01-01

    Retinitis pigmentosa (RP) is an inherited retinal degeneration characterized by nyctalopia, ring scotoma, and bone-spicule pigmentation of the retina. So far, no effective therapy has been found for RP. As a possible molecular etiology of RP, retina-specific gene deficits are most likely involved, but little has been identified in terms of intracellular mechanisms leading to retinal photoreceptor cell death at post-translational levels. In order to find an effective therapy for RP, we must look for underlying common mechanisms that are responsible for the development of RP, instead of designing a specific therapy for each of the RP types with different causes. Therefore, in the present study, several animal models with different causes of RP were studied, including (1)Royal College of Surgeons (RCS) rats with a deficit of retinal pigment epithelium (RPE) function caused by rhodopsin mutation; (2) P23H rats, (3) S334ter rats, (4) photo stress rats, (5) retinal degeneration (rd) mice with a deficit of phosphodiesterase(PDE) function; and (6) cancer-associated retinopathy (CAR) model rats with a deficit of recoverin-dependent photoreceptor adaptation function. In each of these models, the following assessments were made in order to elucidate common pathological mechanisms among the models: (1) retinal function assessed by electroretinogram (ERG), (2) retinal morphology, (3) retinoid analysis, (4) rhodopsin regeneration, (5) rhodopsin phosphorylation and dephosphorylation, and (6) cytosolic cGMP levels. We found that unregulated photoreceptor adaptation processes caused by an imbalance of rhodopsin phosphorylation and dephosphorylation caused retinal dysfunction leading to photoreceptor cell death. As possible candidate drugs for normalizing these retinal dysfunctions and stopping further retinal degeneration, nilvadipine, a Ca channel blocker, retinoid derivatives, and anthocyanine were chosen and tested to determine their effect on the above animal models with

  5. Cellular Reparative Mechanisms of Mesenchymal Stem Cells for Retinal Diseases

    Directory of Open Access Journals (Sweden)

    Suet Lee Shirley Ding

    2017-07-01

    Full Text Available The use of multipotent mesenchymal stem cells (MSCs has been reported as promising for the treatment of numerous degenerative disorders including the eye. In retinal degenerative diseases, MSCs exhibit the potential to regenerate into retinal neurons and retinal pigmented epithelial cells in both in vitro and in vivo studies. Delivery of MSCs was found to improve retinal morphology and function and delay retinal degeneration. In this review, we revisit the therapeutic role of MSCs in the diseased eye. Furthermore, we reveal the possible cellular mechanisms and identify the associated signaling pathways of MSCs in reversing the pathological conditions of various ocular disorders such as age-related macular degeneration (AMD, retinitis pigmentosa, diabetic retinopathy, and glaucoma. Current stem cell treatment can be dispensed as an independent cell treatment format or with the combination of other approaches. Hence, the improvement of the treatment strategy is largely subjected by our understanding of MSCs mechanism of action.

  6. GPU Pro advanced rendering techniques

    CERN Document Server

    Engel, Wolfgang

    2010-01-01

    This book covers essential tools and techniques for programming the graphics processing unit. Brought to you by Wolfgang Engel and the same team of editors who made the ShaderX series a success, this volume covers advanced rendering techniques, engine design, GPGPU techniques, related mathematical techniques, and game postmortems. A special emphasis is placed on handheld programming to account for the increased importance of graphics on mobile devices, especially the iPhone and iPod touch.Example programs and source code can be downloaded from the book's CRC Press web page. 

  7. Fundus changes in central retinal vein occlusion.

    Science.gov (United States)

    Hayreh, Sohan Singh; Zimmerman, M Bridget

    2015-01-01

    To investigate systematically the retinal and optic disk changes in central retinal vein occlusion (CRVO) and their natural history. This study comprised 562 consecutive patients with CRVO (492 nonischemic [NI-CRVO] and 89 ischemic CRVO [I-CRVO] eyes) seen within 3 months of onset. Ophthalmic evaluation at initial and follow-up visits included recording visual acuity, visual fields, and detailed anterior segment and fundus examinations and fluorescein fundus angiography. Retinal and subinternal limiting membrane hemorrhages and optic disk edema in I-CRVO were initially more marked (P retinal epithelial pigment degeneration, serous macular detachment, and retinal perivenous sheathing developed at a higher rate in I-CRVO than that in NI-CRVO (P retinal venous engorgement than NI-CRVO (P = 0.003). Fluorescein fundus angiography showed significantly more fluorescein leakage, retinal capillary dilatation, capillary obliteration, and broken capillary foveal arcade (P < 0.0001) in I-CRVO than NI-CRVO. Resolution time of CRVO was longer for I-CRVO than NI-CRVO (P < 0.0001). Characteristics and natural history of fundus findings in the two types of CRVO are different.

  8. [Surgical managment of retinal detachment].

    Science.gov (United States)

    Haritoglou, C; Wolf, A

    2015-05-01

    The detachment of the neurosensory retina from the underlying retinal pigment epithelium can be related to breaks of the retina allowing vitreous fluid to gain access to the subretinal space, to exudative changes of the choroid such as tumours or inflammatory diseases or to excessive tractional forces exerted by interactions of the collagenous vitreous and the retina. Tractional retinal detachment is usually treated by vitrectomy and exudative detachment can be addressed by treatment of the underlying condition in many cases. In rhegmatogenous retinal detachment two different surgical procedures, vitrectomy and scleral buckling, can be applied for functional and anatomic rehabilitation of our patients. The choice of the surgical procedure is not really standardised and often depends on the experience of the surgeon and other more ocular factors including lens status, the number of retinal breaks, the extent of the detachment and the amount of preexisting PVR. Using both techniques, anatomic success rates of over 90 % can be achieved. Especially in young phakic patients scleral buckling offers the true advantage to prevent the progression of cataract formation requiring cataract extraction and intraocular lens implantation. Therefore, scleral buckling should be considered in selected cases as an alternative surgical option in spite of the very important technical refinements in modern vitrectomy techniques. Georg Thieme Verlag KG Stuttgart · New York.

  9. Retinitis pigmentosa

    NARCIS (Netherlands)

    Hartong, Dyonne T.; Berson, Eliot L.; Dryja, Thaddeus P.

    2006-01-01

    Hereditary degenerations of the human retina are genetically heterogeneous, with well over 100 genes implicated so far. This Seminar focuses on the subset of diseases called retinitis pigmentosa, in which patients typically lose night vision in adolescence, side vision in young adulthood, and centra

  10. Retinal Optical Coherence Tomography Imaging

    Science.gov (United States)

    Drexler, Wolfgang; Fujimoto, James G.

    The eye is essentially transparent, transmitting light with only minimal optical attenuation and scattering providing easy optical access to the anterior segment as well as the retina. For this reason, ophthalmic and especially retinal imaging has been not only the first but also most successful clinical application for optical coherence tomography (OCT). This chapter focuses on the development of OCT technology for retinal imaging. OCT has significantly improved the potential for early diagnosis, understanding of retinal disease pathogenesis, as well as monitoring disease progression and response to therapy. Development of ultrabroad bandwidth light sources and high-speed detection techniques has enabled significant improvements in ophthalmic OCT imaging performance, demonstrating the potential of three-dimensional, ultrahigh-resolution OCT (UHR OCT) to perform noninvasive optical biopsy of the living human retina, i.e., the in vivo visualization of microstructural, intraretinal morphology in situ approaching the resolution of conventional histopathology. Significant improvements in axial resolution and speed not only enable three-dimensional rendering of retinal volumes but also high-definition, two-dimensional tomograms, topographic thickness maps of all major intraretinal layers, as well as volumetric quantification of pathologic intraretinal changes. These advances in OCT technology have also been successfully applied in several animal models of retinal pathologies. The development of light sources emitting at alternative wavelengths, e.g., around #1,060 nm, not only enabled three-dimensional OCT imaging with enhanced choroidal visualization but also improved OCT performance in cataract patients due to reduced scattering losses in this wavelength region. Adaptive optics using deformable mirror technology, with unique high stroke to correct higher-order ocular aberrations, with specially designed optics to compensate chromatic aberration of the human eye, in

  11. Regenerative Therapy for Retinal Disorders

    Directory of Open Access Journals (Sweden)

    Narsis Daftarian

    2010-01-01

    Full Text Available Major advances in various disciplines of basic sciences including embryology, molecular and cell biology, genetics, and nanotechnology, as well as stem cell biology have opened new horizons for regenerative therapy. The unique characteristics of stem cells prompt a sound understanding for their use in modern regenerative therapies. This review article discusses stem cells, developmental stages of the eye field, eye field transcriptional factors, and endogenous and exogenous sources of stem cells. Recent studies and challenges in the application of stem cells for retinal pigment epithelial degeneration models will be summarized followed by obstacles facing regenerative therapy.

  12. Pigmented paravenous chorioretinal atrophy with Coat′s like response

    Directory of Open Access Journals (Sweden)

    Manish Tandon

    2013-01-01

    Full Text Available Pigmented paravenous chorioretinal atrophy (PPCRA is an uncommon retinal disorder of unknown etiology that is neither well understood nor classified. We report an atypical case of PPCRA, associated with Coat′s like response (CLR in a 64-year-old man of Asian origin. Both the eyes were involved, though asymmetrically.

  13. 多能干细胞分化来源视网膜色素上皮细胞移植治疗视网膜变性研究进展%The research progress toward clinical transplantation of pluripotent stem cell-derived retinal pigmented epithelial cells

    Institute of Scientific and Technical Information of China (English)

    邓雯丽; 向萍; 金子兵

    2014-01-01

    Retinal pigmented epithelial (RPE) cell is essential to maintain retinal function. RPE loss or dysfunction is the leading cause of incurable blindness worldwide. RPE cell replacement has been one of the most promising approaches to restore vision for these patients. With rapid progress of stem cell biology, great efforts have been made to induce functional RPE cells from pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Disease-specific RPE cells differentiated from patient iPS cells are greatly expected to elucidate mechanism of pathogenesis and personalized therapies for retinal degenerative diseases. Additionally, transplantation of induced RPE into subretinal space has shown encouraging remedies in both animal models and clinical trials. In this review, we focus on PSC-derived RPE in field of regenerative medicine and to summarize methods for RPE cell production and delivering .%视网膜色素上皮(RPE)对视觉功能的维持起着至关重要的作用。视网膜变性是全球不可治愈性致盲疾病的重要原因,它由视网膜色素上皮功能失常所引起。因此,视网膜色素上皮移植是视网膜变性患者恢复视力的一种最有前景的手段之一。随着干细胞技术的快速发展,从多能干细胞(PSC)到有功能的视网膜色素上皮细胞的体外分化诱导技术已经成熟,其中包括胚胎干细胞(ESCs)和诱导多能干细胞(iPSCs)等。此外,从患者特异性iPSCs分化而来的RPE更能用于阐明发病机理并有针对性地个体治疗。更值得一提的是,经诱导得到RPE的移植不论在动物模型中,还是在临床试验里都已经得到了可喜的治疗效果。本文回顾PSC来源RPE干预治疗视网膜变性的最新研究进展。

  14. Mechanisms of Retinal Damage from Chronic Laser Radiation.

    Science.gov (United States)

    1981-07-01

    W.K.: The effects of the pineal gland on light-induced retinal photoreceptor damage. Exp. Eye Res. 28:37-44, 1979. 17. Hollyfield, Joe G., Rayborn...co-iI workers in 196612. Noell reported that irreversible retinal damage occurs in normal laboratory rats exposed continuously to an illuminated...light than with either red or blue light. In fact, the action spectrum of the damage paralleled the action spectrum of the ERG. The iris of pigmented rats

  15. Probing how initial retinal configuration controls photochemical dynamics in retinal proteins

    Directory of Open Access Journals (Sweden)

    Sheves M.

    2013-03-01

    Full Text Available The effects of the initial retinal configuration and the active isomerization coordinate on the photochemistry of retinal proteins (RPs are assessed by comparing photochemical dynamics of two stable retinal ground state configurations (all-trans,15-anti vs. 13-cis,15-syn, within two RPs: Bacteriorhodopsin (BR and Anabaena Sensory Rhodopsin (ASR. Hyperspectral pump-probe spectroscopy shows that photochemistry starting from 13-cis retinal in both proteins is 3-10 times faster than when started in the all-trans state, suggesting that the hastening is ubiquitous to microbial RPs, regardless of their different biological functions and origin. This may also relate to the known disparity of photochemical rates between microbial RPs and visual pigments. Importance and possible underlying mechanisms are discussed as well.

  16. Real-time graphics rendering engine

    CERN Document Server

    Bao, Hujun

    2011-01-01

    ""Real-Time Graphics Rendering Engine"" reveals the software architecture of the modern real-time 3D graphics rendering engine and the relevant technologies based on the authors' experience developing this high-performance, real-time system. The relevant knowledge about real-time graphics rendering such as the rendering pipeline, the visual appearance and shading and lighting models are also introduced. This book is intended to offer well-founded guidance for researchers and developers who are interested in building their own rendering engines. Hujun Bao is a professor at the State Key Lab of

  17. Hardware Accelerated Point Rendering of Isosurfaces

    DEFF Research Database (Denmark)

    Bærentzen, Jakob Andreas; Christensen, Niels Jørgen

    2003-01-01

    an approximate technique for point scaling using distance attenuation which makes it possible to render points stored in display lists or vertex arrays. This enables us to render points quickly using OpenGL. Our comparisons show that point generation is significantly faster than triangle generation...... and that the advantage of rendering points as opposed to triangles increases with the size and complexity of the volumes. To gauge the visual quality of future hardware accelerated point rendering schemes, we have implemented a software based point rendering method and compare the quality to both MC and our OpenGL based...

  18. Cluster parallel rendering based on encoded mesh

    Institute of Scientific and Technical Information of China (English)

    QIN Ai-hong; XIONG Hua; PENG Hao-yu; LIU Zhen; SHI Jiao-ying

    2006-01-01

    Use of compressed mesh in parallel rendering architecture is still an unexplored area, the main challenge of which is to partition and sort the encoded mesh in compression-domain. This paper presents a mesh compression scheme PRMC (Parallel Rendering based Mesh Compression) supplying encoded meshes that can be partitioned and sorted in parallel rendering system even in encoded-domain. First, we segment the mesh into submeshes and clip the submeshes' boundary into Runs, and then piecewise compress the submeshes and Runs respectively. With the help of several auxiliary index tables, compressed submeshes and Runs can serve as rendering primitives in parallel rendering system. Based on PRMC, we design and implement a parallel rendering architecture. Compared with uncompressed representation, experimental results showed that PRMC meshes applied in cluster parallel rendering system can dramatically reduce the communication requirement.

  19. Lighting a candle in the dark: advances in genetics and gene therapy of recessive retinal dystrophies.

    NARCIS (Netherlands)

    Hollander, A.I. den; Black, A.; Bennett, J.; Cremers, F.P.M.

    2010-01-01

    Nonsyndromic recessive retinal dystrophies cause severe visual impairment due to the death of photoreceptor and retinal pigment epithelium cells. These diseases until recently have been considered to be incurable. Molecular genetic studies in the last two decades have revealed the underlying molecul

  20. Pigmented Villonodular Synovitis (PVNS)

    Science.gov (United States)

    ... OverviewWhat is pigmented villonodular synovitis?Pigmented villonodular synovitis (PVNS) is a joint problem that usually affects the ... ankle, elbow, hand or foot.When you have PVNS, the lining of a joint becomes swollen and ...

  1. Oral pigmentation: A review.

    Science.gov (United States)

    Sreeja, C; Ramakrishnan, K; Vijayalakshmi, D; Devi, M; Aesha, I; Vijayabanu, B

    2015-08-01

    Pigmentations are commonly found in the mouth. They represent in various clinical patterns that can range from just physiologic changes to oral manifestations of systemic diseases and malignancies. Color changes in the oral mucosa can be attributed to the deposition of either endogenous or exogenous pigments as a result of various mucosal diseases. The various pigmentations can be in the form of blue/purple vascular lesions, brown melanotic lesions, brown heme-associated lesions, gray/black pigmentations.

  2. Proteomic Analysis of the Vitreous following Experimental Retinal Detachment in Rabbits

    DEFF Research Database (Denmark)

    Mandal, Nakul; Lewis, Geoffrey P; Fisher, Steven K

    2015-01-01

    of the vitreous following experimental retinal detachment using a comparative proteomic based approach. Materials and Methods. Retinal detachment was created in the right eyes of six New Zealand red pigmented rabbits. Sham surgery was undertaken in five other rabbits that were used as controls. After seven days......, and α-1-antiproteinase F. Conclusions. Proteomic investigation of the rabbit vitreous has identified a set of proteins that help further our understanding of the pathogenesis of rhegmatogenous retinal detachment and its complications....

  3. Diagnostic imaging in patients with retinitis pigmentosa.

    Science.gov (United States)

    Mitamura, Yoshinori; Mitamura-Aizawa, Sayaka; Nagasawa, Toshihiko; Katome, Takashi; Eguchi, Hiroshi; Naito, Takeshi

    2012-01-01

    Retinitis pigmentosa (RP) is a progressive inherited retinal disease, and patients with RP have reduced visual function caused by a degeneration of the photoreceptors and retinal pigment epithelium (RPE). At the end stage of RP, the degeneration of the photoreceptors in the fovea reduces central vision, and RP is one of the main causes of acquired blindness in developed countries. Therefore, morphological and functional assessments of the photoreceptors in the macula area can be useful in estimating the residual retinal function in RP patients. Optical coherence tomography (OCT) is a well-established method of examining the retinal architecture in situ. The photoreceptor inner/outer segment (IS/OS) junction is observed as a distinct, highly reflective line by OCT. The presence of the IS/OS junction in the OCT images is essential for normal visual function. Fundus autofluorescence (FAF) results from the accumulation of lipofuscin in the RPE cells and has been used to investigate RPE and retinal function. More than one-half of RP patients have an abnormally high density parafoveal FAF ring (AF ring). The AF ring represents the border between functional and dysfunctional retina. In this review, we shall summarize recent progress on diagnostic imaging in eyes with RP.

  4. Neovascularisation by tattoo pigment

    Directory of Open Access Journals (Sweden)

    Abdul Razack E

    1991-01-01

    Full Text Available Split skin grafting for the removal of a tattoo resulted in the appearance of pigmented papules in the periphery of the grafted skin as well as distal to it on the normal skin. Histologically they showed large vascular laminae containing red blood corpuscles and pigment deposits, a hitherto not documented complication of tattoo pigment.

  5. Overview of plant pigments

    Science.gov (United States)

    Chlorophylls, carotenoids, flavonoids and betalains are four major classes of biological pigments produced in plants. Chlorophylls are the primary pigments responsible for plant green and photosynthesis. The other three are accessary pigments and secondary metabolites that yield non-green colors and...

  6. Binaural Rendering in MPEG Surround

    Directory of Open Access Journals (Sweden)

    Kristofer Kjörling

    2008-04-01

    Full Text Available This paper describes novel methods for evoking a multichannel audio experience over stereo headphones. In contrast to the conventional convolution-based approach where, for example, five input channels are filtered using ten head-related transfer functions, the current approach is based on a parametric representation of the multichannel signal, along with either a parametric representation of the head-related transfer functions or a reduced set of head-related transfer functions. An audio scene with multiple virtual sound sources is represented by a mono or a stereo downmix signal of all sound source signals, accompanied by certain statistical (spatial properties. These statistical properties of the sound sources are either combined with statistical properties of head-related transfer functions to estimate “binaural parameters” that represent the perceptually relevant aspects of the auditory scene or used to create a limited set of combined head-related transfer functions that can be applied directly on the downmix signal. Subsequently, a binaural rendering stage reinstates the statistical properties of the sound sources by applying the estimated binaural parameters or the reduced set of combined head-related transfer functions directly on the downmix. If combined with parametric multichannel audio coders such as MPEG Surround, the proposed methods are advantageous over conventional methods in terms of perceived quality and computational complexity.

  7. Binaural Rendering in MPEG Surround

    Science.gov (United States)

    Breebaart, Jeroen; Villemoes, Lars; Kjörling, Kristofer

    2008-12-01

    This paper describes novel methods for evoking a multichannel audio experience over stereo headphones. In contrast to the conventional convolution-based approach where, for example, five input channels are filtered using ten head-related transfer functions, the current approach is based on a parametric representation of the multichannel signal, along with either a parametric representation of the head-related transfer functions or a reduced set of head-related transfer functions. An audio scene with multiple virtual sound sources is represented by a mono or a stereo downmix signal of all sound source signals, accompanied by certain statistical (spatial) properties. These statistical properties of the sound sources are either combined with statistical properties of head-related transfer functions to estimate "binaural parameters" that represent the perceptually relevant aspects of the auditory scene or used to create a limited set of combined head-related transfer functions that can be applied directly on the downmix signal. Subsequently, a binaural rendering stage reinstates the statistical properties of the sound sources by applying the estimated binaural parameters or the reduced set of combined head-related transfer functions directly on the downmix. If combined with parametric multichannel audio coders such as MPEG Surround, the proposed methods are advantageous over conventional methods in terms of perceived quality and computational complexity.

  8. Stem cell therapy for retinal diseases

    Institute of Scientific and Technical Information of China (English)

    Jose Mauricio Garcia,; Luisa Mendon?a; Rodrigo Brant; Murilo Abud; Caio Regatieri; Bruno Diniz

    2015-01-01

    In this review, we discuss about current knowledgeabout stem cell (SC) therapy in the treatment of retinaldegeneration. Both human embryonic stem cell andinduced pluripotent stem cell has been growth inculture for a long time, and started to be explored inthe treatment of blinding conditions. The Food andDrug Administration, recently, has granted clinical trialsusing SC retinal therapy to treat complex disorders, asStargardt's dystrophy, and patients with geographicatrophy, providing good outcomes. This study'sintent is to overview the critical regeneration of thesubretinal anatomy through retinal pigment epitheliumtransplantation, with the goal of reestablish importantpathways from the retina to the occipital cortex of thebrain, as well as the differentiation from pluripotentquiescent SC to adult retina, and its relationshipwith a primary retinal injury, different techniques oftransplantation, management of immune rejection andtumorigenicity, its potential application in improvingpatients' vision, and, finally, approaching future directionsand challenges for the treatment of several conditions.

  9. Connexin43 in retinal injury and disease.

    Science.gov (United States)

    Danesh-Meyer, Helen V; Zhang, Jie; Acosta, Monica L; Rupenthal, Ilva D; Green, Colin R

    2016-03-01

    Gap junctions are specialized cell-to-cell contacts that allow the direct transfer of small molecules between cells. A single gap junction channel consists of two hemichannels, or connexons, each of which is composed of six connexin protein subunits. Connexin43 is the most ubiquitously expressed isoform of the connexin family and in the retina it is prevalent in astrocytes, Müller cells, microglia, retinal pigment epithelium and endothelial cells. Prior to docking with a neighboring cell, Connexin43 hemichannels have a low open probability as open channels constitute a large, relatively non-specific membrane pore. However, with injury and disease Connexin43 upregulation and hemichannel opening has been implicated in all aspects of secondary damage, especially glial cell activation, edema and loss of vascular integrity, leading to neuronal death. We here review gap junctions and their roles in the retina, and then focus in on Connexin43 gap junction channels in injury and disease. In particular, the effect of pathological opening of gap junction hemichannels is described, and hemichannel mediated loss of vascular integrity explained. This latter phenomenon underlies retinal pigment epithelium loss and is a common feature in several retinal diseases. Finally, Connexin43 channel roles in a number of retinal diseases including macular degeneration, glaucoma and diabetic retinopathy are considered, along with results from related animal models. A final section describes gap junction channel modulation and the ocular delivery of potential therapeutic molecules. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Optical Coherence Tomography of Retinal and Choroidal Tumors

    Directory of Open Access Journals (Sweden)

    Emil Anthony T. Say

    2011-01-01

    Full Text Available Optical coherence tomography (OCT has revolutionized the field of ophthalmology since its introduction 20 years ago. Originally intended primarily for retina specialists to image the macula, it has found its role in other subspecialties that include glaucoma, cornea, and ocular oncology. In ocular oncology, OCT provides axial resolution to approximately 7 microns with cross-sectional images of the retina, delivering valuable information on the effects of intraocular tumors on the retinal architecture. Some effects include retinal edema, subretinal fluid, retinal atrophy, photoreceptor loss, outer retinal thinning, and retinal pigment epithelial detachment. With more advanced technology, OCT now provides imaging deeper into the choroid using a technique called enhanced depth imaging. This allows characterization of the thickness and reflective quality of small (<3 mm thick choroidal lesions including choroidal nevus and melanoma. Future improvements in image resolution and depth will allow better understanding of the mechanisms of visual loss, tumor growth, and tumor management.

  11. Subvisible retinal laser therapy: titration algorithm and tissue response.

    Science.gov (United States)

    Lavinsky, Daniel; Sramek, Christopher; Wang, Jenny; Huie, Philip; Dalal, Roopa; Mandel, Yossi; Palanker, Daniel

    2014-01-01

    Laser therapy for diabetic macular edema and other retinal diseases has been used within a wide range of laser settings: from intense burns to nondamaging exposures. However, there has been no algorithm for laser dosimetry that could determine laser parameters yielding a predictable extent of tissue damage. This multimodal imaging and structural correlation study aimed to verify and calibrate a computational model-based titration algorithm for predictable laser dosimetry ranging from nondamaging to intense coagulative tissue effects. Endpoint Management, an algorithm based on a computational model of retinal photothermal damage, was used to set laser parameters for various levels of tissue effect. The algorithm adjusts both power and pulse duration to vary the expected level of thermal damage at different percentages of a reference titration energy dose. Experimental verification was conducted in Dutch Belted rabbits using a PASCAL Streamline 577 laser system. Titration was performed by adjusting laser power to produce a barely visible lesion at 20 ms pulse duration, which is defined as the nominal (100%) energy level. Tissue effects were then determined for energy levels of 170, 120, 100, 75, 50, and 30% of the nominal energy at 1 hour and 3, 7, 30, and 60 days after treatment. In vivo imaging included fundus autofluorescence, fluorescein angiography, and spectral-domain optical coherence tomography. Morphologic changes in tissue were analyzed using light microscopy, as well as scanning and transmission electron microscopy. One hundred and seventy percent and 120% levels corresponded to moderate and light burns, respectively, with damage to retinal pigment epithelium, photoreceptors, and at highest settings, to the inner retina. 50% to 75% lesions were typically subvisible ophthalmoscopically but detectable with fluorescein angiography and optical coherence tomography. Histology in these lesions demonstrated some selective damage to retinal pigment epithelium and

  12. Rendering Caustics on Non-Lambertian Surfaces

    DEFF Research Database (Denmark)

    Jensen, Henrik Wann

    1997-01-01

    This paper presents a new technique for rendering caustics on non-Lambertian surfaces. The method is based on an extension of the photon map which removes previous restrictions limiting the usage to Lambertian surfaces. We add information about the incoming direction to the photons and this allow...... reduces the rendering time. We have used the method to render caustics on surfaces with reflectance functions varying from Lambertian to glossy specular....

  13. Building Interstellar's black hole: the gravitational renderer

    OpenAIRE

    James, Oliver; Dieckmann, Sylvan; Pabst, Simon; Roberts, Paul-George H.; Thorne, Kip S.

    2015-01-01

    Interstellar is the first feature film to attempt depicting a black hole as it would actually be seen by somebody nearby. A close collaboration between the production's Scientific Advisor and the Visual Effects team led to the development of a new renderer, DNGR (Double Negative Gravitational Renderer) which uses novel techniques for rendering in curved space-time. Following the completion of the movie, the code was adapted for scientific research, leading to new insights into gravitational l...

  14. Image Based Rendering under Varying Illumination

    Institute of Scientific and Technical Information of China (English)

    Wang Chengfeng (王城峰); Hu Zhanyi

    2003-01-01

    A new approach for photorealistic rendering of a class of objects at arbitrary illumination is presented. The approach of the authors relies entirely on image based rendering techniques. A scheme is utilized for re-illumination of objects based on linear combination of low dimensional image representations. The minimum rendering condition of technique of the authors is three sample images under varying illumination of a reference object and a single input image of an interested object. Important properties of this approach are its simplicity, robustness and speediness. Experimental results validate the proposed rendering approach.

  15. RenderToolbox3: MATLAB tools that facilitate physically based stimulus rendering for vision research.

    Science.gov (United States)

    Heasly, Benjamin S; Cottaris, Nicolas P; Lichtman, Daniel P; Xiao, Bei; Brainard, David H

    2014-02-07

    RenderToolbox3 provides MATLAB utilities and prescribes a workflow that should be useful to researchers who want to employ graphics in the study of vision and perhaps in other endeavors as well. In particular, RenderToolbox3 facilitates rendering scene families in which various scene attributes and renderer behaviors are manipulated parametrically, enables spectral specification of object reflectance and illuminant spectra, enables the use of physically based material specifications, helps validate renderer output, and converts renderer output to physical units of radiance. This paper describes the design and functionality of the toolbox and discusses several examples that demonstrate its use. We have designed RenderToolbox3 to be portable across computer hardware and operating systems and to be free and open source (except for MATLAB itself). RenderToolbox3 is available at https://github.com/DavidBrainard/RenderToolbox3.

  16. Color Tuning in Short Wavelength-Sensitive Human and Mouse Visual Pigments: Ab initio Quantum Mechanics/Molecular Mechanics Studies

    Science.gov (United States)

    Altun, Ahmet; Yokoyama, Shozo; Morokuma, Keiji

    2009-01-01

    We have investigated the protonation state and photoabsorption spectrum of Schiff-base (SB) nitrogen bound 11-cis-retinal in human blue and mouse UV cone visual pigments as well as in bovine rhodopsin by hybrid quantum mechanical/molecular mechanical (QM/MM) calculations. We have employed both multireference (MRCISD+Q, MR-SORCI+Q, and MR-DDCI2+Q) and single reference (TD-B3LYP and RI-CC2) QM methods. The calculated ground-state and vertical excitation energies show that UV-sensitive pigments have deprotonated SB nitrogen, while violet-sensitive pigments have protonated SB nitrogen, in agreement with some indirect experimental evidence. A significant blue shift of the absorption maxima of violet-sensitive pigments relative to rhodopsins arises from the increase in bond length alternation of the polyene chain of 11-cis-retinal induced by polarizing fields of these pigments. The main counterion is Glu113 in both violet-sensitive vertebrate pigments and bovine rhodopsin. Neither Glu113 nor the remaining pigment has a significant influence on the first excitation energy of 11-cis-retinal in the UV-sensitive pigments that have deprotonated SB nitrogen. There is no charge transfer between the SB and β-ionone terminals of 11-cis-retinal in the ground and first excited states. PMID:19630373

  17. Effects of hypoxia on the expression of human retinal pigment epithelium cells cultured in vitro%缺氧对体外培养的人视网膜色素上皮细胞热休克蛋白47表达的影响

    Institute of Scientific and Technical Information of China (English)

    曾爱萍; 曾水清; 吕明良; 程扬

    2006-01-01

    目的:探讨缺氧对体外培养的人视网膜色素上皮(retinal pigment epitelium,RPE)细胞热休克蛋白47(heat shock protein 47,HSP47)表达的影响.方法:培养的人RPE细胞分为两组:缺氧组使用化学缺氧诱导剂CoCl2模拟RPE细胞缺氧环境来培养人RPE细胞,对照组置入正常环境中培养.采用半定量RT-PCR检测人RPE细胞中HSP47mRNA的表达,Western blot蛋白印迹分析检测人RPE细胞中蛋白水平.结果:缺氧组AHSP47/Aβ-actin比值为1.46±0.15与对照组比值0.94±0.13比较,差异有显著意义(P<0.05).同时缺氧组HSP47蛋白含量平均A值为728.03±38.15显著高于正常对照组平均A值571.47±26.95,差异有统计学意义(P<0.05).结论:HSP47在缺氧状态下异常高表达.

  18. 腺病毒转导的胞嘧啶脱氨酶基因联合5-氟胞嘧啶对人视网膜色素上皮细胞增殖的抑制作用%The inhibitive effect of adenovirus mediated CD gene and 5-FC on proliferative human retinal pigment epithelial cells

    Institute of Scientific and Technical Information of China (English)

    王文莹; 严密; 张军军

    2003-01-01

    目的研究毒性基因联合前体药物对人视网膜色素上皮(human retinal pigment epithelium, HRPE)细胞增殖的抑制作用,探讨防治增生性玻璃体视网膜病变(proliferative vitreoretinopathy, PVR)的有效方法. 方法分别将不同浓度胞嘧啶脱氨酶(cytosine deaminase, CD)基因、5-氟胞嘧啶(5-fluorocytosine, 5-FC)加入第3代体外培养的HRPE培养液中,以x-半乳糖甘酶(x-galactosidae, X-gal)与基因乳糖操纵子(lac operon,Lac Z)标记CD基因,通过检测HRPE阳性感染率衡量腺病毒的转导效率.甲基四唑盐比色法(methylthiazolyl-terazollium,MTT)检测细胞的增殖抑制率. 结果 CD基因对HRPE的转染率呈剂量依赖性.CD基因转染阳性的HRPE细胞可将5-FC转变为5-氟尿嘧啶(5-fluorouracil,5-FU),有效抑制体外培养的HRPE细胞增殖.本研究中所用的毒性基因联合前体药物治疗系统对RPE细胞生长无明显的旁观效应. 结论腺病毒载体作为高效载体,可选择性将目的基因转入细胞中.为药物防治PVR形成提供了新思路.

  19. Image Based Rendering and Virtual Reality

    DEFF Research Database (Denmark)

    Livatino, Salvatore

    The Presentation concerns with an overview of Image Based Rendering approaches and their use on Virtual Reality, including Virtual Photography and Cinematography, and Mobile Robot Navigation.......The Presentation concerns with an overview of Image Based Rendering approaches and their use on Virtual Reality, including Virtual Photography and Cinematography, and Mobile Robot Navigation....

  20. Physically based rendering: from theory to implementation

    National Research Council Canada - National Science Library

    Pharr, Matt; Humphreys, Greg, Ph. D

    2010-01-01

    ... rendering algorithm variations. This book is not only a textbook for students, but also a useful reference book for practitioners in the field. The second edition has been extended with sections on Metropolis light transport, subsurface scattering, precomputed light transport, and more. Per Christensen Senior Software Developer, RenderMan Products,...

  1. Image Based Rendering and Virtual Reality

    DEFF Research Database (Denmark)

    Livatino, Salvatore

    The Presentation concerns with an overview of Image Based Rendering approaches and their use on Virtual Reality, including Virtual Photography and Cinematography, and Mobile Robot Navigation.......The Presentation concerns with an overview of Image Based Rendering approaches and their use on Virtual Reality, including Virtual Photography and Cinematography, and Mobile Robot Navigation....

  2. Docosahexaenoic acid protects human retinal pigment epithelial cells against oxidative stress-induced apoptosis%二十二碳六烯酸抑制氧化应激状态下人视网膜色素上皮细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    刘越峰; 罗卫民; 张勇; 钟晓东

    2016-01-01

    AIM:To observe the effect of docosahexaenoic acid ( DHA) on H2 O2-induced apoptosis in human retinal pigment epithelium cells and its molecular mechanism .METHODS: Human retinal pigment epithelium cell line ARPE-19 was cultured in vitro, and 12.5 mmol/L H2 O2 was used to mimic the oxidative stress condition .The cells were treated with 30~100μmol/L DHA for 4~24 h.The expression of heme oxygenase-1 (HO-1) at mRNA and protein levels was detected by real-time PCR and Western blot , respectively .The enzymic activity of HO-1 was measured by colorimetry . Production of reactive oxygen species ( ROS) was determined by fluorescent probe .Activation of NF-E2-related factor 2 (Nrf2) was examined by immunofluorescence method .Apoptosis of ARPE-19 cells was analyzed by flow cytometry .RE-SULTS:The mRNA and protein expression and the enzymic activity of HO-1 were significantly increased in the ARPE-19 cells after DHA treatment .Meanwhile , nuclear translocation of Nrf 2 was also observed .Apoptosis appeared and ROS was produced upon H2O2 incubation.In contrast, DHA at 100 μmol/L significantly abrogated H2O2-induced apoptosis and ROS production.Furthermore, silencing of HO-1 by specific siRNA, or treatment with ZnPP, an inhibitor of HO-1, partly counteracted the protective effect against H 2 O2-induced apoptosis and ROS production .CONCLUSION: DHA protects retinal pigment epithelial cells against oxidative stress via induction of heme oxygenase -1 expression after Nrf2 activation .%目的:观察二十二碳六烯酸( docosahexaenoic acid ,DHA)对外源性H2 O2诱导人视网膜色素上皮细胞凋亡的影响及分子机制。方法:体外培养人视网膜色素上皮细胞系ARPE-19,加入终浓度为12.5 mol/L的H2 O2诱导氧化应激,随后用30~100μmol/L DHA作用细胞4~24 h;real-time PCR和Western blot分别检测血红素氧合酶-1(heme oxygenase-1,HO-1) mRNA和蛋白的表达;比色法分析HO-1酶活性;荧光

  3. Moisture movements in render on brick wall

    DEFF Research Database (Denmark)

    Hansen, Kurt Kielsgaard; Munch, Thomas Astrup; Thorsen, Peter Schjørmann

    2003-01-01

    A three-layer render on brick wall used for building facades is studied in the laboratory. The vertical render surface is held in contact with water for 24 hours simulating driving rain while it is measured with non-destructive X-ray equipment every hour in order to follow the moisture front...... through the render and into the brick. The test specimen is placed between the source and the detector. The test specimens are all scanned before they are exposed to water. In that way the loss of counts from the dry scan to the wet scan qualitatively shows the presence of water. The results show nearly...... no penetration of water through the render and into the brick, and the results are independent of the start condition of the test specimens. Also drying experiments are performed. The results show a small difference in the rate of drying, in favour of the bricks without render....

  4. Physically based rendering from theory to implementation

    CERN Document Server

    Pharr, Matt

    2010-01-01

    "Physically Based Rendering, 2nd Edition" describes both the mathematical theory behind a modern photorealistic rendering system as well as its practical implementation. A method - known as 'literate programming'- combines human-readable documentation and source code into a single reference that is specifically designed to aid comprehension. The result is a stunning achievement in graphics education. Through the ideas and software in this book, you will learn to design and employ a full-featured rendering system for creating stunning imagery. This book features new sections on subsurface scattering, Metropolis light transport, precomputed light transport, multispectral rendering, and much more. It includes a companion site complete with source code for the rendering system described in the book, with support for Windows, OS X, and Linux. Code and text are tightly woven together through a unique indexing feature that lists each function, variable, and method on the page that they are first described.

  5. Optimization-Based Wearable Tactile Rendering.

    Science.gov (United States)

    Perez, Alvaro G; Lobo, Daniel; Chinello, Francesco; Cirio, Gabriel; Malvezzi, Monica; San Martin, Jose; Prattichizzo, Domenico; Otaduy, Miguel A

    2016-10-20

    Novel wearable tactile interfaces offer the possibility to simulate tactile interactions with virtual environments directly on our skin. But, unlike kinesthetic interfaces, for which haptic rendering is a well explored problem, they pose new questions about the formulation of the rendering problem. In this work, we propose a formulation of tactile rendering as an optimization problem, which is general for a large family of tactile interfaces. Based on an accurate simulation of contact between a finger model and the virtual environment, we pose tactile rendering as the optimization of the device configuration, such that the contact surface between the device and the actual finger matches as close as possible the contact surface in the virtual environment. We describe the optimization formulation in general terms, and we also demonstrate its implementation on a thimble-like wearable device. We validate the tactile rendering formulation by analyzing its force error, and we show that it outperforms other approaches.

  6. Retinal structure in young patients aged 10 years or less with Best vitelliform macular dystrophy

    DEFF Research Database (Denmark)

    Schatz, Patrik; Sharon, Dror; Al-Hamdani, Sermed

    2016-01-01

    ranged from subtle thickening at the level of the retinal pigment epithelium-photoreceptor interdigitation line, to subretinal fluid and precipitate-like changes at the level of the photoreceptor outer segments, and further to choroidal neovascularization. The photoreceptor inner segment ellipsoid layer...... of the retinal pigment epithelium-photoreceptor interdigitation line. The photoreceptor inner segment seems to be unaffected unless choroidal neovascularization develops, which seems promising regarding future gene therapy....

  7. Stem Cell Therapies in Retinal Disorders

    Directory of Open Access Journals (Sweden)

    Aakriti Garg

    2017-02-01

    Full Text Available Stem cell therapy has long been considered a promising mode of treatment for retinal conditions. While human embryonic stem cells (ESCs have provided the precedent for regenerative medicine, the development of induced pluripotent stem cells (iPSCs revolutionized this field. iPSCs allow for the development of many types of retinal cells, including those of the retinal pigment epithelium, photoreceptors, and ganglion cells, and can model polygenic diseases such as age-related macular degeneration. Cellular programming and reprogramming technology is especially useful in retinal diseases, as it allows for the study of living cells that have genetic variants that are specific to patients’ diseases. Since iPSCs are a self-renewing resource, scientists can experiment with an unlimited number of pluripotent cells to perfect the process of targeted differentiation, transplantation, and more, for personalized medicine. Challenges in the use of stem cells are present from the scientific, ethical, and political realms. These include transplant complications leading to anatomically incorrect placement, concern for tumorigenesis, and incomplete targeting of differentiation leading to contamination by different types of cells. Despite these limitations, human ESCs and iPSCs specific to individual patients can revolutionize the study of retinal disease and may be effective therapies for conditions currently considered incurable.

  8. Recent Advances towards the Clinical Application of Stem Cells for Retinal Regeneration

    Directory of Open Access Journals (Sweden)

    G. Astrid Limb

    2012-10-01

    Full Text Available Retinal degenerative diseases constitute a major cause of irreversible blindness in the world. Stem cell-based therapies offer hope for these patients at risk of or suffering from blindness due to the deterioration of the neural retina. Various sources of stem cells are currently being investigated, ranging from human embryonic stem cells to adult-derived induced pluripotent stem cells as well as human Müller stem cells, with the first clinical trials to investigate the safety and tolerability of human embryonic stem cell-derived retinal pigment epithelium cells having recently commenced. This review aims to summarize the latest advances in the development of stem cell strategies for the replacement of retinal neurons and their supportive cells, the retinal pigment epithelium (RPE affected by retinal degenerative conditions. Particular emphasis will be given to the advances in stem cell transplantation and the challenges associated with their translation into clinical practice.

  9. Lighting a candle in the dark: advances in genetics and gene therapy of recessive retinal dystrophies.

    Science.gov (United States)

    den Hollander, Anneke I; Black, Aaron; Bennett, Jean; Cremers, Frans P M

    2010-09-01

    Nonsyndromic recessive retinal dystrophies cause severe visual impairment due to the death of photoreceptor and retinal pigment epithelium cells. These diseases until recently have been considered to be incurable. Molecular genetic studies in the last two decades have revealed the underlying molecular causes in approximately two-thirds of patients. The mammalian eye has been at the forefront of therapeutic trials based on gene augmentation in humans with an early-onset nonsyndromic recessive retinal dystrophy due to mutations in the retinal pigment epithelium-specific protein 65kDa (RPE65) gene. Tremendous challenges still lie ahead to extrapolate these studies to other retinal disease-causing genes, as human gene augmentation studies require testing in animal models for each individual gene and sufficiently large patient cohorts for clinical trials remain to be identified through cost-effective mutation screening protocols.

  10. Retinal stem cells and regeneration of vision system.

    Science.gov (United States)

    Yip, Henry K

    2014-01-01

    The vertebrate retina is a well-characterized model for studying neurogenesis. Retinal neurons and glia are generated in a conserved order from a pool of mutlipotent progenitor cells. During retinal development, retinal stem/progenitor cells (RPC) change their competency over time under the influence of intrinsic (such as transcriptional factors) and extrinsic factors (such as growth factors). In this review, we summarize the roles of these factors, together with the understanding of the signaling pathways that regulate eye development. The information about the interactions between intrinsic and extrinsic factors for retinal cell fate specification is useful to regenerate specific retinal neurons from RPCs. Recent studies have identified RPCs in the retina, which may have important implications in health and disease. Despite the recent advances in stem cell biology, our understanding of many aspects of RPCs in the eye remains limited. PRCs are present in the developing eye of all vertebrates and remain active in lower vertebrates throughout life. In mammals, however, PRCs are quiescent and exhibit very little activity and thus have low capacity for retinal regeneration. A number of different cellular sources of RPCs have been identified in the vertebrate retina. These include PRCs at the retinal margin, pigmented cells in the ciliary body, iris, and retinal pigment epithelium, and Müller cells within the retina. Because PRCs can be isolated and expanded from immature and mature eyes, it is possible now to study these cells in culture and after transplantation in the degenerated retinal tissue. We also examine current knowledge of intrinsic RPCs, and human embryonic stems and induced pluripotent stem cells as potential sources for cell transplant therapy to regenerate the diseased retina.

  11. Production of Retinal Cells from Confluent Human iPS Cells.

    Science.gov (United States)

    Reichman, Sacha; Goureau, Olivier

    2016-01-01

    Human induced pluripotent stem (hiPS) cells could be used as an unlimited source of retinal cells for the treatment of retinal degenerative diseases. Although much progress has been made in the differentiation of pluripotent stem cells towards different retinal lineages, the production of retinal cells from hiPS cells for therapeutic approaches require the development of easy and standardized protocols. In this chapter, we describe a simple and effective protocol for retinal differentiation of hiPS cells bypassing embryoid body formation and the use of exogenous molecules and substrates. In 2 weeks, confluent hiPS cells cultured in pro-neural medium can generate both retinal pigmented epithelial cells and self-forming neural retina-like structures containing retinal progenitor cells. These progenitors can be differentiated into all retinal cell types, including retinal ganglion cells and precursors of photoreceptors, which could find important applications in regenerative medicine. This differentiation system and the resulting hiPS-derived retinal cells will also offer opportunity to study the molecular and cellular mechanisms underlying human retinal development, and the establishment of in vitro models of human retinal degenerative diseases.

  12. 3D Rendering - Techniques and Challenges

    Directory of Open Access Journals (Sweden)

    Ekta Walia

    2010-04-01

    Full Text Available Computer generated images and animations are getting more and more common. They are used in many different contexts such as movies,mobiles, medical visualization, architectural visualization and CAD. Advanced ways of describing surface and light source properties are important to ensure that artists are able to create realistic and stylish looking images. Even when using advanced rendering algorithms such as ray tracing, time required for shading may contribute towards a large part of the image creation time. Therefore both performance and flexibility is important in a rendering system. This paper gives a comparative study of various 3D Rendering techniques and their challenges in a complete and systematic manner.

  13. Retinal stem/progenitor cells in the ciliary marginal zone complete retinal regeneration: a study of retinal regeneration in a novel animal model.

    Science.gov (United States)

    Miyake, Ayumi; Araki, Masasuke

    2014-07-01

    Our research group has extensively studied retinal regeneration in adult Xenopus laevis. However, X. laevis does not represent a suitable model for multigenerational genetics and genomic approaches. Instead, Xenopus tropicalis is considered as the ideal model for these studies, although little is known about retinal regeneration in X. tropicalis. In the present study, we showed that a complete retina regenerates at approximately 30 days after whole retinal removal. The regenerating retina was derived from the stem/progenitor cells in the ciliary marginal zone (CMZ), indicating a novel mode of vertebrate retinal regeneration, which has not been previously reported. In a previous study, we showed that in X. laevis, retinal regeneration occurs primarily through the transdifferentiation of retinal pigmented epithelial (RPE) cells. RPE cells migrate to the retinal vascular membrane and reform a new epithelium, which then differentiates into the retina. In X. tropicalis, RPE cells also migrated to the vascular membrane, but transdifferentiation was not evident. Using two tissue culture models of RPE tissues, it was shown that in X. laevis RPE culture neuronal differentiation and reconstruction of the retinal three-dimensional (3-D) structure were clearly observed, while in X. tropicalis RPE culture neither ßIII tubulin-positive cells nor 3-D retinal structure were seen. These results indicate that the two Xenopus species are excellent models to clarify the cellular and molecular mechanisms of retinal regeneration, as these animals have contrasting modes of regeneration; one mode primarily involves RPE cells and the other mode involves stem/progenitor cells in the CMZ.

  14. FAST CROWD RENDERING IN COMPUTER GAMES

    Directory of Open Access Journals (Sweden)

    Kaya OĞUZ

    2010-06-01

    Full Text Available Computer games, with the speed advancements of graphical processors, are coming closer to the quality of cinema industry. Contrary to offline rendering of the scenes in a motion picture, computer games should be able to render at 30 frames per second. Therefore, CPU and memory performance are sought by using various techniques. This paper is about using instancing feature of contemporary graphical processors along with level of detail techniques which has been in use for a very long time. Using instancing, 15,000 instances were successfully rendered at 30 frames per second using a very low %10 CPU usage. The application can render 40,000 instances at 13 frames per second.

  15. Visibility-Aware Direct Volume Rendering

    Institute of Scientific and Technical Information of China (English)

    Wai-Ho Mak; Yingcai Wu; Ming-Yuen Chan; Huamin Qu

    2011-01-01

    Direct volume rendering (DVR) is a powerful visualization technique which allows users to effectively explore and study volumetric datasets. Different transparency settings can be flexibly assigned to different structures such that some valuable information can be revealed in direct volume rendered images (DVRIs). However, end-users often feel that some risks are always associated with DVR because they do not know whether any important information is missing from the transparent regions of DVRIs. In this paper, we investigate how to semi-automatically generate a set of DVRIs and also an animation which can reveal information missed in the original DVRIs and meanwhile satisfy some image quality criteria such as coherence. A complete framework is developed to tackle various problems related to the generation and quality evaluation of visibility-aware DVRIs and animations. Our technique can reduce the risk of using direct volume rendering and thus boost the confidence of users in volume rendering systems.

  16. ARC Code TI: SLAB Spatial Audio Renderer

    Data.gov (United States)

    National Aeronautics and Space Administration — SLAB is a software-based, real-time virtual acoustic environment rendering system being developed as a tool for the study of spatial hearing. SLAB is designed to...

  17. Layered Textures for Image-Based Rendering

    Institute of Scientific and Technical Information of China (English)

    en-Cheng Wang; ui-Yu Li; in Zheng; n-Hua Wu

    2004-01-01

    An extension to texture mapping is given in this paper for improving the efficiency of image-based rendering. For a depth image with an orthogonal displacement at each pixel, it is decomposed by the displacement into a series of layered textures (LTs) with each one having the same displacement for all its texels. Meanwhile,some texels of the layered textures are interpolated for obtaining a continuous 3D approximation of the model represented in the depth image. Thus, the plane-to-plane texture mapping can be used to map these layered textures to produce novel views and the advantages can be obtained as follows: accelerating the rendering speed,supporting the 3D surface details and view motion parallax, and avoiding the expensive task of hole-filling in the rendering stage. Experimental results show the new method can produce high-quality images and run faster than many famous image-based rendering techniques.

  18. Composed Scattering Model for Direct Volume Rendering

    Institute of Scientific and Technical Information of China (English)

    蔡文立; 石教英

    1996-01-01

    Based on the equation of transfer in transport theory of optical physics,a new volume rendering model,called composed scattering model(CSM),is presented.In calculating the scattering term of the equation,it is decomposed into volume scattering intensity and surface scattering intensity,and they are composed with the boundary detection operator as the weight function.This proposed model differs from the most current volume rendering models in the aspect that in CSM segmentation and illumination intensity calculation are taken as two coherent parts while in existing models they are regarded as two separate ones.This model has been applied to the direct volume rendering of 3D data sets obtained by CT and MRI.The resultant images show not only rich details but also clear boundary surfaces.CSM is demonstrated to be an accurate volume rendering model suitable for CT and MRI data sets.

  19. Cell-Based Therapy for Degenerative Retinal Disease.

    Science.gov (United States)

    Zarbin, Marco

    2016-02-01

    Stem cell-derived retinal pigment epithelium (RPE) and photoreceptors (PRs) have restored vision in preclinical models of human retinal degenerative disease. This review discusses characteristics of stem cell therapy in the eye and the challenges to clinical implementation that are being confronted today. Based on encouraging results from Phase I/II trials, the first Phase II clinical trials of stem cell-derived RPE transplantation are underway. PR transplant experiments have demonstrated restoration of visual function in preclinical models of retinitis pigmentosa and macular degeneration, but also indicate that no single approach is likely to succeed in overcoming PR loss in all cases. A greater understanding of the mechanisms controlling synapse formation as well as the immunoreactivity of transplanted retinal cells is urgently needed.

  20. Prospectives for gene therapy of retinal degenerations.

    Science.gov (United States)

    Thumann, Gabriele

    2012-08-01

    Retinal degenerations encompass a large number of diseases in which the retina and associated retinal pigment epithelial (RPE) cells progressively degenerate leading to severe visual disorders or blindness. Retinal degenerations can be divided into two groups, a group in which the defect has been linked to a specific gene and a second group that has a complex etiology that includes environmental and genetic influences. The first group encompasses a number of relatively rare diseases with the most prevalent being Retinitis pigmentosa that affects approximately 1 million individuals worldwide. Attempts have been made to correct the defective gene by transfecting the appropriate cells with the wild-type gene and while these attempts have been successful in animal models, human gene therapy for these inherited retinal degenerations has only begun recently and the results are promising. To the second group belong glaucoma, age-related macular degeneration (AMD) and diabetic retinopathy (DR). These retinal degenerations have a genetic component since they occur more often in families with affected probands but they are also linked to environmental factors, specifically elevated intraocular pressure, age and high blood sugar levels respectively. The economic and medical impact of these three diseases can be assessed by the number of individuals affected; AMD affects over 30 million, DR over 40 million and glaucoma over 65 million individuals worldwide. The basic defect in these diseases appears to be the relative lack of a neurogenic environment; the neovascularization that often accompanies these diseases has suggested that a decrease in pigment epithelium-derived factor (PEDF), at least in part, may be responsible for the neurodegeneration since PEDF is not only an effective neurogenic and neuroprotective agent but also a potent inhibitor of neovascularization. In the last few years inhibitors of vascularization, especially antibodies against vascular endothelial cell

  1. Fine structure of the retinal pigment epithelium and cones of Antarctic fish Notohenia coriiceps Richardson in light and dark-conditions Ultraestrutra do epitélio pigmentar da retina e dos cones do peixe Antártico Notothenia coriiceps Richardson submetido à luz e ao escuro

    Directory of Open Access Journals (Sweden)

    Lucélia Donatti

    2007-03-01

    Full Text Available The Antarctic fish Notothenia coriiceps Richardson, 1844 lives in an environment of daily and annual photic variation and retina cells have to adjust morphologically to environmental luminosity. After seven day dark or seven day light acclimation of two groups of fish, retinas were extracted and processed for light and transmission electron microscopy. In seven day dark adapted, retina pigment epithelium melanin granules were aggregated at the basal region of cells, and macrophages were seen adjacent to the apical microvilli, between the photoreceptors. In seven day light adapted epithelium, melanin granules were inside the apical microvilli of epithelial cells and macrophages were absent. The supranuclear region of cones adapted to seven day light had less electron dense cytoplasm, and an endoplasmic reticulum with broad tubules. The mitochondria in the internal segment of cones adapted to seven day light were larger, and less electron dense. The differences in the morphology of cones and pigment epithelial cells indicate that N. coriiceps has retinal structural adjustments presumably optimizing vision in different light conditions.O peixe Antártico Notothenia coriiceps Richardson, 1844 habita meios com variações fóticas diária e anual e as células da retina se adaptam morfologicamente a esta luminosidade ambiental. Dois grupos de peixes foram aclimatados durante sete dias à luz constante ou ao escuro constante. Após secção medular, as retinas foram extraídas e processadas para microscopia de luz e microscopia eletrônica de transmissão. No epitélio pigmentar da retina adaptado sete dias ao escuro, os pigmentos de melanina agregam-se na base coroidal das células epiteliais pigmentares e macrófagos são encontrados no interior do processos apicais entre as células fotorreceptoras. No epitélio adaptado sete dias à luz os pigmentos de melanina se dispõem ao longo das projeções apicais das células epiteliais pigmentares e os

  2. 体外诱导大鼠骨髓间充质干细胞向视网膜色素上皮细胞分化的可行性研究%Feasibility study of differentiation of invitro induced rat bone marrow-derived mesenchymal stem cells into retinal pigment epithelial cells

    Institute of Scientific and Technical Information of China (English)

    高斐; 董方田

    2009-01-01

    Objective To investigate the feasibility of differentiation of invitro induced rat bone marrow-derived mesenchymal stem cells(rMSCs) into retinal pigment epithelial (RPE) cells.Methods The rMSCs from Brwon-Norway (BN) rats were isolated and cultured by adherent screening method.RPE cells lysate made by repeated freeze-thawing was put into the rMSCs culture system to identify whether the induced cells could express characteristic label cytokeratin(CK)and S-100 simultaneously or not.Results The growth rate of rMSCs induced by RPE cells lysate was slower and protuberant burr surrounded the fusiform cells.The results of immunoblotting and double immunofluorescence showed that partial induced cells expressed CK and S-100 simultaneously.The result of flow cytometry indicated that 14.1% induced cells expressed CK and S-100 simultaneously.Conclusion Induced by RPE cells lysate,rMSCs can differentiate into RPE cells.%目的 探讨体外诱导大鼠骨髓间充质干细胞(rMSCs)向视网膜色素上皮(RPE)细胞分化的可行性.方法 采用贴壁筛选法分离、培养Brown-Norway(BN)rMSCs.将反复冻融制成的BN大鼠RPE细胞裂解液加入到rMSCs培养体系中,鉴定被诱导的细胞是否同时表达RPE细胞的特征性标记物细胞角蛋白(CK)与S-100.结果 经RPE细胞裂解液诱导的rMSCs生长速度减慢,细胞呈长梭形,周边有毛刺样突起.免疫印迹法和双重免疫荧光标记显示部分经诱导的细胞同时表达CK与S-100.流式细胞术显示14.1%的细胞能够同时表达CK与S-100.结论 rMSCs经RPE细胞裂解液诱导后能够向RPE细胞方向分化.

  3. Rendering and Compositing Infrastructure Improvements to VisIt for Insitu Rendering

    Energy Technology Data Exchange (ETDEWEB)

    Loring, Burlen [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Ruebel, Oliver [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2016-01-28

    Compared to posthoc rendering, insitu rendering often generates larger numbers of images, as a result rendering performance and scalability are critical in the insitu setting. In this work we present improvements to VisIt's rendering and compositing infrastructure that deliver increased performance and scalability in both posthoc and insitu settings. We added the capability for alpha blend compositing and use it with ordered compositing when datasets have disjoint block domain decomposition to optimize the rendering of transparent geometry. We also made improvements that increase overall efficiency by reducing communication and data movement and have addressed a number of performance issues. We structured our code to take advantage of SIMD parallelization and use threads to overlap communication and compositing. We tested our improvements on a 20 core workstation using 8 cores to render geometry generated from a $256^3$ cosmology dataset and on a Cray XC31 using 512 cores to render geometry generated from a $2000^2 \\times 800$ plasma dataset. Our results show that ordered compositing provides a speed up of up to $4 \\times$ over the current sort first strategy. The other improvements resulted in modest speed up with one notable exception where we achieve up to $40 \\times$ speed up of rendering and compositing of opaque geometry when both opaque and transparent geometry are rendered together. We also investigated the use of depth peeling, but found that the implementation provided by VTK is substantially slower,both with and without GPU acceleration, than a local camera order sort.

  4. Brain Image Representation and Rendering: A Survey

    Directory of Open Access Journals (Sweden)

    Mudassar Raza

    2012-09-01

    Full Text Available Brain image representation and rendering processes are basically used for evaluation, development and investigation consent experimental examination and formation of brain images of a variety of modalities that includes the major brain types like MEG, EEG, PET, MRI, CT or microscopy. So, there is a need to conduct a study to review the existing work in this area. This paper provides a review of different existing techniques and methods regarding the brain image representation and rendering. Image Rendering is the method of generating an image by means of a model, through computer programs. The basic purpose of brain image representation and rendering processes is to analyze the brain images precisely in order to effectively diagnose and examine the diseases and problems. The basic objective of this study is to evaluate and discuss different techniques and approaches proposed in order to handle different brain imaging types. The paper provides a short overview of different methods, in the form of advantages and limitations, presented in the prospect of brain image representation and rendering along with their sub categories proposed by different authors.

  5. Equalizer: a scalable parallel rendering framework.

    Science.gov (United States)

    Eilemann, Stefan; Makhinya, Maxim; Pajarola, Renato

    2009-01-01

    Continuing improvements in CPU and GPU performances as well as increasing multi-core processor and cluster-based parallelism demand for flexible and scalable parallel rendering solutions that can exploit multipipe hardware accelerated graphics. In fact, to achieve interactive visualization, scalable rendering systems are essential to cope with the rapid growth of data sets. However, parallel rendering systems are non-trivial to develop and often only application specific implementations have been proposed. The task of developing a scalable parallel rendering framework is even more difficult if it should be generic to support various types of data and visualization applications, and at the same time work efficiently on a cluster with distributed graphics cards. In this paper we introduce a novel system called Equalizer, a toolkit for scalable parallel rendering based on OpenGL which provides an application programming interface (API) to develop scalable graphics applications for a wide range of systems ranging from large distributed visualization clusters and multi-processor multipipe graphics systems to single-processor single-pipe desktop machines. We describe the system architecture, the basic API, discuss its advantages over previous approaches, present example configurations and usage scenarios as well as scalability results.

  6. Standardized rendering from IR surveillance motion imagery

    Science.gov (United States)

    Prokoski, F. J.

    2014-06-01

    Government agencies, including defense and law enforcement, increasingly make use of video from surveillance systems and camera phones owned by non-government entities.Making advanced and standardized motion imaging technology available to private and commercial users at cost-effective prices would benefit all parties. In particular, incorporating thermal infrared into commercial surveillance systems offers substantial benefits beyond night vision capability. Face rendering is a process to facilitate exploitation of thermal infrared surveillance imagery from the general area of a crime scene, to assist investigations with and without cooperating eyewitnesses. Face rendering automatically generates greyscale representations similar to police artist sketches for faces in surveillance imagery collected from proximate locations and times to a crime under investigation. Near-realtime generation of face renderings can provide law enforcement with an investigation tool to assess witness memory and credibility, and integrate reports from multiple eyewitnesses, Renderings can be quickly disseminated through social media to warn of a person who may pose an immediate threat, and to solicit the public's help in identifying possible suspects and witnesses. Renderings are pose-standardized so as to not divulge the presence and location of eyewitnesses and surveillance cameras. Incorporation of thermal infrared imaging into commercial surveillance systems will significantly improve system performance, and reduce manual review times, at an incremental cost that will continue to decrease. Benefits to criminal justice would include improved reliability of eyewitness testimony and improved accuracy of distinguishing among minority groups in eyewitness and surveillance identifications.

  7. Dynamic eye phantom for retinal oximetry measurements

    Science.gov (United States)

    Lemaillet, Paul; Ramella-Roman, Jessica C.

    2009-11-01

    Measurements of oxygen saturation and flow in the retina can yield information about eye health and the onset of eye pathologies such as diabetic retinopathy. Recently, we developed a multiaperture camera that uses the division of the retinal image into several wavelength-sensitive subimages to compute retinal oxygen saturation. The calibration of such instruments is particularly difficult due to the layered structure of the eye and the lack of alternative measurement techniques. For this purpose, we realize an in vitro model of the human eye composed of a lens, the retina vessel, and three layers: the choroid, the retinal pigmented epithelium, and the sclera. The retinal vessel is modeled with a microtube connected to a micropump and a hemoglobin reservoir in a closed circulatory system. Hemoglobin oxygenation in the vessel could be altered using a reversible fuel cell. The sclera is represented by a Spectralon slab. The optical properties of the other layers are mimicked using titanium dioxide as a scatterer, ink as an absorber, and epoxy as a supporting structure. The optical thickness of each layer of the eye phantom is matched to each respective eye layer.

  8. A dark and constitutively active mutant of the tiger salamander UV pigment.

    Science.gov (United States)

    Kono, Masahiro; Crouch, Rosalie K; Oprian, Daniel D

    2005-01-18

    A triple mutant (F86L/T93P/S118T; bovine rhodopsin numbering) of the tiger salamander UV cone pigment appears to be trapped in an open conformation that is metarhodopsin-II-like. The pigment is able to activate transducin in the dark, and the ligand-free apoprotein is also able to activate transducin constitutively. The pigment permits protons and chloride ions from solution access to the active site as it displays a pH- and NaCl-dependent absorption spectrum not observed with the wild-type pigment. However, the wild-type properties of light-dependent activity and a pH-independent absorption spectrum are recovered upon reconstitution of the triple mutant with 11-cis-9-demethyl retinal. These results suggest that binding the native chromophore cannot deactivate the protein because of steric interactions between the protein, possibly residue 118, and the 9-methyl group of the chromophore. Furthermore, the absorption spectrum of the 9-demethyl retinal regenerated pigment exhibits a band broader and with lower extinction at the absorption maximum than either the human blue or salamander UV wild-type pigments generated with the same retinal analogue. The broad spectrum appears to be comprised of two or more species and can be well-fit by a sum of scaled spectra of the two wild-type pigments. Binding the chromophore appears to trap the pigment in two or more conformations. The triple mutant reported here represents the first example of a dark-active cone pigment and constitutively active cone opsin.

  9. Pigments in Thermophilic fungi

    OpenAIRE

    Somasundaram, T.; Rao, Sanjay SR; Maheshwari,R.

    1986-01-01

    UV and visible absorption spectra of thermophilic fungi were obtained by photoacoustic spectroscopy. Based on these data as well as on the chem. properties and IR spectra, it is suggested that the pigments may be hydroxylated polycyclic quinones.

  10. Adaptive Rendering Based on Visual Acuity Equations

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    A new method of adaptable rendering for interaction in Virtual Environment(VE) through different visual acuity equations is proposed. An acuity factor equation of luminance vision is first given. Secondly, five equations which calculate the visual acuity through visual acuity factors are presented, and adaptive rendering strategy based on different visual acuity equations is given. The VE system may select one of them on the basis of the host's load, hereby select LOD for each model which would be rendered. A coarser LOD is selected where the visual acuity is lower, and a better LOD is used where it is higher. This method is tested through experiments and the experimental results show that it is effective.

  11. Rendering Falling Leaves on Graphics Hardware

    Directory of Open Access Journals (Sweden)

    Marcos Balsa

    2008-04-01

    Full Text Available There is a growing interest in simulating natural phenomena in computer graphics applications. Animating natural scenes in real time is one of the most challenging problems due to the inherent complexity of their structure, formed by millions of geometric entities, and the interactions that happen within. An example of natural scenario that is needed for games or simulation programs are forests. Forests are difficult to render because the huge amount of geometric entities and the large amount of detail to be represented. Moreover, the interactions between the objects (grass, leaves and external forces such as wind are complex to model. In this paper we concentrate in the rendering of falling leaves at low cost. We present a technique that exploits graphics hardware in order to render thousands of leaves with different falling paths in real time and low memory requirements.

  12. Photosynthetic Pigments in Diatoms

    OpenAIRE

    Paulina Kuczynska; Malgorzata Jemiola-Rzeminska; Kazimierz Strzalka

    2015-01-01

    Photosynthetic pigments are bioactive compounds of great importance for the food, cosmetic, and pharmaceutical industries. They are not only responsible for capturing solar energy to carry out photosynthesis, but also play a role in photoprotective processes and display antioxidant activity, all of which contribute to effective biomass and oxygen production. Diatoms are organisms of a distinct pigment composition, substantially different from that present in plants. Apart from light-harvestin...

  13. 人脂肪间充质干细胞向视网膜色素上皮样细胞的诱导分化及其在体应用研究%Study on human adipose mesenchymal stem cells differentiating into retinal pigment epithelial-like cells and its in vivo application

    Institute of Scientific and Technical Information of China (English)

    郭凯; 罗燕; 李涛; 田景毅; 孙伟; 林少芬; 唐仕波

    2015-01-01

    视网膜的不良反应.%Background Stem cell transplantation represents a promising treatment option for patients suffering from degenerative disorders.Accumulating evidences indicate that mesenchymal stem cells (MSCs) are able to differentiate into retinal pigment epithelial (RPE)-like cells.However,MSCs are difficult to obtain.Human adipose mesenchymal stem cells (ADSCs) are proved to have similar properties to MSCs,but relevant study is less.Objective This study was to assess the feasibility of human ADSCs differentiating into RPE-like cells and the safety of its application in vivo.Methods The third generation of human ADSCs were incubated into 6-well plate,and 100 ng/ml epithelial growth factor,50 μ mol/L taurine and 5×10-7 mol/L retinoic acid were added into the medium 12 hours after cultured to induce the cells,and conventional cultured cells were used as the control group.Induced cells were traced with PKH26,and Pan-cytoke ratin (Pan-CK) monoclonal antibody was used to identify the cells under the fluorescence microscope.Induced RPE-like cell suspension of 1 μl was intravetreally injected in the right eyes of 6 BALB/c mice,and equal volume of PBS was used in the same way in another 6 mice.The animals were sacrificed 1 month after injection,and the retinal morphology was examined by histopathology under the optical microscope.The ultrastructure of retinal ganglion cells (RGCs) was examined by the transmission electron microscope.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Cultured human ADSCs grew well with the slender polygone shape.Cell membranes showed the red fluorescence for PKH26 after induced.In addition,Pan-CK was expressed in the cell membranes with the red fluorescence in the induced cells,but the response was absent in the control cells.One month after intravitreal injection,induced cells located on the retinal surface,and the retinal morphology was clear

  14. Photosynthetic Pigments in Diatoms

    Directory of Open Access Journals (Sweden)

    Paulina Kuczynska

    2015-09-01

    Full Text Available Photosynthetic pigments are bioactive compounds of great importance for the food, cosmetic, and pharmaceutical industries. They are not only responsible for capturing solar energy to carry out photosynthesis, but also play a role in photoprotective processes and display antioxidant activity, all of which contribute to effective biomass and oxygen production. Diatoms are organisms of a distinct pigment composition, substantially different from that present in plants. Apart from light-harvesting pigments such as chlorophyll a, chlorophyll c, and fucoxanthin, there is a group of photoprotective carotenoids which includes β-carotene and the xanthophylls, diatoxanthin, diadinoxanthin, violaxanthin, antheraxanthin, and zeaxanthin, which are engaged in the xanthophyll cycle. Additionally, some intermediate products of biosynthetic pathways have been identified in diatoms as well as unusual pigments, e.g., marennine. Marine algae have become widely recognized as a source of unique bioactive compounds for potential industrial, pharmaceutical, and medical applications. In this review, we summarize current knowledge on diatom photosynthetic pigments complemented by some new insights regarding their physico-chemical properties, biological role, and biosynthetic pathways, as well as the regulation of pigment level in the cell, methods of purification, and significance in industries.

  15. Blender cycles lighting and rendering cookbook

    CERN Document Server

    Iraci, Bernardo

    2013-01-01

    An in-depth guide full of step-by-step recipes to explore the concepts behind the usage of Cycles. Packed with illustrations, and lots of tips and tricks; the easy-to-understand nature of the book will help the reader understand even the most complex concepts with ease.If you are a digital artist who already knows your way around Blender, and you want to learn about the new Cycles' rendering engine, this is the book for you. Even experts will be able to pick up new tips and tricks to make the most of the rendering capabilities of Cycles.

  16. Volume Rendering for Curvilinear and Unstructured Grids

    Energy Technology Data Exchange (ETDEWEB)

    Max, N; Williams, P; Silva, C; Cook, R

    2003-03-05

    We discuss two volume rendering methods developed at Lawrence Livermore National Laboratory. The first, cell projection, renders the polygons in the projection of each cell. It requires a global visibility sort in order to composite the cells in back to front order, and we discuss several different algorithms for this sort. The second method uses regularly spaced slice planes perpendicular to the X, Y, or Z axes, which slice the cells into polygons. Both methods are supplemented with anti-aliasing techniques to deal with small cells that might fall between pixel samples or slice planes, and both have been parallelized.

  17. GPU Pro 5 advanced rendering techniques

    CERN Document Server

    Engel, Wolfgang

    2014-01-01

    In GPU Pro5: Advanced Rendering Techniques, section editors Wolfgang Engel, Christopher Oat, Carsten Dachsbacher, Michal Valient, Wessam Bahnassi, and Marius Bjorge have once again assembled a high-quality collection of cutting-edge techniques for advanced graphics processing unit (GPU) programming. Divided into six sections, the book covers rendering, lighting, effects in image space, mobile devices, 3D engine design, and compute. It explores rasterization of liquids, ray tracing of art assets that would otherwise be used in a rasterized engine, physically based area lights, volumetric light

  18. Digital color acquisition, perception, coding and rendering

    CERN Document Server

    Fernandez-Maloigne, Christine; Macaire, Ludovic

    2013-01-01

    In this book the authors identify the basic concepts and recent advances in the acquisition, perception, coding and rendering of color. The fundamental aspects related to the science of colorimetry in relation to physiology (the human visual system) are addressed, as are constancy and color appearance. It also addresses the more technical aspects related to sensors and the color management screen. Particular attention is paid to the notion of color rendering in computer graphics. Beyond color, the authors also look at coding, compression, protection and quality of color images and videos.

  19. Haptic rendering for simulation of fine manipulation

    CERN Document Server

    Wang, Dangxiao; Zhang, Yuru

    2014-01-01

    This book introduces the latest progress in six degrees of freedom (6-DoF) haptic rendering with the focus on a new approach for simulating force/torque feedback in performing tasks that require dexterous manipulation skills. One of the major challenges in 6-DoF haptic rendering is to resolve the conflict between high speed and high fidelity requirements, especially in simulating a tool interacting with both rigid and deformable objects in a narrow space and with fine features. The book presents a configuration-based optimization approach to tackle this challenge. Addressing a key issue in man

  20. 水蛭渗滤液对凝血酶诱导的人视网膜色素上皮细胞周期的影响%The effects of leech percolate on cell dividing cycle of human retinal pigment epithelium cells induced by thrombin.

    Institute of Scientific and Technical Information of China (English)

    郑燕林; 左玉霞; 马素红

    2008-01-01

    Objective To investigate the effects of leech percolate on cell division cycle of culturedhuman retinal pigment epithelium (hRPE)cells induced by thrombin.Exploring mechanism of leech percolateon preventing and curing PVR.Methods (1)Human retinal pigment epithelium(hRPE)cells were cultured invitro with digest culture method. (2)The selected concentrations of leech percolate (128mg/ml,64mg/ml,32mg/ml,16mg/ml and 8mg/ml)were based on the previous experiments.With or without 0.5NIHU/mlthrombin, hRPE cells were treated with leech percolate by various concentrations for 24hrs, and the effects ofcell division cycle of leech percolate were studied with Flow Cytometer (FCM). (3)With or without0.5NIHU/ml thrombin, hRPE cells were treated with 64mg/ml leech percolate at different times(6hr, 12hr,24hrand 48hr), and the effects of cell division cycle of leech percolate were treated with FCM.Results (1)Therewas a general tendency that leech percolate of the selected concentrations arrested hRPE cells on G0/G1 phaseand 64mg/mi leech percolate inhibited 77.9% hRPE cells on G0/G1 phase and made hRPE cells on S phaseless especially. (2)During the period of 6hrs and 48hrs the longer the treat time of 64mg/ml leech percolate, themore the RPE cells arrested on G0/G1 phase.Conclusion The results approve leech percolate can inhibitproliferation of human RPE cells cultured in vitro.Its mechanism may be that leech percolate could act on celldivision cycle and arrest RPE cells on G0/G1 phase to make proliferation power of hRPE cells lower.%目的 研究水蛭渗滤液对凝血酶诱导的人视网膜色素上皮细胞周期的影响,从而探讨中药水蛭用于防治PVR的作用机制.方法 (1)采用消化培养法,对人视网膜色素上皮细胞进行体外传代培养. (2)在前期实验基础上选择128mg/ml、64mg/ml、32mg/ml、16mg/ml、8mg/ml浓度的水蛭渗滤液,用流式细胞仪测定细胞周期,观察水蛭渗滤液单独或与0.5NIHU/ml凝血酶共同作用