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.

Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq

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Whitmore, S. Scott; Wagner, Alex H.; DeLuca, Adam P.; Drack, Arlene V.; Stone, Edwin M.; Tucker, Budd A.; Zeng, Shemin; Braun, Terry A.; Mullins, Robert F.; Scheetz, Todd E.

2014-01-01

Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell-types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with

Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq.

Science.gov (United States)

Whitmore, S Scott; Wagner, Alex H; DeLuca, Adam P; Drack, Arlene V; Stone, Edwin M; Tucker, Budd A; Zeng, Shemin; Braun, Terry A; Mullins, Robert F; Scheetz, Todd E

2014-12-01

Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with

Bcl-2, Bax, and c-Fos expression correlates to RPE cell apoptosis induced by UV-light and daunorubicin

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Liang, Y G; Jorgensen, A G; Kaestel, C G

2000-01-01

PURPOSE. The aim of this study was to determine the role of Bcl-2, Bcl-X L, Bax, and c-Fos in regulation of apoptosis, induced by ultraviolet-light A (UV-A) and daunorubicin (DNR), in retinal pigment epithelium (RPE) cells grown on bovine extracellular matrix (ECM)-coated or uncoated plastic dishes....... METHODS. Apoptosis in confluent RPE cells cultured on ECM-coated or uncoated dishes was induced by UV-A or DNR. Apoptosis was detected by 7-amino-actinomycin D labeling followed by flow cytometry and by terminal deoxy-transferase mediated X-dUTP nick end labeling (TUNEL). Cellular expression of Bcl-2, Bcl......-X L, Bax, and c-Fos was determined by the use of antibodies and flow cytometry, Western blot analysis, and immunocytochemical staining. RESULTS. Both UV-A and DNR induce apoptosis in human RPE cells in vitro. Human fetal RPE cells grown on ECM-coated dishes were significantly more resistant to UV...

Effects of aflibercept on primary RPE cells: toxicity, wound healing, uptake and phagocytosis.

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Klettner, Alexa; Tahmaz, Nihat; Dithmer, Michaela; Richert, Elisabeth; Roider, Johann

2014-10-01

Anti-VEGF treatment is the therapy of choice in age-related macular degeneration, and is also applied in diabetic macular oedema or retinal vein occlusion. Recently, the fusion protein, aflibercept, has been approved for therapeutic use. In this study, we investigate the effects of aflibercept on primary RPE cells. Primary RPE cells were prepared from freshly slaughtered pigs' eyes. The impact of aflibercept on cell viability was investigated with MTT and trypan blue exclusion assay. The influence of aflibercept on wound healing was assessed with a scratch assay. Intracellular uptake of aflibercept was investigated in immunohistochemistry and its influence on phagocytosis with a phagocytosis assay using opsonised latex beads. Aflibercept displays no cytotoxicity on RPE cells but impairs its wound healing ability. It is taken up into RPE cells and can be intracellularly detected for at least 7 days. Intracellular aflibercept impairs the phagocytic capacity of RPE cells. Aflibercept interferes with the physiology of RPE cells, as it is taken up into RPE cells, which is accompanied by a reduction of the phagocytic ability. Additionally, it impairs the wound healing capacity of RPE cells. These effects on the physiology of RPE cells may indicate possible side effects. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

A2E Suppresses Regulatory Function of RPE Cells in Th1 Cell Differentiation Via Production of IL-1β and Inhibition of PGE2.

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Shi, Qian; Wang, Qiu; Li, Jing; Zhou, Xiaohui; Fan, Huimin; Wang, Fenghua; Liu, Haiyun; Sun, Xiangjun; Sun, Xiaodong

2015-12-01

Inflammatory status of RPE cells induced by A2E is essential in the development of AMD. Recent research indicated T-cell immunity was involved in the pathological progression of AMD. This study was designed to investigate how A2E suppresses immunoregulatory function of RPE cells in T-cell immunity in vitro. Mouse RPE cells or human ARPE19 cells were stimulated with A2E, and co-cultured with naïve T cells under Th1, Th2, Th17, and regulatory T cell (Treg) polarization conditions. The intracellular cytokines or transcript factors of the induced T-cells subset were detected with flow cytometer and qRT-PCR. The ROS levels were detected, and the factors and possible pathways involved in the A2E-laden RPE cells were analyzed through neutralization antibody of IL-1β and inhibitors of related pathways. The A2E reduced regulatory function of RPE cells in Treg differentiation. The A2E-laden RPE cells promoted polarization of Th1 cells in vitro, but not Th2 or Th17 differentiation. The A2E induced RPE cells to release inflammatory cytokines and ROS, but PGE2 production was inhibited. Through neutralization of IL-1β or inhibition of COX2-PGE2 pathways, A2E-laden RPE cells expressed reduced effect in inducing Th1 cells. The A2E inhibited regulatory function of RPE cells in suppressing Th1 cell immunity in vitro through production of IL-1β and inhibition of PGE2. Our data indicate that A2E could suppress immunoregulatory function of RPE cells and adaptive immunity might play a role in the immune pathogenesis of AMD.

The anterior lens capsule used as support material in RPE cell-transplantation

DEFF Research Database (Denmark)

Nicolini, J; Kiilgaard, Jens Folke; Wiencke, A K

2000-01-01

To investigate the use of an ocular basement membrane as support material for transplanted porcine RPE cells.......To investigate the use of an ocular basement membrane as support material for transplanted porcine RPE cells....

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.

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

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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.

Methodologies for analysis of patterning in the mouse RPE sheet

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Boatright, Jeffrey H.; Dalal, Nupur; Chrenek, Micah A.; Gardner, Christopher; Ziesel, Alison; Jiang, Yi; Grossniklaus, Hans E.

2015-01-01

Purpose Our goal was to optimize procedures for assessing shapes, sizes, and other quantitative metrics of retinal pigment epithelium (RPE) cells and contact- and noncontact-mediated cell-to-cell interactions across a large series of flatmount RPE images. Methods The two principal methodological advances of this study were optimization of a mouse RPE flatmount preparation and refinement of open-access software to rapidly analyze large numbers of flatmount images. Mouse eyes were harvested, and extra-orbital fat and muscles were removed. Eyes were fixed for 10 min, and dissected by puncturing the cornea with a sharp needle or a stab knife. Four radial cuts were made with iridectomy scissors from the puncture to near the optic nerve head. The lens, iris, and the neural retina were removed, leaving the RPE sheet exposed. The dissection and outcomes were monitored and evaluated by video recording. The RPE sheet was imaged under fluorescence confocal microscopy after staining for ZO-1 to identify RPE cell boundaries. Photoshop, Java, Perl, and Matlab scripts, as well as CellProfiler, were used to quantify selected parameters. Data were exported into Excel spreadsheets for further analysis. Results A simplified dissection procedure afforded a consistent source of images that could be processed by computer. The dissection and flatmounting techniques were illustrated in a video recording. Almost all of the sheet could be routinely imaged, and substantial fractions of the RPE sheet (usually 20–50% of the sheet) could be analyzed. Several common technical problems were noted and workarounds developed. The software-based analysis merged 25 to 36 images into one and adjusted settings to record an image suitable for large-scale identification of cell-to-cell boundaries, and then obtained quantitative descriptors of the shape of each cell, its neighbors, and interactions beyond direct cell–cell contact in the sheet. To validate the software, human- and computer

Zeaxanthin Inhibits Hypoxia-Induced VEGF Secretion by RPE Cells through Decreased Protein Levels of Hypoxia-Inducible Factors-1α

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Richard Rosen

2015-01-01

Full Text Available Hypoxia is the most important stimulus leading to upregulation of VEGF in the retina and this is caused by accumulation of hypoxia-inducible factors-1α (HIF-1α protein. The effects of zeaxanthin, a natural phytochemical, on the VEGF and HIF-1α expression in the primary culture of human retinal pigment epithelial (RPE cells were studied. An in vitro RPE cell hypoxia model was established by placing cells under 1% oxygen pressure or by adding cobalt chloride (CoCl2 to the culture medium. RPE cells and conditioned media were collected from cultures treated with and without zeaxanthin under normoxic and hypoxic conditions. VEGF and HIF-1α protein and RNA levels were measured by ELISA kits and RT-PCR, respectively. Hypoxia caused a significant increase of VEGF expression and accumulation of HIF-1α in RPE cells. Zeaxanthin at 50–150 μM significantly inhibited the expression of VEGF and accumulation of HIF-1α protein caused by hypoxia but did not affect expression of VEGF and HIF-1α under normoxic conditions. This is the first report on the effect of zeaxanthin on VEGF and HIF-1α levels in cultured RPE cells and suggests that zeaxanthin may have potential value in the prevention and treatment of various retinal diseases associated with vascular leakage and neovascularization.

Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

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Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

2017-10-01

Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

PlGF gene knockdown in human retinal pigment epithelial cells.

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Akrami, Hassan; Soheili, Zahra-Soheila; Sadeghizadeh, Majid; Ahmadieh, Hamid; Rezaeikanavi, Mozhgan; Samiei, Shahram; Khalooghi, Keynoush

2011-04-01

To evaluate the knockdown of placental growth factor (PlGF) gene expression in human retinal pigment epithelium (RPE) cells and its effect on cell proliferation, apoptosis and angiogenic potential of RPE cells. Human RPE cells were isolated by dispase I solution and cultured in DMEM/F12 supplemented with 10% fetal calf serum (FCS). A small interfering RNA (siRNA) corresponding to PlGF mRNA and a scrambled siRNA (scRNA) were introduced into the cells. Cell proliferation and cell death were examined by ELISA. PlGF mRNA and protein were quantified by real-time polymerase chain reaction (PCR) and western blot. The levels of gene expression for human retinal pigment epithelium-specific protein 65 kDa (RPE65), cellular retinaldehyde-binding protein (CRALBP) and tyrosinase were examined by real-time PCR. The angiogenic activity of RPE cell-derived conditioned media was assayed by a tube formation assay using human umbilical vein endothelial cells (HUVECs). At a final siRNA concentration of 20 pmol/ml, the transfection efficiency was about 80%. The amount of PlGF transcripts was reduced to 10% after 36 h of incubation, and the amount of PlGF protein in culture supernatant was significantly decreased. Suppression of PlGF gene had no effect on RPE cell proliferation and survival, and there were no notable changes in the transcript levels of RPE65, CRALBP or tyrosinase for the cultures treated by siRNA cognate to PlGF. Vascular tube formation was efficiently reduced in HUVECs. Our findings present PlGF as a key modulator of angiogenic potential in RPE cells of the human retina.

Canine and human visual cortex intact and responsive despite early retinal blindness from RPE65 mutation.

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Geoffrey K Aguirre

2007-06-01

Full Text Available RPE65 is an essential molecule in the retinoid-visual cycle, and RPE65 gene mutations cause the congenital human blindness known as Leber congenital amaurosis (LCA. Somatic gene therapy delivered to the retina of blind dogs with an RPE65 mutation dramatically restores retinal physiology and has sparked international interest in human treatment trials for this incurable disease. An unanswered question is how the visual cortex responds after prolonged sensory deprivation from retinal dysfunction. We therefore studied the cortex of RPE65-mutant dogs before and after retinal gene therapy. Then, we inquired whether there is visual pathway integrity and responsivity in adult humans with LCA due to RPE65 mutations (RPE65-LCA.RPE65-mutant dogs were studied with fMRI. Prior to therapy, retinal and subcortical responses to light were markedly diminished, and there were minimal cortical responses within the primary visual areas of the lateral gyrus (activation amplitude mean +/- standard deviation [SD] = 0.07% +/- 0.06% and volume = 1.3 +/- 0.6 cm(3. Following therapy, retinal and subcortical response restoration was accompanied by increased amplitude (0.18% +/- 0.06% and volume (8.2 +/- 0.8 cm(3 of activation within the lateral gyrus (p < 0.005 for both. Cortical recovery occurred rapidly (within a month of treatment and was persistent (as long as 2.5 y after treatment. Recovery was present even when treatment was provided as late as 1-4 y of age. Human RPE65-LCA patients (ages 18-23 y were studied with structural magnetic resonance imaging. Optic nerve diameter (3.2 +/- 0.5 mm was within the normal range (3.2 +/- 0.3 mm, and occipital cortical white matter density as judged by voxel-based morphometry was slightly but significantly altered (1.3 SD below control average, p = 0.005. Functional magnetic resonance imaging in human RPE65-LCA patients revealed cortical responses with a markedly diminished activation volume (8.8 +/- 1.2 cm(3 compared to controls

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

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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.

Effect of SOCS1 overexpression on RPE cell activation by proinflammatory cytokines.

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Bazewicz, Magdalena; Draganova, Dafina; Makhoul, Maya; Chtarto, Abdel; Elmaleh, Valerie; Tenenbaum, Liliane; Caspers, Laure; Bruyns, Catherine; Willermain, François

2016-09-06

The purpose of this study was to investigate the in vitro effect of Suppressor Of Cytokine Signaling 1 (SOCS1) overexpression in retinal pigment epithelium (RPE) cells on their activation by pro-inflammatory cytokines IFNγ, TNFα and IL-17. Retinal pigment epithelium cells (ARPE-19) were stably transfected with the control plasmid pIRES2-AcGFP1 or the plasmid pSOCS1-IRES2-AcGFP1. They were stimulated by IFNγ (150ng/ml), TNFα (30ng/ml) or IL-17 (100ng/ml). The levels of SOCS1 mRNA were measured by real-time PCR. Signal Transducer and Activator of Transcription 1 (STAT1) phosphorylation and IκBα expression were analysed by western Blot (WB). IL-8 secretion was analysed by ELISA and expression of MHCII molecules and ICAM-1/CD54 by flow cytometry. Our data show that SOCS1 mRNA overexpression in RPE cells prevents IFNγ-induced SOCS1 mRNA increase and IFNγ-mediated STAT1 phosphorylation. Moreover, SOCS1 overexpression in RPE cells inhibits IFNγ-induced decrease of IL-8 secretion and prevents IFNγ-induced MHC II and ICAM1/CD54 upregulation. However, SOCS1 overexpression does not affect TNFα-induced IκBα degradation nor block TNFα-induced or IL-17-induced IL-8 secretion. On the contrary, IL-17-induced secretion is increased by SOCS1 overexpression. In conclusion, SOCS1 overexpression in RPE cells inhibits some IFNγ-mediated responses that lead to uveitis development. This notion raises the possibility that SOCS1 overexpression could be a novel target for treating non-infectious uveitis. However, some proinflammatory effects of TNFα and IL-17 stimulation on RPE are not blocked by SOCS1 overexpression. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

RPE cell surface proteins in normal and dystrophic rats

International Nuclear Information System (INIS)

Clark, V.M.; Hall, M.O.

1986-01-01

Membrane-bound proteins in plasma membrane enriched fractions from cultured rat RPE were analyzed by two-dimensional gel electrophoresis. Membrane proteins were characterized on three increasingly specific levels. Total protein was visualized by silver staining. A maximum of 102 separate proteins were counted in silver-stained gels. Glycoproteins were labeled with 3H-glucosamine or 3H-fucose and detected by autoradiography. Thirty-eight fucose-labeled and 61-71 glucosamine-labeled proteins were identified. All of the fucose-labeled proteins were labeled with glucosamine-derived radioactivity. Proteins exposed at the cell surface were labeled by lactoperoxidase-catalyzed radioiodination prior to preparation of membranes for two-dimensional analysis. Forty separate 125I-labeled surface proteins were resolved by two-dimensional electrophoresis/autoradiography. Comparison with the glycoprotein map showed that a number of these surface labeled proteins were glycoproteins. Two-dimensional maps of total protein, fucose-labeled, and glucosamine-labeled glycoproteins, and 125I-labeled surface proteins of membranes from dystrophic (RCS rdy-p+) and normal (Long Evans or RCS rdy+p+) RPE were compared. No differences in the total protein or surface-labeled proteins were observed. However, the results suggest that a 183K glycoprotein is more heavily glycosylated with glucosamine and fucose in normal RPE membranes as compared to membranes from dystrophic RPE

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

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Karen G Shadrach

Full Text Available 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.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.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.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 stress is a known factor affecting this disease.

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.

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...

Subretinal posterior pole injury induces selective proliferation of RPE cells in the periphery in in vivo studies in pigs

DEFF Research Database (Denmark)

Kiilgaard, Jens Folke; Prause, Jan U; Prause, Michala

2007-01-01

PURPOSE: To study topographical differences in porcine retinal pigment epithelial (RPE) cell proliferation (1) in vivo, after experimental central surgical subretinal injury, and (2) in vitro. METHODS: Domestic pigs underwent either experimental RPE debridement (n = 5), subretinal amniotic membrane...... cells. This observation was true of both types of experimental surgery performed. In vitro, RPE isolates from the pre-equatorial region consistently yielded higher cell densities than did RPE cell isolates from more central parts of the epithelium. This was apparent through the three first passages...

Comparative study of human embryonic stem cells (hESC and human induced pluripotent stem cells (hiPSC as a treatment for retinal dystrophies

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Marina Riera

2016-01-01

Full Text Available Retinal dystrophies (RD are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC-based therapies. We differentiated RPE from human embryonic stem cells (hESCs or human-induced pluripotent stem cells (hiPSCs and transplanted them into the subretinal space of the Royal College of Surgeons (RCS rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD.

Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies

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Riera, Marina; Fontrodona, Laura; Albert, Silvia; Ramirez, Diana Mora; Seriola, Anna; Salas, Anna; Muñoz, Yolanda; Ramos, David; Villegas-Perez, Maria Paz; Zapata, Miguel Angel; Raya, Angel; Ruberte, Jesus; Veiga, Anna; Garcia-Arumi, Jose

2016-01-01

Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD. PMID:27006969

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

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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...... 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......, MHC class I and II, and of cyclin D of the PBLs. CONCLUSIONS: These results are the first to indicate that RPE cells impede generation of activated T cells by interfering with the induction of high-affinity IL-2R-alphabetagamma, IL-2 production, and the expression of CD71 and cyclin A....

Induced Retro-Differentiation of Human Retinal Pigment Epithelial Cells on PolyHEMA.

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Nazemroaya, Fatemeh; Soheili, Zahra-Soheila; Samiei, Shahram; Deezagi, Abdolkhalegh; Ahmadieh, Hamid; Davari, Malihe; Heidari, Razeih; Bagheri, Abouzar; Darvishalipour-Astaneh, Shamila

2017-10-01

Retinal pigment epithelium (RPE) cells represent a great potential to rescue degenerated cells of the damaged retina. Activation of the virtually plastic properties of RPE cells may aid in recovery of retinal degenerative disorders without the need for entire RPE sheet transplantation. Poly (2-hydroxyethyl methacrylate)(PolyHEMA) is one of the most important hydrogels in the biomaterials world. This hydrophobic polymer does not normally support attachment of mammalian cells. In the current study we investigated the effect of PolyHEMA as a cell culture substrate on the growth, differentiation, and plasticity of hRPE cells. hRPE cells were isolated from neonatal human globes and cultured on PolyHEMA and polystyrene substrates (as controls) in 24-well culture plates. DMEM/F12 was supplemented with 10% fetal bovine serum (FBS) and/or 30% human amniotic fluid (HAF) for cultured cells on polystyrene and PolyHEMA coated vessels. Morphology, rate of cell proliferation and cell death, MTT assay, immunocytochemistry and Real-Time RT-PCR were performed to investigate the effects of PolyHEMA on the growth and differentiation of cultured hRPE cells. Proliferation rate of the cells that had been cultured on PolyHEMA was reduced; PolyHEMA did not induce cell death in the hRPE cultures. hRPE cells cultured on PolyHEMA formed many giant spheroid colonies. The giant colonies were re-cultured and the presence of retinal progenitor markers and markers of hRPE cells were detected in cell cultures on PolyHEMA. PolyHEMA seems to be promising for both maintenance and de-differentiation of hRPE cells and expansion of the retinal progenitor cells from the cultures that are originated from hRPE cells. J. Cell. Biochem. 118: 3080-3089, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Repressed SIRT1/PGC-1α pathway and mitochondrial disintegration in iPSC-derived RPE disease model of age-related macular degeneration.

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Golestaneh, Nady; Chu, Yi; Cheng, Shuk Kei; Cao, Hong; Poliakov, Eugenia; Berinstein, Daniel M

2016-12-20

Study of age related macular degeneration (AMD) has been hampered by lack of human models that represent the complexity of the disease. Here we have developed a human in vitro disease model of AMD to investigate the underlying AMD disease mechanisms. Generation of iPSCs from retinal pigment epithelium (RPE) of AMD donors, age-matched normal donors, skin fibroblasts of a dry AMD patient, and differentiation of iPSCs into RPE (AMD RPE-iPSC-RPE, normal RPE-iPSC-RPE and AMD Skin-iPSC-RPE, respectively). Immunostaining, cell viability assay and reactive oxygen species (ROS) production under oxidative stress conditions, electron microscopy (EM) imaging, ATP production and glycogen concentration assays, quantitative real time PCR, western blot, karyotyping. The AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE present functional impairment and exhibit distinct disease phenotypes compared to RPE-iPSC-RPE generated from normal donors (Normal RPE-iPSC-RPE). The AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE show increased susceptibility to oxidative stress and produced higher levels of reactive oxygen species (ROS) under stress in accordance with recent reports. The susceptibility to oxidative stress-induced cell death in AMD RPE-iPSC-RPE and Skin-iPSC-RPE was consistent with inability of the AMD RPE-iPSC-RPE and Skin-iPSC-RPE to increase SOD2 expression under oxidative stress. Phenotypic analysis revealed disintegrated mitochondria, accumulation of autophagosomes and lipid droplets in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE. Mitochondrial activity was significantly lower in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE compared to normal cells and glycogen concentration was significantly increased in the diseased cells. Furthermore, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a regulator of mitochondrial biogenesis and function was repressed, and lower expression levels of NAD-dependent deacetylase sirtuin1 (SIRT1) were found in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE

Human retinal pigment epithelial cells inhibit proliferation and IL2R expression of activated T cells

DEFF Research Database (Denmark)

Kaestel, Charlotte G; Jørgensen, Annette; Nielsen, Mette

2002-01-01

-Thymidine incorporation assay, respectively. T cells and RPE cells were cultured directly together or in a transwell system for determination of the effect of cell contact. The importance of cell surface molecules was examined by application of a panel of blocking antibodies (CD2, CD18, CD40, CD40L, CD54, CD58......) in addition to use of TCR negative T cell lines. The expression of IL2R-alpha -beta and -gamma chains of activated T cells was analysed by flow cytometry after incubation of T cells alone or with RPE cells. Human RPE cells were found to inhibit the proliferation of activated T cells by a cell contact......-beta and -gamma chain expression within 24 hr after removal from the coculture. It is concluded that the cultured human adult and foetal RPE cells inhibit the proliferation of activated T cells by a process that does not involve apoptosis. It depends on cell contact but the involved surface molecules were...

Effects of the Macular Carotenoid Lutein in Human Retinal Pigment Epithelial Cells

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Xiaoming Gong

2017-12-01

Full Text Available Retinal pigment epithelial (RPE cells are central to retinal health and homoeostasis. Oxidative stress-induced damage to the RPE occurs as part of the pathogenesis of age-related macular degeneration and neovascular retinopathies (e.g., retinopathy of prematurity, diabetic retinopathy. The xanthophyll carotenoids, lutein and zeaxanthin, are selectively taken up by the RPE, preferentially accumulated in the human macula, and transferred to photoreceptors. These macular xanthophylls protect the macula (and the broader retina via their antioxidant and photo-protective activities. This study was designed to investigate effects of various carotenoids (β-carotene, lycopene, and lutein on RPE cells subjected to either hypoxia or oxidative stress, in order to determine if there is effect specificity for macular pigment carotenoids. Using human RPE-derived ARPE-19 cells as an in vitro model, we exposed RPE cells to various concentrations of the specific carotenoids, followed by either graded hypoxia or oxidative stress using tert-butyl hydroperoxide (tBHP. The results indicate that lutein and lycopene, but not β-carotene, inhibit cell growth in undifferentiated ARPE-19 cells. Moreover, cell viability was decreased under hypoxic conditions. Pre-incubation of ARPE-19 cells with lutein or lycopene protected against tBHP-induced cell loss and cell co-exposure of lutein or lycopene with tBHP essentially neutralized tBHP-dependent cell death at tBHP concentrations up to 500 μM. Our findings indicate that lutein and lycopene inhibit the growth of human RPE cells and protect the RPE against oxidative stress-induced cell loss. These findings contribute to the understanding of the protective mechanisms attributable to retinal xanthophylls in eye health and retinopathies.

Ultraviolet (UV and Hydrogen Peroxide Activate Ceramide-ER Stress-AMPK Signaling Axis to Promote Retinal Pigment Epithelium (RPE Cell Apoptosis

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Jin Yao

2013-05-01

Full Text Available Ultraviolet (UV radiation and reactive oxygen species (ROS impair the physiological functions of retinal pigment epithelium (RPE cells by inducing cell apoptosis, which is the main cause of age-related macular degeneration (AMD. The mechanism by which UV/ROS induces RPE cell death is not fully addressed. Here, we observed the activation of a ceramide-endoplasmic reticulum (ER stress-AMP activated protein kinase (AMPK signaling axis in UV and hydrogen peroxide (H2O2-treated RPE cells. UV and H2O2 induced an early ceramide production, profound ER stress and AMPK activation. Pharmacological inhibitors against ER stress (salubrinal, ceramide production (fumonisin B1 and AMPK activation (compound C suppressed UV- and H2O2-induced RPE cell apoptosis. Conversely, cell permeable short-chain C6 ceramide and AMPK activator AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide mimicked UV and H2O2’s effects and promoted RPE cell apoptosis. Together, these results suggest that UV/H2O2 activates the ceramide-ER stress-AMPK signaling axis to promote RPE cell apoptosis.

A human apoB100 transgenic mouse expresses human apoB100 in the RPE and develops features of early AMD

DEFF Research Database (Denmark)

Fujihara, Masashi; Bartels, Emil; Nielsen, Lars B

2009-01-01

changes consistent with early human AMD including loss of basal infoldings and accumulation of cytoplasmic vacuoles in the RPE, and basal laminar deposits containing long-spacing collagen and heterogeneous debris in Bruch membrane of apoB100 mice. In apoB100 mice given a high-fat diet, basal linear...... transgenic for a human genomic fragment encoding the full length human apoB ("apoB100" mice) and litter-mate control mice were given a normal chow or high-fat diet for 12 months. Mice were evaluated for human apoB mRNA expression in the RPE/choroid and liver by RT-qPCR. Phenotypic changes associated......-like deposits were identified in 12-month-old mice. Linear regression analysis showed that the genotype (human apoB transgene) was a stronger influencing factor than high-fat diet in producing AMD-like lesions used in this study. Human apoB100 transgenic mice overexpress apoB in RPE and, with time, develop...

Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells.

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Liu, Ji; Lu, Wennan; Guha, Sonia; Baltazar, Gabriel C; Coffey, Erin E; Laties, Alan M; Rubenstein, Ronald C; Reenstra, William W; Mitchell, Claire H

2012-07-15

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in lysosomal acidification has been difficult to determine. We demonstrate here that CFTR contributes more to the reacidification of lysosomes from an elevated pH than to baseline pH maintenance. Lysosomal alkalinization is increasingly recognized as a factor in diseases of accumulation, and we previously showed that cAMP reacidified alkalinized lysosomes in retinal pigmented epithelial (RPE) cells. As the influx of anions to electrically balance proton accumulation may enhance lysosomal acidification, the contribution of the cAMP-activated anion channel CFTR to lysosomal reacidification was probed. The antagonist CFTR(inh)-172 had little effect on baseline levels of lysosomal pH in cultured human RPE cells but substantially reduced the reacidification of compromised lysosomes by cAMP. Likewise, CFTR activators had a bigger impact on cells whose lysosomes had been alkalinized. Knockdown of CFTR with small interfering RNA had a larger effect on alkalinized lysosomes than on baseline levels. Inhibition of CFTR in isolated lysosomes altered pH. While CFTR and Lamp1 were colocalized, treatment with cAMP did not increase targeting of CFTR to the lysosome. The inhibition of CFTR slowed lysosomal degradation of photoreceptor outer segments while activation of CFTR enhanced their clearance from compromised lysosomes. Activation of CFTR acidified RPE lysosomes from the ABCA4(-/-) mouse model of recessive Stargardt's disease, whose lysosomes are considerably alkalinized. In summary, CFTR contributes more to reducing lysosomal pH from alkalinized levels than to maintaining baseline pH. Treatment to activate CFTR may thus be of benefit in disorders of accumulation associated with lysosomal alkalinization.

Retinal Pigmented Epithelial Cells Obtained from Human Induced Pluripotent Stem Cells Possess Functional Visual Cycle Enzymes in Vitro and in Vivo*

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Maeda, Tadao; Lee, Mee Jee; Palczewska, Grazyna; Marsili, Stefania; Tesar, Paul J.; Palczewski, Krzysztof; Takahashi, Masayo; Maeda, Akiko

2013-01-01

Differentiated retinal pigmented epithelial (RPE) cells have been obtained from human induced pluripotent stem (hiPS) cells. However, the visual (retinoid) cycle in hiPS-RPE cells has not been adequately examined. Here we determined the expression of functional visual cycle enzymes in hiPS-RPE cells compared with that of isolated wild-type mouse primary RPE (mpRPE) cells in vitro and in vivo. hiPS-RPE cells appeared morphologically similar to mpRPE cells. Notably, expression of certain visual cycle proteins was maintained during cell culture of hiPS-RPE cells, whereas expression of these same molecules rapidly decreased in mpRPE cells. Production of the visual chromophore, 11-cis-retinal, and retinosome formation also were documented in hiPS-RPE cells in vitro. When mpRPE cells with luciferase activity were transplanted into the subretinal space of mice, bioluminance intensity was preserved for >3 months. Additionally, transplantation of mpRPE into blind Lrat−/− and Rpe65−/− mice resulted in the recovery of visual function, including increased electrographic signaling and endogenous 11-cis-retinal production. Finally, when hiPS-RPE cells were transplanted into the subretinal space of Lrat−/− and Rpe65−/− mice, their vision improved as well. Moreover, histological analyses of these eyes displayed replacement of dysfunctional RPE cells by hiPS-RPE cells. Together, our results show that hiPS-RPE cells can exhibit a functional visual cycle in vitro and in vivo. These cells could provide potential treatment options for certain blinding retinal degenerative diseases. PMID:24129572

Age-related changes in the retinal pigment epithelium (RPE.

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Xiaorong Gu

Full Text Available Age-related changes in the retina are often accompanied by visual impairment but their mechanistic details remain poorly understood.Proteomic studies were pursued toward a better molecular understanding of retinal pigment epithelium (RPE aging mechanisms. RPE cells were isolated from young adults (3-4 month-old and old (24-25 month-old F344BN rats, and separated into subcellular fractions containing apical microvilli (MV and RPE cell bodies (CB lacking their apical microvilli. Proteins were extracted in detergent, separated by SDS-PAGE, digested in situ with trypsin and analyzed by LC MS/MS. Select proteins detected in young and old rat RPE were further studied using immunofluorescence and Western blot analysis.A total of 356 proteins were identified in RPE MV from young and 378 in RPE MV from old rats, 48% of which were common to each age group. A total of 897 proteins were identified in RPE CB from young rats and 675 in old CB, 56% of which were common to each age group. Several of the identified proteins, including proteins involved in response to oxidative stress, displayed both quantitative and qualitative changes in overall abundance during RPE aging. Numerous proteins were identified for the first time in the RPE. One such protein, collectrin, was localized to the apical membrane of apical brush border of proximal tubules where it likely regulates several amino acid transporters. Elsewhere, collectrin is involved in pancreatic β cell proliferation and insulin secretion. In the RPE, collectrin expression was significantly modulated during RPE aging. Another age-regulated, newly described protein was DJ-1, a protein extensively studied in brain where oxidative stress-related functions have been described.The data presented here reveals specific changes in the RPE during aging, providing the first protein database of RPE aging, which will facilitate future studies of age-related retinal diseases.

Two-photon excited autofluorescence imaging of human retinal pigment epithelial cells

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Han, Meng; Blindewald-Wittich, Almut; Holz, Frank G.; Giese, Günter; Niemz, Markolf H.; Snyder, Sarah; Sun, Hui; Yu, Jiayi; Agopov, Michael; La Schiazza, Olivier; Bille, Josef F.

2006-01-01

Degeneration of retinal pigment epithelial (RPE) cells severely impairs the visual function of retina photoreceptors. However, little is known about the events that trigger the death of RPE cells at the subcellular level. Two-photon excited autofluorescence (TPEF) imaging of RPE cells proves to be well suited to investigate both the morphological and the spectral characteristics of the human RPE cells. The dominant fluorophores of autofluorescence derive from lipofuscin (LF) granules that accumulate in the cytoplasm of the RPE cells with increasing age. Spectral TPEF imaging reveals the existence of abnormal LF granules with blue shifted autofluorescence in RPE cells of aging patients and brings new insights into the complicated composition of the LF granules. Based on a proposed two-photon laser scanning ophthalmoscope, TPEF imaging of the living retina may be valuable for diagnostic and pathological studies of age related eye diseases.

Galectin-3 induces clustering of CD147 and integrin-β1 transmembrane glycoprotein receptors on the RPE cell surface.

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Claudia S Priglinger

Full Text Available Proliferative vitreoretinopathy (PVR is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers

Galectin-3 Induces Clustering of CD147 and Integrin-β1 Transmembrane Glycoprotein Receptors on the RPE Cell Surface

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Priglinger, Claudia S.; Szober, Christoph M.; Priglinger, Siegfried G.; Merl, Juliane; Euler, Kerstin N.; Kernt, Marcus; Gondi, Gabor; Behler, Jennifer; Geerlof, Arie; Kampik, Anselm; Ueffing, Marius; Hauck, Stefanie M.

2013-01-01

Proliferative vitreoretinopathy (PVR) is a blinding disease frequently occurring after retinal detachment surgery. Adhesion, migration and matrix remodeling of dedifferentiated retinal pigment epithelial (RPE) cells characterize the onset of the disease. Treatment options are still restrained and identification of factors responsible for the abnormal behavior of the RPE cells will facilitate the development of novel therapeutics. Galectin-3, a carbohydrate-binding protein, was previously found to inhibit attachment and spreading of retinal pigment epithelial cells, and thus bares the potential to counteract PVR-associated cellular events. However, the identities of the corresponding cell surface glycoprotein receptor proteins on RPE cells are not known. Here we characterize RPE-specific Gal-3 containing glycoprotein complexes using a proteomic approach. Integrin-β1, integrin-α3 and CD147/EMMPRIN, a transmembrane glycoprotein implicated in regulating matrix metalloproteinase induction, were identified as potential Gal-3 interactors on RPE cell surfaces. In reciprocal immunoprecipitation experiments we confirmed that Gal-3 associated with CD147 and integrin-β1, but not with integrin-α3. Additionally, association of Gal-3 with CD147 and integrin-β1 was observed in co-localization analyses, while integrin-α3 only partially co-localized with Gal-3. Blocking of CD147 and integrin-β1 on RPE cell surfaces inhibited binding of Gal-3, whereas blocking of integrin-α3 failed to do so, suggesting that integrin-α3 is rather an indirect interactor. Importantly, Gal-3 binding promoted pronounced clustering and co-localization of CD147 and integrin-β1, with only partial association of integrin-α3. Finally, we show that RPE derived CD147 and integrin-β1, but not integrin-α3, carry predominantly β-1,6-N-actyl-D-glucosamine-branched glycans, which are high-affinity ligands for Gal-3. We conclude from these data that extracellular Gal-3 triggers clustering of CD147 and

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...... privilege of the eye, but due to contradictory published results, it is unclear whether RPE cells express this molecule. The purpose of this study was to investigate the expression of FasL in RPE cells in vitro and in vivo. Cultured human fetal and adult RPE cells were examined by flow cytometry, Western...... 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...

Human embryonic stem cell-derived cells rescue visual function in dystrophic RCS rats.

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Lund, Raymond D; Wang, Shaomei; Klimanskaya, Irina; Holmes, Toby; Ramos-Kelsey, Rebeca; Lu, Bin; Girman, Sergej; Bischoff, N; Sauvé, Yves; Lanza, Robert

2006-01-01

Embryonic stem cells promise to provide a well-characterized and reproducible source of replacement tissue for human clinical studies. An early potential application of this technology is the use of retinal pigment epithelium (RPE) for the treatment of retinal degenerative diseases such as macular degeneration. Here we show the reproducible generation of RPE (67 passageable cultures established from 18 different hES cell lines); batches of RPE derived from NIH-approved hES cells (H9) were tested and shown capable of extensive photoreceptor rescue in an animal model of retinal disease, the Royal College of Surgeons (RCS) rat, in which photoreceptor loss is caused by a defect in the adjacent retinal pigment epithelium. Improvement in visual performance was 100% over untreated controls (spatial acuity was approximately 70% that of normal nondystrophic rats) without evidence of untoward pathology. The use of somatic cell nuclear transfer (SCNT) and/or the creation of banks of reduced complexity human leucocyte antigen (HLA) hES-RPE lines could minimize or eliminate the need for immunosuppressive drugs and/or immunomodulatory protocols.

TNF-α mediates choroidal neovascularization by upregulating VEGF expression in RPE through ROS-dependent β-catenin activation.

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Wang, Haibo; Han, Xiaokun; Wittchen, Erika S; Hartnett, M Elizabeth

2016-01-01

Inflammation, oxidative stress, and angiogenesis have been proposed to interact in age-related macular degeneration. It has been postulated that external stimuli that cause oxidative stress can increase production of vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells. In this study, we tested the hypothesis that the inflammatory cytokine, tumor necrosis factor alpha (TNF-α), contributed to choroidal neovascularization (CNV) by upregulating VEGF in RPE through intracellular reactive oxygen species (ROS)-dependent signaling and sought to understand the mechanisms involved. In a murine laser-induced CNV model, 7 days after laser treatment and intravitreal neutralizing mouse TNF-α antibody or isotype immunoglobulin G (IgG) control, the following measurements were made: 1) TNF-α protein and VEGF protein in RPE/choroids with western blot, 2) CNV volume in RPE/choroidal flatmounts, and 3) semiquantification of oxidized phospholipids stained with E06 antibody within CNV with immunohistochemistry (IHC). In cultured human RPE cells treated with TNF-α or PBS control, 1) ROS generation was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence assay, and 2) NOX4 protein and VEGF protein or mRNA were measured with western blot or quantitative real-time PCR in cells pretreated with apocynin or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, VAS 2870, or transfected with p22phox siRNA, and each was compared to its appropriate control. Western blots of phosphorylated p65 (p-p65), total p65 and β-actin, and quantitative real-time PCR of VEGF mRNA were measured in human RPE cells treated with TNF-α and pretreatment with the nuclear factor kappa B inhibitor, Bay 11-7082 or control. Western blots of β-catenin, VEGF, and p22phox and coimmunoprecipitation of β-catenin and T-cell transcriptional factor were performed in human RPE cells treated with TNF-α following pretreatment with �

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...

Analysis of the RPE sheet in the rd10 retinal degeneration model

Energy Technology Data Exchange (ETDEWEB)

Jiang, Yi [Los Alamos National Laboratory

2011-01-04

The normal RPE sheet in the C57Bl/6J mouse is subclassified into two major tiling patterns: A regular generally hexagonal array covering most of the surface and a 'soft network' near the ciliary body made of irregularly shaped cells. Physics models predict these two patterns based on contractility and elasticity of the RPE cell, and strength of cellular adhesion between cells. We hypothesized and identified major changes in RPE regular hexagonal tiling pattern in rdl0 compared to C57BL/6J mice. RPE sheet damage was extensive but occurred in rd10 later than expected, after most retinal degeneration. RPE sheet changes occur in zones with a bullseye pattern. In the posterior zone around the optic nerve RPE cells take on larger irregular and varied shapes to form an intact monolayer. In mid periphery, there is a higher than normal density of cells that progress into involuted layers of RPE under the retina. The periphery remains mostly normal until late stages of degeneration. The number of neighboring cells varies widely depending on zone and progression. RPE morphology continues to deteriorate long after the photoreceptors have degenerated. The RPE cells are bystanders to the rd10 degeneration within photo receptors, and the collateral damage to the RPE sheet resembles stimulation of migration or chemotaxis. Quantitative measures of the tiling patterns and histopathology detected here, scripted in a pipeline written in Perl and Cell Profiler (an open source Matlab plugin), are directly applicable to RPE sheet images from noninvasive fundus autofluorescence (FAF), adaptive optics confocal scanning laser ophthalmoscope (AO-cSLO), and spectral domain optical coherence tomography (SD-OCT) of patients with early stage AMD or RP.

Human amniotic fluid promotes retinal pigmented epithelial cells' trans-differentiation into rod photoreceptors and retinal ganglion cells.

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Ghaderi, Shima; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Davari, Maliheh; Jahromi, Fatemeh Sanie; Samie, Shahram; Rezaie-Kanavi, Mozhgan; Pakravesh, Jalil; Deezagi, Abdolkhalegh

2011-09-01

To evaluate the effect of human amniotic fluid (HAF) on retinal pigmented epithelial cells growth and trans-differentiation into retinal neurons, retinal pigmented epithelium (RPE) cells were isolated from neonatal human cadaver eye globes and cultured in Dulbecco's modified Eagle's medium-F12 supplemented with 10% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using FBS-containing or HAF-containing media. Amniotic fluid samples were received from pregnant women in the first trimester of gestation. Cell proliferation and death enzyme-linked immunosorbent assays were performed to assess the effect of HAF on RPE cell growth. Trans-differentiation into rod photoreceptors and retinal ganglion cells was also studied using immunocytochemistry and real-time polymerase chain reaction techniques. Primary cultures of RPE cells were successfully established under FBS-containing or HAF-containing media leading to rapid cell growth and proliferation. When RPE cells were moved to in vitro culture system, they began to lose their differentiation markers such as pigmentation and RPE65 marker and trans-differentiated neural-like cells followed by spheroid colonies pertaining to stem/progenitor cells were morphologically detected. Immunocytochemistry (ICC) analysis of HAF-treated cultures showed a considerable expression of Rhodopsin gene (30% Rhodopsin-positive cells) indicating trans-differentiation of RPE cells to rod photoreceptors. Real-time polymerase chain reaction revealed an HAF-dose-dependant expression of Thy-1 gene (RGC marker) and significant promoting effect of HAF on RGCs generation. The data presented here suggest that HAF possesses invaluable stimulatory effect on RPE cells growth and trans-differentiation into retinal neurons. It can be regarded as a newly introduced enriched supplement in serum-free kinds of media used in neuro-retinal regeneration studies.

Exogenous NAD(+) decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy.

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

2016-05-31

Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD(+)) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD(+) administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD(+) administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD(+) administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD(+) against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD(+) administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD(+) administration might be potential value for the treatment of AMD.

Exogenous NAD+ decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy

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Zhu, Ying; Zhao, Ke-ke; Tong, Yao; Zhou, Ya-li; Wang, Yi-xiao; Zhao, Pei-quan; Wang, Zhao-yang

2016-01-01

Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD+) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD+ administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD+ administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD+ administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD+ against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD+ administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD+ administration might be potential value for the treatment of AMD. PMID:27240523

Behavior of a Spontaneously Arising Human Retinal Pigment Epithelial Cell Line Cultivated on Thin Alginate Film.

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Najafabadi, Hoda Shams; Soheili, Zahra-Soheila; Ganji, Shahla Mohammad

2015-01-01

A cell line spontaneously derived from human retinal pigment epithelium (hRPE) was cultured on alginate film gelatinized with different concentrations of neurobasal cell culture medium (NCCM) to assess its growth and morphological behavior on this naturally occurring polysaccharide. Neonatal human globes were used to isolate hRPE cells. They were cultured in Dulbecco's modified Eagle's-medium-and-Ham's-F12-medium-(DMEM/F12) supplemented with 10% fetal bovine serum (FBS). Cultures were continuously studied using phase contrast microscopy. After the nineth passage, cells were characterized through immunocytochemical analysis for Oct4, Chx10, and Pax6 and Ki67 markers. In each well of a 6-well microplate, 1 and 2% weight/volume (w/v) alginate in deionized water was added and gelatinized using 1× and 10× NCCM. hRPE cells were cultured at a density of 2 × 105 cells/well in alginate-coated microplates. After 5 days, hRPE colonies were harvested and re-plated on polystyrene substrates. Morphology and growth of hRPE cultures were determined during the next 2 weeks. The first few passages of the cultures were purely hRPE cells that revealed typical morphological features of the pigmented epithelium. They made spaces, devoid of cells, between hRPE cell monolayer and fill in the unoccupied spaces. They grew faster than native RPE cells and rapidly overgrew. Immunocytochemical test revealed that the founded cells expressed Chx10, Pax6, Ki67 and Oct4. The hRPE cells survived unlimitedly on alginate film and formed giant adjoining colonies. After re-plating, hRPE colonies adhered quickly on polystyrene and displayed native hRPE morphological features. Alginate film can support the survival and growth of hRPE cells and induce the cells to re-organize in tissue-like structures.

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

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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.

Subretinal Implantation of Retinal Pigment Epithelial Cells Derived From Human Embryonic Stem Cells: Improved Survival When Implanted as a Monolayer

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Diniz, Bruno; Thomas, Padmaja; Thomas, Biju; Ribeiro, Ramiro; Hu, Yuntao; Brant, Rodrigo; Ahuja, Ashish; Zhu, Danhong; Liu, Laura; Koss, Michael; Maia, Mauricio; Chader, Gerald; Hinton, David R.; Humayun, Mark S.

2013-01-01

Purpose. To evaluate cell survival and tumorigenicity of human embryonic stem cell–derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM). Methods. Sixty-nine rats (38 male, 31 female) were surgically implanted with CS (n = 33) or PM (n = 36). Cohort subsets were killed at 1, 6, and 12 months after surgery. Both ocular tissues and systemic organs (brain, liver, kidneys, spleen, heart, and lungs) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker), anti-TRA-1-85 (human cell marker), anti-Ki67 (proliferation marker), anti-CD68 (macrophage), and anti-cytokeratin (epithelial marker). Results. The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P = 0.006) and the presence of a monolayer (P < 0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group. Conclusions. hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects. PMID:23833067

Gremlin promotes retinal pigmentation epithelial (RPE) cell proliferation, migration and VEGF production via activating VEGFR2-Akt-mTORC2 signaling.

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Liu, Yuan; Chen, Zhijun; Cheng, Haixia; Chen, Juan; Qian, Jing

2017-01-03

Retinopathy of prematurity (ROP) is characterized by late-phase pathologic retinal vasoproliferation. Gremlin is a novel vascular endothelial growth factors (VEGF) receptor 2 (VEGFR2) agonist and promotes angiogenic response. We demonstrated that gremlin expression was significantly increased in retinas of ROP model mice, which was correlated with VEGF upregulation. In retinal pigmentation epithelial (RPE) cells, gremlin activated VEGFR2-Akt-mTORC2 (mammalian target of rapamycin complex 2) signaling, and promoted cell proliferation, migration and VEGF production. VEGFR inhibition (by SU5416) or shRNA knockdown almost abolished gremlin-mediated pleiotropic functions in RPE cells. Further, pharmacological inhibition of Akt-mTOR, or shRNA knockdown of key mTORC2 component (Rictor or Sin1) also attenuated gremlin-exerted activities in RPE cells. We conclude that gremlin promotes RPE cell proliferation, migration and VEGF production possibly via activating VEGFR2-Akt-mTORC2 signaling. Gremlin could be a novel therapeutic target of ROP or other retinal vasoproliferation diseases.

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

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Huang, Xionggao; Wei, Yantao; Ma, Haizhi; Zhang, Shaochong

2012-01-01

Highlights: ► Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. ► Rac1 is activated in vitreous-transformed RPE cells. ► Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. ► Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. ► 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-transformed human RPE cells undergo cytoskeletal rearrangements via Rac1 GTPase-dependent pathways that modulate LIMK1 and

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

X-ray microprobe analysis of the retina and RPE in sheep with ovine ceroid-lipofuscinosis

International Nuclear Information System (INIS)

Samuelson, D.A.; Armstrong, D.; Jolly, R.

1990-01-01

Ovine ceroid-lipofuscinosis (OCL) is one animal model for the human condition, and because autofluorescent lipopigments are prominent in the brain and eye, it may also prove useful as a model for aging. For example, a progressive decline in electrical recording from brain and retina are observed in both aging and OCL. Samples of retinal and retinal pigment epithelial (RPE) tissues were obtained from a young control. 2 animals with OCL and a normal aged sheep. Specimens were cryo-fractured and examined by scanning electron microscopy/x-ray microanalysis. Measurements made of 6 individual cells in the ganglion layer of OCL specimens, the remainder of the retina, and RPE showed age-related changes in zinc, iron, and copper which were associated with lipopigment accumulation in the RPE. There was marked decrease in phosphate, sulfur, and manganese levels, as photoreceptor cells and their outer segments are lost in the disease process. This is the first report of metal analysis in the retina and RPE in a disease entity, and as a function of normal aging

Multi-nucleate retinal pigment epithelium cells of the human macula exhibit a characteristic and highly specific distribution.

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Starnes, Austin C; Huisingh, Carrie; McGwin, Gerald; Sloan, Kenneth R; Ablonczy, Zsolt; Smith, R Theodore; Curcio, Christine A; Ach, Thomas

2016-01-01

The human retinal pigment epithelium (RPE) is reportedly 3% bi-nucleated. The importance to human vision of multi-nucleated (MN)-RPE cells could be clarified with more data about their distribution in central retina. Nineteen human RPE-flatmounts (9 ≤ 51 years, 10 > 80 years) were imaged at 12 locations: 3 eccentricities (fovea, perifovea, near periphery) in 4 quadrants (superior, inferior, temporal, nasal). Image stacks of lipofuscin-attributable autofluorescence and phalloidin labeled F-actin cytoskeleton were obtained using a confocal fluorescence microscope. Nuclei were devoid of autofluorescence and were marked using morphometric software. Cell areas were approximated by Voronoi regions. Mean number of nuclei per cell among eccentricity/quadrant groups and by age were compared using Poisson and binominal regression models. A total of 11,403 RPE cells at 200 locations were analyzed: 94.66% mono-, 5.31% bi-, 0.02% tri-nucleate, and 0.01% with 5 nuclei. Age had no effect on number of nuclei. There were significant regional differences: highest frequencies of MN-cells were found at the perifovea (9.9%) and near periphery (6.8%). The fovea lacked MN-cells almost entirely. The nasal quadrant had significantly more MN-cells compared to other quadrants, at all eccentricities. This study demonstrates MN-RPE cells in human macula. MN-cells may arise due to endoreplication, cell fusion, or incomplete cell division. The topography of MN-RPE cells follows the topography of photoreceptors; with near-absence at the fovea (cones only) and high frequency at perifovea (highest rod density). This distribution might reflect specific requirements of retinal metabolism or other mechanisms addressable in further studies.

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.

Mechanism of RPE cell death in α-crystallin deficient mice: a novel and critical role for MRP1-mediated GSH efflux.

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Parameswaran G Sreekumar

Full Text Available Absence of α-crystallins (αA and αB in retinal pigment epithelial (RPE cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1 MRP1 mediates GSH and GSSG efflux in RPE cells; 2 MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3 the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

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Foltz, LP; Clegg, DO

2017-01-01

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embry...

Noninvasive near infrared autofluorescence imaging of retinal pigment epithelial cells in the human retina using adaptive optics.

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Liu, Tao; Jung, HaeWon; Liu, Jianfei; Droettboom, Michael; Tam, Johnny

2017-10-01

The retinal pigment epithelial (RPE) cells contain intrinsic fluorophores that can be visualized using infrared autofluorescence (IRAF). Although IRAF is routinely utilized in the clinic for visualizing retinal health and disease, currently, it is not possible to discern cellular details using IRAF due to limits in resolution. We demonstrate that the combination of adaptive optics (AO) with IRAF (AO-IRAF) enables higher-resolution imaging of the IRAF signal, revealing the RPE mosaic in the living human eye. Quantitative analysis of visualized RPE cells in 10 healthy subjects across various eccentricities demonstrates the possibility for in vivo density measurements of RPE cells, which range from 6505 to 5388 cells/mm 2 for the areas measured (peaking at the fovea). We also identified cone photoreceptors in relation to underlying RPE cells, and found that RPE cells support on average up to 18.74 cone photoreceptors in the fovea down to an average of 1.03 cone photoreceptors per RPE cell at an eccentricity of 6 mm. Clinical application of AO-IRAF to a patient with retinitis pigmentosa illustrates the potential for AO-IRAF imaging to become a valuable complementary approach to the current landscape of high resolution imaging modalities.

Probable Chemical Hypoxia Effects on Progress of CNV Through Induction of Promoter CpG Demethylation and Overexpression of IL17RC in Human RPE Cells.

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Alivand, Mohammad Reza; Sabouni, Farzaneh; Soheili, Zahra-Soheila

2016-09-01

To survey the changes of promoter CpG methylation status and mRNA expression of IL17RC (interleukin 17 receptor C) gene in retinal pigment epithelium (RPE) cells under chemical hypoxia condition for choroidal neovascularization (CNV) modeling in vitro. RPE cells were cultured in both untreated as a control group and treated by cobalt chloride media as a hypoxia group for various concentrations (100-150μM) and times (24-36 hrs.) To confirm chemical hypoxia condition, mRNA expression of HIF (Hypoxia Inducible Factor) -1α, -2α, and Vascular Endothelial Growth Factor (VEGF) was compared between two groups by Real-time PCR. Also, in normoxia and hypoxia conditions, IL17RC expression changes and promoter CpG methylation status were evaluated by Real-time PCR and methylation-specific PCR (MSP) techniques, respectively. Overexpression of HIF-1α, HIF-2α, and VEGF was significant in hypoxia versus normoxia conditions. Our data showed overexpression of IL17RC (2.1- to 6.3-fold) and decreasing of its promoter methylation in comparison with hypoxia and normoxia conditions. It was found that there are significant association between promoter methylation status and expression of IL17RC in chemical hypoxia condition. Therefore, methylation of IL17RC could play as a marker in CNV and degeneration of RPE cells in vitro. Additionally, HIF-α and methylation phenomena may be considered as critical targets for blocking in angiogenesis of age-related degeneration in future studies.

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.

Chemokine Expression in Retinal Pigment Epithelial ARPE-19 Cells in Response to Coculture with Activated T Cells

DEFF Research Database (Denmark)

Juel, Helene Bæk; Faber, Carsten; Udsen, Maja

2012-01-01

Purpose. To investigate the effects of T-cell–derived cytokines on gene and protein expression of chemokines in a human RPE cell line (ARPE-19). Methods. We used an in vitro coculture system in which the RPE and CD3/CD28–activated T-cells were separated by a membrane. RPE cell expression of chemo......Purpose. To investigate the effects of T-cell–derived cytokines on gene and protein expression of chemokines in a human RPE cell line (ARPE-19). Methods. We used an in vitro coculture system in which the RPE and CD3/CD28–activated T-cells were separated by a membrane. RPE cell expression...

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

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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.

LC-MS/MS Based Quantitation of ABC and SLC Transporter Proteins in Plasma Membranes of Cultured Primary Human Retinal Pigment Epithelium Cells and Immortalized ARPE19 Cell Line.

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Pelkonen, Laura; Sato, Kazuki; Reinisalo, Mika; Kidron, Heidi; Tachikawa, Masanori; Watanabe, Michitoshi; Uchida, Yasuo; Urtti, Arto; Terasaki, Tetsuya

2017-03-06

The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier between neural retina and choroid. The RPE has several important vision supporting functions, such as transport mechanisms that may also modify pharmacokinetics in the posterior eye segment. Expression of plasma membrane transporters in the RPE cells has not been quantitated. The aim of this study was to characterize and compare transporter protein expression in the ARPE19 cell line and hfRPE (human fetal RPE) cells by using quantitative targeted absolute proteomics (QTAP). Among 41 studied transporters, 16 proteins were expressed in hfRPE and 13 in ARPE19 cells. MRP1, MRP5, GLUT1, 4F2hc, TAUT, CAT1, LAT1, and MATE1 proteins were detected in both cell lines within 4-fold differences. MPR7, OAT2 and RFC1 were detected in the hfRPE cells, but their expression levels were below the limit of quantification in ARPE19 cells. PCFT was detected in both studied cell lines, but the expression was over 4-fold higher in hfRPE cells. MCT1, MCT4, MRP4, and Na + /K + ATPase were upregulated in the ARPE19 cell line showing over 4-fold differences in the quantitative expression values. Expression levels of 25 transporters were below the limit of quantification in both cell models. In conclusion, we present the first systematic and quantitative study on transporter protein expression in the plasma membranes of ARPE19 and hfRPE cells. Overall, transporter expression in the ARPE19 and hfRPE cells correlated well and the absolute expression levels were similar, but not identical. The presented quantitative expression levels could be a useful basis for further studies on drug permeation in the outer blood-retinal barrier.

Intracameral voriconazole: In vitro safety for human ocular cells

International Nuclear Information System (INIS)

Kernt, M.; Kampik, A.

2009-01-01

Fungal keratitis is a sight-threatening infection of the cornea. It sometimes leads to loss of the eye. Despite an expanding range of fungal pathogens, there are only few therapeutic agents for its treatment available. Voriconazole is a second-generation synthetic triazole with a broad action against yeasts and molds. The current study investigates the safety of voriconazole for intracameral application in a cell culture model. Endothelial toxicity of voriconazole was evaluated in cultured human corneas. Possible toxic effects of voriconazole (10 μg/mL-10 mg/mL) in corneal endothelial cells (CEC), primary human trabecular meshwork cells (TMC), and primary human retinal pigment epithelium (RPE) cells were evaluated after 24 h and under conditions of inflammatory stress by treatment with tumor-necrosis-factor alpha (TNF-α), lipopolysaccharides (LPS), or interleukin-6 (IL-6) and hydrogen peroxide. Toxicity was evaluated by tetrazolium dye-reduction assay, and cell viability was quantified by a microscopic live-dead assay. No corneal endothelial toxicity could be detected after 30 days of treatment with 250 μg/mL of voriconazole. Concentrations up to 1 mg/mL had no influence on CEC, TMC, or RPE cell proliferation, or on cell viability when administered for 24 h. Hydrogen peroxide exposure did not increase cellular toxicity of voriconazole at concentrations from 10 to 250 μg/mL. After preincubation with TNF-α, LPS, or IL-6 for 24 h and subsequent voriconazole treatment for 24 h, no significant decrease in proliferation or viability was observed. This study showed no significant toxicity for voriconazole on CEC, TMC, RPE cells, or human corneal endothelium when administered in therapeutic concentrations up to 250 μg/mL

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...

N-Acetylcysteine Amide Protects Against Oxidative Stress–Induced Microparticle Release From Human Retinal Pigment Epithelial Cells

Science.gov (United States)

Carver, Kyle A.; Yang, Dongli

2016-01-01

Purpose Oxidative stress is a major factor involved in retinal pigment epithelium (RPE) apoptosis that underlies AMD. Drusen, extracellular lipid- and protein-containing deposits, are strongly associated with the development of AMD. Cell-derived microparticles (MPs) are small membrane-bound vesicles shed from cells. The purpose of this study was to determine if oxidative stress drives MP release from RPE cells, to assess whether these MPs carry membrane complement regulatory proteins (mCRPs: CD46, CD55, and CD59), and to evaluate the effects of a thiol antioxidant on oxidative stress–induced MP release. Methods Retinal pigment epithelium cells isolated from human donor eyes were cultured and treated with hydrogen peroxide (H2O2) to induce oxidative stress. Isolated MPs were fixed for transmission electron microscopy or processed for component analysis by flow cytometry, Western blot analysis, and confocal microscopy. Results Transmission electron microscopy showed that MPs ranged in diameter from 100 to 1000 nm. H2O2 treatment led to time- and dose-dependent elevations in MPs with externalized phosphatidylserine and phosphatidylethanolamine, known markers of MPs. These increases were strongly correlated to RPE apoptosis. Oxidative stress significantly increased the release of mCRP-positive MPs, which were prevented by a thiol antioxidant, N-acetylcysteine amide (NACA). Conclusions This is the first evidence that oxidative stress induces cultured human RPE cells to release MPs that carry mCRPs on their surface. The levels of released MPs are strongly correlated with RPE apoptosis. N-acetylcysteine amide prevents oxidative stress–induced effects. Our findings indicate that oxidative stress reduces mCRPs on the RPE surface through releasing MPs. PMID:26842754

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

Science.gov (United States)

Sanie-Jahromi, Fatemeh; Ahmadieh, Hamid; Soheili, Zahra-Soheila; Davari, Maliheh; Ghaderi, Shima; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Deezagi, Abdolkhalegh; Pakravesh, Jalil; Bagheri, Abouzar

2012-04-10

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. 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. 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.

Retinal pigment epithelium culture;a potential source of retinal stem cells.

Science.gov (United States)

Akrami, Hassan; Soheili, Zahra-Soheila; Khalooghi, Keynoush; Ahmadieh, Hamid; Rezaie-Kanavi, Mojgan; Samiei, Shahram; Davari, Malihe; Ghaderi, Shima; Sanie-Jahromi, Fatemeh

2009-07-01

To establish human retinal pigment epithelial (RPE) cell culture as a source for cell replacement therapy in ocular diseases. Human cadaver globes were used to isolate RPE cells. Each globe was cut into several pieces of a few millimeters in size. After removing the sclera and choroid, remaining tissues were washed in phosphate buffer saline and RPE cells were isolated using dispase enzyme solution and cultured in Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 supplemented with 10% fetal calf serum. Primary cultures of RPE cells were established and spheroid colonies related to progenitor/stem cells developed in a number of cultures. The colonies included purely pigmented or mixed pigmented and non-pigmented cells. After multiple cellular passages, several types of photoreceptors and neural-like cells were detected morphologically. Cellular plasticity in RPE cell cultures revealed promising results in terms of generation of stem/progenitor cells from human RPE cells. Whether the spheroids and neural-like retinal cells were directly derived from retinal stem cells or offspring of trans-differentiating or de-differentiating RPE cells remains to be answered.

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

NARCIS (Netherlands)

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

2017-01-01

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

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

NARCIS (Netherlands)

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

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

Study of Short-Pulsed Laser Retinal Injury Mechanisms By Time-Resolved Imaging of Photomechanical Transients in RPE

National Research Council Canada - National Science Library

Lin, Charles

2000-01-01

We studied RPE cell damage mechanism for laser duration from 100 femtosec to 5 microsec, and we have investigated the dependence of threshold fluence for cell damage on the laser spot size on the RPE...

17-AAG, a Hsp90 inhibitor, attenuates the hypoxia-induced expression of SDF-1alpha and ILK in mouse RPE cells.

Science.gov (United States)

Wang, Ye Qing; Zhang, Xiao Mei; Wang, Xiao Dan; Wang, Bin Jie; Wang, Wei

2010-03-01

The aim of this study was to investigate the changes of SDF-1alpha and ILK expression in mouse retinal pigment epithelium (RPE) cells in response to hypoxia, and the effect of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (Hsp90) inhibitor, on the hypoxia-induced expression of SDF-1alpha and ILK. RPE cells were cultured with 200 micromol/L cobalt chloride (CoCl(2)) for different times (1, 3, 6, 12, 24, 72 h) to imitate chemical hypoxia. Pretreatment of 17-AAG was 1 h prior to hypoxic insult. Cellular viability after 17-AAG treatment was assessed by MTT assay, and the changes of SDF-1alpha and ILK expression were examined by RT-PCR and Western blot. Up-regulation of SDF-1alpha and ILK expression in response to hypoxia was observed. One hour pretreatment of 17-AAG could remarkably decreased the hypoxia-induced SDF-1alpha and ILK expression in vitro. Our results indicated that SDF-1alpha and ILK involved in the hypoxic response of RPE cells, and 1 h pretreatment of 17-AAG had an inhibitive effect on the hypoxia-induced SDF-1alpha and ILK expression.

Role of macrophage migration inhibitory factor (MIF) in the effects of oxidative stress on human retinal pigment epithelial cells.

Science.gov (United States)

Ko, Ji-Ae; Sotani, Yasuyuki; Ibrahim, Diah Gemala; Kiuchi, Yoshiaki

2017-10-01

Proliferative vitreoretinopathy (PVR) is the major cause of treatment failure in individuals who undergo surgery for retinal detachment. The epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells contributes to the pathogenesis of PVR. Oxidative stress is thought to play a role in the progression of retinal diseases including PVR. We have now examined the effects of oxidative stress on the EMT and related processes in the human RPE cell line. We found that H 2 O 2 induced the contraction of RPE cells in a three-dimensional collagen gel. Analysis of a cytokine array revealed that H 2 O 2 specifically increased the release of macrophage migration inhibitory factor (MIF) from RPE cells. Reverse transcription-polymerase chain reaction and immunoblot analyses showed that H 2 O 2 increased the expression of MIF in RPE cells. Immunoblot and immunofluorescence analyses revealed that H 2 O 2 upregulated the expression of α-SMA and vimentin and downregulated that of ZO-1 and N-cadherin. Consistent with these observations, the transepithelial electrical resistance of cell was reduced by exposure to H 2 O 2 . The effects of oxidative stress on EMT-related and junctional protein expression as well as on transepithelial electrical resistance were inhibited by antibodies to MIF, but they were not mimicked by treatment with recombinant MIF. Finally, analysis with a profiling array for mitogen-activated protein kinase signalling revealed that H 2 O 2 specifically induced the phosphorylation of p38 mitogen-activated protein kinase. Our results thus suggest that MIF may play a role in induction of the EMT and related processes by oxidative stress in RPE cells and that it might thereby contribute to the pathogenesis of PVR. Proliferative vitreoretinopathy is a major complication of rhegmatogenous retinal detachment, and both oxidative stress and induction of the EMT in RPE cells are thought to contribute to the pathogenesis of this condition. We have now

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

Directory of Open Access Journals (Sweden)

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.

Hyperglycaemia exacerbates choroidal neovascularisation in mice via the oxidative stress-induced activation of STAT3 signalling in RPE cells.

Directory of Open Access Journals (Sweden)

Xia Li

Full Text Available Choroidal neovascularisation (CNV that occurs as a result of age-related macular degeneration (AMD causes severe vision loss among elderly patients. The relationship between diabetes and CNV remains controversial. However, oxidative stress plays a critical role in the pathogenesis of both AMD and diabetes. In the present study, we investigated the influence of diabetes on experimentally induced CNV and on the underlying molecular mechanisms of CNV. CNV was induced via photocoagulation in the ocular fundi of mice with streptozotocin-induced diabetes. The effect of diabetes on the severity of CNV was measured. An immunofluorescence technique was used to determine the levels of oxidative DNA damage by anti-8-hydroxy-2-deoxyguanosine (8-OHdG antibody, the protein expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3 and vascular endothelial growth factor (VEGF, in mice with CNV. The production of reactive oxygen species (ROS in retinal pigment epithelial (RPE cells that had been cultured under high glucose was quantitated using the 2',7'-dichlorofluorescein diacetate (DCFH-DA method. p-STAT3 expression was examined using Western blot analysis. RT-PCR and ELISA processes were used to detect VEGF expression. Hyperglycaemia exacerbated the development of CNV in mice. Oxidative stress levels and the expression of p-STAT3 and VEGF were highly elevated both in mice and in cultured RPE cells. Treatment with the antioxidant compound N-acetyl-cysteine (NAC rescued the severity of CNV in diabetic mice. NAC also inhibited the overexpression of p-STAT3 and VEGF in CNV and in RPE cells. The JAK-2/STAT3 pathway inhibitor AG490 blocked VEGF expression but had no effect on the production of ROS in vitro. These results suggest that hyperglycaemia promotes the development of CNV by inducing oxidative stress, which in turn activates STAT3 signalling in RPE cells. Antioxidant supplementation helped attenuate the development of CNV

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

Science.gov (United States)

Zhu, DanHong; Sreekumar, Parameswaran G; Hinton, David R; Kannan, Ram

2010-03-31

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

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...

Resveratrol Protects Against Ultraviolet A-Mediated Inhibition of the Phagocytic Function of Human Retinal Pigment Epithelial Cells Via Large-Conductance Calcium-Activated Potassium Channels

Directory of Open Access Journals (Sweden)

Shwu-Jiuan Sheu

2009-07-01

Full Text Available This study was undertaken to examine the protective effect of resveratrol on human retinal pigment epithelial (RPE cell phagocytosis against ultraviolet irradiation damage. Cultured RPE cells were exposed to ultraviolet A (UVA, 20 minutes irradiation, and treated with meclofenamic acid (30μM, 20 minutes, paxilline (100 μM, 20 minutes or resveratrol (10μM, 20 minutes. Meclofenamic acid and resveratrol were given after exposure to UVA. Pretreatment with meclofenamic acid, resveratrol or paxilline before UVA irradiation was also performed. Fluorescent latex beads were then fed for 4 hours and the phagocytotic function was assessed by flow cytometry. UVA irradiation inhibited the phagocytic function of human RPE cells. The large-conductance calcium-activated potassium channel activator meclofenamic acid ameliorated the damage caused by UVA irradiation. Pretreatment with resveratrol acid also provided protection against damage caused by UVA. Posttreatment with meclofenamic acid offered mild protection, whereas resveratrol did not. In conclusion, the red wine flavonoid resveratrol ameliorated UVA-mediated inhibition of human RPE phagocytosis. The underlying mechanism might involve the large-conductance calcium-activated potassium channels.

Patient-specific mutations impair BESTROPHIN1’s essential role in mediating Ca2+-dependent Cl- currents in human RPE

Energy Technology Data Exchange (ETDEWEB)

Li, Yao [Jonas Children’s Vision Care, and Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology and Pathology & Cell Biology, Edward S. Harkness Eye Institute, New York Presbyterian Hospital/Columbia University, New York, United States; Zhang, Yu [Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, United States; Xu, Yu [Jonas Children’s Vision Care, and Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology and Pathology & Cell Biology, Edward S. Harkness Eye Institute, New York Presbyterian Hospital/Columbia University, New York, United States; Department of Ophthalmology, Xinhua Hospital affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China; Kittredge, Alec [Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, United States; Ward, Nancy [Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, United States; Chen, Shoudeng [Molecular Imaging Center, Department of Experimental Medicine, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China; Tsang, Stephen H. [Jonas Children’s Vision Care, and Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology and Pathology & Cell Biology, Edward S. Harkness Eye Institute, New York Presbyterian Hospital/Columbia University, New York, United States; Yang, Tingting [Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, United States

2017-10-24

Mutations in the human BEST1 gene lead to retinal degenerative diseases displaying progressive vision loss and even blindness. BESTROPHIN1, encoded by BEST1, is predominantly expressed in retinal pigment epithelium (RPE), but its physiological role has been a mystery for the last two decades. Using a patient-specific iPSC-based disease model and interdisciplinary approaches, we comprehensively analyzed two distinct BEST1 patient mutations, and discovered mechanistic correlations between patient clinical phenotypes, electrophysiology in their RPEs, and the structure and function of BESTROPHIN1 mutant channels. Our results revealed that the disease-causing mechanism of BEST1 mutations is centered on the indispensable role of BESTROPHIN1 in mediating the long speculated Ca2+-dependent Cl- current in RPE, and demonstrate that the pathological potential of BEST1 mutations can be evaluated and predicted with our iPSC-based ‘disease-in-a-dish’ approach. Moreover, we demonstrated that patient RPE is rescuable with viral gene supplementation, providing a proof-of-concept for curing BEST1-associated diseases.

That Escalated Quickly—Planning to Ignore RPE Can Backfire

Directory of Open Access Journals (Sweden)

Maik Bieleke

2017-09-01

Full Text Available Ratings of perceived exertion (RPE are routinely assessed in exercise science and RPE is substantially associated with physiological criterion measures. According to the psychobiological model of endurance, RPE is a central limiting factor in performance. While RPE is known to be affected by psychological manipulations, it remains to be examined whether RPE can be self-regulated during static muscular endurance exercises to enhance performance. In this experiment, we investigate the effectiveness of the widely used and recommended self-regulation strategy of if-then planning (i.e., implementation intentions in down-regulating RPE and improving performance in a static muscular endurance task. 62 female students (age: M = 23.7 years, SD = 4.0 were randomly assigned to an implementation intention or a control condition and performed a static muscular endurance task. They held two intertwined rings as long as possible while avoiding contacts between the rings. In the implementation intention condition, participants had an if-then plan: “If the task becomes too strenuous for me, then I ignore the strain and tell myself: Keep going!” Every 25 ± 10 s participants reported their RPE along with their perceived pain. Endurance performance was measured as time to failure, along with contact errors as a measure of performance quality. No differences emerged between implementation intention and control participants regarding time to failure and performance quality. However, mixed-effects model analyses revealed a significant Time-to-Failure × Condition interaction for RPE. Compared to the control condition, participants in the implementation intention condition reported substantially greater increases in RPE during the second half of the task and reached higher total values of RPE before task termination. A similar but weaker pattern evinced for perceived pain. Our results demonstrate that RPE during an endurance task can be self-regulated with if

Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future

Directory of Open Access Journals (Sweden)

Mingyue Luo

2018-01-01

Full Text Available As a constituent of blood-retinal barrier and retinal outer segment (ROS scavenger, retinal pigmented epithelium (RPE is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE (hRPESC-RPE has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE (hESC-RPE can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE (iPSC-RPE is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received iPSC-RPE transplant, which is a hallmark of iPSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE (SC-RPE especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.

Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future.

Science.gov (United States)

Luo, Mingyue; Chen, Youxin

2018-01-01

As a constituent of blood-retinal barrier and retinal outer segment (ROS) scavenger, retinal pigmented epithelium (RPE) is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE (hRPESC-RPE) has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE (hESC-RPE) can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE (iPSC-RPE) is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received iPSC-RPE transplant, which is a hallmark of iPSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE (SC-RPE) especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.

7,8-Dihydroxyflavone ameliorates high-glucose induced diabetic apoptosis in human retinal pigment epithelial cells by activating TrkB.

Science.gov (United States)

Yu, Xiaoyi; Liu, Qiuhong; Wang, Xiaochuan; Liu, Hong; Wang, Yan

2018-01-01

In diabetic retinopathy, prolonged high-level blood glucose induced significant impairments among various retinal tissues, including retinal pigment epithelial (RPE) cells. In an in vitro model of human RPE cells, we evaluated whether 7,8-Dihydroxyflavone (DHF) may effectively prevent high glucose-induced diabetic apoptosis among human RPE cells. ARPE-19 cells, a Human RPE cell line, were treated with d-glucose (50 mM) to induce apoptosis in vitro. Prior to glucose, ARPE-19 cells were pre-incubated with various concentrations of DHF. The effect of DHF on d-glucose-induced apoptosis was examined by TUNEL assay, in a concentration-dependent manner. The biological effects of DHF on Caspase-9 (Casp-9) and TrkB signaling pathways in d-glucose-injured ARPE-19 cells were evaluated by qRT-PCR and western blot (WB) assays. A TrkB antagonist, K252a, was also applied in DHF and d-glucose treated ARPE-19 cells. Possible effect of K252a blocking TrkB signaling pathway, thus reversing DHF-modulated apoptosis prevention was also examined by TUNEL and WB assays. DHF ameliorated d-glucose-induced diabetic apoptosis in ARPE-19 cells. Apoptotic factor Casp-9, at both mRNA and protein levels, were drastically inhibited by DHF in d-glucose-injured ARPE-19 cells. Also, DHF activated TrkB signaling pathway through phosphorylation. K252a dramatically reversed the preventive effect of DHF on d-glucose-induced apoptosis in ARPE-19 cells. Further investigation showed that K252a functioned through de-activating or de-phosphorylating TrkB signaling pathway. This work demonstrates that DHF, through activation of TrkB signaling pathway, has a preventive function in d-glucose-induced apoptosis in PRE cells in diabetic retinopathy. Copyright © 2017. Published by Elsevier Inc.

Voltage-dependent ion channels in the mouse RPE: comparison with Norrie disease mice.

Science.gov (United States)

Wollmann, Guido; Lenzner, Steffen; Berger, Wolfgang; Rosenthal, Rita; Karl, Mike O; Strauss, Olaf

2006-03-01

We studied electrophysiological properties of cultured retinal pigment epithelial (RPE) cells from mouse and a mouse model for Norrie disease. Wild-type RPE cells revealed the expression of ion channels known from other species: delayed-rectifier K(+) channels composed of Kv1.3 subunits, inward rectifier K(+) channels, Ca(V)1.3 L-type Ca(2+) channels and outwardly rectifying Cl(-) channels. Expression pattern and the ion channel characteristics current density, blocker sensitivity, kinetics and voltage-dependence were compared in cells from wild-type and Norrie mice. Although no significant differences were observed, our study provides a base for future studies on ion channel function and dysfunction in transgenic mouse models.

Proteasome inhibitors activate autophagy involving inhibition of PI3K-Akt-mTOR pathway as an anti-oxidation defense in human RPE cells.

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Bingrong Tang

Full Text Available The two major intracellular protein degradation systems, the ubiquitin-proteasome system (UPS and autophagy, work collaboratively in many biological processes including development, apoptosis, aging, and countering oxidative injuries. We report here that, in human retinal pigment epithelial cells (RPE, ARPE-19 cells, proteasome inhibitors, clasto-lactacystinβ-lactone (LA or epoxomicin (Epo, at non-lethal doses, increased the protein levels of autophagy-specific genes Atg5 and Atg7 and enhanced the conversion of microtubule-associated protein light chain (LC3 from LC3-I to its lipidative form, LC3-II, which was enhanced by co-addition of the saturated concentration of Bafilomycin A1 (Baf. Detection of co-localization for LC3 staining and labeled-lysosome further confirmed autophagic flux induced by LA or Epo. LA or Epo reduced the phosphorylation of the protein kinase B (Akt, a downstream target of phosphatidylinositol-3-kinases (PI3K, and mammalian target of rapamycin (mTOR in ARPE-19 cells; by contrast, the induced changes of autophagy substrate, p62, showed biphasic pattern. The autophagy inhibitor, Baf, attenuated the reduction in oxidative injury conferred by treatment with low doses of LA and Epo in ARPE-19 cells exposed to menadione (VK3 or 4-hydroxynonenal (4-HNE. Knockdown of Atg7 with siRNA in ARPE-19 cells reduced the protective effects of LA or Epo against VK3. Overall, our results suggest that treatment with low levels of proteasome inhibitors confers resistance to oxidative injury by a pathway involving inhibition of the PI3K-Akt-mTOR pathway and activation of autophagy.

The novel triterpenoid RTA 408 protects human retinal pigment epithelial cells against H2O2-induced cell injury via NF-E2-related factor 2 (Nrf2 activation

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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.

Controlled surface morphology and hydrophilicity of polycaprolactone toward human retinal pigment epithelium cells

International Nuclear Information System (INIS)

Shahmoradi, Saleheh; Yazdian, Fatemeh; Tabandeh, Fatemeh; Soheili, Zahra-Soheila; Hatamian Zarami, Ashraf Sadat; Navaei-Nigjeh, Mona

2017-01-01

Applying scaffolds as a bed to enhance cell proliferation and even differentiation is one of the treatment of retina diseases such as age-related macular degeneration (AMD) which deteriorating photoreceptors and finally happening blindness. In this study, aligned polycaprolactone (PCL) nanofibers were electrospun and at different conditions and their characteristics were measured by scanning electron microscope (SEM) and contact angle. Response surface methodology (RSM) was used to optimize the diameter of fabricated nanofibers. Two factors as solution concentration and voltage value were considered as independent variables and their effects on nanofibers' diameters were evaluated by central composite design and the optimum conditions were obtained as 0.12 g/mL and 20 kV, respectively. In order to decrease the hydrophobicity of PCL, the surface of the fabricated scaffolds was modified by alkaline hydrolysis method. Contact time of the scaffolds and alkaline solution and concentration of alkaline solution were optimized using Box Behnken design and (120 min and 5 M were the optimal, respectively). Contact angle measurement showed the high hydrophilicity of treated scaffolds (with contact angle 7.48°). Plasma surface treatment was applied to compare the effect of using two kinds of surface modification methods simultaneously on hydrolyzed scaffolds. The RPE cells grown on scaffolds were examined by immunocytochemistry (ICC), MTT and continuous inspection of cellular morphology. Interestingly, Human RPE cells revealed their characteristic morphology on hydrolyzed scaffold well. As a result, we introduced a culture substrate with low diameter (185.8 nm), high porosity (82%) and suitable hydrophilicity (with contact angle 7.48 degree) which can be promising for hRPE cell transplantation. - Highlights: • Dimethylformamide (DMF) has significant effect on reduction of fibers' diameter. • Having high hydrophilicity by alkaline hydrolysis • Suitable

Controlled surface morphology and hydrophilicity of polycaprolactone toward human retinal pigment epithelium cells

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Shahmoradi, Saleheh; Yazdian, Fatemeh [Department of Life Science Engineering, Faculty of New sciences and Technologies, University of Tehran, Tehran (Iran, Islamic Republic of); Tabandeh, Fatemeh, E-mail: taban_f@nigeb.ac.ir [Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran (Iran, Islamic Republic of); Soheili, Zahra-Soheila [Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran (Iran, Islamic Republic of); Hatamian Zarami, Ashraf Sadat [Department of Life Science Engineering, Faculty of New sciences and Technologies, University of Tehran, Tehran (Iran, Islamic Republic of); Navaei-Nigjeh, Mona [Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

2017-04-01

Applying scaffolds as a bed to enhance cell proliferation and even differentiation is one of the treatment of retina diseases such as age-related macular degeneration (AMD) which deteriorating photoreceptors and finally happening blindness. In this study, aligned polycaprolactone (PCL) nanofibers were electrospun and at different conditions and their characteristics were measured by scanning electron microscope (SEM) and contact angle. Response surface methodology (RSM) was used to optimize the diameter of fabricated nanofibers. Two factors as solution concentration and voltage value were considered as independent variables and their effects on nanofibers' diameters were evaluated by central composite design and the optimum conditions were obtained as 0.12 g/mL and 20 kV, respectively. In order to decrease the hydrophobicity of PCL, the surface of the fabricated scaffolds was modified by alkaline hydrolysis method. Contact time of the scaffolds and alkaline solution and concentration of alkaline solution were optimized using Box Behnken design and (120 min and 5 M were the optimal, respectively). Contact angle measurement showed the high hydrophilicity of treated scaffolds (with contact angle 7.48°). Plasma surface treatment was applied to compare the effect of using two kinds of surface modification methods simultaneously on hydrolyzed scaffolds. The RPE cells grown on scaffolds were examined by immunocytochemistry (ICC), MTT and continuous inspection of cellular morphology. Interestingly, Human RPE cells revealed their characteristic morphology on hydrolyzed scaffold well. As a result, we introduced a culture substrate with low diameter (185.8 nm), high porosity (82%) and suitable hydrophilicity (with contact angle 7.48 degree) which can be promising for hRPE cell transplantation. - Highlights: • Dimethylformamide (DMF) has significant effect on reduction of fibers' diameter. • Having high hydrophilicity by alkaline hydrolysis • Suitable

Three-dimensional neuroepithelial culture from human embryonic stem cells and its use for quantitative conversion to retinal pigment epithelium.

Directory of Open Access Journals (Sweden)

Yu Zhu

Full Text Available A goal in human embryonic stem cell (hESC research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.

In vitro differentiation of adipose-tissue-derived mesenchymal stem cells into neural retinal cells through expression of human PAX6 (5a) gene.

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Rezanejad, Habib; Soheili, Zahra-Soheila; Haddad, Farhang; Matin, Maryam M; Samiei, Shahram; Manafi, Ali; Ahmadieh, Hamid

2014-04-01

The neural retina is subjected to various degenerative conditions. Regenerative stem-cell-based therapy holds great promise for treating severe retinal degeneration diseases, although many drawbacks remain to be overcome. One important problem is to gain authentically differentiated cells for replacement. Paired box 6 protein (5a) (PAX6 (5a)) is a highly conserved master control gene that has an essential role in the development of the vertebrate visual system. Human adipose-tissue-derived stem cell (hADSC) isolation was performed by using fat tissues and was confirmed by the differentiation potential of the cells into adipocytes and osteocytes and by their surface marker profile. The coding region of the human PAX6 (5a) gene isoform was cloned and lentiviral particles were propagated in HEK293T. The differentiation of hADSCs into retinal cells was characterized by morphological characteristics, quantitative real-time reverse transcription plus the polymerase chain reaction (qPCR) and immunocytochemistry (ICC) for some retinal cell-specific and retinal pigmented epithelial (RPE) cell-specific markers. hADSCs were successfully isolated. Flow cytometric analysis of surface markers indicated the high purity (~97 %) of isolated hADSCs. After 30 h of post-transduction, cells gradually showed the characteristic morphology of neuronal cells and small axon-like processes emerged. qPCR and ICC confirmed the differentiation of some neural retinal cells and RPE cells. Thus, PAX6 (5a) transcription factor expression, together with medium supplemented with fibronectin, is able to induce the differentiation of hADSCs into retinal progenitors, RPE cells and photoreceptors.

Loss of Extracellular Signal-Regulated Kinase 1/2 in the Retinal Pigment Epithelium Leads to RPE65 Decrease and Retinal Degeneration.

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Pyakurel, Aswin; Balmer, Delphine; Saba-El-Leil, Marc K; Kizilyaprak, Caroline; Daraspe, Jean; Humbel, Bruno M; Voisin, Laure; Le, Yun Z; von Lintig, Johannes; Meloche, Sylvain; Roduit, Raphaël

2017-12-15

Recent work suggested that the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) is increased in the retinal pigment epithelium (RPE) of age-related macular degeneration (ARMD) patients and therefore could be an attractive therapeutic target. Notably, ERK1/2 pathway inhibitors are used in cancer therapy, with severe and noncharacterized ocular side effects. To decipher the role of ERK1/2 in RPE cells, we conditionally disrupted the Erk1 and Erk2 genes in mouse RPE. The loss of ERK1/2 activity resulted in a significant decrease in the level of RPE65 expression, a decrease in ocular retinoid levels concomitant with low visual function, and a rapid disorganization of RPE cells, ultimately leading to retinal degeneration. Our results identify the ERK1/2 pathway as a direct regulator of the visual cycle and a critical component of the viability of RPE and photoreceptor cells. Moreover, our results caution about the need for a very fine adjustment of kinase inhibition in cancer or ARMD treatment in order to avoid ocular side effects. Copyright © 2017 Pyakurel et al.

Does the Timing of Measurement Alter Session-RPE in Boxers?

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Uchida, Marco C; Teixeira, Luis F M; Godoi, Vladmir J; Marchetti, Paulo H; Conte, Marcelo; Coutts, Aaron J; Bacurau, Reury F P

2014-01-01

The purpose of this study was to compare the influence of measuring the overall session rating of perceived exertion (session-RPE) at 10 vs. 30 minutes following exercise. Eight boxers completed three different standardized training sessions of different intensities (easy, moderate and hard) in a matchedpairs, randomized research design. Exercise intensity was assessed during each bout by measuring heart rate, blood lactate concentration and session-RPE. To assess the effect of measurement timing on session-RPE, RPE data were collected either 10 or 30 minutes post-exercise. There was no significant effect of measurement time on session-RPE values following easy (10 minutes: session-RPE = 1.3 ± 1.0 Arbitrary Unit (AU), %Heart Rate Reserve (HRR) = 49.5 ± 11.1, and ∆Blood lactate = -2.3 ± 16.3%; 30 minutes: session-RPE = 1.7 ± 1.0 AU, %HRR = 51.3 ± 10.8, and ∆Blood lactate = 0.7 ± 25.2%), moderate (10 minutes: session-RPE = 2.7 ± 1.6 AU, %HRR = 67.2 ± 10.8, and ∆Blood lactate = 2.2 ± 19%; 30 minutes: session-RPE = 2.5 ± 0.9 AU, %HRR = 67.2 ± 5.9, and ∆Blood lactate = 24.5 ± 17.1%) and hard (10 minutes: session-RPE = 5.7 ± 1.0 AU, %HRR = 88.1 ± 6.3, and ∆Blood lactate = 146.3 ± 87.9%; 30 minutes: session-RPE = 5.8 ± 1.9 AU, %HRR> = 83.3 ± 8.0, and ∆Blood lactate = 91.6 ± 39%) sessions. In conclusion, our findings suggest that session-RPE can be used in boxing training routines across a range of intensities and accurate measurements can be determined as early as 10 minutes after exercise. Key PointsIt is difficult to quantify and monitoring the external training load in martial arts (e.g. Aikido, Kung Fu, Judo) and physical combat sports (e.g. Boxing, Muay Thai), session RPE method appears to be a reliable method to quantifying training load in those sports.For many athletes it is impractical to wait 30 minutes after training session to provide a session-RPE. The present findings show that collecting ses-sion-RPE measures at 10 min

Morphological and functional rescue in RCS rats after RPE cell line transplantation at a later stage of degeneration.

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Wang, Shaomei; Lu, Bin; Girman, Sergej; Holmes, Toby; Bischoff, Nicolas; Lund, Raymond D

2008-01-01

It is well documented that grafting of cells in the subretinal space of Royal College of Surgeons (RCS) rats limits deterioration of vision and loss of photoreceptors if performed early in postnatal life. What is unclear is whether cells introduced later, when photoreceptor degeneration is already advanced, can still be effective. This possibility was examined in the present study, using the human retinal pigment epithelial cell line, ARPE-19. Dystrophic RCS rats (postnatal day [P] 60) received subretinal injection of ARPE-19 cells (2 x 10(5)/3 microL/eye). Spatial frequency was measured by recording optomotor responses at P100 and P150, and luminance threshold responses were recorded from the superior colliculus at P150. Retinas were stained with cresyl violet, retinal cell-specific markers, and a human nuclear marker. Control animals were injected with medium alone. Animals comparably treated with grafts at P21 were available for comparison. All animals were treated with immunosuppression. Later grafts preserved both spatial frequency and threshold responses over the control and delayed photoreceptor degeneration. There were two to three layers of rescued photoreceptors even at P150, compared with a scattered single layer in sham and untreated control retinas. Retinal cell marker staining showed an orderly array of the inner retinal lamination. The morphology of the second-order neurons was better preserved around the grafted area than in regions distant from graft. Sham injection had little effect in rescuing the photoreceptors. RPE cell line transplants delivered later in the course of degeneration can preserve not only the photoreceptors and inner retinal lamination but also visual function in RCS rats. However, early intervention can achieve better rescue.

Progranulin increases phagocytosis by retinal pigment epithelial cells in culture.

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Murase, Hiromi; Tsuruma, Kazuhiro; Kuse, Yoshiki; Shimazawa, Masamitsu; Hara, Hideaki

2017-12-01

Retinal pigment epithelium (RPE) cells take part in retinal preservation, such as phagocytizing the shed photoreceptor outer segments (POS), every day. The incomplete phagocytic function accelerates RPE degeneration and formation of the toxic by-product lipofuscin. Excessive lipofuscin accumulation is characteristic of various blinding diseases in the human eye. Progranulin is a cysteine-rich protein that has multiple biological activities, and it has a high presence in the retina. Progranulin has been recognized to be involved in macrophage phagocytosis in the brain. The purpose of this study is to determine whether progranulin influences phagocytosis by RPE cells. All experiments were performed on primary human RPE (hRPE) cells in culture. pHrodo was used to label the isolated porcine POS, and quantification of pHrodo fluorescence was used to determine the degree of phagocytosis. Western blotting and immunohistochemistry of key proteins involved in phagocytosis were used to clarify the mechanism of progranulin. Progranulin increased RPE phagocytosis in hydrogen peroxide-treated and nontreated RPE cells. The phosphorylated form of Mer tyrosine kinase, which is important for POS internalization, was significantly increased in the progranulin-exposed cells. This increase was attenuated by SU11274, an inhibitor of hepatic growth factor receptor. Under the oxidative stress condition, exposure to progranulin led to an approximately twofold increase in integrin alpha-v, which is associated with the first step in recognition of POS by RPE cells. These results suggest that progranulin could be an effective stimulator for RPE phagocytosis and could repair RPE function. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Fundus albipunctatus associated with compound heterozygous mutations in RPE65

DEFF Research Database (Denmark)

Schatz, Patrik; Preising, Markus; Lorenz, Birgit

2011-01-01

To describe a family with an 18-year-old woman with fundus albipunctatus and compound heterozygous mutations in RPE65 whose unaffected parents and 1 female sibling harbored single heterozygous RPE65 mutations.......To describe a family with an 18-year-old woman with fundus albipunctatus and compound heterozygous mutations in RPE65 whose unaffected parents and 1 female sibling harbored single heterozygous RPE65 mutations....

In vivo imaging of the retinal pigment epithelial cells

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Morgan, Jessica Ijams Wolfing

The retinal pigment epithelial (RPE) cells form an important layer of the retina because they are responsible for providing metabolic support to the photoreceptors. Techniques to image the RPE layer include autofluorescence imaging with a scanning laser ophthalmoscope (SLO). However, previous studies were unable to resolve single RPE cells in vivo. This thesis describes the technique of combining autofluorescence, SLO, adaptive optics (AO), and dual-wavelength simultaneous imaging and registration to visualize the individual cells in the RPE mosaic in human and primate retina for the first time in vivo. After imaging the RPE mosaic non-invasively, the cell layer's structure and regularity were characterized using quantitative metrics of cell density, spacing, and nearest neighbor distances. The RPE mosaic was compared to the cone mosaic, and RPE imaging methods were confirmed using histology. The ability to image the RPE mosaic led to the discovery of a novel retinal change following light exposure; 568 nm exposures caused an immediate reduction in autofluorescence followed by either full recovery or permanent damage in the RPE layer. A safety study was conducted to determine the range of exposure irradiances that caused permanent damage or transient autofluorescence reductions. Additionally, the threshold exposure causing autofluorescence reduction was determined and reciprocity of radiant exposure was confirmed. Light exposures delivered by the AOSLO were not significantly different than those delivered by a uniform source. As all exposures tested were near or below the permissible light levels of safety standards, this thesis provides evidence that the current light safety standards need to be revised. Finally, with the retinal damage and autofluorescence reduction thresholds identified, the methods of RPE imaging were modified to allow successful imaging of the individual cells in the RPE mosaic while still ensuring retinal safety. This thesis has provided a

USING SESSION RPE TO MONITOR DIFFERENT METHODS OF RESISTANCE EXERCISE

Directory of Open Access Journals (Sweden)

Alison D. Egan

2006-06-01

Full Text Available The purpose of this study was to compare session rating of perceived exertion for different resistance training techniques in the squat exercise. These techniques included traditional resistance training, super slow, and maximal power training. Fourteen college-age women (Mean ± SD; age = 22 ± 3 years; height = 1.68 ± 0. 07 m completed three experimental trials in a randomized crossover design. The traditional resistance training protocol consisted of 6 sets of 6 repetitions of squats using 80% of 1-RM. The super slow protocol consisted of 6 sets of 6 repetitions using 55% of 1-RM. The maximal power protocol consisted of 6 sets of 6 repetitions using 30% of 1-RM. Rating of perceived exertion (RPE measures were obtained following each set using Borg's CR-10 scale. In addition, a session RPE value was obtained 30 minutes following each exercise session. When comparing average RPE and session RPE, no significant difference was found. However, power training had significantly lower (p < 0.05 average and session RPE (4.50 ± 1.9 and 4.5 ± 2.1 compared to both super slow training (7.81 ± 1.75 and 7.43 ± 1.73 and traditional training (7.33 ± 1.52 and 7.13 ± 1.73. The results indicate that session RPE values are not significantly different from the more traditional methods of measuring RPE during exercise bouts. It does appear that the resistance training mode that is used results in differences in perceived exertion that does not relate directly to the loading that is used. Using session RPE provides practitioners with the same information about perceived exertion as the traditional RPE measures. Taking a single measure following a training session would appear to be much easier than using multiple measures of RPE throughout a resistance training workout. However, practitioners should also be aware that the RPE does not directly relate to the relative intensity used and appears to be dependent on the mode of resistance exercise that is used

Role for nectin-1 in herpes simplex virus 1 entry and spread in human retinal pigment epithelial cells

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Tiwari, Vaibhav; Oh, Myung-Jin; Kovacs, Maria; Shukla, Shripaad Y.; Valyi-Nagy, Tibor; Shukla, Deepak

2009-01-01

Herpes simplex virus 1 (HSV-1) demonstrates a unique ability to infect a variety of host cell types. Retinal pigment epithelial (RPE) cells form the outermost layer of the retina and provide a potential target for viral invasion and permanent vision impairment. Here we examine the initial cellular and molecular mechanisms that facilitate HSV-1 invasion of human RPE cells. High-resolution confocal microscopy demonstrated initial interaction of green fluorescent protein (GFP)-tagged virions with filopodia-like structures present on cell surfaces. Unidirectional movement of the virions on filopodia to the cell body was detected by live cell imaging of RPE cells, which demonstrated susceptibility to pH-dependent HSV-1 entry and replication. Use of RT-PCR indicated expression of nectin-1, herpes virus entry mediator (HVEM) and 3-O-sulfotransferase-3 (as a surrogate marker for 3-O-sulfated heparan sulfate). HVEM and nectin-1 expression was subsequently verified by flow cytometry. Nectin-1 expression in murine retinal tissue was also demonstrated by immunohistochemistry. Antibodies against nectin-1, but not HVEM, were able to block HSV-1 infection. Similar blocking effects were seen with a small interfering RNA construct specifically directed against nectin-1, which also blocked RPE cell fusion with HSV-1 glycoprotein-expressing Chinese hamster ovary (CHO-K1) cells. Anti-nectin-1 antibodies and F-actin depolymerizers were also successful in blocking the cytoskeletal changes that occur upon HSV-1 entry into cells. Our findings shed new light on the cellular and molecular mechanisms that help the virus to enter the cells of the inner eye. PMID:18803666

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

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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.

Survival Improvement in Human Retinal Pigment Epithelial Cells via Fas Receptor Targeting by miR-374a.

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Tasharrofi, Nooshin; Kouhkan, Fatemeh; Soleimani, Masoud; Soheili, Zahra-Sheila; Kabiri, Mahboubeh; Mahmoudi Saber, Mohaddeseh; Dorkoosh, Farid Abedin

2017-12-01

Oxidative conditions of the eye could contribute to retinal cells loss through activating the Fas-L/Fas pathway. This phenomenon is one of the leading causes of some ocular diseases like age-related macular degeneration (AMD). By targeting proteins at their mRNA level, microRNAs (miRNAs) can regulate gene expression and cell function. The aim of the present study is to investigate Fas targeting by miR-374a and find whether it can inhibit Fas-mediated apoptosis in primary human retinal pigment epithelial (RPE) cells under oxidative stress. So, the primary human RPE cells were transfected with pre-miR-374a pLEX construct using polymeric carrier and were exposed to H 2 O 2 (200 μM) as an oxidant agent for induction of Fas expression. Fas expression at mRNA and protein level was evaluated by quantitative real-time PCR and Western blot analysis, respectively. These results revealed that miR-374a could prevent Fas upregulation under oxidative conditions. Moreover, Luciferase activity assay confirmed that Fas could be a direct target of miR-374a. The cell viability studies demonstrated that caspase-3 activity was negligible in miR-374a treated cells compared to the controls. Our data suggest miR-374a is a negative regulator of Fas death receptor which is able to enhance the cell survival and protect RPE cells against oxidative conditions. J. Cell. Biochem. 118: 4854-4861, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Construction of a cDNA library from human retinal pigment epithelial cells challenged with rod outer segments.

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Cavaney, D M; Rakoczy, P E; Constable, I J

1995-05-01

To study genes expressed by retinal pigment epithelial (RPE) cells during phagocytosis and digestion of rod outer segments (ROS), a complementary (c)DNA library was produced using an in-vitro model. The cDNA library can be used to study molecular changes which contribute to the development of diseases due to a failure in outer segment phagocytosis and digestion by RPE cells. Here we demonstrate a way to study genes and their functions using a molecular biological approach and describing the first step involved in this process, the construction of a cDNA library. Human RPE cells obtained from the eyes of a seven-year-old donor were cultured and challenged with bovine ROS. The culture was harvested and total RNA was extracted. Complementary DNA was transcribed from the messenger (m)RNA and was directionally cloned into the LambdaGEM-4 bacteriophage vector successfully. Some clones were picked and the DNA extracted, to determine the size of the inserts as a measure of the quality of the library. Molecular biology and cell culture are important tools to be used in eye research, especially in areas where tissue is limiting and animal models are not available. We now have a ROS challenged RPE cDNA library which will be used to identify genes responsible for degrading phagocytosed debris within the retinal pigment epithelium.

New medium used in the differentiation of human pluripotent stem cells to retinal cells is comparable to fetal human eye tissue.

Science.gov (United States)

Wang, Xiaobing; Xiong, Kai; Lin, Cong; Lv, Lei; Chen, Jing; Xu, Chongchong; Wang, Songtao; Gu, Dandan; Zheng, Hua; Yu, Hurong; Li, Yan; Xiao, Honglei; Zhou, Guomin

2015-06-01

Human pluripotent stem cells (hPSCs) have the potential to differentiate along the retinal lineage. However, most induction systems are dependent on multiple small molecular compounds such as Dkk-1, Lefty-A, and retinoic acid. In the present study, we efficiently differentiated hPSCs into retinal cells using a retinal differentiation medium (RDM) without the use of small molecular compounds. This novel differentiation system recapitulates retinal morphogenesis in humans, i.e. hPSCs gradually differentiate into optic vesicle-shaped spheres, followed by optic cup-shaped spheres and, lastly, retinal progenitor cells. Furthermore, at different stages, hPSC-derived retinal cells mirror the transcription factor expression profiles seen in their counterparts during human embryogenesis. Most importantly, hinge epithelium was found between the hPSC-derived neural retina (NR) and retinal pigment epithelium (RPE). These data suggest that our culture system provides a new method for generating hPSC-derived retinal cells that, for the first time, might be used in human transplantation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Efficient gene delivery to primary human retinal pigment epithelial cells: The innate and acquired properties of vectors.

Science.gov (United States)

Tasharrofi, Nooshin; Kouhkan, Fatemeh; Soleimani, Masoud; Soheili, Zahra-Soheila; Atyabi, Fatemeh; Akbari Javar, Hamid; Abedin Dorkoosh, Farid

2017-02-25

The purpose of this study is designing non-viral gene delivery vectors for transfection of the primary human retinal pigment epithelial cells (RPE). In the design process of gene delivery vectors, considering physicochemical properties of vectors alone does not seem to be enough since they interact with constituents of the surrounding environment and hence gain new characteristics. Moreover, due to these interactions, their cargo can be released untimely or undergo degradation before reaching to the target cells. Further, the characteristics of cells itself can also influence the transfection efficacy. For example, the non-dividing property of RPE cells can impede the transfection efficiency which in most studies was ignored by using immortal cell lines. In this study, vectors with different characteristics differing in mixing orders of pDNA, PEI polymer, and PLGA/PEI or PLGA nanoparticles were prepared and characterized. Then, their characteristics and efficacy in gene delivery to RPE cells in the presence of vitreous or fetal bovine serum (FBS) were evaluated. All formulations showed no cytotoxicity and were able to protect pDNA from premature release and degradation in extracellular media. Also, the adsorption of vitreous or serum proteins onto the surface of vectors changed their properties and hence cellular uptake and transfection efficacy. Copyright © 2016 Elsevier B.V. All rights reserved.

Memory, reconsolidation and extinction in Lymnaea require the soma of RPeD1.

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Sangha, Susan; Varshney, Nishi; Fras, Mary; Smyth, Kim; Rosenegger, David; Parvez, Kashif; Sadamoto, Hisayo; Lukowiak, Ken

2004-01-01

The central pattern generator (CPG) that drives aerial respiratory behaviour in Lymnaea consists of 3 neurons. One of these, RPeD1--the cell that initiates activity in the circuit, plays an absolutely necessary role as a site for memory formation, memory reconsolidation, and extinction. Using an operant conditioning training procedure that results in a long-term non-declarative memory (LTM), we decrease the occurrence of aerial respiratory behaviour. Since snails can still breathe cutaneously learning this procedure is not harmful. Concomitant with behavioural memory are changes in the spiking activity of RPeD1. Going beyond neural correlates of memory we directly show that RPeD1 is a necessary site for LTM formation. Expanding on this finding we show that this neuron is also a necessary site for memory reconsolidation and 'Pavlovian' extinction. As far as we can determine, this is the first time a single neuron has been shown to be a necessary site for these different aspects memory. RPeD1 is thus a key neuron mediating different hierarchical aspects of memory. We are now in a position to determine the necessary neuronal, molecular and proteomic events in this neuron that are causal to memory formation, reconsolidation and extinction.

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

Differential behavioral outcomes following neonatal versus fetal human retinal pigment epithelial cell striatal implants in parkinsonian rats

DEFF Research Database (Denmark)

Russ, Kaspar; Flores, Joseph; Brudek, Tomasz

2017-01-01

Following the failure of a Phase II clinical study evaluating human retinal pigment epithelial (hRPE) cell implants as a potential treatment option for Parkinson's disease, speculation has centered on implant function and survival as possible contributors to the therapeutic outcomes. We recently ...

Human stem cell-derived retinal epithelial cells activate complement via collectin 11 in response to stress

DEFF Research Database (Denmark)

Fanelli, Giorgia; Gonzalez-Cordero, Anai; Gardner, Peter J

2017-01-01

induced-pluripotent stem cell (iPSC)-derived RPE cells, particularly with regard to the complement pathway. We focused on collectin-11 (CL-11), a pattern recognition molecule that can trigger complement activation in renal epithelial tissue. We found evidence of constitutive and hypoxia-induced expression......, failed to activate complement. The presence of CL-11 in healthy murine and human retinal tissues confirmed the biological relevance of CL-11. Our data describe a new trigger mechanism of complement activation that could be important in disease pathogenesis and therapeutic interventions....

Functional recovery after experimental RPE debridement, mfERG studies in a porcine model

DEFF Research Database (Denmark)

Sørensen, Nina Buus; Lassota, Nathan; Kyhn, Maria Voss

2013-01-01

BACKGROUND: The correlation between histologically identified regeneration of retinal pigment epithelium (RPE) and functional outcome measured by multifocal electroretinography (mfERG) following surgical debridement is examined in a porcine model. In humans, visual acuity is reduced in diseases......, and by brushing the Bruch's membrane with a silicone catheter. Immediately following surgery (baseline) and after 2 and 6 weeks respectively, the animals were examined by mfERG, fundus photographs (FPs), fluorescein angiograms (FAs), and histopathology. RESULTS: The mfERG P1 amplitude was decreased 2 weeks (T2....... CONCLUSION: This is the first study to show that the histological regeneration of hypopigmented RPE correlates to a return of the retinal function, measured by mfERG....

Induced pluripotent stem cell-based therapy for age-related macular degeneration.

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Bracha, Peter; Moore, Nicholas A; Ciulla, Thomas A

2017-09-01

In age-related macular degeneration (AMD), stem cells could possibly replace or regenerate disrupted pathologic retinal pigment epithelium (RPE), and produce supportive growth factors and cytokines such as brain-derived neurotrophic factor.  Induced pluripotent stem cells (iPSCs)-derived RPE was first subretinally transplanted in a neovascular AMD patient in 2014. Areas covered: Induced PSCs are derived from the introduction of transcription factors to adult cells under specific cell culture conditions, followed by differentiation into RPE cells. Induced PSC-derived RPE cells exhibit ion transport, membrane potential, polarized VEGF secretion and gene expression that is similar to native RPE. Despite having similar in vitro function, morphology, immunostaining and microscopic analysis, it remains to be seen if iPSC-derived RPE can replicate the myriad of in vivo functions, including immunomodulatory effects, of native RPE cells.  Historically, adjuvant RPE transplantation during CNV resections were technically difficult and complicated by immune rejection. Autologous iPSCs are hypothesized to reduce the risk of immune rejection, but their production is time-consuming and expensive.  Alternatively, allogenic transplantation using human leukocyte antigen (HLA)-matched iPSCs, similar to HLA-matched organ transplantation, is currently being investigated. Expert opinion: Challenges to successful transplantation with iPSCs include surgical technique, a pathologic subretinal microenvironment, possible immune rejection, and complications of immunosuppression.

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

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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.

Evidence for RPE65-independent vision in the cone-dominated zebrafish retina.

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Schonthaler, Helia B; Lampert, Johanna M; Isken, Andrea; Rinner, Oliver; Mader, Andreas; Gesemann, Matthias; Oberhauser, Vitus; Golczak, Marcin; Biehlmaier, Oliver; Palczewski, Krzysztof; Neuhauss, Stephan C F; von Lintig, Johannes

2007-10-01

An enzyme-based cyclic pathway for trans to cis isomerization of the chromophore of visual pigments (11-cis-retinal) is intrinsic to vertebrate cone and rod vision. This process, called the visual cycle, is mostly characterized in rod-dominated retinas and essentially depends on RPE65, an all-trans to 11-cis-retinoid isomerase. Here we analysed the role of RPE65 in zebrafish, a species with a cone-dominated retina. We cloned zebrafish RPE65 and showed that its expression coincided with photoreceptor development. Targeted gene knockdown of RPE65 resulted in morphologically altered rod outer segments and overall reduced 11-cis-retinal levels. Cone vision of RPE65-deficient larvae remained functional as demonstrated by behavioural tests and by metabolite profiling for retinoids. Furthermore, all-trans retinylamine, a potent inhibitor of the rod visual cycle, reduced 11-cis-retinal levels of control larvae to a similar extent but showed no additive effects in RPE65-deficient larvae. Thus, our study of zebrafish provides in vivo evidence for the existence of an RPE65-independent pathway for the regeneration of 11-cis-retinal for cone vision.

Mechanisms of selective delivery of xanthophylls to retinal pigment epithelial cells by human lipoproteins.

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Thomas, Sara E; Harrison, Earl H

2016-10-01

The xanthophylls, lutein and zeaxanthin, are dietary carotenoids that selectively accumulate in the macula of the eye providing protection against age-related macular degeneration. To reach the macula, carotenoids cross the retinal pigment epithelium (RPE). Xanthophylls and β-carotene mostly associate with HDL and LDL, respectively. HDL binds to cells via a scavenger receptor class B1 (SR-B1)-dependent mechanism, while LDL binds via the LDL receptor. Using an in-vitro, human RPE cell model (ARPE-19), we studied the mechanisms of carotenoid uptake into the RPE by evaluating kinetics of cell uptake when delivered in serum or isolated LDL or HDL. For lutein and β-carotene, LDL delivery resulted in the highest rates and extents of uptake. In contrast, HDL was more effective in delivering zeaxanthin and meso-zeaxanthin leading to the highest rates and extents of uptake of all four carotenoids. Inhibitors of SR-B1 suppressed zeaxanthin delivery via HDL. Results show a selective HDL-mediated uptake of zeaxanthin and meso-zeaxanthin via SR-B1 and a LDL-mediated uptake of lutein. This demonstrates a plausible mechanism for the selective accumulation of zeaxanthin greater than lutein and xanthophylls over β-carotene in the retina. We found no evidence of xanthophyll metabolism to apocarotenoids or lutein conversion to meso-zeaxanthin. Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.

Quantitative Fundus Autofluorescence in Best Vitelliform Macular Dystrophy: RPE Lipofuscin is not Increased in Non-Lesion Areas of Retina.

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Sparrow, Janet R; Duncker, Tobias; Woods, Russell; Delori, François C

2016-01-01

Since the lipofuscin of retinal pigment epithelial (RPE) cells has been implicated in the pathogenesis of Best vitelliform macular dystrophy, we quantified fundus autofluorescence (quantitative fundus autofluorescence, qAF) as an indirect measure of RPE lipofuscin levels. Mean non-lesion qAF was found to be within normal limits for age. By spectral domain optical coherence tomography (SD-OCT) vitelliform lesions presented as fluid-filled subretinal detachments containing reflective material. We discuss photoreceptor outer segment debris as the source of the intense fluorescence of these lesions and loss of anion channel functioning as an explanation for the bullous photoreceptor-RPE detachment. Unexplained is the propensity of the disease for central retina.

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.

Combination of retinal pigment epithelium cell-conditioned medium and photoreceptor outer segments stimulate mesenchymal stem cell differentiation toward a functional retinal pigment epithelium cell phenotype.

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Huang, Chen; Zhang, Jing; Ao, Mingxin; Li, Ying; Zhang, Chun; Xu, Yonggen; Li, Xuemin; Wang, Wei

2012-02-01

Recent studies have suggested that bone marrow-derived mesenchymal stem cells (BMMSCs) are capable of retinal tissue-specific differentiation but not retinal pigment epithelium (RPE) cell-specific differentiation. Photoreceptor outer segments (POS) contribute to RPE development and maturation. However, there has been no standard culture system that fosters the differentiation of BMMSCs into mature RPE cells in vitro. In this study, we investigated if the soluble factors from RPE cells and POS could differentiate BMMSCs into cells having a phenotype characteristic of RPE cells. Rat BMMSCs were separately co-cultured with RPE cells, or they were exposed to either control medium, RPE cell-conditioned medium (RPECM), POS, or a combination of RPECM and POS (RPECM-POS). After 7 days, the cells were analyzed for morphology and the expression of RPE markers (cytokeratin 8, CRALBP, and RPE65) to assess the RPE differentiation. Significantly higher pigment accumulation and increased protein expression of the three markers were seen in cells cultured in RPECM-POS than in other treated cultures. Furthermore, the RPECM-POS-treated cultures displayed ultrastructural features typical of RPE cells, expressed RPE cell functional proteins, and had the capability to phagocytose POS. Together, theses results suggest the combination of RPECM and POS stimulate BMMSCs differentiation toward a functional RPE phenotype. Our results provide the foundation for a new route to RPE regenerative therapy involving BMMSCs. Future work isolating the active agent in RPECM and POS would be useful in therapies for RPE diseases or in developing appropriately pre-differentiated BMMSCs for tissue-engineered RPE reconstruction. Copyright © 2011 Wiley Periodicals, Inc.

SPECIFIC ROLE OF LYMPHATIC MARKER PODOPLANIN IN RETINAL PIGMENT EPITHELIAL CELLS

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Grimaldo, S.; Garcia, M.; Zhang, H.; Chen, L.

2015-01-01

Podoplanin is a small transmembrane glycoprotein widely known to be a marker for lymphatic endothelial cells. In this study, we identify a novel localization of podoplanin in the retinal pigment epithelium (RPE), a cellular monolayer critically involved in the visual process. Using a small interfering RNA (siRNA)-mediated gene silencing approach, we have also demonstrated, for the first time, that podoplanin depletion in human RPE cells leads to a marked reduction of cell aggregates and tight junctions. Additionally, the podoplanin-depleted cells also exhibit a significantly lower rate of proliferation. These data together indicate that podoplanin plays a crucial role in RPE cell functions. Further investigation on this factor may reveal novel mechanisms and therapeutic strategies for RPE-related eye diseases, such as proliferative retinopathy and age-related macular degeneration. PMID:21226415

Anti-apoptotic effects of Curcuma longa L. extract and its curcuminoids against blue light-induced cytotoxicity in A2E-laden human retinal pigment epithelial cells.

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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.

Isolation, Characterization, and Establishment of Spontaneously Immortalized Cell Line HRPE-2S With Stem Cell Properties.

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Shams Najafabadi, Hoda; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Ranaei Pirmardan, Ehsan; Masoumi, Maryam

2017-10-01

The retinal pigment epithelium is a monolayer of highly specialized pigmented cells located between the neural retina and the Bruch's membrane of the choroid. RPE cells play a crucial role in the maintenance and function of the underlying photoreceptors. This study introduces a spontaneously arising human retinal pigment epithelial cell line, HRPE-2S, which was isolated from primary RPE cell culture of 2 days old male donor. We characterized morphology and functional properties of the new cell line. The immortalized cell line was maintained in culture for more than 70 passages and 240 divisions. The average doubling time of the cells was approximately 22 h and got freezed at 26th passage. The cell line expressed RPE-specific markers RPE65 and cell junction protein ZO1 as an epithelial cell marker. It also expressed CHX10, PAX6, Nestin, SOX2 as stem and retinal progenitor cell markers. Ki67 as a marker of cell proliferation was expressed in all HRPE-2S cells. It represented typical epithelial cobblestone morphology and did not phenotypically change through several passages. Stem cell-like aggregations (neurospheres) were observed in SEM microscopy. The cells represented high mitotic index. They could be viable under hypoxic conditions and serum deprivation. According to functional studies, the cell line exhibited stem cell-like behaviors with particular emphasis on its self-renewal capacity. LDH isoenzymes expression pattern confirmed the same cellular source for both of the HRPE-2S cells and primary RPE cells. Characteristics of HRPE-2S cells promise it as an in vitro model for RPE stem cell-based researches. J. Cell. Physiol. 232: 2626-2640, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Session-RPE Method for Training Load Monitoring: Validity, Ecological Usefulness, and Influencing Factors

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Monoem Haddad

2017-11-01

Full Text Available Purpose: The aim of this review is to (1 retrieve all data validating the Session-rating of perceived exertion (RPE-method using various criteria, (2 highlight the rationale of this method and its ecological usefulness, and (3 describe factors that can alter RPE and users of this method should take into consideration.Method: Search engines such as SPORTDiscus, PubMed, and Google Scholar databases in the English language between 2001 and 2016 were consulted for the validity and usefulness of the session-RPE method. Studies were considered for further analysis when they used the session-RPE method proposed by Foster et al. in 2001. Participants were athletes of any gender, age, or level of competition. Studies using languages other than English were excluded in the analysis of the validity and reliability of the session-RPE method. Other studies were examined to explain the rationale of the session-RPE method and the origin of RPE.Results: A total of 950 studies cited the Foster et al. study that proposed the session RPE-method. 36 studies have examined the validity and reliability of this proposed method using the modified CR-10.Conclusion: These studies confirmed the validity and good reliability and internal consistency of session-RPE method in several sports and physical activities with men and women of different age categories (children, adolescents, and adults among various expertise levels. This method could be used as “standing alone” method for training load (TL monitoring purposes though some recommend to combine it with other physiological parameters as heart rate.

Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

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Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. (Univ. of Southern California, Los Angeles (United States)); Pandey, S. (Doheny Eye Inst., Los Angeles, CA (United States))

1991-05-01

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha} protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

Protective effect of bone morphogenetic protein 6 on RPE cells injury caused by H2O2

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Li Chen

2016-01-01

Full Text Available AIM:To investigate the effect of bone morphogenetic protein 6(BMP-6on cellular morphology, proliferation and apoptosis of retinal pigment epithelial cells(ARPE-19incubated in hydrogen peroxide(H2O2. METHODS:ARPE-19 cells were cultured conventionally and divided into four groups. One group was untreated as blank group, the other three groups were incubated in 75μm/L H2O2, 150ng/mLBMP-6 or75μm/L H2O2+150ng/mL BMP-6. All the groups were incubated for 3h, 6h, 9h and 12h. We tested the cell viabilitity by MTT. We used flow cytometry to test the cell cycle and cell apoptosis.RESULTS:H2O2 significantly decreased the cell activity in time-dependent manner. The activity of cells with BMP-6+H2O2 was higher H2O2 group, and the differences between the two groups at 3h and 6h were significant(P2O2, while the cells with BMP-6 were less cell detachment and apoptosis. CONCLUSION:BMP-6 has protective effects on RPE cells from oxidative stress in certain extent.

Controlled surface morphology and hydrophilicity of polycaprolactone toward human retinal pigment epithelium cells.

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Shahmoradi, Saleheh; Yazdian, Fatemeh; Tabandeh, Fatemeh; Soheili, Zahra-Soheila; Hatamian Zarami, Ashraf Sadat; Navaei-Nigjeh, Mona

2017-04-01

Applying scaffolds as a bed to enhance cell proliferation and even differentiation is one of the treatment of retina diseases such as age-related macular degeneration (AMD) which deteriorating photoreceptors and finally happening blindness. In this study, aligned polycaprolactone (PCL) nanofibers were electrospun and at different conditions and their characteristics were measured by scanning electron microscope (SEM) and contact angle. Response surface methodology (RSM) was used to optimize the diameter of fabricated nanofibers. Two factors as solution concentration and voltage value were considered as independent variables and their effects on nanofibers' diameters were evaluated by central composite design and the optimum conditions were obtained as 0.12g/mL and 20kV, respectively. In order to decrease the hydrophobicity of PCL, the surface of the fabricated scaffolds was modified by alkaline hydrolysis method. Contact time of the scaffolds and alkaline solution and concentration of alkaline solution were optimized using Box Behnken design and (120min and 5M were the optimal, respectively). Contact angle measurement showed the high hydrophilicity of treated scaffolds (with contact angle 7.48°). Plasma surface treatment was applied to compare the effect of using two kinds of surface modification methods simultaneously on hydrolyzed scaffolds. The RPE cells grown on scaffolds were examined by immunocytochemistry (ICC), MTT and continuous inspection of cellular morphology. Interestingly, Human RPE cells revealed their characteristic morphology on hydrolyzed scaffold well. As a result, we introduced a culture substrate with low diameter (185.8nm), high porosity (82%) and suitable hydrophilicity (with contact angle 7.48 degree) which can be promising for hRPE cell transplantation. Copyright © 2016. Published by Elsevier B.V.

Survival and Functionality of hESC-Derived Retinal Pigment Epithelium Cells Cultured as a Monolayer on Polymer Substrates Transplanted in RCS Rats.

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Thomas, Biju B; Zhu, Danhong; Zhang, Li; Thomas, Padmaja B; Hu, Yuntao; Nazari, Hossein; Stefanini, Francisco; Falabella, Paulo; Clegg, Dennis O; Hinton, David R; Humayun, Mark S

2016-05-01

To determine the safety, survival, and functionality of human embryonic stem cell-derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats. Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n = 46) received vitronectin-coated parylene membrane without cells (rMSPM+VN), group 2 (n = 59) received rCPCB-RPE1 implants, and group 3 (n = 13) served as the control group. Animals that are selected based on optical coherence tomography screening were subjected to visual function assays using optokinetic (OKN) testing and superior colliculus (SC) electrophysiology. At approximately 25 weeks of age (21 weeks after surgery), the eyes were examined histologically for cell survival, phagocytosis, and local toxicity. Eighty-seven percent of the rCPCB-RPE1-implanted animals showed hESC-RPE survivability. Significant numbers of outer nuclear layer cells were rescued in both group 1 (rMSPM+VN) and group 2 (rCPCB-RPE1) animals. A significantly higher ratio of rod photoreceptor cells to cone photoreceptor cells was found in the rCPCB-RPE1-implanted group. Animals with rCPCB-RPE1 implant showed hESC-RPE cells containing rhodopsin-positive particles in immunohistochemistry, suggesting phagocytic function. Superior colliculus mapping data demonstrated that a significantly higher number of SC sites responded to light stimulus at a lower luminance threshold level in the rCPCB-RPE1-implanted group. Optokinetic data suggested both implantation groups showed improved visual acuity. These results demonstrate the safety, survival, and functionality of the hESC-RPE monolayer transplantation in an RPE dysfunction rat model.

Collectin-11 Is an Important Modulator of Retinal Pigment Epithelial Cell Phagocytosis and Cytokine Production.

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Dong, Xia; Wu, Weiju; Ma, Liang; Liu, Chengfei; Bhuckory, Mohajeet B; Wang, Liping; Nandrot, Emeline F; Xu, Heping; Li, Ke; Liu, Yizhi; Zhou, Wuding

2017-01-01

In this paper, we report previously unknown roles for collectin-11 (CL-11, a soluble C-type lectin) in modulating the retinal pigment epithelial (RPE) cell functions of phagocytosis and cytokine production. We found that CL-11 and its carbohydrate ligand are expressed in both the murine and human neural retina; these resemble each other in terms of RPE and photoreceptor cells. Functional analysis of murine RPE cells showed that CL-11 facilitates the opsonophagocytosis of photoreceptor outer segments and apoptotic cells, and also upregulates IL-10 production. Mechanistic analysis revealed that calreticulin on the RPE cells is required for CL-11-mediated opsonophagocytosis whereas signal-regulatory protein α and mannosyl residues on the cells are involved in the CL-11-mediated upregulation of IL-10 production. This study is the first to demonstrate the role of CL-11 and the molecular mechanisms involved in modulating RPE cell phagocytosis and cytokine production. It provides a new insight into retinal health and disease and has implications for other phagocytic cells. © 2017 S. Karger AG, Basel.

Altered bioenergetics and enhanced resistance to oxidative stress in human retinal pigment epithelial cells from donors with age-related macular degeneration

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Deborah A. Ferrington

2017-10-01

Full Text Available Age-related macular degeneration (AMD is the leading cause of blindness among older adults. It has been suggested that mitochondrial defects in the retinal pigment epithelium (RPE underlies AMD pathology. To test this idea, we developed primary cultures of RPE to ask whether RPE from donors with AMD differ in their metabolic profile compared with healthy age-matched donors. Analysis of gene expression, protein content, and RPE function showed that these cultured cells replicated many of the cardinal features of RPE in vivo. Using the Seahorse Extracellular Flux Analyzer to measure bioenergetics, we observed RPE from donors with AMD exhibited reduced mitochondrial and glycolytic function compared with healthy donors. RPE from AMD donors were also more resistant to oxidative inactivation of these two energy-producing pathways and were less susceptible to oxidation-induced cell death compared with cells from healthy donors. Investigation of the potential mechanism responsible for differences in bioenergetics and resistance to oxidative stress showed RPE from AMD donors had increased PGC1α protein as well as differential expression of multiple genes in response to an oxidative challenge. Based on our data, we propose that cultured RPE from donors phenotyped for the presence or absence of AMD provides an excellent model system for studying “AMD in a dish”. Our results are consistent with the ideas that (i a bioenergetics crisis in the RPE contributes to AMD pathology, and (ii the diseased environment in vivo causes changes in the cellular profile that are retained in vitro.

Defining the Human Macula Transcriptome and Candidate Retinal Disease Genes UsingEyeSAGE

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Rickman, Catherine Bowes; Ebright, Jessica N.; Zavodni, Zachary J.; Yu, Ling; Wang, Tianyuan; Daiger, Stephen P.; Wistow, Graeme; Boon, Kathy; Hauser, Michael A.

2009-01-01

Purpose To develop large-scale, high-throughput annotation of the human macula transcriptome and to identify and prioritize candidate genes for inherited retinal dystrophies, based on ocular-expression profiles using serial analysis of gene expression (SAGE). Methods Two human retina and two retinal pigment epithelium (RPE)/choroid SAGE libraries made from matched macula or midperipheral retina and adjacent RPE/choroid of morphologically normal 28- to 66-year-old donors and a human central retina longSAGE library made from 41- to 66-year-old donors were generated. Their transcription profiles were entered into a relational database, EyeSAGE, including microarray expression profiles of retina and publicly available normal human tissue SAGE libraries. EyeSAGE was used to identify retina- and RPE-specific and -associated genes, and candidate genes for retina and RPE disease loci. Differential and/or cell-type specific expression was validated by quantitative and single-cell RT-PCR. Results Cone photoreceptor-associated gene expression was elevated in the macula transcription profiles. Analysis of the longSAGE retina tags enhanced tag-to-gene mapping and revealed alternatively spliced genes. Analysis of candidate gene expression tables for the identified Bardet-Biedl syndrome disease gene (BBS5) in the BBS5 disease region table yielded BBS5 as the top candidate. Compelling candidates for inherited retina diseases were identified. Conclusions The EyeSAGE database, combining three different gene-profiling platforms including the authors’ multidonor-derived retina/RPE SAGE libraries and existing single-donor retina/RPE libraries, is a powerful resource for definition of the retina and RPE transcriptomes. It can be used to identify retina-specific genes, including alternatively spliced transcripts and to prioritize candidate genes within mapped retinal disease regions. PMID:16723438

Grafting of ARPE-19 and Schwann cells to the subretinal space in RCS rats.

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Wang, Shaomei; Lu, Bin; Wood, Patrick; Lund, Raymond D

2005-07-01

To study the distribution of the human retinal pigment epithelium (hRPE) cell line ARPE-19 and human Schwann (hSC) cells grafted to the subretinal space of the Royal College of Surgeon (RCS) rat and the relation of graft cell distribution to photoreceptor rescue. Cell suspensions of both donor types were injected into the subretinal space of 3-week-old dystrophic RCS rats through a transscleral approach, human fibroblast and medium were used as control grafts. All animals were maintained on oral cyclosporine. At 1, 2, 4, 6, 15, 28, and 36 weeks after grafting, animals were killed. Human cell-specific markers were used to localize donor cells. Both donor cell types, as revealed by antibodies survived for a substantial time. Their distribution was very different: hRPE cells formed a large clump early on and, with time, spread along the host RPE in a layer one to two cells deep, whereas hSCs formed many smaller clumps, mainly in the subretinal space. Both cells rescued photoreceptors beyond the area of donor cell distribution. The number of surviving cells declined with time. Both hRPE and hSC grafts can survive and rescue photoreceptors for a substantial time after grafting. The number of both donor cell types declined with time, which could be an immune-related problem and/or due to other factors intrinsic to the host RCS retina. The fact that rescue occurred beyond the area of donor cell distribution suggests that diffusible factors are involved, raising the possibility that the two cell types function in a similar manner to rescue photoreceptors.

Human Umbilical Cord Mesenchymal Stem Cells: Subpopulations and Their Difference in Cell Biology and Effects on Retinal Degeneration in RCS Rats.

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Wang, L; Li, P; Tian, Y; Li, Z; Lian, C; Ou, Q; Jin, C; Gao, F; Xu, J-Y; Wang, J; Wang, F; Zhang, J; Zhang, J; Li, W; Tian, H; Lu, L; Xu, G-T

2017-01-01

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are potential candidates for treating retinal degeneration (RD). To further study the biology and therapeutic effects of the hUC-MSCs on retinal degeneration. Two hUC-MSC subpopulations, termed hUC-MSC1 and hUC-MSC2, were isolated by single-cell cloning method and their therapeutic functions were compared in RCS rat, a RD model. Although both subsets satisfied the basic requirements for hUC-MSCs, they were significantly different in morphology, proliferation rate, differentiation capacity, phenotype and gene expression. Furthermore, only the smaller, fibroblast-like, faster growing subset hUC-MSC1 displayed stronger colony forming potential as well as adipogenic and osteogenic differentiation capacities. When the two subsets were respectively transplanted into the subretinal spaces of RCS rats, both subsets survived, but only hUC-MSC1 expressed RPE cell markers Bestrophin and RPE65. More importantly, hUC-MSC1 showed stronger rescue effect on the retinal function as indicated by the higher b-wave amplitude on ERG examination, thicker retinal nuclear layer, and decreased apoptotic photoreceptors. When both subsets were treated with interleukin-6, mimicking the inflammatory environment when the cells were transplanted into the eyes with degenerated retina, hUC-MSC1 expressed much higher levels of trophic factors in comparison with hUC-MSC2. The data here, in addition to prove the heterogeneity of hUC-MSCs, confirmed that the stronger therapeutic effects of hUC-MSC1 were attributed to its stronger anti-apoptotic effect, paracrine of trophic factors and potential RPE cell differentiation capacity. Thus, the subset hUC-MSC1, not the other subset or the ungrouped hUC-MSCs should be used for effective treatment of RD. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

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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

RPE and RPO in the revised Swiss radiation protection legislation; RPE und RPO in der revidierten schweizerischen Strahlenschutzgesetzgebung

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Jahn, S.G. [Eidgenoessisches Nuklearsicherheitsinspektorat (ENSI), Brugg (Switzerland); Stritt, N.; Bogni, S. [Bundesamt fuer Gesundheit (BAG), Bern (Switzerland); Hammans, M. [Schweizerische Unfallversicherungsanstalt (SUVA), Luzern (Switzerland); Duerst, B. [Departement fuer Verteidigung, Bevoelkerungsschutz und Sport (VBS), Bern (Switzerland); Kuehne, G. [Paul-Scherrer-Institut (PSI), Wuerenlingen (Switzerland); Besancon, A. [Institut de Radiophysique (IRA), Lausanne (Switzerland)

2013-07-01

Currently the Swiss 'Ordinance on Radiation Protection' and the 'Ordinance about Education and Training in RP' are undergoing a revision process. Concerning responsibilities and obligations as well as the capabilities and skills in RP the new ordinances are supposed to take account on the new EU Basic Safety Standard on the one hand and on the other hand to resume the approved approaches on RP in Switzerland. An overview of the planned content of the revised ordinances will be given in this report. In the Swiss system the education, training and retraining of RPO has a higher priority. Particularly it will be described which groups of persons assume the roles of RPE and therefore the definition of RPE in the new Swiss legislation is needless. (orig.)

Novel Epigenetic Controlling of Hypoxia Pathway Related to Overexpression and Promoter Hypomethylation of TET1 and TET2 in RPE Cells.

Science.gov (United States)

Alivand, Mohammad Reza; Soheili, Zahra-Soheila; Pornour, Majid; Solali, Saeed; Sabouni, Farzaneh

2017-10-01

CpG methylation of DNA takes part in a specific epigenetic memory that plays crucial roles in the differentiation and abnormality of the cells. The methylation pattern aberration of genomes is affected in three ways, namely DNA methyltransferase (DNMT), ten-eleven translocation (TET), and methyl-binding domain (MBD) proteins. Of these, TET enzymes have recently been demonstrated to be master modifier enzymes in the DNA methylation process. Additionally, recent studies emphasize that not only epigenetic phenomena play a role in controlling hypoxia pathway, but the hypoxia condition also triggers hypomethylation of genomes that may help with the expression of hypoxia pathway genes. In this study, we suggested that TET1 and TET2 could play a role in the demethylation of genomes under chemical hypoxia conditions. Herein, the evaluating methylation status and mRNA expression of mentioned genes were utilized through real-time PCR and methylation-specific PCR (MSP), respectively. Our results showed that TET1 and TET2 genes were overexpressed (P < 0.05) under chemical hypoxia conditions in Retinal Pigment Epithelial (RPE) cells, whereas the promoter methylation status of them were hypomethylated in the same condition. Therefore, chemical hypoxia not only causes overexpression of TET1 and TET2 but also could gradually do promoter demethylation of same genes. This is the first study to show the relationship between epigenetics and the expression of mentioned genes related to hypoxia pathways. Furthermore, it seems that these associations in RPE cells are subjected to chemical hypoxia as a mechanism that could play a crucial role in methylation pattern changes of hypoxia-related diseases such as cancer and ischemia. J. Cell. Biochem. 118: 3193-3204, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

International Nuclear Information System (INIS)

Li, Chao-Peng; Yao, Jin; Tao, Zhi-Fu; Li, Xiu-Miao; Jiang, Qin; Yan, Biao

2013-01-01

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

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.

Microphthalmia-associated transcription factor as the molecular target of cadmium toxicity in human melanocytes

Energy Technology Data Exchange (ETDEWEB)

Chantarawong, Wipa [Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai (Japan); Inter Departmental Multidisciplinary Graduate Program in Bioscience, Faculty of Science, Kasetsart University, Bangkok (Thailand); Takeda, Kazuhisa; Sangartit, Weerapon; Yoshizawa, Miki [Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai (Japan); Pradermwong, Kantimanee [Department of Zoology, Faculty of Science, Kasetsart University, Bangkok (Thailand); Shibahara, Shigeki, E-mail: shibahar@med.tohoku.ac.jp [Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai (Japan)

2014-11-28

Highlights: • In human melanocytes, cadmium decreases the expression of MITF-M and tyrosinase and their mRNAs. • In human melanoma cells, cadmium decreases the expression of MITF-M protein and tyrosinase mRNA. • Expression of MITF-H is less sensitive to cadmium toxicity in melanocyte-linage cells. • Cadmium does not decrease the expression of MITF-H in retinal pigment epithelial cells. • MITF-M is the molecular target of cadmium toxicity in melanocytes. - Abstract: Dietary intake of cadmium is inevitable, causing age-related increase in cadmium accumulation in many organs, including hair, choroid and retinal pigment epithelium (RPE). Cadmium has been implicated in the pathogenesis of hearing loss and macular degeneration. The functions of cochlea and retina are maintained by melanocytes and RPE, respectively, and the differentiation of these pigment cells is regulated by microphthalmia-associated transcription factor (MITF). In the present study, we explored the potential toxicity of cadmium in the cochlea and retina by using cultured human melanocytes and human RPE cell lines. MITF consists of multiple isoforms, including melanocyte-specific MITF-M and widely expressed MITF-H. Levels of MITF-M protein and its mRNA in human epidermal melanocytes and HMV-II melanoma cells were decreased significantly by cadmium. In parallel with the MITF reduction, mRNA levels of tyrosinase, the key enzyme of melanin biosynthesis that is regulated by MITF-M, were also decreased. In RPE cells, however, the levels of total MITF protein, constituting mainly MITF-H, were not decreased by cadmium. We thus identify MITF-M as the molecular target of cadmium toxicity in melanocytes, thereby accounting for the increased risk of disability from melanocyte malfunction, such as hearing and vision loss among people with elevated cadmium exposure.

Microphthalmia-associated transcription factor as the molecular target of cadmium toxicity in human melanocytes

International Nuclear Information System (INIS)

Chantarawong, Wipa; Takeda, Kazuhisa; Sangartit, Weerapon; Yoshizawa, Miki; Pradermwong, Kantimanee; Shibahara, Shigeki

2014-01-01

Highlights: • In human melanocytes, cadmium decreases the expression of MITF-M and tyrosinase and their mRNAs. • In human melanoma cells, cadmium decreases the expression of MITF-M protein and tyrosinase mRNA. • Expression of MITF-H is less sensitive to cadmium toxicity in melanocyte-linage cells. • Cadmium does not decrease the expression of MITF-H in retinal pigment epithelial cells. • MITF-M is the molecular target of cadmium toxicity in melanocytes. - Abstract: Dietary intake of cadmium is inevitable, causing age-related increase in cadmium accumulation in many organs, including hair, choroid and retinal pigment epithelium (RPE). Cadmium has been implicated in the pathogenesis of hearing loss and macular degeneration. The functions of cochlea and retina are maintained by melanocytes and RPE, respectively, and the differentiation of these pigment cells is regulated by microphthalmia-associated transcription factor (MITF). In the present study, we explored the potential toxicity of cadmium in the cochlea and retina by using cultured human melanocytes and human RPE cell lines. MITF consists of multiple isoforms, including melanocyte-specific MITF-M and widely expressed MITF-H. Levels of MITF-M protein and its mRNA in human epidermal melanocytes and HMV-II melanoma cells were decreased significantly by cadmium. In parallel with the MITF reduction, mRNA levels of tyrosinase, the key enzyme of melanin biosynthesis that is regulated by MITF-M, were also decreased. In RPE cells, however, the levels of total MITF protein, constituting mainly MITF-H, were not decreased by cadmium. We thus identify MITF-M as the molecular target of cadmium toxicity in melanocytes, thereby accounting for the increased risk of disability from melanocyte malfunction, such as hearing and vision loss among people with elevated cadmium exposure

Multi-level communication of human retinal pigment epithelial cells via tunneling nanotubes.

Directory of Open Access Journals (Sweden)

Dierk Wittig

Full Text Available BACKGROUND: Tunneling nanotubes (TNTs may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis. METHODOLOGY AND PRINCIPAL FINDINGS: Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca²⁺ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells. CONCLUSIONS AND SIGNIFICANCE: Our observations indicate that

Induction of oxidative and nitrosative stresses in human retinal pigment epithelial cells by all-trans-retinal

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Zhu, Xue [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, Jiangsu Province (China); Wang, Ke, E-mail: wangke@jsinm.org [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, Jiangsu Province (China); Zhang, Kai [Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi 214063, Jiangsu Province (China); Zhou, Fanfan [Faculty of Pharmacy, University of Sydney, New South Wales 2006 (Australia); Zhu, Ling [Save Sight Institute, University of Sydney, New South Wales 2000 (Australia)

2016-10-15

Delayed clearance of free form all-trans-retinal (atRAL) is estimated be the key cause of retinal pigment epithelium (RPE) cells injury during the pathogenesis of retinopathies such as age-related macular degeneration (AMD), however, the underlying molecular mechanisms are far from clear. In this study, we investigated the cytotoxicity effect and underlying molecular mechanism of atRAL on human retinal pigment epithelium ARPE-19 cells. The results indicated that atRAL could cause cell dysfunction by inducing oxidative and nitrosative stresses in ARPE-19 cells. The oxidative stress induced by atRAL was mediated through up-regulation of reactive oxygen species (ROS) generation, activating mitochondrial-dependent and MAPKs signaling pathways, and finally resulting in apoptosis of ARPE-19 cells. The NADPH oxidase inhibitor apocynin could partly attenuated ROS generation, indicating that NADPH oxidase activity was involved in atRAL-induced oxidative stress in ARPE-19 cells. The nitrosative stress induced by atRAL was mainly reflected in increasing nitric oxide (NO) production, enhancing iNOS, ICAM-1 and VCAM-1 expressions, and promoting monocyte adhesion. Furthermore, above effects could be dramatically blocked by using a nuclear factor kappa B (NF-κB) inhibitor SN50, indicated that atRAL-induced oxidative and nitrosative stresses were mediated by NF-κB. The results provide better understanding of atRAL-induced toxicity in human RPE cells. - Highlights: • atRAL induces oxidative stress-mediated apoptosis in ARPE-19 cells. • atRAL induces oxidative stress-mediated inflammation in ARPE-19 cells. • NF-κB is involved in atRAL-induced oxidative and nitrosative stresses.

Induction of oxidative and nitrosative stresses in human retinal pigment epithelial cells by all-trans-retinal

International Nuclear Information System (INIS)

Zhu, Xue; Wang, Ke; Zhang, Kai; Zhou, Fanfan; Zhu, Ling

2016-01-01

Delayed clearance of free form all-trans-retinal (atRAL) is estimated be the key cause of retinal pigment epithelium (RPE) cells injury during the pathogenesis of retinopathies such as age-related macular degeneration (AMD), however, the underlying molecular mechanisms are far from clear. In this study, we investigated the cytotoxicity effect and underlying molecular mechanism of atRAL on human retinal pigment epithelium ARPE-19 cells. The results indicated that atRAL could cause cell dysfunction by inducing oxidative and nitrosative stresses in ARPE-19 cells. The oxidative stress induced by atRAL was mediated through up-regulation of reactive oxygen species (ROS) generation, activating mitochondrial-dependent and MAPKs signaling pathways, and finally resulting in apoptosis of ARPE-19 cells. The NADPH oxidase inhibitor apocynin could partly attenuated ROS generation, indicating that NADPH oxidase activity was involved in atRAL-induced oxidative stress in ARPE-19 cells. The nitrosative stress induced by atRAL was mainly reflected in increasing nitric oxide (NO) production, enhancing iNOS, ICAM-1 and VCAM-1 expressions, and promoting monocyte adhesion. Furthermore, above effects could be dramatically blocked by using a nuclear factor kappa B (NF-κB) inhibitor SN50, indicated that atRAL-induced oxidative and nitrosative stresses were mediated by NF-κB. The results provide better understanding of atRAL-induced toxicity in human RPE cells. - Highlights: • atRAL induces oxidative stress-mediated apoptosis in ARPE-19 cells. • atRAL induces oxidative stress-mediated inflammation in ARPE-19 cells. • NF-κB is involved in atRAL-induced oxidative and nitrosative stresses.

Session-RPE for quantifying load of different youth taekwondo training sessions.

Science.gov (United States)

Lupo, Corrado; Capranica, Laura; Cortis, Cristina; Guidotti, Flavia; Bianco, Antonino; Tessitore, Antonio

2017-03-01

The session rating of perceived exertion (session-RPE) proved to be a valuable method to quantify the internal training load (ITL) in taekwondo. However, no study validated this method in youth taekwondo athletes performing different training sessions. Thus this study aimed at evaluating the reliability of the session-RPE to monitor the ITL of prepubescent taekwondo athletes during pre-competitive (PC) and competitive (C) training sessions. Five female (age: 12.0±0.7 y; height: 1.54±0.08 m; body mass: 48.8±7.3 kg) and four male (age: 12.0±0.8 yrs; height: 1.55±0.07 m; body mass: 47.3±5.3 kg) taekwondo athletes were monitored during 100 individual sessions (PC: N.=33; C: N.=67). The Edwards' HR method was used as reference measure of ITL; the CR-10 RPE scale was administered at 1- and 30-minutes from the end of each session. No difference for gender emerged. The ITLs of C (Edwards: 228±40 arbitrary units, AU) resulted higher than that of PC (192±26 AU; P=0.04). Although all training typologies and data collections achieved significant correlations between Edwards' and session-RPE methods, a large relationship (r =0.71, Psessions evaluated at 30 minutes of the recovery phases. Findings support coaches of prepubescent taekwondo athletes to successfully use session-RPE to monitor the ITL of different training typologies. However, PC training evaluated at 30 minutes of the recovery phase represents the best condition for a highly reliable ITL perception.

Loss of Melanin by Eye Retinal Pigment Epithelium Cells Is Associated with Its Oxidative Destruction in Melanolipofuscin Granules.

Science.gov (United States)

Dontsov, A E; Sakina, N L; Ostrovsky, M A

2017-08-01

The effect of superoxide radicals on melanin destruction and degradation of melanosomes isolated from cells of retinal pigment epithelium (RPE) of the human eye was studied. We found that potassium superoxide causes destruction of melanin in melanosomes of human and bovine RPE, as well as destruction of melanin from the ink bag of squid, with the formation of fluorescent decay products having an emission maximum at 520-525 nm. The initial kinetics of the accumulation of the fluorescent decay products is linear. Superoxide radicals lead simultaneously to a decrease in the number of melanosomes and to a decrease in concentration of paramagnetic centers in them. Complete degradation of melanosomes leads to the formation of a transparent solution containing dissolved proteins and melanin degradation products that do not exhibit paramagnetic properties. To completely degrade one melanosome of human RPE, 650 ± 100 fmol of superoxide are sufficient. The concentration of paramagnetic centers in a melanolipofuscin granule of human RPE is on average 32.5 ± 10.4% (p melanin undergoing a destruction process in these granules. RPE cells also contain intermediate granules that have an EPR signal with a lower intensity than that of melanolipofuscin granules, but higher than that of lipofuscin granules. This signal is due to the presence of residual melanin in these granules. Irradiation of a mixture of melanosomes with lipofuscin granules with blue light (450 nm), in contrast to irradiation of only melanosomes, results in the appearance of fluorescent melanin degradation products. We suggest that one of the main mechanisms of age-related decrease in melanin concentration in human RPE cells is its destruction in melanolipofuscin granules under the action of superoxide radicals formed during photoinduced oxygen reduction by lipofuscin fluorophores.

Oxidative Stress Induces Senescence in Cultured RPE Cells.

Science.gov (United States)

Aryan, Nona; Betts-Obregon, Brandi S; Perry, George; Tsin, Andrew T

2016-01-01

The aim of this research is to determine whether oxidative stress induces cellular senescence in human retinal pigment epithelial cells. Cultured ARPE19 cells were subjected to different concentrations of hydrogen peroxide to induce oxidative stress. Cells were seeded into 24-well plates with hydrogen peroxide added to cell medium and incubated at 37°C + 5% CO2 for a 90-minute period [at 0, 300, 400 and 800 micromolar (MCM) hydrogen peroxide]. The number of viable ARPE19 cells were recorded using the Trypan Blue Dye Exclusion Method and cell senescence was measured by positive staining for senescence-associated beta-galactosidase (SA-beta-Gal) protein. Without hydrogen peroxide treatment, the number of viable ARPE19 cells increased significantly from 50,000 cells/well to 197,000 within 72 hours. Treatment with hydrogen peroxide reduced this level of cell proliferation significantly (to 52,167 cells at 400 MCM; to 49,263 cells at 800 MCM). Meanwhile, cells with a high level of positive senescence-indicator SA-Beta-Gal-positive staining was induced by hydrogen peroxide treatment (from a baseline level of 12% to 80% at 400 MCM and at 800 MCM). Our data suggests that oxidative stress from hydrogen peroxide treatment inhibited ARPE19 cell proliferation and induced cellular senescence.

Session-RPE for quantifying the load of different youth basketball training sessions.

Science.gov (United States)

Lupo, C; Tessitore, A; Gasperi, L; Gomez, Mar

2017-03-01

The aim of the study was to evaluate youth basketball training, verifying the reliability of the session-RPE method in relation to session duration (session) categories. Six male youth basketball players (age, 16.5±0.5 years; height, 195.5±6.75 cm; body mass, 93.9±10.9 kg; and body mass index, 23.6±2.8 kg.m -2 ) were monitored (HR, type and duration of workouts) during 15 (66 individual) training sessions (80±26 minutes). Edwards' HR method was used as a reference measure of internal training load (ITL); the CR-10 RPE scale was administered 30 minutes after the end of each session. The results obtained showed that all comparisons between different session durations and workout portions revealed effects in term of Edwards' ITLs except for warm-up portions. Moderate to strong relationships between Edwards' and session- RPE methods emerged for all sessions (r = .85, P sessions (r range = .79 - .95, P session durations (session-RPE to monitor the ITL, regardless of session durations and workout portions.

Performance measurement of a new concept reciprocating piston expander (RPE using a newly developed small-scale dynamometer unit

Directory of Open Access Journals (Sweden)

Mohamad MNA

2017-01-01

Full Text Available This paper presents the progress of a small-scale dynamometer prototype development for performance measurement of a reciprocating piston expander (RPE. Since the available dynamometer systems in the market are limited to specific applications that require for the customization, their price normally very expensive. Since the current study on the RPE required a dynamometer unit, therefore, a new and cheaper dynamometer prototype that was suitable for RPE application has been developed. Using air as RPE working fluid, a case study has been carried out to measure its performance at different inlet fluid conditions, i.e., within 20°C–140°C and 3–5 bars. The results observed that the performance of RPE was proportionally increased to the increased of inlet fluid pressure and temperature. The maximum brake power produced was 27 Watt when the RPE operated at 140°C, 5 bars and the speed of 820 rpm. It also revealed that the changes in the pressure of inlet fluid can give significant change on the performance of the RPE due to its direct relation to the RPE actual rotating force. Although the RPE and dynamometer seem good being adapted to each other, both of them require some improvements to ensure both systems well operated and reliable.

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

Directory of Open Access Journals (Sweden)

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.

TGF-β-stimulated aberrant expression of class III β-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

International Nuclear Information System (INIS)

Chung, Eun Jee; Chun, Ji Na; Jung, Sun-Ah; Cho, Jin Won; Lee, Joon H.

2011-01-01

Highlights: ► TGF-β induces aberrant expression of βIII in RPE cells via the ERK pathway. ► TGF-β increases O-GlcNAc modification of βIII in RPE cells. ► Mature RPE cells have the capacity to express a neuron-associated gene by TGF-β. -- Abstract: The class III β-tubulin isotype (β 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 β-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-β (TGF-β) 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-β on the aberrant expression of class III β-tubulin and the intracellular signaling pathway mediating these changes. TGF-β-induced aberrant expression and O-linked-β-N-acetylglucosamine (O-GlcNac) modification of class III β-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-β also stimulated phosphorylation of ERK. TGF-β-induced aberrant expression of class III β-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-β stimulated aberrant expression of class III β-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-β stimulation and provide useful information towards understanding the pathogenesis of proliferative vitreoretinal diseases.

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

Energy Technology Data Exchange (ETDEWEB)

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

Fisetin induces apoptosis through mitochondrial apoptosis pathway in human uveal melanoma cells.

Science.gov (United States)

Wang, Kai; Hu, Dan-Ning; Lin, Hui-Wen; Yang, Wei-En; Hsieh, Yi-Hsien; Chien, Hsiang-Wen; Yang, Shun-Fa

2018-05-01

Fisetin, a diatery flavonoid, been reported that possess anticancer effects in various cancers. The purpose of the study was to investigate the antitumor effects of fisetin in cultured uveal melanoma cell lines and compared with normal retinal pigment epithelial (RPE) cells. MTT assay was used for evaluating cytotoxic effects of fisetin. Flow cytometry study was used for the determination of apoptosis. JC-1 fluorescent reader was used to determine mitochondrial transmembrane potential changes. The results shown that fisetin dose-dependently decreased the cell viability of uveal melanoma cells but not influenced the cell viability of RPE cells. Apoptosis of uveal melanoma cells was induced by fisetin efficiently. Fisetin inhibited antiapoptotic Bcl-2 family proteins and damaged the mitochondrial transmembrane potential. The levels of proapoptotic Bcl-2 proteins, cytochrome c, and various caspase activities were increased by fisetin. In conclusion, fisetin induces apoptosis of uveal melanoma cells selectively and may be a promising agent to be explored for the treatment of uveal melanoma. © 2018 Wiley Periodicals, Inc.

Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells.

OpenAIRE

Jiang, M; Pandey, S; Tran, V T; Fong, H K

1991-01-01

The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein alpha subunits (G alpha) including Gs alpha, Gi-1 alpha, Gi-2 alpha, Gi-3 alpha, and Gz alpha (or Gx alpha), where Gs and Gi are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensi...

Lycopene inhibits ICAM-1 expression and NF-κB activation by Nrf2-regulated cell redox state in human retinal pigment epithelial cells.

Science.gov (United States)

Yang, Po-Min; Wu, Zhi-Zhen; Zhang, Yu-Qi; Wung, Being-Sun

2016-06-15

Age-related macular degeneration (AMD) is one of the most common diseases leading to blindness in elderly people. The progression of AMD may be prevented through anti-inflammation and antioxidation in retinal pigment epithelium (RPE) cells. Lycopene, a carotenoid, has been shown to possess both antioxidative and anti-inflammatory properties. This research was conducted to detail the mechanisms of these effects of lycopene-treated RPE cells. We exposed ARPE-19 cells to TNFα after pretreatment with lycopene, and measured monocyte adhesion, ICAM-1 expression, NF-κB nuclear translocation, and transcriptional activity. Cell viability was assayed with Alamar Blue. The cell redox state was tested by glutathione (GSH) and reactive oxygen species (ROS) levels. The importance of the Nrf2 pathway was tested in nuclear translocation, promoter reporter assay, and siRNA. Lycopene could reduce TNF-α-induced monocyte adhesion and H2O2- induced cell damage in RPE cells. Furthermore, lycopene inhibits ICAM-1 expression and abolishes NF-κB activation for up to 12h in TNFα-treated RPE cells. Lycopene upregulates Nrf2 levels in nuclear extracts and increases the transactivity of antioxidant response elements. The use of Nrf2 siRNA blocks the inhibitory effect of lycopene in TNF-α-induced ICAM-1 expression and NF-κB activation. Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the de novo synthesis of GSH. We found that lycopene increases intracellular GSH levels and GCL expression. Following lycopene treatment, TNF-α-induced ROS production was abolished. The Nrf2-regulated antioxidant property plays a pivotal role in the anti-inflammatory mechanism underlying the inhibition of NF-κB activation in lycopene-treated ARPE-19 cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Isolation and characterization of a spontaneously immortalized bovine retinal pigmented epithelial cell line

Directory of Open Access Journals (Sweden)

Griffiths T Daniel

2009-05-01

Full Text Available Abstract Background The Retinal Pigmented Epithelium (RPE is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD, Retinitis Pigmentosa (RP, Fundus Flavimaculatus (FFM, Best's disease and Age-related Macular Degeneration (AMD. We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases. Results The tRPE cell line has been passaged up to 150 population doublings and was shown to be morphologically similar to primary cells. They have been characterized to be of RPE origin by reverse transcriptase PCR and immunocytochemistry using the RPE-specific genes RPE65 and CRALBP and RPE-specific proteins RPE65 and Bestrophin. The tRPE cells are also immunoreactive to vimentin, cytokeratin and zonula occludens-1 antibodies. Chromosome analysis indicates a normal diploid number. The tRPE cells do not grow in suspension or in soft agar. After 3H thymidine incorporation, the cells do not appear to divide appreciably after confluency. Conclusion The tRPE cells are immortal, but still exhibit contact inhibition, serum dependence, monolayer growth and secrete an extra-cellular matrix. They retain the in-vivo morphology, gene expression and cell polarity. Additionally, the cells endocytose exogenous melanin, A2E and purified lipofuscin granules. This cell line may be a useful in-vitro research model for retinal

Identification of intracellular phospholipases A2 in the human eye: involvement in phagocytosis of photoreceptor outer segments

DEFF Research Database (Denmark)

Kolko, Miriam; Wang, Jinmei; Zhan, Chen

2007-01-01

PURPOSE: To identify intracellular phospholipases A(2) (PLA(2)) in the human retina and to explore the role of these enzymes in human retinal pigment epithelium (RPE) phagocytosis of photoreceptor outer segments (POS). METHODS: PCR amplification and Western blot analysis were used to identify m......)-VIA activity was found to be specifically increased 12 hours after ARPE-19 cells were fed with POS. Finally, RPE phagocytosis was inhibited by the iPLA(2)-VIA inhibitor bromoenol lactone. CONCLUSIONS: Various intracellular PLA(2) subtypes are present in the human retina. iPLA(2)-VIA may play...

Validity and reliability of the session-RPE method for quantifying training load in karate athletes.

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Tabben, M; Tourny, C; Haddad, M; Chaabane, H; Chamari, K; Coquart, J B

2015-04-24

To test the construct validity and reliability of the session rating of perceived exertion (sRPE) method by examining the relationship between RPE and physiological parameters (heart rate: HR and blood lactate concentration: [La --] ) and the correlations between sRPE and two HR--based methods for quantifying internal training load (Banister's method and Edwards's method) during karate training camp. Eighteen elite karate athletes: ten men (age: 24.2 ± 2.3 y, body mass: 71.2 ± 9.0 kg, body fat: 8.2 ± 1.3% and height: 178 ± 7 cm) and eight women (age: 22.6 ± 1.2 y, body mass: 59.8 ± 8.4 kg, body fat: 20.2 ± 4.4%, height: 169 ± 4 cm) were included in the study. During training camp, subjects participated in eight karate--training sessions including three training modes (4 tactical--technical, 2 technical--development, and 2 randori training), during which RPE, HR, and [La -- ] were recorded. Significant correlations were found between RPE and physiological parameters (percentage of maximal HR: r = 0.75, 95% CI = 0.64--0.86; [La --] : r = 0.62, 95% CI = 0.49--0.75; P training load ( r = 0.65--0.95; P reliability of the same intensity across training sessions (Cronbach's α = 0.81, 95% CI = 0.61--0.92). This study demonstrates that the sRPE method is valid for quantifying internal training load and intensity in karate.

Retinal pigment epithelial cell multinucleation in the aging eye - a mechanism to repair damage and maintain homoeostasis.

Science.gov (United States)

Chen, Mei; Rajapakse, Dinusha; Fraczek, Monika; Luo, Chang; Forrester, John V; Xu, Heping

2016-06-01

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age-related retinal degenerative disorders particularly age-related macular degeneration. During aging RPE cells decline in number, suggesting an age-dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose-dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted 'postmitotic' status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long-standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age-related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments

International Nuclear Information System (INIS)

Ranaei Pirmardan, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Mowla, Seyed Javad; Ezzati, Razie; Naseri, Marzieh

2016-01-01

Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells’ functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells’ (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. - Highlights: • Isolation of a spontaneously generated retinal pigmented epithelium cell line is reported. • The cells express some of the retinal progenitor cell markers in addition to the RPE markers. • The aforesaid cell line is highly transfecable and considerably infectable by AAV2. • These results confirm the great RPE plasticity and its invaluable potential in research studies.

Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments

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Ranaei Pirmardan, Ehsan [Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Soheili, Zahra-Soheila [Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran (Iran, Islamic Republic of); Samiei, Shahram [Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran (Iran, Islamic Republic of); Ahmadieh, Hamid [Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran (Iran, Islamic Republic of); Mowla, Seyed Javad [Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ezzati, Razie [Department of Molecular Medicine, National Institute of Genetic Engineering and Biotechnology, Tehran (Iran, Islamic Republic of); Naseri, Marzieh [Department of Molecular Medicine, Faculty of Advanced Technology, Iran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

2016-10-01

Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells’ functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells’ (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. - Highlights: • Isolation of a spontaneously generated retinal pigmented epithelium cell line is reported. • The cells express some of the retinal progenitor cell markers in addition to the RPE markers. • The aforesaid cell line is highly transfecable and considerably infectable by AAV2. • These results confirm the great RPE plasticity and its invaluable potential in research studies.

Suppression of topoisomerase IIα expression and function in human cells decreases chromosomal radiosensitivity

International Nuclear Information System (INIS)

Terry, Samantha Y.A.; Riches, Andrew C.; Bryant, Peter E.

2009-01-01

The mechanism behind chromatid break formation is as yet unclear, although it is known that DNA double-strand breaks (DSBs) are the initiating lesions. Chromatid breaks formed in cells in the G2-phase of the cell-cycle disappear ('rejoin') as a function of time between radiation exposure and cell fixation. However, the kinetics of disappearance of chromatid breaks does not correspond to those of DSB rejoining, leading us to seek alternative models. We have proposed that chromatid breaks could be formed indirectly from DSB and that the mechanism involves topoisomerase IIα. In support of this hypothesis we have recently shown that frequencies of radiation-induced chromatid breaks are lower in two variant human promyelocytic leukaemic cell lines with reduced topoisomerase IIα expression. Here we report that suppression of topoisomerase IIα in human hTERT-RPE1 cells, either by its abrogation using specific siRNA or by inhibition of its catalytic activity with the inhibitor ICRF-193, causes a reduction in frequency of chromatid breaks in radiation-exposed cells. The findings support our hypothesis for the involvement of topoisomerase IIα in the formation of radiation-induced chromatid breaks, and could help explain inter-individual variation in human chromosomal radiosensitivity; elevation of which has been linked with cancer susceptibility.

Shape Analysis of the Peripapillary RPE Layer in Papilledema and Ischemic Optic Neuropathy

Science.gov (United States)

Kupersmith, Mark J.; Rohlf, F. James

2011-01-01

Purpose. Geometric morphometrics (GM) was used to analyze the shape of the peripapillary retinal pigment epithelium–Bruch's membrane (RPE/BM) layer imaged on the SD-OCT 5-line raster in normal subjects and in patients with papilledema and ischemic optic neuropathy. Methods. Three groups of subjects were compared: 30 normals, 20 with anterior ischemic optic neuropathy (AION), and 25 with papilledema and intracranial hypertension. Twenty equidistant semilandmarks were digitized on OCT images of the RPE/BM layer spanning 2500 μm on each side of the neural canal opening (NCO). The data were analyzed using standard GM techniques, including a generalized least-squares Procrustes superimposition, principal component analysis, thin-plate spline (to visualize deformations), and permutation statistical analysis to evaluate differences in shape variables. Results. The RPE/BM layer in normals and AION have a characteristic V shape pointing away from the vitreous; the RPE/BM layer in papilledema has an inverted U shape, skewed nasally inward toward the vitreous. The differences were statistically significant. There was no significant difference in shapes between normals and AION. Pre- and posttreatment OCTs, in select cases of papilledema, showed that the inverted U-shaped RPE/BM moved posteriorly into a normal V shape as the papilledema resolved with weight loss or shunting. Conclusions. The shape difference in papilledema, absent in AION, cannot be explained by disc edema alone. The difference is a consequence of both the translaminar pressure gradient and the material properties of the peripapillary sclera. GM offers a novel way of statistically assessing shape differences of the peripapillary optic nerve head. PMID:21896851

Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments.

Science.gov (United States)

Ranaei Pirmardan, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Mowla, Seyed Javad; Ezzati, Razie; Naseri, Marzieh

2016-10-01

Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells' functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells' (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. Copyright © 2016 Elsevier Inc. All rights reserved.

ROCK-1 mediates diabetes-induced retinal pigment epithelial and endothelial cell blebbing: Contribution to diabetic retinopathy.

Science.gov (United States)

Rothschild, Pierre-Raphaël; Salah, Sawsen; Berdugo, Marianne; Gélizé, Emmanuelle; Delaunay, Kimberley; Naud, Marie-Christine; Klein, Christophe; Moulin, Alexandre; Savoldelli, Michèle; Bergin, Ciara; Jeanny, Jean-Claude; Jonet, Laurent; Arsenijevic, Yvan; Behar-Cohen, Francine; Crisanti, Patricia

2017-08-18

In diabetic retinopathy, the exact mechanisms leading to retinal capillary closure and to retinal barriers breakdown remain imperfectly understood. Rho-associated kinase (ROCK), an effector of the small GTPase Rho, involved in cytoskeleton dynamic regulation and cell polarity is activated by hyperglycemia. In one year-old Goto Kakizaki (GK) type 2 diabetic rats retina, ROCK-1 activation was assessed by its cellular distribution and by phosphorylation of its substrates, MYPT1 and MLC. In both GK rat and in human type 2 diabetic retinas, ROCK-1 is activated and associated with non-apoptotic membrane blebbing in retinal vessels and in retinal pigment epithelium (RPE) that respectively form the inner and the outer barriers. Activation of ROCK-1 induces focal vascular constrictions, endoluminal blebbing and subsequent retinal hypoxia. In RPE cells, actin cytoskeleton remodeling and membrane blebs in RPE cells contributes to outer barrier breakdown. Intraocular injection of fasudil, significantly reduces both retinal hypoxia and RPE barrier breakdown. Diabetes-induced cell blebbing may contribute to ischemic maculopathy and represent an intervention target.

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

Session-RPE for quantifying the load of different youth basketball training sessions

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C Lupo

2016-12-01

Full Text Available The aim of the study was to evaluate youth basketball training, verifying the reliability of the session-RPE method in relation to session duration (< and ≥ 80 minutes and workout typology (reduced and high warm-up, conditioning, technical, tactical, game portions within a single session categories. Six male youth basketball players (age, 16.5±0.5 years; height, 195.5±6.75 cm; body mass, 93.9±10.9 kg; and body mass index, 23.6±2.8 kg.m-2 were monitored (HR, type and duration of workouts during 15 (66 individual training sessions (80±26 minutes. Edwards’ HR method was used as a reference measure of internal training load (ITL; the CR-10 RPE scale was administered 30 minutes after the end of each session. The results obtained showed that all comparisons between different session durations and workout portions revealed effects in term of Edwards’ ITLs except for warm-up portions. Moderate to strong relationships between Edwards’ and session- RPE methods emerged for all sessions (r = .85, P < .001, player’s sessions (r range = .79 - .95, P < .001, session durations (< 80 minutes: r = .67, P < .001; ≥ 80 minutes: r = .75, P < .001, and workout portions (r range = .78 - .89, P range = .002 - < .001. The findings indicated that coaches of youth basketball players can successfully use session-RPE to monitor the ITL, regardless of session durations and workout portions.

Bone Marrow–Derived Cells Home to and Regenerate Retinal Pigment Epithelium after Injury

Science.gov (United States)

Harris, Jeffrey R.; Brown, Gary A. J.; Jorgensen, Marda; Kaushal, Shalesh; Ellis, E. Ann; Grant, Maria B.; Scott, Edward W.

2013-01-01

Purpose To determine whether hematopoietic stem and progenitor cells (HSCs/HPCs) can home to and regenerate the retinal pigment epithelium (RPE) after induced injury. Methods Enriched HSCs/HPCs from green fluorescent protein (gfp) transgenic mice were transplanted into irradiated recipient mice to track bone marrow–derived cells. Physical damage was induced by breaching Bruch’s membrane and inducing vascular endothelial growth factor A (VEGFa) expression to promote neovascularization. RPE damage was also induced by sodium iodate injection (40 mg/kg) into wild-type or albino C57Bl/6 mice. Cell morphology, gfp expression, the presence of the Y chromosome, and the presence of melanosomes were used to determine whether the injured RPE was being repaired by the donor bone marrow. Results Injury to the RPE recruits HSC/HPC–derived cells to incorporate into the RPE layer and differentiate into an RPE phenotype. A portion of the HSCs/HPCs adopt RPE morphology, express melanosomes, and integrate into the RPE without cell fusion. Conclusions HSCs/HPCs can migrate to the RPE layer after physical or chemical injury and regenerate a portion of the damaged cell layer. PMID:16639022

Acute and session RPE responses during resistance training: Bouts ...

African Journals Online (AJOL)

Journal Home > Vol 21, No 1 (2009) > ... On separate days in a counterbalanced order, subjects performed 3 sets of each exercise to volitional failure at a ... total work and acute RPE were significantly greater (p=0.01) for LI for all exercises.

TRANSPORT OF THIOL-CONJUGATES OF INORGANIC MERCURY IN HUMAN RETINAL PIGMENT EPITHELIAL CELLS

Science.gov (United States)

Bridges, Christy C.; Battle, Jamie R.; Zalups, Rudolfs K.

2007-01-01

Inorganic mercury (Hg2+) is a prevalent environmental contaminant to which exposure to can damage rod photoreceptor cells and compromise scotopic vision. The retinal pigment epithelium (RPE) likely plays a role in the ocular toxicity associated with Hg2+ exposure in that it mediates transport of substances to the photoreceptor cells. In order for Hg2+ to access photoreceptor cells, it must be first be taken up by the RPE, possibly by mechanisms involving transporters of essential nutrients. In other epithelia, Hg2+, when conjugated to cysteine (Cys) or homocysteine (Hcy), gains access to the intracellular compartment of the target cells via amino acid and organic anion transporters. Accordingly, the purpose of the current study was to test the hypothesis that Cys and Hcy S-conjugates of Hg2+ utilize amino acid transporters to gain access into RPE cells. Time- and temperature-dependence, saturation kinetics, and substrate-specificity of the transport of Hg2+, was assessed in ARPE-19 cells exposed to the following S-conjugates of Hg2+: Cys (Cys-S-Hg-S-Cys), Hcy (Hcy-S-Hg-S-Hcy), N-acetylcysteine (NAC-S-Hg-S-NAC) or glutathione (GSH-S-Hg-S-GSH). We discovered that only Cys-S-Hg-S-Cys and Hcy-S-Hg-S-Hcy were taken up by these cells. This transport was Na+-dependent and was inhibited by neutral and cationic amino acids. RT-PCR analyses identified systems B0,+ and ASC in ARPE-19 cells. Overall, our data suggest that Cys-S-Hg-S-Cys and Hcy-S-Hg-S-Hcy are taken up into ARPE-19 cells by Na-dependent amino acid transporters, possibly systems B0,+ and ASC. These amino acid transporters may play a role in the retinal toxicity observed following exposure to mercury. PMID:17467761

Transport of thiol-conjugates of inorganic mercury in human retinal pigment epithelial cells

International Nuclear Information System (INIS)

Bridges, Christy C.; Battle, Jamie R.; Zalups, Rudolfs K.

2007-01-01

Inorganic mercury (Hg 2+ ) is a prevalent environmental contaminant to which exposure to can damage rod photoreceptor cells and compromise scotopic vision. The retinal pigment epithelium (RPE) likely plays a role in the ocular toxicity associated with Hg 2+ exposure in that it mediates transport of substances to the photoreceptor cells. In order for Hg 2+ to access photoreceptor cells, it must first be taken up by the RPE, possibly by mechanisms involving transporters of essential nutrients. In other epithelia, Hg 2+ , when conjugated to cysteine (Cys) or homocysteine (Hcy), gains access to the intracellular compartment of the target cells via amino acid and organic anion transporters. Accordingly, the purpose of the current study was to test the hypothesis that Cys and Hcy S-conjugates of Hg 2+ utilize amino acid transporters to gain access into RPE cells. Time- and temperature-dependence, saturation kinetics, and substrate-specificity of the transport of Hg 2+ , was assessed in ARPE-19 cells exposed to the following S-conjugates of Hg 2+ : Cys (Cys-S-Hg-S-Cys), Hcy (Hcy-S-Hg-S-Hcy), N-acetylcysteine (NAC-S-Hg-S-NAC) or glutathione (GSH-S-Hg-S-GSH). We discovered that only Cys-S-Hg-S-Cys and Hcy-S-Hg-S-Hcy were taken up by these cells. This transport was Na + -dependent and was inhibited by neutral and cationic amino acids. RT-PCR analyses identified systems B 0,+ and ASC in ARPE-19 cells. Overall, our data suggest that Cys-S-Hg-S-Cys and Hcy-S-Hg-S-Hcy are taken up into ARPE-19 cells by Na-dependent amino acid transporters, possibly systems B 0,+ and ASC. These amino acid transporters may play a role in the retinal toxicity observed following exposure to mercury

rpe v5: an emulator for reduced floating-point precision in large numerical simulations

Science.gov (United States)

Dawson, Andrew; Düben, Peter D.

2017-06-01

This paper describes the rpe (reduced-precision emulator) library which has the capability to emulate the use of arbitrary reduced floating-point precision within large numerical models written in Fortran. The rpe software allows model developers to test how reduced floating-point precision affects the result of their simulations without having to make extensive code changes or port the model onto specialized hardware. The software can be used to identify parts of a program that are problematic for numerical precision and to guide changes to the program to allow a stronger reduction in precision.The development of rpe was motivated by the strong demand for more computing power. If numerical precision can be reduced for an application under consideration while still achieving results of acceptable quality, computational cost can be reduced, since a reduction in numerical precision may allow an increase in performance or a reduction in power consumption. For simulations with weather and climate models, savings due to a reduction in precision could be reinvested to allow model simulations at higher spatial resolution or complexity, or to increase the number of ensemble members to improve predictions. rpe was developed with a particular focus on the community of weather and climate modelling, but the software could be used with numerical simulations from other domains.

A comparison of some organizational characteristics of the mouse central retina and the human macula.

Science.gov (United States)

Volland, Stefanie; Esteve-Rudd, Julian; Hoo, Juyea; Yee, Claudine; Williams, David S

2015-01-01

Mouse models have greatly assisted our understanding of retinal degenerations. However, the mouse retina does not have a macula, leading to the question of whether the mouse is a relevant model for macular degeneration. In the present study, a quantitative comparison between the organization of the central mouse retina and the human macula was made, focusing on some structural characteristics that have been suggested to be important in predisposing the macula to stresses leading to degeneration: photoreceptor density, phagocytic load on the RPE, and the relative thinness of Bruch's membrane. Light and electron microscopy measurements from retinas of two strains of mice, together with published data on human retinas, were used for calculations and subsequent comparisons. As in the human retina, the central region of the mouse retina possesses a higher photoreceptor cell density and a thinner Bruch's membrane than in the periphery; however, the magnitudes of these periphery to center gradients are larger in the human. Of potentially greater relevance is the actual photoreceptor cell density, which is much greater in the mouse central retina than in the human macula, underlying a higher phagocytic load for the mouse RPE. Moreover, at eccentricities that correspond to the peripheral half of the human macula, the rod to cone ratio is similar between mouse and human. Hence, with respect to photoreceptor density and phagocytic load of the RPE, the central mouse retina models at least the more peripheral part of the macula, where macular degeneration is often first evident.

acute and session rPe responses during resistance training: Bouts ...

African Journals Online (AJOL)

tive measure of aerobic exercise intensity. While correlating well ... ing intensity in aerobic-type exercise. 7 ... While original work dealt with RPE during an exercise bout, ..... Borg G. Perceived exertion as an indicator of somatic stress. Scan J.

The Use of Session RPE to Monitor the Intensity of Weight Training in Older Women: Acute Responses to Eccentric, Concentric, and Dynamic Exercises

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Sandro S. Ferreira

2014-01-01

Full Text Available The rating of perceived exertion (RPE is ability to detect and interpret organic sensations while performing exercises. This method has been used to measure the level of effort that is felt during weight-training at a given intensity. The purpose of this investigation was to compare session RPE values with those of traditional RPE measurements for different weight-training muscle actions, performed together or separately. Fourteen women with no former weight-training experience were recruited for the investigation. All participants completed five sessions of exercise: familiarization, maximum force, concentric-only (CONC-only, eccentric-only (ECC-only, and dynamic (DYN = CONC + ECC. The traditional RPE method was measured after each series of exercises, and the session RPE was measured 30 min after the end of the training session. The statistical analyses used were the paired t-test, one-way analysis of variance, and repeated measures analysis of variance. Significant differences between traditional RPE and session RPE for DYN, CONC, and ECC exercises were not found. This investigation demonstrated that session RPE is similar to traditional RPE in terms of weight-training involving concentric, eccentric, or dynamic muscle exercises, and that it can be used to prescribe and monitor weight-training sessions in older subjects.

Xanthophylls are preferentially taken up compared with beta-carotene by retinal cells via a SRBI-dependent mechanism.

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During, Alexandrine; Doraiswamy, Sundari; Harrison, Earl H

2008-08-01

The purpose of this study was to investigate the mechanisms by which carotenoids [xanthophylls vs. beta-carotene(beta-C)] are taken up by retinal pigment epithelial (RPE) cells. The human RPE cell line, ARPE-19, was used. When ARPE-19 cells were fully differentiated (7-9 weeks), the xanthophylls lutein (LUT) and zeaxanthin (ZEA) were taken up by cells to an extent 2-fold higher than beta-C (P xanthophylls versus the carotene by a process that appears to be entirely SR-BI-dependent for ZEA and partly so for beta-C. This mechanism may explain, in part, the preferential accumulation of xanthophylls in the macula of the retina.

Differential cytotoxic effects of 7-dehydrocholesterol-derived oxysterols on cultured retina-derived cells: Dependence on sterol structure, cell type, and density.

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Pfeffer, Bruce A; Xu, Libin; Porter, Ned A; Rao, Sriganesh Ramachandra; Fliesler, Steven J

2016-04-01

Tissue accumulation of 7-dehydrocholesterol (7DHC) is a hallmark of Smith-Lemli-Opitz Syndrome (SLOS), a human inborn error of the cholesterol (CHOL) synthesis pathway. Retinal 7DHC-derived oxysterol formation occurs in the AY9944-induced rat model of SLOS, which exhibits a retinal degeneration characterized by selective loss of photoreceptors and associated functional deficits, Müller cell hypertrophy, and engorgement of the retinal pigment epithelium (RPE) with phagocytic inclusions. We evaluated the relative effects of four 7DHC-derived oxysterols on three retina-derived cell types in culture, with respect to changes in cellular morphology and viability. 661W (photoreceptor-derived) cells, rMC-1 (Müller glia-derived) cells, and normal diploid monkey RPE (mRPE) cells were incubated for 24 h with dose ranges of either 7-ketocholesterol (7kCHOL), 5,9-endoperoxy-cholest-7-en-3β,6α-diol (EPCD), 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), or 4β-hydroxy-7-dehydrocholesterol (4HDHC); CHOL served as a negative control (same dose range), along with appropriate vehicle controls, while staurosporine (Stsp) was used as a positive cytotoxic control. For 661W cells, the rank order of oxysterol potency was: EPCD > 7kCHOL > DHCEO > 4HDHC ≈ CHOL. EC50 values were higher for confluent vs. subconfluent cultures. 661W cells exhibited much higher sensitivity to EPCD and 7kCHOL than either rMC-1 or mRPE cells, with the latter being the most robust when challenged, either at confluence or in sub-confluent cultures. When tested on rMC-1 and mRPE cells, EPCD was again an order of magnitude more potent than 7kCHOL in compromising cellular viability. Hence, 7DHC-derived oxysterols elicit differential cytotoxicity that is dose-, cell type-, and cell density-dependent. These results are consistent with the observed progressive, photoreceptor-specific retinal degeneration in the rat SLOS model, and support the hypothesis that 7DHC-derived oxysterols are causally linked to that

Acquisition of T regulatory function in cathepsin L-inhibited T cells by eye-derived CTLA-2alpha during inflammatory conditions.

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Sugita, Sunao; Horie, Shintaro; Nakamura, Orie; Maruyama, Kazuichi; Takase, Hiroshi; Usui, Yoshihiko; Takeuchi, Masaru; Ishidoh, Kazumi; Koike, Masato; Uchiyama, Yasuo; Peters, Christoph; Yamamoto, Yoshimi; Mochizuki, Manabu

2009-10-15

Pigment epithelium isolated from the eye possesses immunosuppressive properties such as regulatory T (Treg) cell induction; e.g., cultured retinal pigment epithelium (RPE) converts CD4(+) T cells into Treg cells in vitro. RPE constitutively expresses a novel immunosuppressive factor, CTLA-2alpha, which is a cathepsin L (CathL) inhibitor, and this molecule acts via RPE to induce Treg cells. To clarify CTLA-2alpha's role in the T cell response to RPE in ocular inflammation, we used the experimental autoimmune uveitis (EAU) animal model to examine this new immunosuppressive property of RPE. In EAU models, TGF-beta, but not IFN-gamma inflammatory cytokines, promotes the up-regulation of the expression of CTLA-2alpha in RPE. Similarly, CTLA-2alpha via RPE was able to promote TGF-beta production by the CD4(+) T cells. The RPE-exposed T cells (RPE-induced Treg cells) greatly produced TGF-beta and suppressed bystander effector T cells. There was less expression of CathL by the RPE-exposed T cells, and CathL-inhibited T cells were able to acquire the Treg phenotype. Moreover, CathL-deficient mice spontaneously produced Treg cells, with the increase in T cells potentially providing protection against ocular inflammation. More importantly, CD4(+) T cells from EAU in CathL knockout mice or rCTLA-2alpha from EAU animals were found to contain a high population of forkhead box p3(+) T cells. In both EAU models, there was significant suppression of the ocular inflammation. These results indicate that RPE secretes CTLA-2alpha, thereby enabling the bystander T cells to be converted into Treg cells via TGF-beta promotion.

Differentiation/Purification Protocol for Retinal Pigment Epithelium from Mouse Induced Pluripotent Stem Cells as a Research Tool.

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Yuko Iwasaki

Full Text Available To establish a novel protocol for differentiation of retinal pigment epithelium (RPE with high purity from mouse induced pluripotent stem cells (iPSC.Retinal progenitor cells were differentiated from mouse iPSC, and RPE differentiation was then enhanced by activation of the Wnt signaling pathway, inhibition of the fibroblast growth factor signaling pathway, and inhibition of the Rho-associated, coiled-coil containing protein kinase signaling pathway. Expanded pigmented cells were purified by plate adhesion after Accutase® treatment. Enriched cells were cultured until they developed a cobblestone appearance with cuboidal shape. The characteristics of iPS-RPE were confirmed by gene expression, immunocytochemistry, and electron microscopy. Functions and immunologic features of the iPS-RPE were also evaluated.We obtained iPS-RPE at high purity (approximately 98%. The iPS-RPE showed apical-basal polarity and cellular structure characteristic of RPE. Expression levels of several RPE markers were lower than those of freshly isolated mouse RPE but comparable to those of primary cultured RPE. The iPS-RPE could form tight junctions, phagocytose photoreceptor outer segments, express immune antigens, and suppress lymphocyte proliferation.We successfully developed a differentiation/purification protocol to obtain mouse iPS-RPE. The mouse iPS-RPE can serve as an attractive tool for functional and morphological studies of RPE.

Protective Effects of Resveratrol against UVA-Induced Damage in ARPE19 Cells

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Chi-Ming Chan

2015-03-01

Full Text Available Ultraviolet radiation, especially UVA, can penetrate the lens, reach the retina, and induce oxidative stress to retinal pigment epithelial (RPE cells. Even though it is weakly absorbed by protein and DNA, it may trigger the production of reactive oxygen species (ROS and generate oxidative injury; oxidative injury to the retinal pigment epithelium has been implicated to play a contributory role in age-related macular degeneration (AMD. Studies showed that resveratrol, an abundant and active component of red grapes, can protect several cell types from oxidative stress. In this study, adult RPE cells being treated with different concentrations of resveratrol were used to evaluate the protective effect of resveratrol on RPE cells against UVA-induced damage. Cell viability assay showed that resveratrol reduced the UVA-induced decrease in RPE cell viability. Through flow cytometry analysis, we found that the generation of intracellular H2O2 induced by UVA irradiation in RPE cells could be suppressed by resveratrol in a concentration-dependent manner. Results of Western blot analysis demonstrated that resveratrol lowered the activation of UVA-induced extracellular signal-regulated kinase, c-jun-NH2 terminal kinase and p38 kinase in RPE cells. In addition, there was also a reduction in UVA-induced cyclooxygenase-2 (COX-2 expression in RPE cells pretreated with resveratrol. Our observations suggest that resveratrol is effective in preventing RPE cells from being damaged by UVA radiation, and is worth considering for further development as a chemoprotective agent for the prevention of early AMD.

Protective Effects of Resveratrol against UVA-Induced Damage in ARPE19 Cells

Science.gov (United States)

Chan, Chi-Ming; Huang, Cheng-Hua; Li, Hsin-Ju; Hsiao, Chien-Yu; Su, Ching-Chieh; Lee, Pei-Lan; Hung, Chi-Feng

2015-01-01

Ultraviolet radiation, especially UVA, can penetrate the lens, reach the retina, and induce oxidative stress to retinal pigment epithelial (RPE) cells. Even though it is weakly absorbed by protein and DNA, it may trigger the production of reactive oxygen species (ROS) and generate oxidative injury; oxidative injury to the retinal pigment epithelium has been implicated to play a contributory role in age-related macular degeneration (AMD). Studies showed that resveratrol, an abundant and active component of red grapes, can protect several cell types from oxidative stress. In this study, adult RPE cells being treated with different concentrations of resveratrol were used to evaluate the protective effect of resveratrol on RPE cells against UVA-induced damage. Cell viability assay showed that resveratrol reduced the UVA-induced decrease in RPE cell viability. Through flow cytometry analysis, we found that the generation of intracellular H2O2 induced by UVA irradiation in RPE cells could be suppressed by resveratrol in a concentration-dependent manner. Results of Western blot analysis demonstrated that resveratrol lowered the activation of UVA-induced extracellular signal-regulated kinase, c-jun-NH2 terminal kinase and p38 kinase in RPE cells. In addition, there was also a reduction in UVA-induced cyclooxygenase-2 (COX-2) expression in RPE cells pretreated with resveratrol. Our observations suggest that resveratrol is effective in preventing RPE cells from being damaged by UVA radiation, and is worth considering for further development as a chemoprotective agent for the prevention of early AMD. PMID:25775159

The effect of culture density and proliferation rate on the expression of ouabain-sensitive Na/K ATPase pumps in cultured human retinal pigment epithelium

International Nuclear Information System (INIS)

Burke, J.M.; Jaffe, G.J.; Brzeski, C.M.

1991-01-01

The number and activity of ouabain-sensitive Na/K ATPase pumps expressed by many cell types in vitro, including human retinal pigment epithelial cells (RPE), have been shown to decline with increasing culture density. Cell proliferation also declined as cultures became dense so it was unclear if pump number was modulated by cell proliferation or culture confluency. By exposing RPE cultures to various feeding regimens, using culture medium containing or lacking serum, it was possible to produce RPE cultures with a range of culture densities and growth rates. These were analyzed for proliferative activity by quantifying [ 3 H]thymidine incorporation and for Na/K ATPase pump number by measuring specific [ 3 H]ouabain binding. The results suggest that pump number is modulated by culture density and, further, that the density-dependent regulation of pump number requires serum. Although density-dependent modulation of culture growth is also serum requiring, cell proliferation and pump number did not appear to be related; cultures of similar density which differed significantly in growth rate had similar numbers of pumps. The view that elevated numbers of pumps were not necessarily found in proliferating cells was further supported by qualitative examination of radioautographs of cells dually labeled with [ 3 H]thymidine and [ 3 H]ouabain. Cycling cells which had [ 3 H]thymidine-labeled nuclei did not have notably higher labeling with [ 3 H]ouabain. However, [ 3 H]ouabain labeling, as an indicator of pump site number and distribution, did vary among cells in an RPE population and also within individual cells. This latter observation suggests that unpolarized RPE cells in sparse cultures may have regionally different requirements for ionic regulation

RBP-Jκ-dependent Notch signaling enhances retinal pigment epithelial cell proliferation in transgenic mice.

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Schouwey, K; Aydin, I T; Radtke, F; Beermann, F

2011-01-20

The Notch signaling pathway is an ubiquitous cell-cell interaction mechanism, which is essential in controlling processes like cell proliferation, cell fate decision, differentiation or stem cell maintenance. Recent data have shown that Notch signaling is RBP-Jκ-dependent in melanocytes, being required for survival of these pigment cells that are responsible for coloration of the skin and hairs in mammals. In addition, Notch is believed to function as an oncogene in melanoma, whereas it is a tumor suppressor in mouse epidermis. In this study, we addressed the implication of the Notch signaling in the development of another population of pigment cells forming the retinal pigment epithelium (RPE) in mammalian eyes. The constitutive activity of Notch in Tyrp1::NotchIC/° transgenic mice enhanced RPE cell proliferation, and the resulting RPE-derived pigmented tumor severely affected the overall eye structure. This RPE cell proliferation is dependent on the presence of the transcription factor RBP-Jκ, as it is rescued in mice lacking RBP-Jκ in the RPE. In conclusion, Notch signaling in the RPE uses the canonical pathway, which is dependent on the transcription factor RBP-Jκ. In addition, it is of importance for RPE development, and constitutive Notch activity leads to hyperproliferation and benign tumors of these pigment cells.

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.

Loss of Hfe Leads to Progression of Tumor Phenotype in Primary Retinal Pigment Epithelial Cells

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Gnana-Prakasam, Jaya P.; Veeranan-Karmegam, Rajalakshmi; Coothankandaswamy, Veena; Reddy, Sushma K.; Martin, Pamela M.; Thangaraju, Muthusamy; Smith, Sylvia B.; Ganapathy, Vadivel

2013-01-01

Purpose. Hemochromatosis is a disorder of iron overload arising mostly from mutations in HFE. HFE is expressed in retinal pigment epithelium (RPE), and Hfe−/− mice develop age-related iron accumulation and retinal degeneration associated with RPE hyperproliferation. Here, the mechanism underlying the hyperproliferative phenotype in RPE was investigated. Methods. Cellular senescence was monitored by β-galactosidase activity. Gene expression was monitored by real-time PCR. Survivin was analyzed by Western blot and immunofluorescence. Migration and invasion were monitored using appropriate kits. Glucose transporters (GLUTs) were monitored by 3-O-methyl-D-glucose uptake. Histone deacetylases (HDACs) were studied by monitoring catalytic activity and acetylation status of histones H3/H4. Results. Hfe−/− RPE cells exhibited slower senescence rate and higher survivin expression than wild type cells. Hfe−/− cells migrated faster and showed greater glucose uptake and increased expression of GLUTs. The expression of HDACs and DNA methyltransferase (DNMTs) also was increased. Similarly, RPE cells from hemojuvelin (Hjv)-knockout mice, another model of hemochromatosis, also had increased expression of GLUTs, HDACs, and DNMTs. The expression of Slc5a8 was decreased in Hfe−/− RPE cells, but treatment with a DNA methylation inhibitor restored the transporter expression, indicating involvement of DNA methylation in the silencing of Slc5a8 in Hfe−/− cells. Conclusions. RPE cells from iron-overloaded mice exhibit several features of tumor cells: decreased senescence, enhanced migration, increased glucose uptake, and elevated levels of HDACs and DNMTs. These features are seen in Hfe−/− RPE cells as well as in Hjv−/− RPE cells, providing a molecular basis for the hyperproliferative phenotype of Hfe−/− and Hjv−/− RPE cells. PMID:23169885

Retinal Pigment Epithelial Cell Culture and Cooperation of L-carnitine in Reducing Stress Induced Cellular Damage

International Nuclear Information System (INIS)

Shamsi, Farrukh A.; Al-Rajhi, Ali A.; Athmanathan, S.; Boulton, M.; Chaudhry, Imtiaz A.

2006-01-01

Purpose was to show that L-carnitine (LC) is capable of reducing non-oxidative stress in the retinal pigment epithelial cells (RPE) of the human eye. The RPE cells were cultured from donor eyes, obtained immediately after post-mortem. The interaction between bovine serum albumin (BSA) and non-oxidative (sodium hydroxide and methyl methane sulphonate) stress-inducers was observed by recording the change in the absorption profiles of the interacting molecules after incubation in light for 5 hours and after treatment with LC. The isolated and cultured RPE cells from the human eyes were treated with sodium hydroxide or methyl methane sulphonate and/or LC for 5 hours under light, and the qualitative effect on cell morphology after treatment was analyzed by staining cells with Giemsa and visualization by light microscopy. The cell morphology was also qualitatively analyzed by scanning electron microscopy (SEM). L-carnitine and stress-inducers interact with BSA and bring about changes in the spectral profile of the interacted molecules. Light microscopy as well as SEM show that the changes in the cellular morphology, induced by 100 uM concentrations of non-oxidative stress-inducers, are considerably reduced in the presence of 100 uM LC. However, L-carnitine alone does not cause any qualitative damage to the cell morphology during incubation under similar conditions. The results give a preliminary indication that LC has ability to reduce the changes brought about by the non-oxidative stress-inducers in the RPF cells in culture. (author)

Incorporation of Human Recombinant Tropoelastin into Silk Fibroin Membranes with the View to Repairing Bruch’s Membrane

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Audra M. A. Shadforth

2015-09-01

Full Text Available Bombyx mori silk fibroin membranes provide a potential delivery vehicle for both cells and extracellular matrix (ECM components into diseased or injured tissues. We have previously demonstrated the feasibility of growing retinal pigment epithelial cells (RPE on fibroin membranes with the view to repairing the retina of patients afflicted with age-related macular degeneration (AMD. The goal of the present study was to investigate the feasibility of incorporating the ECM component elastin, in the form of human recombinant tropoelastin, into these same membranes. Two basic strategies were explored: (1 membranes prepared from blended solutions of fibroin and tropoelastin; and (2 layered constructs prepared from sequentially cast solutions of fibroin, tropoelastin, and fibroin. Optimal conditions for RPE attachment were achieved using a tropoelastin-fibroin blend ratio of 10 to 90 parts by weight. Retention of tropoelastin within the blend and layered constructs was confirmed by immunolabelling and Fourier-transform infrared spectroscopy (FTIR. In the layered constructs, the bulk of tropoelastin was apparently absorbed into the initially cast fibroin layer. Blend membranes displayed higher elastic modulus, percentage elongation, and tensile strength (p < 0.01 when compared to the layered constructs. RPE cell response to fibroin membranes was not affected by the presence of tropoelastin. These findings support the potential use of fibroin membranes for the co-delivery of RPE cells and tropoelastin.

Size-dependent internalisation of folate-decorated nanoparticles via the pathways of clathrin and caveolae-mediated endocytosis in ARPE-19 cells.

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Langston Suen, Wai-Leung; Chau, Ying

2014-04-01

We aim to quantify the effect of size and degree of folate loading of folate-decorated polymeric nanoparticles (NPs) on the kinetics of cellular uptake and the selection of endocytic pathways in retinal pigment epithelium (RPE) cells. In this study, methoxy-poly(ethylene glycol)-b-polycaprolactone (mPEG-b-PCL) and folate-functionalized PEG-b-PCL were synthesized for assembling into nanoparticles with sizes ranging from 50 nm to 250 nm. These nanoparticles were internalized into ARPE-19 (human RPE cell line) via receptor-mediated endocytosis. A two-step endocytosis process mathematical model was adopted to quantify binding affinity and uptake kinetics of nanoparticles in RPE cells in uptake and inhibition studies. Nanoparticles with 100% folate loading have highest binding affinity and uptake rate in RPE cells. Maximum uptake rate (Vmax) of nanoparticles increased as the size of particles decreased from 250 nm to 50 nm. Endocytic pathway study was studied by using chlorpromazine and methyl-β-cyclodextran (MβCD), which are clathrin- and caveolae-mediated endocytosis inhibitors, respectively. Both chlorpromazine and MβCD inhibited the uptake of folate-decorated nanoparticles. Inhibition constant (Ki) and maximum uptake rate (Vmax) revealed that 50 nm and 120 nm folate-decorated nanoparticles were found to be internalized via both clathrin- and caveolae-mediated endocytosis. The 250 nm folate-decorated nanoparticles, however, were only internalized via caveolae-mediated pathway. Increased uptake rate of folate-decorated NPs into RPE cells is observed with increasing degree of folate modification. These NPs utilize both clathrin- and caveolae-mediated receptor-mediated endocytosis pathways to enter RPE cells upon size variation. The 50 nm NPs are internalized the fastest, with clathrin-mediated endocytosis as the preferred route. Uptake of 250 nm particles is the slowest and is dominated by caveolae-mediated endocytosis. © 2013 Royal Pharmaceutical

Xanthophylls are preferentially taken up compared with β-carotene by retinal cells via a SRBI-dependent mechanism*1

Science.gov (United States)

During, Alexandrine; Doraiswamy, Sundari; Harrison, Earl H.

2008-01-01

The purpose of this study was to investigate the mechanisms by which carotenoids [xanthophylls vs. β-carotene(β-C)] are taken up by retinal pigment epithelial (RPE) cells. The human RPE cell line, ARPE-19, was used. When ARPE-19 cells were fully differentiated (7–9 weeks), the xanthophylls lutein (LUT) and zeaxanthin (ZEA) were taken up by cells to an extent 2-fold higher than β-C (P xanthophylls versus the carotene by a process that appears to be entirely SR-BI-dependent for ZEA and partly so for β-C. This mechanism may explain, in part, the preferential accumulation of xanthophylls in the macula of the retina. PMID:18424859

RPE and Velocity Relationships for the Back Squat, Bench Press, and Deadlift in Powerlifters.

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Helms, Eric R; Storey, Adam; Cross, Matt R; Brown, Scott R; Lenetsky, Seth; Ramsay, Hamish; Dillen, Carolina; Zourdos, Michael C

2017-02-01

Helms, ER, Storey, A, Cross, MR, Browm, SR, Lenetsky, S, Ramsay, H, Dillen, C, and Zourdos, MC. RPE and velocity relationships for the back squat, bench press, and deadlift in powerlifters. J Strength Cond Res 31(2): 292-297, 2017-The purpose of this study was to compare average concentric velocity (ACV) and rating of perceived exertion (RPE) based on repetitions in reserve on the squat, bench press, and deadlift. Fifteen powerlifters (3 women and 12 men, mean age 28.4 ± 8.5 years) worked up to a one repetition maximum (1RM) on each lift. Rating of perceived exertion was recorded on all sets, and the ACV was recorded for all sets performed at 80% of estimated 1RM and higher, up to 1RM. Rating of perceived exertion at 1RM on squat, bench press, and deadlift was 9.6 ± 0.5, 9.7 ± 0.4, and 9.6 ± 0.5, respectively and was not significantly different (p > 0.05). The ACV at 1RM on squat, bench press and deadlift was 0.23 ± 0.05, 0.10 ± 0.04, and 0.14 ± 0.05 m·second, respectively. Squat was faster than both bench press and deadlift (p > 0.001), and deadlift was faster than bench press (p = 0.05). Very strong relationships (r = 0.88-0.91) between percentage 1RM and RPE were observed on each lift. The ACV showed strong (r = -0.79 to -0.87) and very strong (r = -0.90 to 92) inverse relationships with RPE and percentage 1RM on each lift, respectively. We conclude that RPE may be a useful tool for prescribing intensity for squat, bench press, and deadlift in powerlifters, in addition to traditional methods such as percentage of 1RM. Despite high correlations between percentage 1RM and ACV, a "velocity load profile" should be developed to prescribe intensity on an individual basis with appropriate accuracy.

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.

RPE65 gene: multiplex PCR and mutation screening in patients from ...

Indian Academy of Sciences (India)

Unknown

The RPE65 protein is believed to play an important role in the metabolism of vitamin A in the ... PCR and mutation screening in patients from India with retinal degenerative diseases. ..... Bennett J. 2001 Gene therapy restores vision in a canine.

The effect of culture density and proliferation rate on the expression of ouabain-sensitive Na/K ATPase pumps in cultured human retinal pigment epithelium

Energy Technology Data Exchange (ETDEWEB)

Burke, J.M.; Jaffe, G.J.; Brzeski, C.M. (Medical College of Wisconsin, Milwaukee (USA))

1991-06-01

The number and activity of ouabain-sensitive Na/K ATPase pumps expressed by many cell types in vitro, including human retinal pigment epithelial cells (RPE), have been shown to decline with increasing culture density. Cell proliferation also declined as cultures became dense so it was unclear if pump number was modulated by cell proliferation or culture confluency. By exposing RPE cultures to various feeding regimens, using culture medium containing or lacking serum, it was possible to produce RPE cultures with a range of culture densities and growth rates. These were analyzed for proliferative activity by quantifying ({sup 3}H)thymidine incorporation and for Na/K ATPase pump number by measuring specific ({sup 3}H)ouabain binding. The results suggest that pump number is modulated by culture density and, further, that the density-dependent regulation of pump number requires serum. Although density-dependent modulation of culture growth is also serum requiring, cell proliferation and pump number did not appear to be related; cultures of similar density which differed significantly in growth rate had similar numbers of pumps. The view that elevated numbers of pumps were not necessarily found in proliferating cells was further supported by qualitative examination of radioautographs of cells dually labeled with ({sup 3}H)thymidine and ({sup 3}H)ouabain. Cycling cells which had ({sup 3}H)thymidine-labeled nuclei did not have notably higher labeling with ({sup 3}H)ouabain. However, ({sup 3}H)ouabain labeling, as an indicator of pump site number and distribution, did vary among cells in an RPE population and also within individual cells. This latter observation suggests that unpolarized RPE cells in sparse cultures may have regionally different requirements for ionic regulation.

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 β 1 subunit of Na + , K + -ATPase plays an important role in this mechanism because homotypic β 1 -β 1 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 β 2 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 β 2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the β 2 subunit. qPCR results showed a time-dependent increase in the level of β 2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the β 2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α 2 subunit in that domain. Our results demonstrate that the apical polarization of Na + , K + -ATPase in RPE cells depends on the expression of the β 2 subunit.

Validity and reliability of the session-RPE method for quantifying training in Australian football: a comparison of the CR10 and CR100 scales.

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Scott, Tannath J; Black, Cameron R; Quinn, John; Coutts, Aaron J

2013-01-01

The purpose of this study was to examine and compare the criterion validity and test-retest reliability of the CR10 and CR100 rating of perceived exertion (RPE) scales for team sport athletes that undertake high-intensity, intermittent exercise. Twenty-one male Australian football (AF) players (age: 19.0 ± 1.8 years, body mass: 83.92 ± 7.88 kg) participated the first part (part A) of this study, which examined the construct validity of the session-RPE (sRPE) method for quantifying training load in AF. Ten male athletes (age: 16.1 ± 0.5 years) participated in the second part of the study (part B), which compared the test-retest reliability of the CR10 and CR100 RPE scales. In part A, the validity of the sRPE method was assessed by examining the relationships between sRPE, and objective measures of internal (i.e., heart rate) and external training load (i.e., distance traveled), collected from AF training sessions. Part B of the study assessed the reliability of sRPE through examining the test-retest reliability of sRPE during 3 different intensities of controlled intermittent running (10, 11.5, and 13 km·h(-1)). Results from part A demonstrated strong correlations for CR10- and CR100-derived sRPE with measures of internal training load (Banisters TRIMP and Edwards TRIMP) (CR10: r = 0.83 and 0.83, and CR100: r = 0.80 and 0.81, p training load (distance, higher speed running and player load) for both the CR10 (r = 0.81, 0.71, and 0.83) and CR100 (r = 0.78, 0.69, and 0.80) were significant (p reliability for both the CR10 (31.9% CV) and CR100 (38.6% CV) RPE scales after short bouts of intermittent running. Collectively, these results suggest both CR10- and CR100-derived sRPE methods have good construct validity for assessing training load in AF. The poor levels of reliability revealed under field testing indicate that the sRPE method may not be sensible to detecting small changes in exercise intensity during brief intermittent running bouts. Despite this limitation

Optimization of Storage Temperature for Cultured ARPE-19 Cells

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Lara Pasovic

2013-01-01

Full Text Available Purpose. The establishment of future retinal pigment epithelium (RPE replacement therapy is partly dependent on the availability of tissue-engineered RPE cells, which may be enhanced by the development of suitable storage methods for RPE. This study investigates the effect of different storage temperatures on the viability, morphology, and phenotype of cultured RPE. Methods. ARPE-19 cells were cultured under standard conditions and stored in HEPES-buffered MEM at nine temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, and 37°C for seven days. Viability and phenotype were assessed by a microplate fluorometer and epifluorescence microscopy, while morphology was analyzed by scanning electron microscopy. Results. The percentage of viable cells preserved after storage was highest in the 16°C group (48.7%±9.8%; P<0.01 compared to 4°C, 8°C, and 24°C–37°C; P<0.05 compared to 12°C. Ultrastructure was best preserved at 12°C, 16°C, and 20°C. Expression of actin, ZO-1, PCNA, caspase-3, and RPE65 was maintained after storage at 16°C compared to control cells that were not stored. Conclusion. Out of nine temperatures tested between 4°C and 37°C, storage at 12°C, 16°C, and 20°C was optimal for maintenance of RPE cell viability, morphology, and phenotype. The preservation of RPE cells is critically dependent on storage temperature.

Protective Effect of Combined Caffeic Acid Phenethyl Ester and Bevacizumab Against Hydrogen Peroxide-Induced Oxidative Stress in Human RPE Cells.

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Dinc, Erdem; Ayaz, Lokman; Kurt, Akif Hakan

2017-12-01

This study aimed to evaluate the protective effects of caffeic acid phenethyl ester (CAPE) and combined CAPE-bevacizumab against oxidative stress induced by hydrogen peroxide (H 2 O 2 ) in human retinal pigment epithelium. ARPE-19 cells were pretreated with 5, 10, and 30 μM CAPE alone and in combination with bevacizumab for 3 h, then exposed to H 2 O 2 for 16 h. Cell viability was evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Vascular endothelial growth factor (VEGF) protein levels in the medium were measured using a human VEGF ELISA kit. Total antioxidant status (TAS) and total oxidant status (TOS) were measured in ARPE-19 cells using the test kit from Rel Assay. Expression levels of VEGF, Bax, Bcl-2, cytochrome c, apoptotic protease activating factor-1 (apaf-1), and caspase-3 were determined using reverse transcription polymerase chain reaction. Pretreatment of ARPE-19 cells with 30 μM CAPE and combined CAPE-bevacizumab reduced H 2 O 2 mediated cell death. H 2 O 2 -induced oxidative stress increased TOS and VEGF production, which was significantly inhibited by CAPE and the CAPE-bevacizumab combination. VEGF, Bax, cytochrome c, apaf-1, and caspase-3 gene expressions were significantly decreased in cells pretreated with 5, 10, and 30 μM CAPE and combined CAPE-bevacizumab compared to the H 2 O 2 group. In addition, Bcl-2 expression was significantly increased in both the CAPE and CAPE-bevacizumab combination groups compared to the H 2 O 2 group. CAPE has a protective effect on ARPE-19 cells against oxidative stress, and VEGF protein level and expression can be decreased by incubation with different concentrations of CAPE. These results demonstrate that CAPE suppresses the mitochondria-mediated apoptosis in ARPE-19 cells under oxidative stress. In addition, the use of CAPE in combination with bevacizumab has an additive effect.

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.

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

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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.

Cholesterol enhances amyloid {beta} deposition in mouse retina by modulating the activities of A{beta}-regulating enzymes in retinal pigment epithelial cells

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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

Taurine uptake by human retinal pigment epithelium: implications for the transport of small solutes between the choroid and the outer retina.

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Hillenkamp, Jost; Hussain, Ali A; Jackson, Timothy L; Cunningham, Joanna R; Marshall, John

2004-12-01

To characterize the Michaelis-Menten kinetics of the taurine transporter (TT) in retinal pigment epithelium (RPE) freshly isolated from human donor eyes. To identify the rate limiting compartment in the pathway of taurine delivery from the choroidal blood supply to the outer retina composed by Bruch's-choroid (BC) and the RPE in the human older age group. In human donor samples (4 melanoma-affected eyes, and 14 control eyes; age range, 62-93 years), radiochemical techniques were used to determine the RPE taurine accumulation at various exogenous concentrations. The transport capability of human RPE was obtained from a kinetic analysis of the high-affinity carrier over a substrate concentration of 1 to 60 microM taurine. Uptake of taurine into human RPE at a taurine concentration of 1 microM was independent of donor age (P > 0.05) and averaged at 2.83 +/- 0.27 (SEM) pmol/10 minutes per 6-mm trephine. Taurine transport by human RPE was mediated by a high-affinity carrier of K(m) 50 microM and V(max) of 267 pmol/10 minutes per 5-mm disc. In human donor RPE, uptake of taurine remained viable in the age range 62 to 93 years. Taurine transport rates in the RPE were lower than across the isolated BC complex, and thus the data suggest that the former compartment houses the rate-limiting step in the delivery of taurine to the outer retina.

RPE vs. Percentage 1RM Loading in Periodized Programs Matched for Sets and Repetitions

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Helms, Eric R.; Byrnes, Ryan K.; Cooke, Daniel M.; Haischer, Michael H.; Carzoli, Joseph P.; Johnson, Trevor K.; Cross, Matthew R.; Cronin, John B.; Storey, Adam G.; Zourdos, Michael C.

2018-01-01

Purpose: To investigate differences between rating of perceived exertion (RPE) and percentage one-repetition maximum (1RM) load assignment in resistance-trained males (19–35 years) performing protocols with matched sets and repetitions differentiated by load-assignment. Methods: Participants performed squats then bench press 3x/weeks in a daily undulating format over 8-weeks. Participants were counterbalanced by pre-test 1RM then assigned to percentage 1RM (1RMG, n = 11); load-assignment via percentage 1RMs, or RPE groups (RPEG, n = 10); participant-selected loads to reach target RPE ranges. Ultrasonography determined pre and post-test pectoralis (PMT), and vastus lateralis muscle thickness at 50 (VLMT50) and 70% (VLMT70) femur-length. Results: Bench press (1RMG +9.64 ± 5.36; RPEG + 10.70 ± 3.30 kg), squat (1RMG + 13.91 ± 5.89; RPEG + 17.05 ± 5.44 kg) and their combined-total 1RMs (1RMG + 23.55 ± 10.38; RPEG + 27.75 ± 7.94 kg) increased (p 0.05). Magnitude-based inferences revealed 79, 57, and 72% chances of mean small effect size (ES) advantages for squat; ES 90% confidence limits (CL) = 0.50 ± 0.63, bench press; ES 90% CL = 0.28 ± 0.73, and combined-total; ES 90% CL = 0.48 ± 0.68 respectively, in RPEG. There were 4, 14, and 6% chances 1RMG had a strength advantage of the same magnitude, and 18, 29, and 22% chances, respectively of trivial differences between groups. Conclusions: Both loading-types are effective. However, RPE-based loading may provide a small 1RM strength advantage in a majority of individuals. PMID:29628895

Bioengineered Bruch's-like extracellular matrix promotes retinal pigment epithelial differentiation

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Samuel McLenachan

2017-07-01

Full Text Available In the eye, the retinal pigment epithelium (RPE adheres to a complex protein matrix known as Bruch's membrane (BrM. The aim of this study was to provide enriched conditions for RPE cell culture through the production of a BrM-like matrix. Our hypothesis was that a human RPE cell line would deposit an extracellular matrix (ECM resembling BrM. The composition and structure of ECM deposited by ARPE19 cells (ARPE19-ECM was characterized. To produce ARPE19-ECM, ARPE19 cells were cultured in the presence dextran sulphate. ARPE19-ECM was decellularized using deoxycholate and characterized by immunostaining and western blot analysis. Primary human RPE and induced pluripotent stem cells were seeded onto ARPE19-ECM or geltrex coated surfaces and examined by microscopy or RT-PCR. Culture of ARPE19 cells with dextran sulphate promoted nuclear localization of SOX2, formation of tight junctions and deposition of ECM. ARPE19 cells deposited ECM proteins found in the inner layers of BrM, including fibronectin, vitronectin, collagens IV and V as well as laminin-alpha-5, but not those found in the middle elastic layer (elastin or the outer layers (collagen VI. ARPE19-ECM promoted pigmentation in human RPE and pluripotent stem cell cultures. Expression of RPE65 was significantly increased on ARPE19-ECM compared with geltrex in differentiating pluripotent stem cell cultures. ARPE19 cells deposit ECM with a composition and structure similar to BrM in the retina. Molecular cues present in ARPE19-ECM promote the acquisition and maintenance of the RPE phenotype. Together, these results demonstrate a simple method for generating a BrM-like surface for enriched RPE cell cultures.

Effects of melatonin and its receptor antagonist on retinal pigment epithelial cells against hydrogen peroxide damage

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Rosen, Richard B.; Hu, Dan-Ning; Chen, Min; McCormick, Steven A.; Walsh, Joseph

2012-01-01

Purpose Recently, we reported finding that circulating melatonin levels in age-related macular degeneration patients were significantly lower than those in age-matched controls. The purpose of this study was to investigate the hypothesis that melatonin deficiency may play a role in the oxidative damage of the retinal pigment epithelium (RPE) by testing the protective effect of melatonin and its receptor antagonist on RPE cells exposed to H2O2 damage. Methods Cultured human RPE cells were subjected to oxidative stress induced by 0.5 mM H2O2. Cell viability was measured using the microculture tetrazoline test (MTT) assay. Cells were pretreated with or without melatonin for 24 h. Luzindole (50 μM), a melatonin membrane-receptor antagonist, was added to the culture 1 h before melatonin to distinguish direct antioxidant effects from indirect receptor-dependent effects. All tests were performed in triplicate. Results H2O2 at 0.5 mM decreased cell viability to 20% of control levels. Melatonin showed dose-dependent protective effects on RPE cells against H2O2. Cell viability of RPE cells pretreated with 10−10, 10−8, 10−6, and 10−4 M melatonin for 24 h was 130%, 160%, 187%, and 230% of cells treated with H2O2 alone (all p<0.05). Using cells cultured without H2O2 as the control, cell viability of cells treated with H2O2 after pretreatment with 10−10-10−4 M melatonin was still significantly lower than that of the controls, suggesting that melatonin significantly decreased but did not completely abolish the in vitro cytotoxic effects of H2O2. Luzindole completely blocked melatonin’s protective effects at low concentrations of melatonin (10−10-10−8 M) but not at high concentrations (10−6-10−4 M). Conclusions Melatonin has a partial protective effect on RPE cells against H2O2 damage across a wide range of concentrations (10−10-10−4 M). This protective effect occurs through the activation of melatonin membrane receptors at low concentrations (10−10

Assessment of Safety and Functional Efficacy of Stem Cell-Based Therapeutic Approaches Using Retinal Degenerative Animal Models

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Tai-Chi Lin

2017-01-01

Full Text Available Dysfunction and death of retinal pigment epithelium (RPE and or photoreceptors can lead to irreversible vision loss. The eye represents an ideal microenvironment for stem cell-based therapy. It is considered an “immune privileged” site, and the number of cells needed for therapy is relatively low for the area of focused vision (macula. Further, surgical placement of stem cell-derived grafts (RPE, retinal progenitors, and photoreceptor precursors into the vitreous cavity or subretinal space has been well established. For preclinical tests, assessments of stem cell-derived graft survival and functionality are conducted in animal models by various noninvasive approaches and imaging modalities. In vivo experiments conducted in animal models based on replacing photoreceptors and/or RPE cells have shown survival and functionality of the transplanted cells, rescue of the host retina, and improvement of visual function. Based on the positive results obtained from these animal experiments, human clinical trials are being initiated. Despite such progress in stem cell research, ethical, regulatory, safety, and technical difficulties still remain a challenge for the transformation of this technique into a standard clinical approach. In this review, the current status of preclinical safety and efficacy studies for retinal cell replacement therapies conducted in animal models will be discussed.

Calcium-independent phospholipase A2 regulates retinal pigment epithelium proliferation and may be important in the pathogenesis of retinal diseases

DEFF Research Database (Denmark)

Kolko, M; Kiilgaard, J F; Wang, J

2009-01-01

Calcium-independent phospholipase A2, group VIA (iPLA2-VIA) is involved in cell proliferation. This study aimed to evaluate the role of iPLA2-VIA in retinal pigment epithelium (RPE) cell proliferation and in retinal diseases involving RPE proliferation. A human RPE cell line (ARPE-19) was used...... the expression of iPLA2-VIA in proliferative vitreoretinopathy (PVR). PVR membranes revealed nuclear expression of iPLA2-VIA in the RPE cells which had migrated and participated in the formation of the membranes. Overall, the present results point to an important role of iPLA2-VIA in the regulation of RPE...

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.

Gait and posture analysis in patients with maxillary transverse discrepancy, before and after RPE.

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Mason, Martina; Spolaor, Fabiola; Guiotto, Annamaria; De Stefani, Alberto; Gracco, Antonio; Sawacha, Zimi

2018-03-01

The purpose of this study was to evaluate the effects of the rapid palatal expansion (RPE) on posture and gait analysis in subjects with maxillary transverse discrepancies. Forty-one patients between 6 and 12 years were divided into 3 groups: 10 control subjects (Cs), 16 patients with unilateral posterior crossbite (CbMono), 15 patients with maxillary transverse discrepancy and no crossbite (Nocb). Every subject underwent gait analysis and posturographic examination in order to evaluate the presence of balance alterations before (T0) and after (T4) RPE application. The examinations were performed through a six-cameras stereophotogrammetric system (60-120Hz, BTS S.p.A.) synchronized with two force plates (FP4060, Bertec Corp.). Romberg test was performed on a force plate, and the statokinesiogram and joint kinematics were evaluated. One-way Anova was performed among the variables after evidence of normal distribution (Levene's test for equality of variances) and Kruskal-Wallis test (Ptest was performed, or Kruskal-Wallis test, instead when comparing pre- and post-RPE application within the same group of subjects (P<0.05). Tamane T2 or Bonferroni correction was applied where needed. The posturographic analysis reveal significant differences across the 3 population: 95% power frequency in medio-lateral and antero-posterior direction in T0, median frequency in medio-lateral direction in T0, mean power frequency in medio-lateral direction in T0. Significant differences were also registered in the three-dimensional joints kinematics variables, mainly between Cs and Cbmono in T0 and T4 and between Cbmono and Nocb in T4. A detectable correlation between dental occlusion and body posture is shown in this study that confirms another benefit of the RPE. This was mainly revealed in the dynamic posture where modifications at the mandibular level affect the whole body. Copyright © 2018. Published by Elsevier Masson SAS.

Phototoxicity and cytotoxicity of fullerol in human retinal pigment epithelial cells

International Nuclear Information System (INIS)

Wielgus, Albert R.; Zhao, Baozhong; Chignell, Colin F.; Hu, Dan-Ning; Roberts, Joan E.

2010-01-01

The water-soluble nanoparticle hydroxylated fullerene [fullerol, nano-C 60 (OH) 22-26 ] has several clinical applications including use as a drug carrier to bypass the blood ocular barriers. We have previously found that fullerol is both cytotoxic and phototoxic to human lens epithelial cells (HLE B-3) and that the endogenous antioxidant lutein blocked some of this phototoxicity. In the present study we have found that fullerol induces cytotoxic and phototoxic damage to human retinal pigment epithelial cells. Accumulation of nano-C 60 (OH) 22-26 in the cells was confirmed spectrophotometrically at 405 nm, and cell viability, cell metabolism and membrane permeability were estimated using trypan blue, MTS and LDH assays, respectively. Fullerol was cytotoxic toward hRPE cells maintained in the dark at concentrations higher than 10 μM. Exposure to an 8.5 J.cm -2 dose of visible light in the presence of > 5 μM fullerol induced TBARS formation and early apoptosis, indicating phototoxic damage in the form of lipid peroxidation. Pretreatment with 10 and 20 μM lutein offered some protection against fullerol photodamage. Using time resolved photophysical techniques, we have now confirmed that fullerol produces singlet oxygen with a quantum yield of Φ = 0.05 in D 2 O and with a range of 0.002-0.139 in various solvents. As our previous studies have shown that fullerol also produces superoxide in the presence of light, retinal phototoxic damage may occur through both type I (free radical) and type II (singlet oxygen) mechanisms. In conclusion, ocular exposure to fullerol, particularly in the presence of sunlight, may lead to retinal damage.

Enhanced Detection of Sub-Retinal Pigment Epithelial Cell Layer Deposits in Human and Murine Tissue: Imaging Zinc as a Biomarker for Age-Related Macular Degeneration (An American Ophthalmological Society Thesis).

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van Kuijk, Frederik J G M; McPherson, Scott W; Roehrich, Heidi

2017-08-01

Understanding the apparent paradoxical role of zinc in the pathogenesis and prevention of age-related macular degeneration (AMD) has been limited by the lack of animal models for its detection in sub-retinal epithelial deposits (drusen), a definitive early hallmark of AMD. In-vitro studies using Zinpyr-1 showed drusen contained high levels of zinc, but the probe was not suitable for in-vivo studies. This study compares Zinpyr-1 to ZPP1, a new fluorescein-based probe for zinc, to assess the potential of ZPP1 for in-vivo detection of zinc in drusen. Flat mounts of human sub-RPE tissue using the probes were analyzed by fluorescence and confocal microscopy. Flat mounts of sub-RPE tissue from mice deficient in superoxide dismutase isoform-1 (CuZn-SOD-KO) or isoform-2 (Mn-SOD-RPE-KO) were analyzed with sub-RPE deposits confirmed by histology. Drusen are detected in greater numbers and intensity with ZPP1 compared to Zinpyr-1. Using ZPP1, drusen was detected in a sample from a 46-year old human donor without ocular history, suggesting that ZPP1 might be sensitive enough to detect drusen at an early stage. With CuZn-SOD KO mice, ZPP1 detected sub-RPE deposits at 10 months of age, whereas Zinpyr-1 required 14 months. Detection of sub-RPE deposits by ZPP1 was greatly enhanced compared to Zinpyr-1. This enhanced sensitivity will allow for more insightful analysis of zinc in AMD using human specimens and mouse models. This could result in the development of a sensitive in-vivo probe to enhance research on the role zinc in drusen formation and the early clinical diagnosis of AMD.

Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model.

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Pilgrim, Matthew G; Lengyel, Imre; Lanzirotti, Antonio; Newville, Matt; Fearn, Sarah; Emri, Eszter; Knowles, Jonathan C; Messinger, Jeffrey D; Read, Russell W; Guidry, Clyde; Curcio, Christine A

2017-02-01

Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Apparently functional primary RPE cells, when cultured on 10-μm-thick inserts with 0.4-μm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.

Retinal Cell Death Caused by Sodium Iodate Involves Multiple Caspase-Dependent and Caspase-Independent Cell-Death Pathways

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Jasmin Balmer

2015-07-01

Full Text Available Herein, we have investigated retinal cell-death pathways in response to the retina toxin sodium iodate (NaIO3 both in vivo and in vitro. C57/BL6 mice were treated with a single intravenous injection of NaIO3 (35 mg/kg. Morphological changes in the retina post NaIO3 injection in comparison to untreated controls were assessed using electron microscopy. Cell death was determined by TdT-mediated dUTP-biotin nick end labeling (TUNEL staining. The activation of caspases and calpain was measured using immunohistochemistry. Additionally, cytotoxicity and apoptosis in retinal pigment epithelial (RPE cells, primary retinal cells, and the cone photoreceptor (PRC cell line 661W were assessed in vitro after NaIO3 treatment using the ApoToxGlo™ assay. The 7-AAD/Annexin-V staining was performed and necrostatin (Nec-1 was administered to the NaIO3-treated cells to confirm the results. In vivo, degenerating RPE cells displayed a rounded shape and retracted microvilli, whereas PRCs featured apoptotic nuclei. Caspase and calpain activity was significantly upregulated in retinal sections and protein samples from NaIO3-treated animals. In vitro, NaIO3 induced necrosis in RPE cells and apoptosis in PRCs. Furthermore, Nec-1 significantly decreased NaIO3-induced RPE cell death, but had no rescue effect on treated PRCs. In summary, several different cell-death pathways are activated in retinal cells as a result of NaIO3.

RPE vs. Percentage 1RM Loading in Periodized Programs Matched for Sets and Repetitions

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Eric R. Helms

2018-03-01

Full Text Available Purpose: To investigate differences between rating of perceived exertion (RPE and percentage one-repetition maximum (1RM load assignment in resistance-trained males (19–35 years performing protocols with matched sets and repetitions differentiated by load-assignment.Methods: Participants performed squats then bench press 3x/weeks in a daily undulating format over 8-weeks. Participants were counterbalanced by pre-test 1RM then assigned to percentage 1RM (1RMG, n = 11; load-assignment via percentage 1RMs, or RPE groups (RPEG, n = 10; participant-selected loads to reach target RPE ranges. Ultrasonography determined pre and post-test pectoralis (PMT, and vastus lateralis muscle thickness at 50 (VLMT50 and 70% (VLMT70 femur-length.Results: Bench press (1RMG +9.64 ± 5.36; RPEG + 10.70 ± 3.30 kg, squat (1RMG + 13.91 ± 5.89; RPEG + 17.05 ± 5.44 kg and their combined-total 1RMs (1RMG + 23.55 ± 10.38; RPEG + 27.75 ± 7.94 kg increased (p < 0.05 in both groups as did PMT (1RMG + 1.59 ± 1.33; RPEG +1.90 ± 1.91 mm, VLMT50 (1RMG +2.13 ± 1.95; RPEG + 1.85 ± 1.97 mm and VLMT70 (1RMG + 2.40 ± 2.22; RPEG + 2.31 ± 2.27 mm. Between-group differences were non-significant (p > 0.05. Magnitude-based inferences revealed 79, 57, and 72% chances of mean small effect size (ES advantages for squat; ES 90% confidence limits (CL = 0.50 ± 0.63, bench press; ES 90% CL = 0.28 ± 0.73, and combined-total; ES 90% CL = 0.48 ± 0.68 respectively, in RPEG. There were 4, 14, and 6% chances 1RMG had a strength advantage of the same magnitude, and 18, 29, and 22% chances, respectively of trivial differences between groups.Conclusions: Both loading-types are effective. However, RPE-based loading may provide a small 1RM strength advantage in a majority of individuals.

Myo-inositol uptake by cultured calf retinal pigment epithelial cells: regulation by glucose

International Nuclear Information System (INIS)

Khatami, M.; Rockey, J.H.

1986-01-01

Confluent primary (P-1) or subcultured passage 2 or 3 (P-2, P-3) calf retinal pigment epithelial cells (RPE) were incubated with [ 3 H]-myo-inositol (MI, 100-200 μM) in balanced salt solution (BSS), for 5 to 60 min at 37 0 C. MI uptake into RPE (P-2, 5 days old) was saturable with K/sub m/ of 147 μM and V/sub max/ of 5.5 pmole/min/μg DNA. P-1 or P-2 incubated with 10 μM MI for 40 min accumulated MI against a concentration gradient ([MI]in/[MI]out > 20). Replacement of 150 mM NaCl in BSS by 150 mM choline-Cl reduced the uptake of MI by 87%. MI uptake was inhibited (39%) when cells were incubated in BSS in the absence of Ca Cl 2 . Transport of MI into RPE incubated in the presence of phloridzin, ouabain or 2,4-dinitrophenol (1 mM each) for 10 min was inhibited by 65, 37 and 21%, respectively. α-D-Glucose (20 mM) in the incubation media inhibited MI uptake into primary (or P-2) cultured RPE by 30 or 43% when cells were incubated for 10 or 60 min, respectively. The ability of RPE cells, grown in the presence of 50 mM glucose for 15-25 days, to concentrate MI (40 μM) was reduced up to 41%. Cultured RPE cells accumulated myo-inositol by an active transport system, sensitive to ouabain, DNP and phloridzin. High glucose in the incubation media or in the growth media inhibited the uptake of MI into calf RPE cells

Outer segment phagocytosis by cultured retinal pigment epithelial cells requires Gas6.

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Hall, M O; Prieto, A L; Obin, M S; Abrams, T A; Burgess, B L; Heeb, M J; Agnew, B J

2001-10-01

The function and viability of vertebrate photoreceptors requires the daily phagocytosis of photoreceptor outer segments (OS) by the adjacent retinal pigment epithelium (RPE). We demonstrate here a critical role in this process for Gas6 and by implication one of its receptor protein tyrosine kinases (RTKs), Mertk (Mer). Gas6 specifically and selectively stimulates the phagocytosis of OS by normal cultured rat RPE cells. The magnitude of the response is dose-dependent and shows an absolute requirement for calcium. By contrast the Royal College of Surgeons (RCS) rat RPE cells, in which a mutation in the gene Mertk results in the expression of a truncated, non-functional receptor, does not respond to Gas6. These data strongly suggest that activation of Mertk by its ligand, Gas6, is the specific signaling pathway responsible for initiating the ingestion of shed OS. Moreover, photoreceptor degeneration in the RCS rat retina, which lacks Mertk, and in humans with a mutation in Mertk, strongly suggests that the Gas6/Mertk signaling pathway is essential for photoreceptor viability. We believe that this is the first demonstration of a specific function for Gas6 in the eye. Copyright 2001 Academic Press.

The Expansion of RPE Atrophy after the Inverted ILM Flap Technique for a Chronic Large Macular Hole

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Hisanori Imai

2014-03-01

Full Text Available Purpose: To report a case of the expansion of submacular retinal pigment epithelium (RPE atrophy after using the inverted internal limiting membrane (ILM flap technique for a persisting, large, stage IV macular hole (MH. Case Report: A 79-year-old woman presented with a chronic large MH that remained open despite pars plana vitrectomy (PPV. The surgery was performed twice for the MH closure 14 years earlier. ILM peeling was not performed during the previous surgeries. The best-corrected visual acuity (BCVA with the Landolt ring chart was 0.08 at her visit. The minimum MH diameter was 1,240 μm. Inverted ILM flap technique with 20% SF6 gas tamponade was performed for the MH closure. For the inverted ILM flap technique, 25-gauge PPV and ILM staining with indocyanine green were used. The ILM was peeled off for 2 disc diameters around the MH, but the ILM was not removed completely. The ILM was then inverted and covered the MH. Results: One month after surgery, the MH was closed, accompanied by glial cell proliferation spreading from the inverted ILM flap (as reported before. On the other hand, the area of the submacular RPE atrophy, which was already observed 1 week after surgery, gradually increased in size. BCVA improved to 0.3 six months after the surgery. Conclusions: The inverted ILM flap technique may be promising even for persisting large MH which were not closed in previous surgeries, but long-term observation is needed because the detailed behavior of the inverted ILM and the Müller cells after surgery is not yet known.

Personalized Medicine: Cell and Gene Therapy Based on Patient-Specific iPSC-Derived Retinal Pigment Epithelium Cells.

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Li, Yao; Chan, Lawrence; Nguyen, Huy V; Tsang, Stephen H

2016-01-01

Interest in generating human induced pluripotent stem (iPS) cells for stem cell modeling of diseases has overtaken that of patient-specific human embryonic stem cells due to the ethical, technical, and political concerns associated with the latter. In ophthalmology, researchers are currently using iPS cells to explore various applications, including: (1) modeling of retinal diseases using patient-specific iPS cells; (2) autologous transplantation of differentiated retinal cells that undergo gene correction at the iPS cell stage via gene editing tools (e.g., CRISPR/Cas9, TALENs and ZFNs); and (3) autologous transplantation of patient-specific iPS-derived retinal cells treated with gene therapy. In this review, we will discuss the uses of patient-specific iPS cells for differentiating into retinal pigment epithelium (RPE) cells, uncovering disease pathophysiology, and developing new treatments such as gene therapy and cell replacement therapy via autologous transplantation.

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.

Spontaneous correction of anterior crossbite by RPE anchored on deciduous teeth in the early mixed dentition.

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Rosa, M; Lucchi, P; Mariani, L; Caprioglio, A

2012-09-01

The purpose of this study was to evaluate the effectiveness of Haas RPE anchored on deciduous teeth in the early mixed dentition, for inducing the spontaneous correction of permanent incisor's crossbite, without compliance, without post bite-plane and no involvement of the permanent teeth. The sample group comprised 50 consecutive patients (mean age 8y 5m, SD 2y 1m), 31 males, 19 females. They showed a cross-bite affecting one or more permanent incisors, for a total of 70 teeth. The patients were treated with Haas RPE appliance anchored on second deciduous molars and bonded on deciduous canines. No direct forces were applied on the permanent teeth. Anterior crossbite self-corrected 'spontaneously' in 84% of the cases. Lateral incisors had a higher rate of self-correction than central incisors. All hyper-divergent subjects showed a spontaneous crossbite self-correction. The early maxillary expansion by Haas RPE anchored on deciduous teeth is an efficient and effective procedure to induce the anterior crossbite self-correction in the early mixed dentition without the need of a bite-plane, no involvement of the permanent teeth and without compliance.

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

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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.

Spatiotemporal neural characterization of prediction error valence and surprise during reward learning in humans.

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Fouragnan, Elsa; Queirazza, Filippo; Retzler, Chris; Mullinger, Karen J; Philiastides, Marios G

2017-07-06

Reward learning depends on accurate reward associations with potential choices. These associations can be attained with reinforcement learning mechanisms using a reward prediction error (RPE) signal (the difference between actual and expected rewards) for updating future reward expectations. Despite an extensive body of literature on the influence of RPE on learning, little has been done to investigate the potentially separate contributions of RPE valence (positive or negative) and surprise (absolute degree of deviation from expectations). Here, we coupled single-trial electroencephalography with simultaneously acquired fMRI, during a probabilistic reversal-learning task, to offer evidence of temporally overlapping but largely distinct spatial representations of RPE valence and surprise. Electrophysiological variability in RPE valence correlated with activity in regions of the human reward network promoting approach or avoidance learning. Electrophysiological variability in RPE surprise correlated primarily with activity in regions of the human attentional network controlling the speed of learning. Crucially, despite the largely separate spatial extend of these representations our EEG-informed fMRI approach uniquely revealed a linear superposition of the two RPE components in a smaller network encompassing visuo-mnemonic and reward areas. Activity in this network was further predictive of stimulus value updating indicating a comparable contribution of both signals to reward learning.

The 3' untranslated region of the cyclin B mRNA is not sufficient to enhance the synthesis of cyclin B during a mitotic block in human cells.

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Dominik Schnerch

Full Text Available Antimitotic agents are frequently used to treat solid tumors and hematologic malignancies. However, one major limitation of antimitotic approaches is mitotic slippage, which is driven by slow degradation of cyclin B during a mitotic block. The extent to which cyclin B levels decline is proposed to be governed by an equilibrium between cyclin B synthesis and degradation. It was recently shown that the 3' untranslated region (UTR of the murine cyclin B mRNA contributes to the synthesis of cyclin B during mitosis in murine cells. Using a novel live-cell imaging-based technique allowing us to study synthesis and degradation of cyclin B simultaneously at the single cell level, we tested here the role of the human cyclin B 3'UTR in regulating cyclin B synthesis during mitosis in human cells. We observed that the cyclin B 3'UTR was not sufficient to enhance cyclin B synthesis in human U2Os, HeLa or hTERT RPE-1 cells. A better understanding of how the equilibrium of cyclin B is regulated in mitosis may contribute to the development of improved therapeutic approaches to prevent mitotic slippage in cancer cells treated with antimitotic agents.

Neural retina-specific Aldh1a1 controls dorsal choroidal vascular development via Sox9 expression in retinal pigment epithelial cells.

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Goto, So; Onishi, Akishi; Misaki, Kazuyo; Yonemura, Shigenobu; Sugita, Sunao; Ito, Hiromi; Ohigashi, Yoko; Ema, Masatsugu; Sakaguchi, Hirokazu; Nishida, Kohji; Takahashi, Masayo

2018-04-03

VEGF secreted from retinal pigment epithelial (RPE) cells is responsible for the choroidal vascular development; however, the molecular regulatory mechanism is unclear. We found that Aldh1a1 -/- mice showed choroidal hypoplasia with insufficient vascularization in the dorsal region, although Aldh1a1, an enzyme that synthesizes retinoic acids (RAs), is expressed in the dorsal neural retina, not in the RPE/choroid complex. The level of VEGF in the RPE/choroid was significantly decreased in Aldh1a1 -/- mice, and RA-dependent enhancement of VEGF was observed in primary RPE cells. An RA-deficient diet resulted in dorsal choroidal hypoplasia, and simple RA treatment of Aldh1a1 -/- pregnant females suppressed choroid hypoplasia in their offspring. We also found downregulation of Sox9 in the dorsal neural retina and RPE of Aldh1a1 -/- mice and RPE-specific disruption of Sox9 phenocopied Aldh1a1 -/- choroidal development. These results suggest that RAs produced by Aldh1a1 in the neural retina directs dorsal choroidal vascular development via Sox9 upregulation in the dorsal RPE cells to enhance RPE-derived VEGF secretion. © 2018, Goto et al.

Celecoxib Down-Regulates the Hypoxia-Induced Expression of HIF-1α and VEGF Through the PI3K/AKT Pathway in Retinal Pigment Epithelial Cells

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Yi-zhou Sun

2017-12-01

Full Text Available Background/Aims: The goal of this study was to detect the expression of hypoxia-inducible factor 1α (HIF-1α and vascular endothelial growth factor (VEGF in human retinal pigmented epithelial (RPE cells treated with celecoxib, a selective cyclooxygenase-2 (COX-2 inhibitor, under hypoxic and normoxic conditions and to explore the signaling mechanism involved in regulating the hypoxia-induced expression of HIF-1α and VEGF in RPE cells. Methods: D407 cells were cultured in normoxic or hypoxic conditions, with or without celecoxib or a PI3K inhibitor (LY294002. The anti-proliferative effect of celecoxib was assessed using the MTT assay. RT-PCR, Western blotting and ELISA were performed to detect the levels of PI3K, phosphorylated AKT (p-AKT, HIF-1α, VEGF and COX-2. Results: Celecoxib inhibited the proliferation of RPE cells in a dose-dependent manner. Celecoxib suppressed the expression of VEGF at both the mRNA and protein levels and decreased HIF-1α protein expression. HIF-1α activation was regulated by the PI3K/AKT pathway. The celecoxib-induced down-regulation of HIF-1α and VEGF required the suppression of the hypoxia-induced PI3K/AKT pathway. However, the down-regulation of COX-2 did not occur in cells treated with celecoxib. Conclusions: The antiangiogenic effects of celecoxib in RPE cells under hypoxic conditions resulted from the inhibition of HIF-1α and VEGF expression, which may be partly mediated by a COX-2-independent, PI3K/AKT-dependent pathway.

Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

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Ming-Wai Poon

Full Text Available A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs. Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2 reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%. Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs. This cell type may also have advantages in retinal pigmented epithelial differentiation.

Human Ocular Epithelial Cells Endogenously Expressing SOX2 and OCT4 Yield High Efficiency of Pluripotency Reprogramming.

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Poon, Ming-Wai; He, Jia; Fang, Xiaowei; Zhang, Zhao; Wang, Weixin; Wang, Junwen; Qiu, Fangfang; Tse, Hung-Fat; Li, Wei; Liu, Zuguo; Lian, Qizhou

2015-01-01

A variety of pluripotency reprogramming frequencies from different somatic cells has been observed, indicating cell origin is a critical contributor for efficiency of pluripotency reprogramming. Identifying the cell sources for efficient induced pluripotent stem cells (iPSCs) generation, and defining its advantages or disadvantages on reprogramming, is therefore important. Human ocular tissue-derived conjunctival epithelial cells (OECs) exhibited endogenous expression of reprogramming factors OCT4A (the specific OCT 4 isoform on pluripotency reprogramming) and SOX2. We therefore determined whether OECs could be used for high efficiency of iPSCs generation. We compared the endogenous expression levels of four pluripotency factors and the pluripotency reprograming efficiency of human OECs with that of ocular stromal cells (OSCs). Real-time PCR, microarray analysis, Western blotting and immunostaining assays were employed to compare OECiPSCs with OSCiPSCs on molecular bases of reprogramming efficiency and preferred lineage-differentiation potential. Using the traditional KMOS (KLF4, C-MYC, OCT4 and SOX2) reprogramming protocol, we confirmed that OECs, endogenously expressing reprogramming factors OCT4A and SOX2, yield very high efficiency of iPSCs generation (~1.5%). Furthermore, higher efficiency of retinal pigmented epithelial differentiation (RPE cells) was observed in OECiPSCs compared to OSCiPSCs or skin fibroblast iMR90iPSCs. The findings in this study suggest that conjunctival-derived epithelial (OECs) cells can be easier converted to iPSCs than conjunctival-derived stromal cells (OSCs). This cell type may also have advantages in retinal pigmented epithelial differentiation.

Intraocular distribution of melanin in human, monkey, rabbit, minipig and dog eyes.

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Durairaj, Chandrasekar; Chastain, James E; Kompella, Uday B

2012-05-01

The purpose of this study was to quantify the melanin pigment content in sclera, choroid-RPE, and retina, three tissues encountered during transscleral drug delivery to the vitreous, in human, rabbit, monkey, minipig, and dog models. Strain differences were assessed in NZW × NZR F1 and Dutch belted rabbits and Yucatan and Gottingen minipigs. The choroid-RPE and retina tissues were divided into central (posterior pole area) and peripheral (away from posterior pole) regions while the sclera was analyzed without such division. Melanin content in the tissues was analyzed using a colorimetric assay. In all species the rank order for pigment content was: choroid-RPE >retina ≥ sclera, except in humans, where scleral melanin levels were higher than retina and central choroid. The melanin content in a given tissue differed between species. Further, while the peripheral tissue pigment levels tended to be generally higher compared to the central regions, these differences were significant in human in the case of choroid-RPE and in human, monkey, and dogs in the case of retina. Strain difference was observed only in the central choroid-RPE region of rabbits (NZW × NZR F1 >Dutch Belted). Species, strain, and regional differences exist in the melanin pigment content in the tissues of the posterior segment of the eye, with Gottingen minipig being closest to humans among the animals assessed. These differences in melanin content might contribute to differences in drug binding, delivery, and toxicity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of a class of nonlinear state-space models using RPE techniques

DEFF Research Database (Denmark)

Zhou, W. W.; Blanke, Mogens

1986-01-01

The recursive prediction error methods in state-space form have been efficiently used as parameter identifiers for linear systems, and especially Ljung's innovations filter using a Newton search direction has proved to be quite ideal. In this paper, the RPE method in state-space form is developed...... a quite convincing performance of the filter as combined parameter and state estimator....

Immune Privilege and Eye-Derived T-Regulatory Cells

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Hiroshi Keino

2018-01-01

Full Text Available Certain cellular components of the eye, such as neural retina, are unable to regenerate and replicate after destructive inflammation. Ocular immune privilege provides the eye with immune protection against intraocular inflammation in order to minimize the risk to vision integrity. The eye and immune system use strategies to maintain the ocular immune privilege by regulating the innate and adaptive immune response, which includes immunological ignorance, peripheral tolerance to eye-derived antigens, and intraocular immunosuppressive microenvironment. In this review, we summarize current knowledge regarding the molecular mechanism responsible for the development and maintenance of ocular immune privilege via regulatory T cells (Tregs, which are generated by the anterior chamber-associated immune deviation (ACAID, and ocular resident cells including corneal endothelial (CE cells, ocular pigment epithelial (PE cells, and aqueous humor. Furthermore, we examined the therapeutic potential of Tregs generated by RPE cells that express transforming growth factor beta (TGF-β, cytotoxic T lymphocyte-associated antigen-2 alpha (CTLA-2α, and retinoic acid for autoimmune uveoretinitis and evaluated a new strategy using human RPE-induced Tregs for clinical application in inflammatory ocular disease. We believe that a better understanding of the ocular immune privilege associated with Tregs might offer a new approach with regard to therapeutic interventions for ocular autoimmunity.

Immune Privilege and Eye-Derived T-Regulatory Cells.

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Keino, Hiroshi; Horie, Shintaro; Sugita, Sunao

2018-01-01

Certain cellular components of the eye, such as neural retina, are unable to regenerate and replicate after destructive inflammation. Ocular immune privilege provides the eye with immune protection against intraocular inflammation in order to minimize the risk to vision integrity. The eye and immune system use strategies to maintain the ocular immune privilege by regulating the innate and adaptive immune response, which includes immunological ignorance, peripheral tolerance to eye-derived antigens, and intraocular immunosuppressive microenvironment. In this review, we summarize current knowledge regarding the molecular mechanism responsible for the development and maintenance of ocular immune privilege via regulatory T cells (Tregs), which are generated by the anterior chamber-associated immune deviation (ACAID), and ocular resident cells including corneal endothelial (CE) cells, ocular pigment epithelial (PE) cells, and aqueous humor. Furthermore, we examined the therapeutic potential of Tregs generated by RPE cells that express transforming growth factor beta (TGF- β ), cytotoxic T lymphocyte-associated antigen-2 alpha (CTLA-2 α ), and retinoic acid for autoimmune uveoretinitis and evaluated a new strategy using human RPE-induced Tregs for clinical application in inflammatory ocular disease. We believe that a better understanding of the ocular immune privilege associated with Tregs might offer a new approach with regard to therapeutic interventions for ocular autoimmunity.

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

2009-11-01

Full Text Available 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.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.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.

Profile of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium determined through serial analysis of gene expression (SAGE)

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Sharon, Dror; Blackshaw, Seth; Cepko, Constance L.; Dryja, Thaddeus P.

2002-01-01

We used the serial analysis of gene expression (SAGE) technique to catalogue and measure the relative levels of expression of the genes expressed in the human peripheral retina, macula, and retinal pigment epithelium (RPE) from one or both of two humans, aged 88 and 44 years. The cone photoreceptor contribution to all transcription in the retina was found to be similar in the macula versus the retinal periphery, whereas the rod contribution was greater in the periphery versus the macula. Genes encoding structural proteins for axons were found to be expressed at higher levels in the macula versus the retinal periphery, probably reflecting the large proportion of ganglion cells in the central retina. In comparison with the younger eye, the peripheral retina of the older eye had a substantially higher proportion of mRNAs from genes encoding proteins involved in iron metabolism or protection against oxidative damage and a substantially lower proportion of mRNAs from genes encoding proteins involved in rod phototransduction. These differences may reflect the difference in age between the two donors or merely interindividual variation. The RPE library had numerous previously unencountered tags, suggesting that this cell type has a large, idiosyncratic repertoire of expressed genes. Comparison of these libraries with 100 reported nonocular SAGE libraries revealed 89 retina-specific or enriched genes expressed at substantial levels, of which 14 are known to cause a retinal disease and 53 are RPE-specific genes. We expect that these libraries will serve as a resource for understanding the relative expression levels of genes in the retina and the RPE and for identifying additional disease genes. PMID:11756676

Development of an automated chip culture system with integrated on-line monitoring for maturation culture of retinal pigment epithelial cells

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Mee-Hae Kim

2017-10-01

Full Text Available In cell manufacturing, the establishment of a fully automated, microfluidic, cell culture system that can be used for long-term cell cultures, as well as for process optimization is highly desirable. This study reports the development of a novel chip bioreactor system that can be used for automated long-term maturation cultures of retinal pigment epithelial (RPE cells. The system consists of an incubation unit, a medium supply unit, a culture observation unit, and a control unit. In the incubation unit, the chip contains a closed culture vessel (2.5 mm diameter, working volume 9.1 μL, which can be set to 37 °C and 5% CO2, and uses a gas-permeable resin (poly- dimethylsiloxane as the vessel wall. RPE cells were seeded at 5.0 × 104 cells/cm2 and the medium was changed every day by introducing fresh medium using the medium supply unit. Culture solutions were stored either in the refrigerator or the freezer, and fresh medium was prepared before any medium change by warming to 37 °C and mixing. Automated culture was allowed to continue for 30 days to allow maturation of the RPE cells. This chip culture system allows for the long-term, bubble-free, culture of RPE cells, while also being able to observe cells in order to elucidate their cell morphology or show the presence of tight junctions. This culture system, along with an integrated on-line monitoring system, can therefore be applied to long-term cultures of RPE cells, and should contribute to process control in RPE cell manufacturing.

Choice of Cell Source in Cell-Based Therapies for Retinal Damage due to Age-Related Macular Degeneration: A Review

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Sudhakar John

2013-01-01

Full Text Available Background. Age-related macular degeneration (AMD is a complex disorder that affects primarily the macula involving the retinal pigment epithelium (RPE but also to a certain extent the photoreceptor layer and the retinal neurons. Cell transplantation is a promising option for AMD and clinical trials are underway using different cell types. Methods. We hypothesize that instead of focusing on a particular cell source for concurrent regeneration of all the retinal layers and also to prevent exhaustive research on an array of cell sources for regeneration of each layer, the choice should depend on, precisely, which layer is damaged. Results. Thus, for a damage limited to the retinal pigment epithelial (RPE layer, the choice we suggest would be RPE cells. When the damage extends to rods and cones, the choice would be bone marrow stem cells and when retinal neurons are involved, relatively immature stem cell populations with an inherent capacity to yield neuronal lineage such as hematopoietic stem cells, embryonic stem cells, or induced pluripotent stem cells can be tried. Conclusion. This short review will prove to be a valuable guideline for those working on cell therapy for AMD to plan their future directions of research and therapy for this condition.

The P2Y12 Receptor Antagonist Ticagrelor Reduces Lysosomal pH and Autofluorescence in Retinal Pigmented Epithelial Cells From the ABCA4-/- Mouse Model of Retinal Degeneration

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Wennan Lu

2018-04-01

Full Text Available The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent lysosome-associated organelles of the retinal pigmented epithelial (RPE cells, and may be related to early stages of age-related macular degeneration. While lysosomal enzymes degrade material optimally at acidic pH levels, lysosomal pH is elevated in RPE cells from the ABCA4-/- mouse model of Stargardt’s disease, an early onset retinal degeneration. Lowering lysosomal pH through cAMP-dependent pathways decreases accumulation of autofluorescent material in RPE cells in vitro, but identification of an appropriate receptor is crucial for manipulating this pathway in vivo. As the P2Y12 receptor for ADP is coupled to the inhibitory Gi protein, we asked whether blocking the P2Y12 receptor with ticagrelor could restore lysosomal acidity and reduce autofluorescence in compromised RPE cells from ABCA4-/- mice. Oral delivery of ticagrelor giving rise to clinically relevant exposure lowered lysosomal pH in these RPE cells. Ticagrelor also partially reduced autofluorescence in the RPE cells of ABCA4-/- mice. In vitro studies in ARPE-19 cells using more specific antagonists AR-C69931 and AR-C66096 confirmed the importance of the P2Y12 receptor for lowering lysosomal pH and reducing autofluorescence. These observations identify P2Y12 receptor blockade as a potential target to lower lysosomal pH and clear lysosomal waste in RPE cells.

Protection and sensitization of normal and malignant cells by a naturally occurring compound in a model of photochemical damage

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Lee, Yuan-Hao; Kumar, Neeru; Glickman, Randolph D.

2012-03-01

Certain phytonutrients are known to confer protection and immunosuppression against radiation insults. Radiation-induced reactive oxygen species (ROS) can either lead to the destruction of normal tissue cells, or induce tumor radioresistance by activating ROS scavenging proteins. To identify whether the triterpene phytonutrient, ursolic acid, reduces radiation-induced damage in normal cells and promotes the apoptosis of malignant cells, we investigated the biologic mechanisms and effect of radiation-cell interaction with or without treatment with ursolic acid in human skin melanoma cells (ATCC CRL-11147TM) and transformed human retinal pigment epithelial (hTERT-RPE) cells. UV-VIS light was employed to investigate the efficacy of ursolic acid in altering cellular viability by modulations of p53 and NF-κB p65 signaling. Cell response was investigated by changes in proliferative activity and free radical generation assessed by 2',7'-dichlorofluorescin liquid chromatography. Ursolic acid pretreatment strongly increased the level of p53 and decreased the level of phosphorylated p65 leading to enhanced cell death of skin melanoma cells in response to UV-VIS exposure. In contrast, ursolic acid appeared to downregulate p53 levels without disturbing NF-κB activation along with an increase of oxidative stress in hTERT-RPE cells. These findings indicate that ursolic acid may beneficially increase the radiosensitivity of tumor cells while potentiating a photoprotective effect on benign cells through differential effects on the NF-κB and p53 signaling pathways.

Thrombin induces epithelial-mesenchymal transition and collagen production by retinal pigment epithelial cells via autocrine PDGF-receptor signaling.

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Bastiaans, Jeroen; van Meurs, Jan C; van Holten-Neelen, Conny; Nagtzaam, Nicole M A; van Hagen, P Martin; Chambers, Rachel C; Hooijkaas, Herbert; Dik, Willem A

2013-12-19

De-differentiation of RPE cells into mesenchymal cells (epithelial-mesenchymal transition; EMT) and associated collagen production contributes to development of proliferative vitreoretinopathy (PVR). In patients with PVR, intraocular coagulation cascade activation occurs and may play an important initiating role. Therefore, we examined the effect of the coagulation proteins factor Xa and thrombin on EMT and collagen production by RPE cells. Retinal pigment epithelial cells were stimulated with factor Xa or thrombin and the effect on zonula occludens (ZO)-1, α-smooth muscle actin (α-SMA), collagen, and platelet-derived growth factor (PDGF)-B were determined by real-time quantitative-polymerase chain reaction (RQ-PCR), immunofluorescence microscopy, and HPLC and ELISA for collagen and PDGF-BB in culture supernatants, respectively. PDGF-receptor activation was determined by phosphorylation analysis and inhibition studies using the PDGF-receptor tyrosine kinase inhibitor AG1296. Thrombin reduced ZO-1 gene expression (P production of α-SMA and collagen increased. In contrast to thrombin, factor Xa hardly stimulated EMT by RPE. Thrombin clearly induced PDGF-BB production and PDGF-Rβ chain phosphorylation in RPE. Moreover, AG1296 significantly blocked the effect of thrombin on EMT and collagen production. Our findings demonstrate that thrombin is a potent inducer of EMT by RPE via autocrine activation of PDGF-receptor signaling. Coagulation cascade-induced EMT of RPE may thus contribute to the formation of fibrotic retinal membranes in PVR and should be considered as treatment target in PVR.

Results at 2 Years after Gene Therapy for RPE65-Deficient Leber Congenital Amaurosis and Severe Early-Childhood-Onset Retinal Dystrophy.

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Weleber, Richard G; Pennesi, Mark E; Wilson, David J; Kaushal, Shalesh; Erker, Laura R; Jensen, Lauren; McBride, Maureen T; Flotte, Terence R; Humphries, Margaret; Calcedo, Roberto; Hauswirth, William W; Chulay, Jeffrey D; Stout, J Timothy

2016-07-01

To provide an initial assessment of the safety of a recombinant adeno-associated virus vector expressing RPE65 (rAAV2-CB-hRPE65) in adults and children with retinal degeneration caused by RPE65 mutations. Nonrandomized, multicenter clinical trial. Eight adults and 4 children, 6 to 39 years of age, with Leber congenital amaurosis (LCA) or severe early-childhood-onset retinal degeneration (SECORD). Patients received a subretinal injection of rAAV2-CB-hRPE65 in the poorer-seeing eye, at either of 2 dose levels, and were followed up for 2 years after treatment. The primary safety measures were ocular and nonocular adverse events. Exploratory efficacy measures included changes in best-corrected visual acuity (BCVA), static perimetry central 30° visual field hill of vision (V30) and total visual field hill of vision (VTOT), kinetic perimetry visual field area, and responses to a quality-of-life questionnaire. All patients tolerated subretinal injections and there were no treatment-related serious adverse events. Common adverse events were those associated with the surgical procedure and included subconjunctival hemorrhage in 8 patients and ocular hyperemia in 5 patients. In the treated eye, BCVA increased in 5 patients, V30 increased in 6 patients, VTOT increased in 5 patients, and kinetic visual field area improved in 3 patients. One subject showed a decrease in BCVA and 2 patients showed a decrease in kinetic visual field area. Treatment with rAAV2-CB-hRPE65 was not associated with serious adverse events, and improvement in 1 or more measures of visual function was observed in 9 of 12 patients. The greatest improvements in visual acuity were observed in younger patients with better baseline visual acuity. Evaluation of more patients and a longer duration of follow-up will be needed to determine the rate of uncommon or rare side effects or safety concerns. Copyright © 2016 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

Rescue of compromised lysosomes enhances degradation of photoreceptor outer segments and reduces lipofuscin-like autofluorescence in retinal pigmented epithelial cells.

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Guha, Sonia; Liu, Ji; Baltazar, Gabe; Laties, Alan M; Mitchell, Claire H

2014-01-01

Healthful cell maintenance requires the efficient degradative processing and removal of waste material. Retinal pigmented epithelial (RPE) cells have the onerous task of degrading both internal cellular debris generated through autophagy as well as phagocytosed photoreceptor outer segments. We propose that the inadequate processing material with the resulting accumulation of cellular waste contributes to the downstream pathologies characterized as age-related macular degeneration (AMD). The lysosomal enzymes responsible for clearance function optimally over a narrow range of acidic pH values; elevation of lysosomal pH by compounds like chloroquine or A2E can impair degradative enzyme activity and lead to a lipofuscin-like autofluorescence. Restoring acidity to the lysosomes of RPE cells can enhance activity of multiple degradative enzymes and is therefore a logical target in early AMD. We have identified several approaches to reacidify lysosomes of compromised RPE cells; stimulation of beta-adrenergic, A2A adenosine and D5 dopamine receptors each lowers lysosomal pH and improves degradation of outer segments. Activation of the CFTR chloride channel also reacidifies lysosomes and increases degradation. These approaches also restore the lysosomal pH of RPE cells from aged ABCA4(-/-) mice with chronically high levels of A2E, suggesting that functional signaling pathways to reacidify lysosomes are retained in aged cells like those in patients with AMD. Acidic nanoparticles transported to RPE lysosomes also lower pH and improve degradation of outer segments. In summary, the ability of diverse approaches to lower lysosomal pH and enhance outer segment degradation support the proposal that lysosomal acidification can prevent the accumulation of lipofuscin-like material in RPE cells.

Cell-based therapeutic strategies for replacement and preservation in retinal degenerative diseases

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Jones, Melissa K.; Lu, Bin; Girman, Sergey; Wang, Shaomei

2017-01-01

Cell-based therapeutics offer diverse options for treating retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). AMD is characterized by both genetic and environmental risks factors, whereas RP is mainly a monogenic disorder. Though treatments exist for some patients with neovascular AMD, a majority of retinal degenerative patients have no effective therapeutics, thus indicating a need for universal therapies to target diverse patient populations. Two main cell-based mechanistic approaches are being tested in clinical trials. Replacement therapies utilize cell-derived retinal pigment epithelial (RPE) cells to supplant lost or defective host RPE cells. These cells are similar in morphology and function to native RPE cells and can potentially supplant the responsibilities of RPE in vivo. Preservation therapies utilize supportive cells to aid in visual function and photoreceptor preservation partially by neurotrophic mechanisms. The goal of preservation strategies is to halt or slow the progression of disease and maintain remaining visual function. A number of clinical trials are testing the safety of replacement and preservation cell therapies in patients; however, measures of efficacy will need to be further evaluated. In addition, a number of prevailing concerns with regards to the immune-related response, longevity, and functionality of the grafted cells will need to be addressed in future trials. This review will summarize the current status of cell-based preclinical and clinical studies with a focus on replacement and preservation strategies and the obstacles that remain regarding these types of treatments. PMID:28111323

Identification of a Class of Non-linear State Space Models using RPE Techniques

DEFF Research Database (Denmark)

Zhou, Wei-Wu; Blanke, Mogens

1989-01-01

The RPE (recursive prediction error) method in state-space form is developed in the nonlinear systems and extended to include the exact form of a nonlinearity, thus enabling structure preservation for certain classes of nonlinear systems. Both the discrete and the continuous-discrete versions...... of the algorithm in an innovations model are investigated, and a nonlinear simulation example shows a quite convincing performance of the filter as combined parameter and state estimator...

Subunits of highly Fluorescent Protein R-Phycoerythrin as Probes for Cell Imaging and Single-Molecule Detection

Energy Technology Data Exchange (ETDEWEB)

Isailovic, Dragan [Iowa State Univ., Ames, IA (United States)

2005-01-01

The purposes of our research were: (1) To characterize subunits of highly fluorescent protein R-Phycoerythrin (R-PE) and check their suitability for single-molecule detection (SMD) and cell imaging, (2) To extend the use of R-PE subunits through design of similar proteins that will be used as probes for microscopy and spectral imaging in a single cell, and (3) To demonstrate a high-throughput spectral imaging method that will rival spectral flow cytometry in the analysis of individual cells. We first demonstrated that R-PE subunits have spectroscopic and structural characteristics that make them suitable for SMD. Subunits were isolated from R-PE by high-performance liquid chromatography (HPLC) and detected as single molecules by total internal reflection fluorescence microscopy (TIRFM). In addition, R-PE subunits and their enzymatic digests were characterized by several separation and detection methods including HPLC, capillary electrophoresis, sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) and HPLC-electrospray ionization mass spectrometry (ESI-MS). Favorable absorption and fluorescence of the R-PE subunits and digest peptides originate from phycoerythrobilin (PEB) and phycourobilin (PUB) chromophores that are covalently attached to cysteine residues. High absorption coefficients and strong fluorescence (even under denaturing conditions), broad excitation and emission fluorescence spectra in the visible region of electromagnetic spectrum, and relatively low molecular weights make these molecules suitable for use as fluorescence labels of biomolecules and cells. We further designed fluorescent proteins both in vitro and in vivo (in Escherichia coli) based on the highly specific attachment of PEB chromophore to genetically expressed apo-subunits of R-PE. In one example, apo-alpha and apo-beta R-PE subunits were cloned from red algae Polisiphonia boldii (P. boldii), and expressed in E. coli. Although expressed apo-subunits formed inclusion

Generation of retinal pigmented epithelium from iPSCs derived from the conjunctiva of donors with and without age related macular degeneration.

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Zhouhui Geng

Full Text Available Fidelity in pluripotent stem cell differentiation protocols is necessary for the therapeutic and commercial use of cells derived from embryonic and induced pluripotent stem cells. Recent advances in stem cell technology, especially the widespread availability of a range of chemically defined media, substrates and differentiation components, now allow the design and implementation of fully defined derivation and differentiation protocols intended for replication across multiple research and manufacturing locations. In this report we present an application of these criteria to the generation of retinal pigmented epithelium from iPSCs derived from the conjunctiva of donors with and without age related macular degeneration. Primary conjunctival cells from human donors aged 70-85 years were reprogrammed to derive multiple iPSC lines that were differentiated into functional RPE using a rapid and defined differentiation protocol. The combination of defined iPSC derivation and culture with a defined RPE differentiation protocol, reproducibly generated functional RPE from each donor without requiring protocol adjustments for each individual. This successful validation of a standardized, iPSC derivation and RPE differentiation process demonstrates a practical approach for applications requiring the cost-effective generation of RPE from multiple individuals such as drug testing, population studies or for therapies requiring patient-specific RPE derivations. In addition, conjunctival cells are identified as a practical source of somatic cells for deriving iPSCs from elderly individuals.

Modulation of photochemical damage in normal and malignant cells by naturally occurring compounds.

Science.gov (United States)

Lee, Yuan-Hao; Kumar, Neeru C; Glickman, Randolph D

2012-01-01

Certain phytochemicals, such as the stilbene, resveratrol (RES, found in red grapes and berries), and the triterpenoid, ursolic acid (UA, found in waxy berries and herbs such as rosemary and oregano), have antioxidant, anti-inflammatory and antiproliferative effects. Two human-derived cell lines, hTERT-RPE with a nonmalignant phenotype derived from retinal pigment epithelium, and ATCC CRL-11147 derived from a malignant skin melanoma, were used as in vitro models of photooxidative stress produced by exposure to the broadband output of a 150 W Hg vapor arc lamp at an irradiance of 19-26 mW cm(-2). In untreated cells, UV-VIS broadband light exposure produced a loss of proliferative ability, an activation of NF-κB and an increase in protein carbonyl adducts at 24 h postexposure. Pretreatment of the cells with RES or UA at 1-2 μmsignificantly reduced the amount of phosphorylated NF-κB at 24 h postexposure. RES pretreatment reduced the burden of light-induced protein carbonyl adducts by up to 25% in exposed cells. UA treatment markedly increased the sensitivity of melanoma cells to UV radiation, while conferring some photoprotection to RPE cells. These observations indicate that phytochemicals modulate the cellular response to photochemical stress by interacting with specific cell-signaling pathways. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

Attenuation of iron-binding proteins in ARPE-19 cells reduces their resistance to oxidative stress.

Science.gov (United States)

Karlsson, Markus; Kurz, Tino

2016-09-01

Oxidative stress-related damage to retinal pigment epithelial (RPE) cells is an important feature in the development of age-related macular degeneration. Iron-catalysed intralysosomal production of hydroxyl radicals is considered a major pathogenic factor, leading to lipofuscin formation with ensuing depressed cellular autophagic capacity, lysosomal membrane permeabilization and apoptosis. Previously, we have shown that cultured immortalized human RPE (ARPE-19) cells are extremely resistant to exposure to bolus doses of hydrogen peroxide and contain considerable amounts of the iron-binding proteins metallothionein (MT), heat-shock protein 70 (HSP70) and ferritin (FT). According to previous findings, autophagy of these proteins depresses lysosomal redox-active iron. The aim of this study was to investigate whether up- or downregulation of these proteins would affect the resistance of ARPE-19 cells to oxidative stress. The sensitivity of ARPE-19 cells to H2 O2 exposure was tested following upregulation of MT, HSP70 and/or FT by pretreatment with ZnSO4 , heat shock or FeCl3 , as well as siRNA-mediated downregulation of the same proteins. Upregulation of MT, HSP70 and FT did not improve survival following exposure to H2 O2 . This was interpreted as existence of an already maximal protection. Combined siRNA-mediated attenuation of both FT chains (H and L), or simultaneous downregulation of all three proteins, made the cells significantly more susceptible to oxidative stress confirming the importance of iron-binding proteins. The findings support our hypothesis that the oxidative stress resistance exhibited by RPE cells may be explained by a high autophagic influx of iron-binding proteins that would keep levels of redox-active lysosomal iron low. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

Toll-Like Receptor-3 and Geographic Atrophy in Age-Related Macular Degeneration

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Yang, Zhenglin; Stratton, Charity; Francis, Peter J.; Kleinman, Mark E.; Tan, Perciliz L.; Gibbs, Daniel; Tong, Zongzhong; Chen, Haoyu; Constantine, Ryan; Yang, Xian; Chen, Yuhong; Zeng, Jiexi; Davey, Lisa; Ma, Xiang; Hau, Vincent S.; Wang, Chi; Harmon, Jennifer; Buehler, Jeanette; Pearson, Erik; Patel, Shrena; Kaminoh, Yuuki; Watkins, Scott; Luo, Ling; Zabriskie, Norman A.; Bernstein, Paul S.; Cho, Wongil; Schwager, Andrea; Hinton, David R; Klein, Michael L; Hamon, Sara C.; Simmons, Emily; Yu, Beifeng; Campochiaro, Betsy; Sunness, Janet S.; Campochiaro, Peter; Jorde, Lynn; Parmigiani, Giovanni; Zack, Donald J.; Katsanis, Nicholas; Ambati, Jayakrishna; Zhang, Kang

2008-01-01

BACKGROUND Age-related macular degeneration (AMD) is the most common cause of irreversible visual impairment in the developed world. Advanced AMD is comprised of geographic atrophy (GA) and choroidal neovascularization (CNV). Specific genetic variants that predispose for GA are largely unknown. METHODS We tested (i) for association between the functional toll-like receptor-3 (TLR3) variant rs3775291 (L412F) and AMD in European Americans and (ii) the effect of TLR3 L and F variants on the viability of human retinal pigment epithelium (RPE) cells in vitro and on RPE cell apoptosis in wildtype and Tlr3−/− mice. RESULTS The F variant (or T allele at single nucleotide polymorphism at rs3775291) was associated with protection against GA (P=0.005); this association was replicated in two independent GA case-control series (P=5.43×10−4 and P=0.002, respectively. We observed no association between TLR3 variants and CNV. The rs377291 variant is probably critical to the function of TLR3, because a prototypic TLR3 ligand induced cell death and apoptosis in human RPE cells with the LL genotype to a greater extent than it did RPE cells with the LF genotype. Moreover, the ligand induced more RPE cell death and apoptosis in wild-type than in Tlr3−/− mice. CONCLUSIONS The TLR3 412F variant confers protection against GA, probably by suppressing RPE cell death. Given that double stranded RNA can activate TLR3-mediated apoptosis, our results suggest a possible role for viral dsRNA transcripts in the development of GA and raise awareness of potential toxicity induced by short interfering RNA (siRNA) therapeutics in the eye. PMID:18753640

Cellular models and therapies for age-related macular degeneration

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David L. Forest

2015-05-01

Full Text Available Age-related macular degeneration (AMD is a complex neurodegenerative visual disorder that causes profound physical and psychosocial effects. Visual impairment in AMD is caused by the loss of retinal pigmented epithelium (RPE cells and the light-sensitive photoreceptor cells that they support. There is currently no effective treatment for the most common form of this disease (dry AMD. A new approach to treating AMD involves the transplantation of RPE cells derived from either human embryonic or induced pluripotent stem cells. Multiple clinical trials are being initiated using a variety of cell therapies. Although many animal models are available for AMD research, most do not recapitulate all aspects of the disease, hampering progress. However, the use of cultured RPE cells in AMD research is well established and, indeed, some of the more recently described RPE-based models show promise for investigating the molecular mechanisms of AMD and for screening drug candidates. Here, we discuss innovative cell-culture models of AMD and emerging stem-cell-based therapies for the treatment of this vision-robbing disease.

Progress of stem/progenitor cell-based therapy for retinal degeneration.

Science.gov (United States)

Tang, Zhimin; Zhang, Yi; Wang, Yuyao; Zhang, Dandan; Shen, Bingqiao; Luo, Min; Gu, Ping

2017-05-10

Retinal degeneration (RD), such as age-related macular degeneration (AMD) and retinitis pigmentosa, is one of the leading causes of blindness. Presently, no satisfactory therapeutic options are available for these diseases principally because the retina and retinal pigmented epithelium (RPE) do not regenerate, although wet AMD can be prevented from further progression by anti-vascular endothelial growth factor therapy. Nevertheless, stem/progenitor cell approaches exhibit enormous potential for RD treatment using strategies mainly aimed at the rescue and replacement of photoreceptors and RPE. The sources of stem/progenitor cells are classified into two broad categories in this review, which are (1) ocular-derived progenitor cells, such as retinal progenitor cells, and (2) non-ocular-derived stem cells, including embryonic stem cells, induced pluripotent stem cells, and mesenchymal stromal cells. Here, we discuss in detail the progress in the study of four predominant stem/progenitor cell types used in animal models of RD. A short overview of clinical trials involving the stem/progenitor cells is also presented. Currently, stem/progenitor cell therapies for RD still have some drawbacks such as inhibited proliferation and/or differentiation in vitro (with the exception of the RPE) and limited long-term survival and function of grafts in vivo. Despite these challenges, stem/progenitor cells represent the most promising strategy for RD treatment in the near future.

Some regularities in the transport and radioprotective efficiency of methylated hematoporphyrin in experiments of cell cultures

International Nuclear Information System (INIS)

Yartsev, E.I.; Novosel'tseva, S.D.; Shkirman, S.F.; Arabej, S.M.; Solov'ev, K.N.; Ponomarev, G.V.

1982-01-01

It was shown on a SOC culture that radioprotective effectiveness (RPE) of a methylated derivative of deuteroporphyrin-9 depends upon the level of accumulation there of in cells. The highest RPE was registered with porphyrin administered to a culture medium 8 h before irradiation of cells. By that time, exogenous porphyrin was accumulated by cells to reach a definite stable level. The content of the preparation in cells decreases depending on radiation dose. It was noted that the radioresistance of cells remained increased with considerable porphyrin excretion from there

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

International Nuclear Information System (INIS)

Li, Hui; Li, Min; Xu, Ding; Zhao, Chun; Liu, Guodong; Wang, Fang

2014-01-01

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

Disease susceptibility of the human macula: differential gene transcription in the retinal pigmented epithelium/choroid.

Science.gov (United States)

Radeke, Monte J; Peterson, Katie E; Johnson, Lincoln V; Anderson, Don H

2007-09-01

The discoveries of gene variants associated with macular diseases have provided valuable insight into their molecular mechanisms, but they have not clarified why the macula is particularly vulnerable to degenerative disease. Its predisposition may be attributable to specialized structural features and/or functional properties of the underlying macular RPE/choroid. To examine the molecular basis for the macula's disease susceptibility, we compared the gene expression profile of the human RPE/choroid in the macula with the profile in the extramacular region using DNA microarrays. Seventy-five candidate genes with differences in macular:extramacular expression levels were identified by microarray analysis, of which 29 were selected for further analysis. Quantitative PCR confirmed that 21 showed statistically significant differences in expression. Five genes were expressed at higher levels in the macula. Two showed significant changes in the macular:extramacular expression ratio; another two exhibited changes in absolute expression level, as a function of age or AMD. Several of the differentially expressed genes have potential relevance to AMD pathobiology. One is an RPE cell growth factor (TFPI2), five are extracellular matrix components (DCN, MYOC, OGN, SMOC2, TFPI2), and six are related to inflammation (CCL19, CCL26, CXCL14, SLIT2) and/or angiogenesis (CXCL14, SLIT2, TFPI2, WFDC1). The identification of regional differences in gene expression in the RPE/choroid is a first step in clarifying the macula's propensity for degeneration. These findings lay the groundwork for further studies into the roles of the corresponding gene products in the normal, aged, and diseased macula.

Barrier properties of cultured retinal pigment epithelium.

Science.gov (United States)

Rizzolo, Lawrence J

2014-09-01

The principal function of an epithelium is to form a dynamic barrier that regulates movement between body compartments. Each epithelium is specialized with barrier functions that are specific for the tissues it serves. The apical surface commonly faces a lumen, but the retinal pigment epithelium (RPE) appears to be unique by a facing solid tissue, the sensory retina. Nonetheless, there exists a thin (subretinal) space that can become fluid filled during pathology. RPE separates the subretinal space from the blood supply of the outer retina, thereby forming the outer blood-retinal barrier. The intricate interaction between the RPE and sensory retina presents challenges for learning how accurately culture models reflect native behavior. The challenge is heightened by findings that detail the variation of RPE barrier proteins both among species and at different stages of the life cycle. Among the striking differences is the expression of claudin family members. Claudins are the tight junction proteins that regulate ion diffusion across the spaces that lie between the cells of a monolayer. Claudin expression by RPE varies with species and life-stage, which implies functional differences among commonly used animal models. Investigators have turned to transcriptomics to supplement functional studies when comparing native and cultured tissue. The most detailed studies of the outer blood-retinal barrier have focused on human RPE with transcriptome and functional studies reported for human fetal, adult, and stem-cell derived RPE. Copyright © 2014 Elsevier Ltd. All rights reserved.

Activation of the Small GTPase Rap1 Inhibits Choroidal Neovascularization by Regulating Cell Junctions and ROS Generation in Rats.

Science.gov (United States)

Li, Jiajia; Zhang, Rong; Wang, Caixia; Wang, Xin; Xu, Man; Ma, Jingxue; Shang, Qingli

2018-03-30

Choroidal neovascularization (CNV) is a common vision-threatening complication associated with many  fundus diseases. The retinal pigment epithelial (RPE) cell junction barrier has critical functions in preventing CNV, and oxidative stress can cause compromise of barrier integrity and induce angiogenesis. Rap1, a small guanosine triphosphatase (GTPase), is involved in regulating endothelial and epithelial cell junctions. In this work, we explored the function and mechanism of Rap1 in CNV in vivo. A laser-induced rat CNV model was developed. Rap1 was activated through intravitreal injection of the Rap1 activator 8CPT-2'-O-Me-cAMP (8CPT). At 14 days after laser treatment, CNV size in RPE/choroid flat mounts was measured by fluorescein isothiocyanate-dextran staining. Expression of vascular endothelial growth factor (VEGF) and cell junction proteins in RPE/choroid tissues were analyzed by western blots and quantitative real-time PCR assays. Reactive oxygen species (ROS) in RPE cells were detectedbydichloro-dihydro-fluorescein diacetate assays. The antioxidant apocynin was intraperitoneally injected into rats. Activating Rap1 by 8CPT significantly reduced CNV size and VEGF expression in the rat CNV model. Rap1 activation enhanced protein and mRNA levels of ZO-1 and occludin, two tight junction proteins in the RPE barrier. In addition, reducing ROS generation by injection of apocynin, a NADPH oxidase inhibitor, inhibited CNV formation. Rap1 activation reduced ROS generation and expression of NADPH oxidase 4. Rap1 activation inhibits CNV through regulating barrier integrity and ROS generation of RPE in vivo, and selectively activating Rap1 may be a way to reduce vision loss from CNV.

RPE y velocidad como marcadores de intensidad en el press de banca

OpenAIRE

Bautista, I. J.; Chirosa, I. J.; Chirosa, L. J.; Martín, I.; Revilla, J.

2016-01-01

El propósito de esta investigación fue analizar la relación entre la evolución de la velocidad media (Velmedia) y los valores de la escala OMNI-RES, en 3 intensidades (carga Inicial [CI], máxima potencia [MP] y repetición máxima [RM]). 38 sujetos divididos en dos grupos: Entrenados (G1, n = 19) y No Entrenados (G2, n = 19) realizaron un test incremental de cargas en el ejercicio del press de banca. En la percepción subjetiva del esfuerzo (RPE) no se encontraron diferencias significativas entr...

Quantification of training load using session RPE method and performance in futsal. DOI: 10.5007/1980-0037.2012v14n1p73

Directory of Open Access Journals (Sweden)

Victor Hugo Freitas

2011-12-01

Full Text Available There is a need of studies about training load (TL quantification using session rating of perceived exertion (session-RPE and its relation to athlete’s performance in futsal. The objective of this study was to describe training load behavior using session-RPE method and to analyze futsal athlete´s performance during a preparatory period and its relation to TL. Twelve male futsal players (age 24,92 ± 5,20 year, weight 73,42 ± 5,70 kg, height 175,83 ± 5,05 cm were submitted to 14 weeks of training quantified by the session-RPE method. It was measured maximum volume of oxygen (VO₂max, velocity, agility and vertical jump (VJ before and after the training period and also the VJ every week. Using the Wilcoxon test, it was observed an increase in VO₂max (p=0,004, VJ (p=0,003, velocity (p=0,003 and agility (p=0,002 after the training period. Using the ANOVA to repeated measures (Tukey post-hoc, it was observed a greater training load (TL in the first mesocycle compared to the others and a greater CT in the second compared to the third mesocycle. It was not found a relation between VJ and TL. It was concluded that the session RPE allowed an effective TL quantification through the training period and the athlete’s performance increased along with the progressive TL reduction description. There wasn’t correlation between the TL and performance.

Quantification of training load using session RPE method and performance in futsal. DOI: 10.5007/1980-0037.2012v14n1p73

Directory of Open Access Journals (Sweden)

Maurício Gattas Bara Filho

2012-01-01

Full Text Available There is a need of studies about training load (TL quantification using session rating of perceived exertion (session-RPE and its relation to athlete’s performance in futsal. The objective of this study was to describe training load behavior using session-RPE method and to analyze futsal athlete´s performance during a preparatory period and its relation to TL. Twelve male futsal players (age 24,92 ± 5,20 year, weight 73,42 ± 5,70 kg, height 175,83 ± 5,05 cm were submitted to 14 weeks of training quantified by the session-RPE method. It was measured maximum volume of oxygen (VO₂max, velocity, agility and vertical jump (VJ before and after the training period and also the VJ every week. Using the Wilcoxon test, it was observed an increase in VO₂max (p=0,004, VJ (p=0,003, velocity (p=0,003 and agility (p=0,002 after the training period. Using the ANOVA to repeated measures (Tukey post-hoc, it was observed a greater training load (TL in the first mesocycle compared to the others and a greater CT in the second compared to the third mesocycle. It was not found a relation between VJ and TL. It was concluded that the session RPE allowed an effective TL quantification through the training period and the athlete’s performance increased along with the progressive TL reduction description. There wasn’t correlation between the TL and performance.

Ectopic AP4 expression induces cellular senescence via activation of p53 in long-term confluent retinal pigment epithelial cells.

Science.gov (United States)

Wang, Yiping; Wong, Matthew Man-Kin; Zhang, Xiaojian; Chiu, Sung-Kay

2015-11-15

When cells are grown to confluence, cell-cell contact inhibition occurs and drives the cells to enter reversible quiescence rather than senescence. Confluent retinal pigment epithelial (RPE) cells exhibiting contact inhibition was used as a model in this study to examine the role of overexpression of transcription factor AP4, a highly expressed transcription factor in many types of cancer, in these cells during long-term culture. We generated stable inducible RPE cell clones expressing AP4 or AP4 without the DNA binding domain (DN-AP4) and observed that, when cultured for 24 days, RPE cells with a high level of AP4 exhibit a large, flattened morphology and even cease proliferating; these changes were not observed in DN-AP4-expressing cells or non-induced cells. In addition, AP4-expressing cells exhibited senescence-associated β-galactosidase activity and the senescence-associated secretory phenotype. We demonstrated that the induced cellular senescence was mediated by enhanced p53 expression and that AP4 regulates the p53 gene by binding directly to two of the three E-boxes present on the promoter of the p53 gene. Moreover, we showed that serum is essential for AP4 in inducing p53-associated cellular senescence. Collectively, we showed that overexpression of AP4 mediates cellular senescence involving in activation of p53 in long-term post-confluent RPE cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Inhibition or Stimulation of Autophagy Affects Early Formation of Lipofuscin-Like Autofluorescence in the Retinal Pigment Epithelium Cell

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Lei Lei

2017-03-01

Full Text Available The accumulation of lipofuscin in the retinal pigment epithelium (RPE is dependent on the effectiveness of photoreceptor outer segment material degradation. This study explored the role of autophagy in the fate of RPE lipofuscin degradation. After seven days of feeding with either native or modified rod outer segments, ARPE-19 cells were treated with enhancers or inhibitors of autophagy and the autofluorescence was detected by fluorescence-activated cell sorting. Supplementation with different types of rod outer segments increased lipofuscin-like autofluorescence (LLAF after the inhibition of autophagy, while the induction of autophagy (e.g., application of rapamycin decreased LLAF. The effects of autophagy induction were further confirmed by Western blotting, which showed the conversion of LC3-I to LC3-II, and by immunofluorescence microscopy, which detected the lysosomal activity of the autophagy inducers. We also monitored LLAF after the application of several autophagy inhibitors by RNA-interference and confocal microscopy. The results showed that, in general, the inhibition of the autophagy-related proteins resulted in an increase in LLAF when cells were fed with rod outer segments, which further confirms the effect of autophagy in the fate of RPE lipofuscin degradation. These results emphasize the complex role of autophagy in modulating RPE autofluorescence and confirm the possibility of the pharmacological clearance of RPE lipofuscin by small molecules.

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

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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.

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

International Nuclear Information System (INIS)

Chan, Chi-Ming; Fang, Jia-You; Lin, Hsin-Huang; Yang, Chi-Yea; Hung, Chi-Feng

2009-01-01

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.

The Human Cell Atlas.

Science.gov (United States)

Regev, Aviv; Teichmann, Sarah A; Lander, Eric S; Amit, Ido; Benoist, Christophe; Birney, Ewan; Bodenmiller, Bernd; Campbell, Peter; Carninci, Piero; Clatworthy, Menna; Clevers, Hans; Deplancke, Bart; Dunham, Ian; Eberwine, James; Eils, Roland; Enard, Wolfgang; Farmer, Andrew; Fugger, Lars; Göttgens, Berthold; Hacohen, Nir; Haniffa, Muzlifah; Hemberg, Martin; Kim, Seung; Klenerman, Paul; Kriegstein, Arnold; Lein, Ed; Linnarsson, Sten; Lundberg, Emma; Lundeberg, Joakim; Majumder, Partha; Marioni, John C; Merad, Miriam; Mhlanga, Musa; Nawijn, Martijn; Netea, Mihai; Nolan, Garry; Pe'er, Dana; Phillipakis, Anthony; Ponting, Chris P; Quake, Stephen; Reik, Wolf; Rozenblatt-Rosen, Orit; Sanes, Joshua; Satija, Rahul; Schumacher, Ton N; Shalek, Alex; Shapiro, Ehud; Sharma, Padmanee; Shin, Jay W; Stegle, Oliver; Stratton, Michael; Stubbington, Michael J T; Theis, Fabian J; Uhlen, Matthias; van Oudenaarden, Alexander; Wagner, Allon; Watt, Fiona; Weissman, Jonathan; Wold, Barbara; Xavier, Ramnik; Yosef, Nir

2017-12-05

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

Grown on Novel Microcarriers

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Torsten Falk

2012-01-01

Full Text Available Human retinal pigment epithelial (hRPE cells have been tested as a cell-based therapy for Parkinson’s disease but will require additional study before further clinical trials can be planned. We now show that the long-term survival and neurotrophic potential of hRPE cells can be enhanced by the use of FDA-approved plastic-based microcarriers compared to a gelatin-based microcarrier as used in failed clinical trials. The hRPE cells grown on these plastic-based microcarriers display several important characteristics of hRPE found in vivo: (1 characteristic morphological features, (2 accumulation of melanin pigment, and (3 high levels of production of the neurotrophic factors pigment epithelium-derived factor (PEDF and vascular endothelial growth factor-A (VEGF-A. Growth of hRPE cells on plastic-based microcarriers led to sustained levels (>1 ng/ml of PEDF and VEGF-A in conditioned media for two months. We also show that the expression of VEGF-A and PEDF is reciprocally regulated by activation of the GPR143 pathway. GPR143 is activated by L-DOPA (1 μM which decreased VEGF-A secretion as opposed to the previously reported increase in PEDF secretion. The hRPE microcarriers are therefore novel candidate delivery systems for achieving long-term delivery of the neuroprotective factors PEDF and VEGF-A, which could have a value in neurodegenerative conditions such as Parkinson’s disease.

Indian hedgehog signaling from endothelial cells is required for sclera and retinal pigment epithelium development in the mouse eye.

Science.gov (United States)

Dakubo, Gabriel D; Mazerolle, Chantal; Furimsky, Marosh; Yu, Chuan; St-Jacques, Benoit; McMahon, Andrew P; Wallace, Valerie A

2008-08-01

The development of extraocular orbital structures, in particular the choroid and sclera, is regulated by a complex series of interactions between neuroectoderm, neural crest and mesoderm derivatives, although in many instances the signals that mediate these interactions are not known. In this study we have investigated the function of Indian hedgehog (Ihh) in the developing mammalian eye. We show that Ihh is expressed in a population of non-pigmented cells located in the developing choroid adjacent to the RPE. The analysis of Hh mutant mice demonstrates that the RPE and developing scleral mesenchyme are direct targets of Ihh signaling and that Ihh is required for the normal pigmentation pattern of the RPE and the condensation of mesenchymal cells to form the sclera. Our findings also indicate that Ihh signals indirectly to promote proliferation and photoreceptor specification in the neural retina. This study identifies Ihh as a novel choroid-derived signal that regulates RPE, sclera and neural retina development.

An Induced Pluripotent Stem Cell Patient Specific Model of Complement Factor H (Y402H) Polymorphism Displays Characteristic Features of Age-Related Macular Degeneration and Indicates a Beneficial Role for UV Light Exposure.

Science.gov (United States)

Hallam, Dean; Collin, Joseph; Bojic, Sanja; Chichagova, Valeria; Buskin, Adriana; Xu, Yaobo; Lafage, Lucia; Otten, Elsje G; Anyfantis, George; Mellough, Carla; Przyborski, Stefan; Alharthi, Sameer; Korolchuk, Viktor; Lotery, Andrew; Saretzki, Gabriele; McKibbin, Martin; Armstrong, Lyle; Steel, David; Kavanagh, David; Lako, Majlinda

2017-11-01

Age-related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease; however, these do not exist for the dry form. Complement factor H polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, RPE damage, and visual decline. We have derived and characterized induced pluripotent stem cell (iPSC) lines from two subjects without AMD and low-risk genotype and two patients with advanced AMD and high-risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H, factor I, and factor H-like protein 1. The iPSC RPE cells derived from high-risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy, and deposition of "drüsen"-like deposits. The low- and high-risk RPE cells respond differently to intermittent exposure to UV light, which leads to an improvement in cellular and functional phenotype only in the high-risk AMD-RPE cells. Taken together, our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing. Stem Cells 2017;35:2305-2320. © 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

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.

The human cell atlas

DEFF Research Database (Denmark)

Regev, Aviv; Teichmann, Sarah A.; Lander, Eric S.

2017-01-01

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international...... collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells...... in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early...

Retinol Dehydrogenases Regulate Vitamin A Metabolism for Visual Function

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Bhubanananda Sahu

2016-11-01

Full Text Available The visual system produces visual chromophore, 11-cis-retinal from dietary vitamin A, all-trans-retinol making this vitamin essential for retinal health and function. These metabolic events are mediated by a sequential biochemical process called the visual cycle. Retinol dehydrogenases (RDHs are responsible for two reactions in the visual cycle performed in retinal pigmented epithelial (RPE cells, photoreceptor cells and Müller cells in the retina. RDHs in the RPE function as 11-cis-RDHs, which oxidize 11-cis-retinol to 11-cis-retinal in vivo. RDHs in rod photoreceptor cells in the retina work as all-trans-RDHs, which reduce all-trans-retinal to all-trans-retinol. Dysfunction of RDHs can cause inherited retinal diseases in humans. To facilitate further understanding of human diseases, mouse models of RDHs-related diseases have been carefully examined and have revealed the physiological contribution of specific RDHs to visual cycle function and overall retinal health. Herein we describe the function of RDHs in the RPE and the retina, particularly in rod photoreceptor cells, their regulatory properties for retinoid homeostasis and future therapeutic strategy for treatment of retinal diseases.

Ocular Stem Cell Research from Basic Science to Clinical Application: A Report from Zhongshan Ophthalmic Center Ocular Stem Cell Symposium

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Hong Ouyang

2016-03-01

Full Text Available Stem cells hold promise for treating a wide variety of diseases, including degenerative disorders of the eye. The eye is an ideal organ for stem cell therapy because of its relative immunological privilege, surgical accessibility, and its being a self-contained system. The eye also has many potential target diseases amenable to stem cell-based treatment, such as corneal limbal stem cell deficiency, glaucoma, age-related macular degeneration (AMD, and retinitis pigmentosa (RP. Among them, AMD and glaucoma are the two most common diseases, affecting over 200 million people worldwide. Recent results on the clinical trial of retinal pigment epithelial (RPE cells from human embryonic stem cells (hESCs and induced pluripotent stem cells (iPSCs in treating dry AMD and Stargardt’s disease in the US, Japan, England, and China have generated great excitement and hope. This marks the beginning of the ocular stem cell therapy era. The recent Zhongshan Ophthalmic Center Ocular Stem Cell Symposium discussed the potential applications of various stem cell types in stem cell-based therapies, drug discoveries and tissue engineering for treating ocular diseases.

Alanine/RPE dosimetry in the process of blood irradiation; Dosimetria alanina/RPE en el proceso de irradiacion de sangre

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Chen, F.; Covas, D.T.; Baffa, O. [Departamento de Fisica e Matematica, FFCLRP, Universidade de Sao Paulo, 14040-901 Ribeirao Preto-SP (Brazil)

2000-07-01

A set of eighty dosemeters approximately of Dl- alanine was proportioned to the Hemo center of Hospital das Clinicas of Medicine Faculty at Ribeirao Preto (HC-FRMP) in the Sao Paulo University, with the purpose to realize a quality control of the radiation dose supplied to blood bags. These dosemeters were divided in eight groups and irradiated for a two months period.The dosemeters were adhered to the bags and arranged inside of an acrylic box filled water with dimensions: (40x40x20) cm{sup 3}. The box irradiation was made using two fields parallel-opposed of (40x40) cm{sup 2} at 80 cm distance source-surface, in the Radiotherapy Service of HC-FMRP, with a {sup 60} Co teletherapy unit. The irradiation time was sufficient to supply a dose of 20 Gy approximately in the box center. The RPE measures were realized in a Varian E-4 spectrometer operating in X-band. For the total of dosemeters and for the irradiation volume, the minimum and maximum doses were of 14 Gy and 23 Gy respectively. The mean dose was (18{+-}2) Gy (1 {sigma}), and the variability coefficient 11 %. Around 5 % of the bags received a dose under of the recommended limit by the Brazilian legislation and as consequence, the exposure time was increased. (Author)

Culture of Iris Pigment Epithelial Cells on Expanded-Polytetrafluroethylene (ePTFE Substrates for the Treatment of Age-Related Macular Degeneration

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S Nian

2011-05-01

Full Text Available Introduction: Transplantation of an intact differentiated retinal pigment epithelial (RPE cell layer may provide a means to treat Age-Related Macular Degeneration (AMD. However, harvesting RPE cells can be a technically complicated procedure. Our current work aimed to prepare intact differentiated iris pigment epithelial (IPE cell layers, which are easy to obtain and have the same embryonic origin and similar properties as RPE cells, on ePTFE substrates for transplantation purposes to rescue deteriorated photoreceptors in AMD. Methods: IPE cells isolated from rat eyes were seeded on different substrates, including fibronectin n-heptylamine (HA ePTFE substrates, HA ePTFE substrates, ePTFE substrates and fibronectin tissue culture polystyrene (TCPS as control. Cell number and morphology were assessed at each time interval. The formation of tight junction was examined by immunostaining of junction proteins. Results: An obvious increasing trend of cell number was observed in IPE cells on fibronectin n-heptylamine (HA ePTFE substrate, exhibiting heavy pigmentation and epithelial morphology. At Day 28, tight junction formation was indicated by cell-cell junctional proteins along cell borders. Conclusion: Harvested IPE cells cultured on fibronectin HA-ePTFE substrates can differentiate and form a cell monolayer that may be suitable for transplantation.

Human secretory phospholipase A(2), group IB in normal eyes and in eye diseases

DEFF Research Database (Denmark)

Kolko, Miriam; Prause, Jan U; Bazan, Nicolas G

2007-01-01

, retinitis pigmentosa and glaucoma were evaluated. RESULTS: Expression of hGIB was found in various cells of the eye. The most abundant expression was found in retinal pigment epithelium (RPE) cells, the inner photoreceptor segments, ganglion cells and the corneal endothelium. We explored diseases involving...

Decreased VEGF-A and sustained PEDF expression in a human retinal pigment epithelium cell line cultured under hypothermia

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Masayuki Takeyama

2015-01-01

Full Text Available BACKGROUND: Previous reports have described a decrease in retinal temperature and clinical improvement of wet age-related macular degeneration (AMD after vitrectomy. We hypothesized that the retinal temperature decrease after vitrectomy plays a part in the suppression of wet AMD development. To test this hypothesis, we evaluated the temperature dependence of the expression of vascular endothelial growth factor-A (VEGF-A and in vitro angiogen-esis in retinal pigment epithelium (RPE. RESULTS: We cultured ARPE-19 cells at 37, 35, 33 and 31°C and measured the expression of VEGF-A, VEGF-A splicing variants, and pigment epithelium-derived factor (PEDF. We performed an in vitro tube formation assay. The dehydrogenase activity was also evaluated at each temperature. Expression of VEGF-A significantly decreased with decreased temperature while PEDF expression did not. VEGF165 expression and in vitro angiogenesis also were temperature dependent. The dehydrogenase activity significantly decreased as the culture temperature decreased. CONCLUSIONS: RPE cultured under hypothermia that decreased cellular metabolism also had decreased VEGF-A and sustained PEDF expression, creating an anti-angiogenic environment. This mechanism may be associated with a beneficial effect after vitrectomy in patients with wet AMD.

Dietary antioxidants prevent age-related retinal pigment epithelium actin damage and blindness in mice lacking αvβ5 integrin

Science.gov (United States)

Yu, Chia-Chia; Nandrot, Emeline F.; Dun, Ying; Finnemann, Silvia C.

2011-01-01

In the aging human eye, oxidative damage and accumulation of pro-oxidant lysosomal lipofuscin cause functional decline of the retinal pigment epithelium (RPE), which contributes to age-related macular degeneration. In mice with an RPE-specific phagocytosis defect due to lack of αvβ5 integrin receptors, RPE accumulation of lipofuscin suggests that the age-related blindness we previously described in this model may also result from oxidative stress. Cellular and molecular targets of oxidative stress in the eye remain poorly understood. Here we identify actin among 4-hydroxynonenal (HNE) adducts formed specifically in β5−/− RPE but not neural retina with age. HNE modification directly correlated with loss of resistance of actin to detergent extraction, suggesting cytoskeletal damage in aging RPE. Dietary enrichment with natural antioxidants grapes or marigold extract containing macular pigments lutein/zeaxanthin was sufficient to prevent HNE-adduct formation, actin solubility, lipofuscin accumulation, and age-related cone and rod photoreceptor dysfunction in β5−/− mice. Acute generation of HNE-adducts directly destabilized actin but not tubulin cytoskeletal elements of RPE cells. These findings identify destabilization of the actin cytoskeleton as a consequence of physiological, sublethal oxidative burden of RPE cells in vivo that is associated with age-related blindness and that can be prevented by consuming an antioxidant-rich diet. PMID:22178979

Effect of Changing Weight and Mass on Human Performance in a Lunar Prototype Spacesuit

Science.gov (United States)

Chappell, Steve; Lee, Lesley; Gemhardt, Mike

2010-01-01

Physical effort, compensation, and controllability in a spacesuit can be affected by suit mass and gravity level. Because of limitations in certain reduced-gravity simulators and the finite selection of lunar prototype suits, it is difficult to ascertain how a change in suit mass affects suited human performance. One method of simulating a change in mass is to vary the total gravity-adjusted weight (TGAW), which is defined as the sum of the suit mass and subject mass, multiplied by the gravity level. PURPOSE: To determine if two methods of changing TGAW during parabolic flight - changing suit mass or gravity level - affect subjective ratings of suited human performance equally.METHODS: A custom weight support structure was connected to a lunar prototype spacesuit, allowing the addition of mass to the suit while maintaining a near-constant center of mass. In the varied-weight (VW) series, suit mass (120 kg) was constant at 0.1-g, 0.17-g, and 0.3-g, yielding TGAWs of 196, 333, and 588 N, assuming an 80-kg subject. In the varied-mass (VM) series, gravity level was constant at 0.17-g and suit mass was 89, 120, and 181 kg, yielding TGAWs of 282, 333, and 435 N. The 333 N condition was common to both series. Direct comparison was not possible due to limited adjustability of suit mass and limited options for parabolic profiles. Five astronaut subjects (80.311.8 kg) completed 4 different tasks (walk, bag pickup, lunge, and shoveling) in all conditions and provided ratings of perceived exertion (RPE) and the gravity compensation and performance scale (GCPS) upon completion of each task. RESULTS: Where VM and VW series overlapped, RPE and GCPS trend lines were similar. Mean RPE and GCPS at 333 N was 8.4 and 3.7. Mean RPE and GCPS for VM was 7.8 and 3.8 for 282 N and 9.8 and 4.1 for 435 N. Extrapolation of the VM trend to match VW TGAWs 196 and 588 N predicts an RPE of 6.5 and 12.3 and GCPS of 4.4 and 5.9, whereas the measured VW values for RPE were 8.1 and 9.8 and GCPS were

Enantioselective synthesis of (R)-phenylephrine by Serratia marcescens BCRC10948 cells that homologously express SM_SDR.

Science.gov (United States)

Kuan, Yi-Chia; Xu, Yue-Bin; Wang, Wen-Ching; Yang, Ming-Te

2018-03-01

A short-chain dehydrogenase/reductase from Serratia marcescens BCRC10948, SM_SDR, has been cloned and expressed in Escherichia coli for the bioconversion of 1-(3-hydroxyphenyl)-2-(methylamino) ethanone (HPMAE) to (R)-phenylephrine[(R)-PE]. However, only 5.11mM (R)-PE was obtained from 10mM HPMAE after a 9h conversion in the previous report. To improve the biocatalytic efficiency, the homologous expression of the SM_SDR in S. marcescens BCRC10948 was achieved using the T5 promoter for expression. By using 2% glycerol as carbon source, we found that 8.00±0.15mM of (R)-PE with more than 99% enantiomeric excess was produced from 10mM HPMAE after 12h conversion at 30°C and pH 7.0. More importantly, by using 50mM HPMAE as the substrate, 23.78±0.84mM of (R)-PE was produced after a 12h conversion with the productivity and the conversion yield of 1.98mmol (R)-PE/lh and 47.50%, respectively. The recombinant S. marcescens cells could be recycled 6 times for the production of (R)-PE, and the bioconversion efficiency remained at 85% when compared to that at the first cycle. Our data indicated that a high conversion efficiency of HPMAE to (R)-PE could be achieved using S. marcescens BCRC10948 cells that homologously express the SM_SDR. Copyright © 2017 Elsevier Inc. All rights reserved.

Defective repair of UV-damaged DNA in human tumor and SV40-transformed human cells but not in adenovirus-transformed human cells

International Nuclear Information System (INIS)

Rainbow, A.J.

1989-01-01

The DNA repair capacities of five human tumor cell lines, one SV40-transformed human cell line and one adenovirus-transformed human cell line were compared with that of normal human fibroblasts using a sensitive host cell reactivation (HCR) technique. Unirradiated and UV-irradiated suspensions of adenovirus type 2 (Ad 2) were assayed for their ability to form viral structural antigens (Vag) in the various cell types using immunofluorescent staining. The survival of Vag formation for UV-irradiated Ad 2 was significantly reduced in all the human tumor cell lines and the SV40-transformed human line compared to the normal human fibroblasts, but was apparently normal in the adenovirus-transformed human cells. D 0 values for the UV survival of Ad 2 Vag synthesis in the tumor and virally transformed lines expressed as a percentage of that obtained on normal fibroblast strains were used as a measure of DNA repair capacity. Percent HCR values ranged from 26 to 53% in the tumor cells. These results indicate a deficiency in the repair of UV-induced DNA damage associated with human tumorigenesis and the transformation of human cells by SV40 but not the transformation of human cells by adenovirus. (author)

Pluripotent stem cells: A therapeutic source for age-related macular degeneration

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Sowmya Parameswaran

2017-01-01

Full Text Available Age-related macular degeneration (AMD leads to progressive loss of central vision in the elderly. At a cellular level, there is aging of the retinal pigment epithelial (RPE cells, and accumulation of lipofuscin that interferes with the proper functioning of RPE which eventually leads to apoptosis. Treatment depends on the stage of the disease. Wet AMD which has neovascularization is managed by local therapies such as laser photocoagulation and photodynamic therapy and is managed with injections of antivascular endothelial growth factor-based therapy. Unlike the wet AMD, an effective therapy does not exist for dry AMD and geographic atrophy. Cell replacement therapy has shown promise. This review discusses the opportunities in the various types of cell-based therapy, their limitations, and what is possible for India.

Genome engineering in human cells.

Science.gov (United States)

Song, Minjung; Kim, Young-Hoon; Kim, Jin-Soo; Kim, Hyongbum

2014-01-01

Genome editing in human cells is of great value in research, medicine, and biotechnology. Programmable nucleases including zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases recognize a specific target sequence and make a double-strand break at that site, which can result in gene disruption, gene insertion, gene correction, or chromosomal rearrangements. The target sequence complexities of these programmable nucleases are higher than 3.2 mega base pairs, the size of the haploid human genome. Here, we briefly introduce the structure of the human genome and the characteristics of each programmable nuclease, and review their applications in human cells including pluripotent stem cells. In addition, we discuss various delivery methods for nucleases, programmable nickases, and enrichment of gene-edited human cells, all of which facilitate efficient and precise genome editing in human cells.

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.

Plasma membrane proteomics of human embryonic stem cells and human embryonal carcinoma cells.

NARCIS (Netherlands)

Dormeyer, W.; van Hoof, D.; Braam, S.R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

2008-01-01

Human embryonic stem cells (hESCs) are of immense interest in regenerative medicine as they can self-renew indefinitely and can give rise to any adult cell type. Human embryonal carcinoma cells (hECCs) are the malignant counterparts of hESCs found in testis tumors. hESCs that have acquired

Mutation in the RPE65 gene causing hereditary retinal dystrophy in the Briard dogs: application of a new detection method

Czech Academy of Sciences Publication Activity Database

Bechyňová, Renata; Dostál, Jaromír; Stratil, Antonín; Jílek, F.; Horák, Pavel

2008-01-01

Roč. 53, č. 4 (2008), s. 176-179 ISSN 1212-1819 R&D Projects: GA AV ČR 1QS500450578; GA ČR GD523/03/H076 Institutional research plan: CEZ:AV0Z50450515 Keywords : RPE65 gene * CSNB * dog Subject RIV: EG - Zoology Impact factor: 0.735, year: 2008

Cell therapeutics to treat diseases of the retina

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Natarajan S

2008-11-01

Full Text Available Background: The adult Bone Marrow Stem Cells (BMSCs have distinct advantages over the other types of stem cells. They are multipotent, can be stored for upto 10 years and considered to be one of the best sources of hematopoietic and mesenchymal stem cells in an adult body. Genetically inherited diseases such as Retinitis Pigmentosa and Degenerative diseases such as Age Related Macular Degeneration remain unsolved as no definitive treatment is available to repair the damages caused to the RPE and Photoreceptors as of now. In this scenario, the technique of Bone Marrow aspiration & isolation of Mono Nucleated Cells (MNCs & intra-vitreal injection of a very small volume of MNCs in human retinal disorders has been standardized and is safe and feasible for human studies (Mohanty et al and autotransplantation of RPEs from periphery to affected area are underpractice(Coffey et al. In this study we report our research work on different approaches to the above diseases using cell therapeuticsStudy 1 Materials & methods: Ciliary Pigment Epithelium was harvested from donor eyes from Aditya Jyot Eye Hospital, Mumbai and was taken to and grown at NCRM lab. The cells were grown in the earlier reported methodology of Brenda et al (Science 2004. Results: The CPE derived Retinal stem cells grew well in the lab. However, the practical difficulties of harvesting the same in patients limited our further steps in this study. Study II:? Materials & methods: Cadaver eye RPE cells were harvested and grown using polymer scaffolds after transporting them over 6 to 12 Hrs. The RPEs were grown on conventional methods and in polymer scaffolds and were subjected to RT-PCR. Results: Human RPEs were able to grow without amniotic membrane and the same was proven by RT-PCR. This would make it possible for the peripheral RPEs taken from patients to be stored and later expanded and used for replacing the diseased cells of the central portion of the retina in future, without having

cGAS drives noncanonical-inflammasome activation in age-related macular degeneration.

Science.gov (United States)

Kerur, Nagaraj; Fukuda, Shinichi; Banerjee, Daipayan; Kim, Younghee; Fu, Dongxu; Apicella, Ivana; Varshney, Akhil; Yasuma, Reo; Fowler, Benjamin J; Baghdasaryan, Elmira; Marion, Kenneth M; Huang, Xiwen; Yasuma, Tetsuhiro; Hirano, Yoshio; Serbulea, Vlad; Ambati, Meenakshi; Ambati, Vidya L; Kajiwara, Yuji; Ambati, Kameshwari; Hirahara, Shuichiro; Bastos-Carvalho, Ana; Ogura, Yuichiro; Terasaki, Hiroko; Oshika, Tetsuro; Kim, Kyung Bo; Hinton, David R; Leitinger, Norbert; Cambier, John C; Buxbaum, Joseph D; Kenney, M Cristina; Jazwinski, S Michal; Nagai, Hiroshi; Hara, Isao; West, A Phillip; Fitzgerald, Katherine A; Sadda, SriniVas R; Gelfand, Bradley D; Ambati, Jayakrishna

2018-01-01

Geographic atrophy is a blinding form of age-related macular degeneration characterized by retinal pigmented epithelium (RPE) death; the RPE also exhibits DICER1 deficiency, resultant accumulation of endogenous Alu-retroelement RNA, and NLRP3-inflammasome activation. How the inflammasome is activated in this untreatable disease is largely unknown. Here we demonstrate that RPE degeneration in human-cell-culture and mouse models is driven by a noncanonical-inflammasome pathway that activates caspase-4 (caspase-11 in mice) and caspase-1, and requires cyclic GMP-AMP synthase (cGAS)-dependent interferon-β production and gasdermin D-dependent interleukin-18 secretion. Decreased DICER1 levels or Alu-RNA accumulation triggers cytosolic escape of mitochondrial DNA, which engages cGAS. Moreover, caspase-4, gasdermin D, interferon-β, and cGAS levels were elevated in the RPE in human eyes with geographic atrophy. Collectively, these data highlight an unexpected role of cGAS in responding to mobile-element transcripts, reveal cGAS-driven interferon signaling as a conduit for mitochondrial-damage-induced inflammasome activation, expand the immune-sensing repertoire of cGAS and caspase-4 to noninfectious human disease, and identify new potential targets for treatment of a major cause of blindness.

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo.

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Roux, Clémence; Saviane, Gaëlle; Pini, Jonathan; Belaïd, Nourhène; Dhib, Gihen; Voha, Christine; Ibáñez, Lidia; Boutin, Antoine; Mazure, Nathalie M; Wakkach, Abdelilah; Blin-Wakkach, Claudine; Rouleau, Matthieu

2017-01-01

Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for therapeutic approaches, but their functions remained to be fully characterized. We generated multipotent MSCs derived from huiPS cells (huiPS-MSCs), and focusing on their immunosuppressive activity, we showed that human T-cell activation in coculture with huiPS-MSCs was significantly reduced. We also observed the generation of functional CD4 + FoxP3 + regulatory T (Treg) cells. Further tested in vivo in a model of human T-cell expansion in immune-deficient NSG mice, huiPS-MSCs immunosuppressive activity prevented the circulation and the accumulation of activated human T cells. Intracytoplasmic labeling of cytokines produced by the recovered T cells showed reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines. By contrast, T cells producing IL-10 and FoxP3 + -Treg cells, absent in non-treated animals, were detected in huiPS-MSCs treated mice. For the first time, these results highlight the immunosuppressive activity of the huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo . They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance.

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo

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Clémence Roux

2018-01-01

Full Text Available Despite mesenchymal stromal cells (MSCs are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate. Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS cells, remediating part of these issues, are considered as well as a valuable tool for therapeutic approaches, but their functions remained to be fully characterized. We generated multipotent MSCs derived from huiPS cells (huiPS-MSCs, and focusing on their immunosuppressive activity, we showed that human T-cell activation in coculture with huiPS-MSCs was significantly reduced. We also observed the generation of functional CD4+ FoxP3+ regulatory T (Treg cells. Further tested in vivo in a model of human T-cell expansion in immune-deficient NSG mice, huiPS-MSCs immunosuppressive activity prevented the circulation and the accumulation of activated human T cells. Intracytoplasmic labeling of cytokines produced by the recovered T cells showed reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines. By contrast, T cells producing IL-10 and FoxP3+-Treg cells, absent in non-treated animals, were detected in huiPS-MSCs treated mice. For the first time, these results highlight the immunosuppressive activity of the huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo. They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance.

Alloimmune Responses of Humanized Mice to Human Pluripotent Stem Cell Therapeutics

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Nigel G. Kooreman

2017-08-01

Full Text Available There is growing interest in using embryonic stem cell (ESC and induced pluripotent stem cell (iPSC derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an “allogenized” mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts.

Trophoblast lineage cells derived from human induced pluripotent stem cells

International Nuclear Information System (INIS)

Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

2013-01-01

Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro

Trophoblast lineage cells derived from human induced pluripotent stem cells

Energy Technology Data Exchange (ETDEWEB)

Chen, Ying, E-mail: ying.chen@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Wang, Kai; Chandramouli, Gadisetti V.R. [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Knott, Jason G. [Developmental Epigenetics Laboratory, Department of Animal Science, Michigan State University (United States); Leach, Richard, E-mail: Richard.leach@hc.msu.edu [Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University, 333 Bostwick NE, Grand Rapids, MI 49503 (United States); Department of Obstetrics, Gynecology and Women’s Health, Spectrum Health Medical Group (United States)

2013-07-12

Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

Age- and Gene-Dosage–Dependent Cre-Induced Abnormalities in the Retinal Pigment Epithelium

Science.gov (United States)

He, Lizhi; Marioutina, Mariya; Dunaief, Joshua L.; Marneros, Alexander G.

2015-01-01

To conditionally inactivate genes in the retinal pigment epithelium (RPE) transgenic mouse strains have been developed, in which Cre recombinase (Cre) expression is driven by an RPE-specific gene promoter. The RPE is a quiescent epithelium, and continuous expression of Cre could affect its function. Here, we tested the hypothesis that continuous postnatal Cre expression in the RPE may lead to cellular abnormalities, which may depend on both age and Cre gene dosage. We therefore examined the eyes of homozygous and heterozygous VMD2-Cre mice at various ages. In VMD2-Cre heterozygous mice variable progressive age-dependent RPE abnormalities were noticed, including attenuation of phalloidin and cytoplasmic active β-catenin staining, reduced cell size, and loss of the typical honeycomb pattern of RPE morphology in those RPE cells that stained for Cre. These morphological RPE abnormalities were not noticed in Cre-negative RPE cells in VMD2-Cre or age-matched control mice. In addition, an abnormal number and morphology of cell nuclei were noticed in a subset of Cre-expressing RPE cells in aged heterozygous VMD2-Cre mice, whereas more severe nuclear abnormalities were observed already in young homozygous VMD2-Cre mice. Thus, continuous postnatal expression of Cre causes abnormalities in the RPE in an age- and Cre gene dosage-dependent manner, which needs to be considered in the interpretation of gene targeting studies in the RPE. PMID:24854863

Formation of human hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human-rat chimeras

International Nuclear Information System (INIS)

Sun, Yan; Xiao, Dong; Zhang, Ruo-Shuang; Cui, Guang-Hui; Wang, Xin-Hua; Chen, Xi-Gu

2007-01-01

We took advantage of the proliferative and permissive environment of the developing pre-immune fetus to develop a noninjury human-rat xenograft small animal model, in which the in utero transplantation of low-density mononuclear cells (MNCs) from human umbilical cord blood (hUCB) into fetal rats at 9-11 days of gestation led to the formation of human hepatocyte-like cells (hHLCs) with different cellular phenotypes, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), cytokeratin 19 (CK19), cytokeratin 8 (CK8), cytokeratin 18 (CK18), and albumin (Alb), and with some animals exhibiting levels as high as 10.7% of donor-derived human cells in the recipient liver. More interestingly, donor-derived human cells stained positively for CD34 and CD45 in the liver of 2-month-old rat. Human hepatic differentiation appeared to partially follow the process of hepatic ontogeny, as evidenced by the expression of AFP gene at an early stage and albumin gene at a later stage. Human hepatocytes generated in this model retained functional properties of normal hepatocytes. In this xenogeneic system, the engrafted donor-derived human cells persisted in the recipient liver for at least 6 months after birth. Taken together, these findings suggest that the donor-derived human cells with different cellular phenotypes are found in the recipient liver and hHLCs hold biological activity. This humanized small animal model, which offers an in vivo environment more closely resembling the situations in human, provides an invaluable approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future

c-Myc-Dependent Cell Competition in Human Cancer Cells.

Science.gov (United States)

Patel, Manish S; Shah, Heta S; Shrivastava, Neeta

2017-07-01

Cell Competition is an interaction between cells for existence in heterogeneous cell populations of multicellular organisms. This phenomenon is involved in initiation and progression of cancer where heterogeneous cell populations compete directly or indirectly for the survival of the fittest based on differential gene expression. In Drosophila, cells having lower dMyc expression are eliminated by cell competition through apoptosis when present in the milieu of cells having higher dMyc expression. Thus, we designed a study to develop c-Myc (human homolog) dependent in vitro cell competition model of human cancer cells. Cells with higher c-Myc were transfected with c-myc shRNA to prepare cells with lower c-Myc and then co-cultured with the same type of cells having a higher c-Myc in equal ratio. Cells with lower c-Myc showed a significant decrease in numbers when compared with higher c-Myc cells, suggesting "loser" and "winner" status of cells, respectively. During microscopy, engulfment of loser cells by winner cells was observed with higher expression of JNK in loser cells. Furthermore, elimination of loser cells was prevented significantly, when co-cultured cells were treated with the JNK (apoptosis) inhibitor. Above results indicate elimination of loser cells in the presence of winner cells by c-Myc-dependent mechanisms of cell competition in human cancer cells. This could be an important mechanism in human tumors where normal cells are eliminated by c-Myc-overexpressed tumor cells. J. Cell. Biochem. 118: 1782-1791, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Human induced pluripotent stem cell-derived models to investigate human cytomegalovirus infection in neural cells.

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Leonardo D'Aiuto

Full Text Available Human cytomegalovirus (HCMV infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs, neural progenitor cells (NPCs and neurons suggests that (i iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii Neural stem cells have impaired differentiation when infected by HCMV; (iii NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv most iPS-derived neurons are not permissive to HCMV infection; and (v infected neurons have impaired calcium influx in response to glutamate.

Differential cellular responses by oncogenic levels of c-Myc expression in long-term confluent retinal pigment epithelial cells.

Science.gov (United States)

Wang, Yiping; Cheng, Xiangdong; Samma, Muhammad Kaleem; Kung, Sam K P; Lee, Clement M; Chiu, Sung Kay

2018-06-01

c-Myc is a highly pleiotropic transcription factor known to control cell cycle progression, apoptosis, and cellular transformation. Normally, ectopic expression of c-Myc is associated with promoting cell proliferation or triggering cell death via activating p53. However, it is not clear how the levels of c-Myc lead to different cellular responses. Here, we generated a series of stable RPE cell clones expressing c-Myc at different levels, and found that consistent low level of c-Myc induced cellular senescence by activating AP4 in post-confluent RPE cells, while the cells underwent cell death at high level of c-Myc. In addition, high level of c-Myc could override the effect of AP4 on cellular senescence. Further knockdown of AP4 abrogated senescence-like phenotype in cells expressing low level of c-Myc, and accelerated cell death in cells with medium level of c-Myc, indicating that AP4 was required for cellular senescence induced by low level of c-Myc.