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Sample records for pigmented epithelial cells

  1. Force dependence of phagosome trafficking in retinal pigment epithelial cells

    NASA Astrophysics Data System (ADS)

    Daniel, Rebekah; Koll, Andrew T.; Altman, David

    2014-09-01

    Retinal pigment epithelial (RPE) cells play an integral role in the renewal of photoreceptor disk membranes. As rod and cone cells shed their outer segments, they are phagocytosed and degraded by the RPE, and a failure in this process can result in retinal degeneration. We have studied the role of myosin VI in nonspecific phagocytosis in a human RPE primary cell line (ARPE-19), testing the hypothesis that this motor generates the forces required to traffic phagosomes in these cells. Experiments were conducted in the presence of forces through the use of in vivo optical trapping. Our results support a role for myosin VI in phagosome trafficking and demonstrate that applied forces modulate rates of phagosome trafficking.

  2. Skin as a living coloring book: how epithelial cells create patterns of pigmentation.

    PubMed

    Weiner, Lorin; Fu, Wenyu; Chirico, William J; Brissette, Janice L

    2014-11-01

    The pigmentation of mammalian skin and hair develops through the interaction of two basic cell types - pigment donors and recipients. The pigment donors are melanocytes, which produce and distribute melanin through specialized structures. The pigment recipients are epithelial cells, which acquire melanin and put it to use, collectively yielding the pigmentation visible to the eye. This review will focus on the pigment recipients, the historically less understood cell type. These end-users of pigment are now known to exert a specialized control over the patterning of pigmentation, as they identify themselves as melanocyte targets, recruit pigment donors, and stimulate the transfer of melanin. As such, this review will discuss the evidence that the skin is like a coloring book: the pigment recipients create a 'picture,' a blueprint for pigmentation, which is colorless initially but outlines where pigment should be placed. Melanocytes then melanize the recipients and 'color in' the picture. PMID:25104547

  3. SKIN AS A LIVING COLORING BOOK: HOW EPITHELIAL CELLS CREATE PATTERNS OF PIGMENTATION

    PubMed Central

    Weiner, Lorin; Fu, Wenyu; Chirico, William J.; Brissette, Janice L.

    2014-01-01

    Summary The pigmentation of mammalian skin and hair develops through the interaction of two basic cell types — pigment donors and recipients. The pigment donors are melanocytes, which produce and distribute melanin through specialized structures. The pigment recipients are epithelial cells, which acquire melanin and put it to use, collectively yielding the pigmentation visible to the eye. This review will focus on the pigment recipients, the historically less understood cell type. These end-users of pigment are now known to exert a specialized control over the patterning of pigmentation, as they identify themselves as melanocyte targets, recruit pigment donors, and stimulate the transfer of melanin. As such, this review will discuss the evidence that the skin is like a coloring book: the pigment recipients create a “picture,” a blueprint for pigmentation, which is colorless initially but outlines where pigment should be placed. Melanocytes then melanize the recipients and “color in” the picture. PMID:25104547

  4. Incremental responses to light recorded from pigment epithelial cells and horizontal cells of the cat retina

    PubMed Central

    Steinberg, Roy H.

    1971-01-01

    1. Rod-dependent incremental responses were recorded intracellularly in both pigment epithelial cells and horizontal cells of the cat retina. They were elicited by test flashes which were superimposed on background flashes after a delay. 2. In pigment epithelial cells smaller test responses were produced as background intensity was raised. The incremental sensitivity function was linear for about 1·4 log units, with a slope of 0·86, and the approach of saturation occurred at about 2·5 log td scotopic. 3. The amplitude of pigment epithelial test responses could be estimated from the dark-adapted amplitude—log intensity function obtained with single flashes. Test flashes produced the voltage increment predicted by the slope of this function just above the point on the curve equal to the background intensity. The pigment epithelial response to a test flash, therefore, is the response expected if the background were presented alone and made more intense by the amount of the test flash. 4. Rod-dependent incremental sensitivity functions of horizontal cells closely resembled the ones obtained from pigment epithelial cells. 5. It was concluded that the adaptive effects observed in pigment epithelial cells originated in individual rods. These effects arose from the compressive nature of the dark-adapted amplitude—intensity function. In horizontal cell responses these effects may be modified by the failure of the background response to maintain its initial voltage. PMID:5571955

  5. Phototoxicity of chlorpromazine on retinal pigment epithelial cells.

    PubMed

    Persad, S; Menon, I A; Basu, P K; Carre, F

    1988-01-01

    As it is known that chlorpromazine (CPZ) can bind to melanins as well as cause ocular phototoxicity, we investigated the cytotoxic effects of UV-visible irradiation of melanotic and amelanotic retinal pigment epithelial (RPE) cells in the presence of CPZ. At low concentrations (5 micrograms/ml) of CPZ a photosensitization reaction took place which lysed the cells as measured by the release of 51Cr from cells labelled with chromium. At concentrations of CPZ less than 5 micrograms/ml, no significant cell lysis occurred when the cells were incubated at 37 degrees C in the dark. As the concentration of CPZ was increased to 25 micrograms/ml or more, high percentages of cells were lysed. When the melanotic RPE cells were exposed to different concentrations of CPZ and grown in culture, the cell growth (multiplication) diminished drastically with low concentrations (less than 2 micrograms/ml CPZ). Vitamin E decreased the cell lysis both in the dark and upon irradiation. Oxygen radical scavengers such as glutathione, B-carotene, mannitol, D-penicillamine as well as superoxide dismutase and catalase did not decrease cell lysis. The phototoxic effects of CPZ was found not to be due to stable photoproducts formed during irradiation of CPZ. PMID:3359800

  6. Mesd extrinsically promotes phagocytosis by retinal pigment epithelial cells.

    PubMed

    Chen, Xiuping; Guo, Feiye; LeBlanc, Michelle E; Ding, Ying; Zhang, Chenming; Shakya, Akhalesh; Li, Wei

    2016-08-01

    Phagocytosis is a critical process to maintain tissue homeostasis. In the retina, photoreceptor cells renew their photoexcitability by shedding photoreceptor outer segments (POSs) in a diurnal rhythm. Shed POSs are phagocytosed by retinal pigment epithelial (RPE) cells to prevent debris accumulation, retinal degeneration, and blindness. Phagocytosis ligands are the key to understanding how RPE recognizes shed POSs. Here, we characterized mesoderm development candidate 2 (Mesd or Mesdc2), an endoplasmic reticulum (ER) chaperon for low-density lipoprotein receptor-related proteins (LRPs), to extrinsically promote RPE phagocytosis. The results showed that Mesd stimulated phagocytosis of fluorescence-labeled POS vesicles by D407 RPE cells. Ingested POSs were partially degraded within 3 h in some RPE cells to dispense undegradable fluorophore throughout the cytoplasm. Internalized POSs were colocalized with phagosome biomarker Rab7, suggesting that Mesd-mediated engulfment is involved in a phagocytosis pathway. Mesd also facilitated phagocytosis of POSs by primary RPE cells. Mesd bound to unknown phagocytic receptor(s) on RPE cells. Mesd was detected in the cytoplasm, but not nuclei, of different retinal layers and is predominantly expressed in the ER-free cellular compartment of POSs. Mesd was not secreted into medium from healthy cells but passively released from apoptotic cells with increased membrane permeability. Released Mesd selectively bound to the surface of POS vesicles and apoptotic cells, but not healthy cells. These results suggest that Mesd may be released from and bind to shed POSs to facilitate their phagocytic clearance. PMID:27184668

  7. Effect of curcumin on aging retinal pigment epithelial cells.

    PubMed

    Zhu, Wei; Wu, Yan; Meng, Yi-Fang; Wang, Jin-Yu; Xu, Ming; Tao, Jian-Jun; Lu, Jiong

    2015-01-01

    Age-related macular degeneration (AMD) is now one of the leading causes of blindness in the elderly population. The antioxidative effects of curcumin on aging retinal pigment epithelial (RPE) cells are still unclear. We conducted an in vitro study to investigate the effects of curcumin on aging RPE cells. A pulsed H2O2 exposure aging model was adopted. Aging RPE cells were treated with curcumin 20 µM, 40 µM, and 80 µM. Apoptosis of RPE cells was analyzed by flow cytometry. The intracellular reactive oxygen species concentration was detected using a specific probe and apoptosis-associated proteins were detected by Western blot. Expression of oxidative biomarkers, including superoxide dismutase, maleic dialdehyde, and glutathione, was detected commercially available assay kits. Compared with normal cells, lower cell viability, higher apoptosis rates, and more severe oxidation status were identified in the aging RPE cell model. Curcumin improved cell viability and decreased apoptosis and oxidative stress. Further, curcumin had a significant influence on expression of apoptosis-associated proteins and oxidative stress biomarkers. In conclusion, treatment with curcumin was able to regulate proliferation, oxidative stress, and apoptosis in aging RPE cells. Accordingly, application of curcumin may be a novel strategy to protect against age-related change in AMD. PMID:26445530

  8. ABCF1 extrinsically regulates retinal pigment epithelial cell phagocytosis

    PubMed Central

    Guo, Feiye; Ding, Ying; Caberoy, Nora; Alvarado, Gabriela; Wang, Feng; Chen, Rui; Li, Wei

    2015-01-01

    Phagocytosis of shed photoreceptor outer segments (POSs) by retinal pigment epithelial (RPE) cells is critical to retinal homeostasis and shares many conserved signaling pathways with other phagocytes, including extrinsic regulations. Phagocytotic ligands are the key to cargo recognition, engulfment initiation, and activity regulation. In this study, we identified intracellular protein ATP-binding cassette subfamily F member 1 (ABCF1) as a novel RPE phagocytotic ligand by a new approach of functional screening. ABCF1 was independently verified to extrinsically promote phagocytosis of shed POSs by D407 RPE cells. This finding was further corroborated with primary RPE cells and RPE explants. Internalized POS vesicles were colocalized with a phagosome marker, suggesting that ABCF1-mediated engulfment is through a phagocytic pathway. ABCF1 was released from apoptotic cells and selectively bound to shed POS vesicles and apoptotic cells, possibly via externalized phosphatidylserine. ABCF1 is predominantly expressed in POSs and colocalized with the POS marker rhodopsin, providing geographical convenience for regulation of RPE phagocytosis. Collectively these results suggest that ABCF1 is released from and binds to shed POSs in an autocrine manner to facilitate RPE phagocytosis through a conserved pathway. Furthermore, the new approach is broadly applicable to many other phagocytes and will enable systematic elucidation of their ligands to understand extrinsic regulation and cargo recognition. PMID:25904329

  9. Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation

    PubMed Central

    Taylor, AW; Dixit, S; Yu, J

    2015-01-01

    The eye is an immune privileged tissue with multiple mechanisms of immunosuppression to protect the light gathering tissues from the damage of inflammation. One of theses mechanisms involves retinal pigment epithelial cell suppression of phagosome activation in macrophages. The objective of this work is to determine if the human RPE cell line ARPE-19 is capable of suppressing the activation of the phagolysosome in macrophages in a manner similar to primary RPE. The conditioned media of RPE eyecups, sub-confluent, just confluent cultures, or established confluent cultures of human ARPE-19 cells were generated. These condition media were used to treat macrophages phagocytizing pHrodo bioparticles. After 24 hours incubation the macrophages were imaged by fluorescent microscopy, and fluorescence was measured. The fluorescent intensity is proportional to the amount of bioparticles phagocytized and are in an activated phagolysosome. The conditioned media of in situ mouse RPE eyecups significantly suppressed the activation of phagolysosome. The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation. In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation. The neuropeptides alpha-MSH and NPY were depleted from the conditioned media of established confluent ARPE-19 cell cultures. This depleted conditioned media had diminished suppression of phagolysosome activation while promoting macrophage cell death. In addition, the condition media from cultures of ARPE-19 monolayers wounded with a bisecting scrape was diminished in suppressing phagolysosome activation. This technical report suggests that like primary RPE monolayers, established confluent cultures of ARPE-19 cells produce soluble factors that suppress the activation of macrophages, and can be used to study the molecular mechanisms of retinal immunobiology. In

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

    PubMed Central

    Fronk, Aaron H; Vargis, Elizabeth

    2016-01-01

    The retinal pigment epithelium is an important part of the vertebrate eye, particularly in studying the causes and possible treatment of age-related macular degeneration. The retinal pigment epithelium is difficult to access in vivo due to its location at the back of the eye, making experimentation with age-related macular degeneration treatments problematic. An alternative to in vivo experimentation is cultivating the retinal pigment epithelium in vitro, a practice that has been going on since the 1970s, providing a wide range of retinal pigment epithelial culture protocols, each producing cells and tissue of varying degrees of similarity to natural retinal pigment epithelium. The purpose of this review is to provide researchers with a ready list of retinal pigment epithelial protocols, their effects on cultured tissue, and their specific possible applications. Protocols using human and animal retinal pigment epithelium cells, derived from tissue or cell lines, are discussed, and recommendations for future researchers included. PMID:27493715

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

    PubMed

    Fronk, Aaron H; Vargis, Elizabeth

    2016-01-01

    The retinal pigment epithelium is an important part of the vertebrate eye, particularly in studying the causes and possible treatment of age-related macular degeneration. The retinal pigment epithelium is difficult to access in vivo due to its location at the back of the eye, making experimentation with age-related macular degeneration treatments problematic. An alternative to in vivo experimentation is cultivating the retinal pigment epithelium in vitro, a practice that has been going on since the 1970s, providing a wide range of retinal pigment epithelial culture protocols, each producing cells and tissue of varying degrees of similarity to natural retinal pigment epithelium. The purpose of this review is to provide researchers with a ready list of retinal pigment epithelial protocols, their effects on cultured tissue, and their specific possible applications. Protocols using human and animal retinal pigment epithelium cells, derived from tissue or cell lines, are discussed, and recommendations for future researchers included. PMID:27493715

  12. [Regeneration of the retina using pigment epithelial cell transplantation].

    PubMed

    Abe, Toshiaki

    2002-12-01

    At has been reported that transplantation of appropriate cells, growth factors, and/or extracellular matrix may help the regeneration of damaged tissues or organs. Some growth factors, such as basic fibroblast growth factor(bFGF), have been successfully transferred to patients with ischemic heart disease. Embryonic dopamine neurons were also transplanted into the brains of patients with Parkinson's disease successfully. We have also performed cultured auto iris pigment epithelial cell (IPE) transplantation into the subretinal space after removal of choroidal neovascularization in patients with age-related macular degeneration (AMD). Here, we report the results of auto IPE transplantation in 35 patients, who could be followed for more than 6 months. We also tried to apply cell transplantation to other retinal diseases by managing the transplanted cells as introduced growth factor genes. Auto IPE transplantation was performed after removal of choroidal neovascular membranes (CNV). Visual acuity wes improved by more than 0.2 log MAR in 18 of 35 patients (51.5%), it was unchanged in 11 patients (31.5%), and it was worsened in 6 patients (17%). No significant difference was observed in comparison to patients who underwent CNV removal only. However, unlike the previous reports, we found no patients showing rejection. We also found that the cultured transplanted cells never showed proliferation under the retina or in the vitreous cavity and concluded that cultured auto IPE transplantation can be performed safely without complications. Next, we examined whether cell transplantation can be expanded to other degenerative retinal diseases. One of our results showed that host RPE may play an important role against the transplanted cells in the subretinal regions. When we introduced bFGF gene into the cells, we found synexpression cluster of the genes in the cells. One of the most prominent movements among the genes was lysyl oxidase like-1 gene, which plays an important role

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

    PubMed

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

    2012-09-01

    Dysfunction and loss of retinal pigment epithelium (RPE) are major pathologic changes observed in various retinal degenerative diseases such as aged-related macular degeneration. RPE generated from human pluripotent stem cells can be a good candidate for RPE replacement therapy. Here, we show the differentiation of human embryonic stem cells (hESCs) toward RPE with the generation of spherical neural masses (SNMs), which are pure masses of hESCs-derived neural precursors. During the early passaging of SNMs, cystic structures arising from opened neural tube-like structures showed pigmented epithelial morphology. These pigmented cells were differentiated into functional RPE by neuroectodermal induction and mechanical purification. Most of the differentiated cells showed typical RPE morphologies, such as a polygonal-shaped epithelial monolayer, and transmission electron microscopy revealed apical microvilli, pigment granules, and tight junctions. These cells also expressed molecular markers of RPE, including Mitf, ZO-1, RPE65, CRALBP, and bestrophin. The generated RPE also showed phagocytosis of isolated bovine photoreceptor outer segment and secreting pigment epithelium-derived factor and vascular endothelial growth factor. Functional RPE could be generated from SNM in our method. Because SNMs have several advantages, including the capability of expansion for long periods without loss of differentiation capability, easy storage and thawing, and no need for feeder cells, our method for RPE differentiation may be used as an efficient strategy for generating functional RPE cells for retinal regeneration therapy. PMID:22683799

  14. Yap and Taz regulate retinal pigment epithelial cell fate

    PubMed Central

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

    2015-01-01

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

  15. Proteasome-dependent regulation of signal transduction in retinal pigment epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As in many other types of cells, retinal pigment epithelial (RPE) cells have an active ubiquitin-proteasome pathway (UPP). However, the function of the UPP in RPE remains to be elucidated. The objective of this study is to determine the role of the UPP in controlling the levels and activities of tra...

  16. Retinal pigment epithelial cell necroptosis in response to sodium iodate

    PubMed Central

    Hanus, J; Anderson, C; Sarraf, D; Ma, J; Wang, S

    2016-01-01

    Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly in developed countries. The late stage of dry AMD, or geographic atrophy (GA), is characterized by extensive retinal pigment epithelium (RPE) degeneration. The underlying molecular mechanism for RPE cell death in GA remains unclear. Our previous study has established that RPE cells die predominantly from necroptosis in response to oxidative stress in vitro. Here, we extend our study and aim to characterize the nature of RPE cell death in response to sodium iodate (NaIO3) in vitro and in a NaIO3-induced retina degeneration mouse model. We found that NaIO3 induces RPE necroptosis in vitro by using a combination of molecular hallmarks. By using TUNEL assays, active caspase-3 and HMGB1 immunostaining, we confirmed that photoreceptor cells die mainly from apoptosis and RPE cells die mainly from necroptosis in response to NaIO3 in vivo. RPE necroptosis in this model is also supported by use of the RIPK1 inhibitor, Necrostatin-1. Furthermore, using novel RIPK3-GFP transgenic mouse lines, we detected RIPK3 aggregation, a hallmark of necroptosis, in the RPE cells in vivo after NaIO3 injection. Our findings suggest the necessity of re-evaluating RPE cell death mechanism in AMD models and have the potential to influence therapeutic development for dry AMD, especially GA. PMID:27551542

  17. Retinal pigment epithelial cell necroptosis in response to sodium iodate.

    PubMed

    Hanus, J; Anderson, C; Sarraf, D; Ma, J; Wang, S

    2016-01-01

    Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly in developed countries. The late stage of dry AMD, or geographic atrophy (GA), is characterized by extensive retinal pigment epithelium (RPE) degeneration. The underlying molecular mechanism for RPE cell death in GA remains unclear. Our previous study has established that RPE cells die predominantly from necroptosis in response to oxidative stress in vitro. Here, we extend our study and aim to characterize the nature of RPE cell death in response to sodium iodate (NaIO3) in vitro and in a NaIO3-induced retina degeneration mouse model. We found that NaIO3 induces RPE necroptosis in vitro by using a combination of molecular hallmarks. By using TUNEL assays, active caspase-3 and HMGB1 immunostaining, we confirmed that photoreceptor cells die mainly from apoptosis and RPE cells die mainly from necroptosis in response to NaIO3 in vivo. RPE necroptosis in this model is also supported by use of the RIPK1 inhibitor, Necrostatin-1. Furthermore, using novel RIPK3-GFP transgenic mouse lines, we detected RIPK3 aggregation, a hallmark of necroptosis, in the RPE cells in vivo after NaIO3 injection. Our findings suggest the necessity of re-evaluating RPE cell death mechanism in AMD models and have the potential to influence therapeutic development for dry AMD, especially GA. PMID:27551542

  18. THE PROTEASOME IS A TARGET OF OXIDATIVE DAMAGE IN HUMAN RETINA PIGMENT EPITHELIAL CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Purpose: Dysfunction of the ubiquitin-proteasome pathway (UPP) is associated with several age-related degenerative diseases. The objective of this study is to investigate the effect of oxidative stress on the UPP in retina pigment epithelial cells. Methods: To mimic physiological oxidative stress...

  19. Altered aldose reductase gene regulation in cultured human retinal pigment epithelial cells.

    PubMed Central

    Henry, D N; Del Monte, M; Greene, D A; Killen, P D

    1993-01-01

    Aldose reductase (AR2), a putative "hypertonicity stress protein" whose gene is induced by hyperosmolarity, protects renal medullary cells against the interstitial hyperosmolarity of antidiuresis by catalyzing the synthesis of millimolar concentrations of intracellular sorbitol from glucose. Although AR2 gene induction has been noted in a variety of renal and nonrenal cells subjected to hypertonic stress in vitro, the functional significance of AR2 gene expression in cells not normally exposed to a hyperosmolar milieu is not fully understood. The physiological impact of basal AR2 expression in such cells may be limited to hyperglycemic states in which AR2 promotes pathological polyol accumulation, a mechanism invoked in the pathogenesis of diabetic complications. Since AR2 overexpression in the retinal pigment epithelium has been associated with diabetic retinopathy, the regulation of AR2 gene expression and associated changes in sorbitol and myo-inositol were studied in human retinal pigment epithelial cells in culture. The relative abundance of aldehyde reductase (AR1) and AR2 mRNA was quantitated by filter hybridization of RNA from several human retinal pigment epithelial cell lines exposed to hyperglycemic and hyperosmolar conditions in vitro. AR2 but not AR1 mRNA was significantly increased some 11- to 18-fold by hyperosmolarity in several retinal pigment epithelial cell lines. A single cell line with a 15-fold higher basal level of AR2 mRNA than other cell lines tested demonstrated no significant increase in AR2 mRNA in response to hypertonic stress. This cell line demonstrated accelerated and exaggerated production of sorbitol and depletion of myo-inositol upon exposure to 20 mM glucose. Therefore, abnormal AR2 expression may enhance the sensitivity of cells to the biochemical consequences of hyperglycemia potentiating the development of diabetic complications. Images PMID:8349800

  20. Low Power Laser Irradiation Stimulates the Proliferation of Adult Human Retinal Pigment Epithelial Cells in Culture

    PubMed Central

    Song, Qing; Uygun, Basak; Banerjee, Ipsita; Nahmias, Yaakov; Zhang, Quan; Berthiaume, François; Latina, Mark; Yarmush, Martin L.

    2015-01-01

    We investigated the effects of low power laser irradiation on the proliferation of retinal pigment epithelial (RPE) cells. Adult human RPE cells were artificially pigmented by preincubation with sepia melanin, and exposed to a single sublethal laser pulse (590 nm, 1 µs, <200 mJ/cm2). DNA synthesis, cell number, and growth factor activity in irradiated RPE cells were subsequently monitored. The effect of sublethal laser irradiation on the “wound” healing response of an RPE monolayer in an in vitro scratch assay was also investigated. Single pulsed laser irradiation increased DNA synthesis in pigmented RPE cells measured 6 h post-treatment. In the scratch assay, laser irradiation increased the rates of cell proliferation and wound closure. Conditioned medium, collected 48 h following laser treatment, increased cell proliferation of unirradiated cells. Irradiation increased RPE cell secretion of platelet-derived growth factor (PDGF)-B chain, and increased mRNA levels of several growth factors and their receptors, including PDGF, transforming growth factor-β1, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor, as well as heat shock proteins. This demonstrates, for the first time, that low power single pulsed laser irradiation stimulates the proliferation of RPE cells, and upregulates growth factors that are mitogenic for RPE cells. PMID:26740823

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

    PubMed Central

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

    2011-01-01

    Purpose The purpose was to develop suitable in vitro methods to detect ocular epithelial cell damage when exposed to UV radiation, in an effort to evaluate UV-absorbing ophthalmic biomaterials. Methods Human corneal epithelial cells (HCEC), lens epithelial cells (HLEC), and retinal pigment epithelial cells (ARPE-19) were cultured and Ultraviolet A/Ultraviolet B (UVA/UVB) blocking filters and UVB-only blocking filters were placed between the cells and a UV light source. Cells were irradiated with UV radiations at various energy levels with and without filter protections. Cell viability after exposure was determined using the metabolic dye alamarBlue and by evaluating for changes in the nuclei, mitochondria, membrane permeability, and cell membranes of the cells using the fluorescent dyes Hoechst 33342, rhodamine 123, calcein AM, ethidium homodimer-1, and annexin V. High-resolution images of the cells were taken with a Zeiss 510 confocal laser scanning microscope. Results The alamarBlue assay results of UV-exposed cells without filters showed energy level-dependent decreases in cellular viability. However, UV treated cells with 400 nm LP filter protection showed the equivalent viability to untreated control cells at all energy levels. Also, UV irradiated cells with 320 nm LP filter showed lower cell viability than the unexposed control cells, yet higher viability than UV-exposed cells without filters in an energy level-dependent manner. The confocal microscopy results also showed that UV radiation can cause significant dose-dependent degradations of nuclei and mitochondria in ocular cells. The annexin V staining also showed an increased number of apoptotic cells after UV irradiation. Conclusions The findings suggest that UV-induced HCEC, HLEC, and ARPE-19 cell damage can be evaluated by bioassays that measure changes in the cell nuclei, mitochondria, cell membranes, and cell metabolism, and these assay methods provide a valuable in vitro model for evaluating the

  2. Expression of pigment epithelium‐derived factor and thrombospondin‐1 regulate proliferation and migration of retinal pigment epithelial cells

    PubMed Central

    Farnoodian, Mitra; Kinter, James B.; Yadranji Aghdam, Saeed; Zaitoun, Ismail; Sorenson, Christine M.; Sheibani, Nader

    2015-01-01

    Abstract Age‐related macular degeneration (AMD) is the leading cause of vision loss among elderly. Although the pathogenesis of AMD is associated with retinal pigmented epithelium (RPE) dysfunction and abnormal neovascularization the detailed mechanisms remain unresolved. RPE is a specialized monolayer of epithelial cells with important functions in ocular homeostasis. Pathological RPE damage contributes to major ocular conditions including retinal degeneration and irreversible loss of vision in AMD. RPE cells also assist in the maintenance of the ocular angiogenic balance by production of positive and negative regulatory factors including vascular endothelial growth factor (VEGF), thrombospondin‐1 (TSP1), and pigment epithelium‐derived factor (PEDF). The altered production of PEDF and TSP1, as endogenous inhibitors of angiogenesis and inflammation, by RPE cells have been linked to pathogenesis of AMD and choroidal and retinal neovascularization. However, lack of simple methods for isolation and culture of mouse RPE cells has resulted in limited knowledge regarding the cell autonomous role of TSP1 and PEDF in RPE cell function. Here, we describe a method for routine isolation and propagation of RPE cells from wild‐type, TSP1, and PEDF‐deficient mice, and have investigated their impact on RPE cell function. We showed that expression of TSP1 and PEDF significantly impacted RPE cell proliferation, migration, adhesion, oxidative state, and phagocytic activity with minimal effect on their basal rate of apoptosis. Together, our results indicated that the expression of PEDF and TSP1 by RPE cells play crucial roles not only in regulation of ocular vascular homeostasis but also have significant impact on their cellular function. PMID:25602019

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

    PubMed

    Han, Meng; Bindewald-Wittich, Almut; Holz, Frank G; Giese, Guenter; 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. PMID:16526877

  4. Protection of photoreceptor cells from phototoxicity by transplanted retinal pigment epithelial cells expressing different neurotrophic factors.

    PubMed

    Abe, Toshiaki; Saigo, Yoko; Hojo, Masayoshi; Kano, Tetsuya; Wakusawa, Ryosuke; Tokita, Yumi; Tamai, Makoto

    2005-01-01

    Transplantation of cells or tissues and the intravitreal injection of neurotrophic factors are two methods that have been used to treat retinal diseases. The purpose of this study was to examine the effects of combining both methods: the transplantation of retinal pigment epithelial (RPE) cells expressing different neurotrophic factors. The neutrophic factors were Axokine, brain derived-neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF). The enhanced green fluorescence protein (eGFP) gene was used as a reporter gene. These genes were transduced into RPE cells by lipofection, selected by antibiotics, and transplanted into the subretinal space of 108 rats. The rats were examined at 1 week and 3 months after the transplantation to determine whether the transduced cells were present, were expressing the protein, and were able to protect photoreceptors against phototoxicity. The survival of the transplanted cells was monitored by the presence of eGFP. The degree of protection was determined by the thickness of the outer nuclear layer. Our results showed that the degree of photoreceptor protection was different for the different types of neurotrophic factors at 1 week. After 3 months, the number of surviving transplanted cell was markedly reduced, and protection was observed only with the BDNF-transduced RPE cells. A significant degree of rescue was also observed by BDNF-transduced RPE cells in the nontransplanted area of the retina at both the early and late times. Lymphocytic infiltration was not detected in the vitreous, retina, and choroid at any time. We conclude that the transplantation of BDNF-transduced RPE cells can reduce the photoreceptor damage induced by phototoxicity in the transplanted area and weakly in the nontransplanted area. PMID:16454354

  5. Regulation of angiogenin expression and epithelial-mesenchymal transition by HIF-1α signaling in hypoxic retinal pigment epithelial cells.

    PubMed

    Lai, Kairan; Luo, Chenqi; Zhang, Xiaobo; Ye, Panpan; Zhang, Yidong; He, Jiliang; Yao, Ke

    2016-09-01

    Choroidal neovascularization (CNV) is a major cause of vision loss in many retinal diseases. Hypoxia is determined to be a key inducer of CNV and hypoxia-inducible factor-1 (HIF-1) is an important transcription factor. Epithelial-mesenchymal transition (EMT) and the synthesis of proangiogenic cytokines make great contributions to the development of CNV. In the present study, the role of HIF-1α signaling in the regulation of angiogenin (ANG) expression and EMT in hypoxic retinal pigment epithelial cells was investigated. A significant elevation expression of ANG expression level in a mouse model of laser-induced CNV was demonstrated. In a hypoxic model of ARPE-19, an increased expression level of ANG and induction of EMT accompanied with stabilization and nucleus translocation of HIF-1α. Blockage of HIF-1α signaling resulted in inhibition of high expression of ANG and EMT features. The direct interaction between HIF-1α and ANG promoter region was identified by ChIP-qPCR. The association of RNase 4 mRNA level with HIF-1α signaling was also clarified in APRE-19. Moreover, the exogenous ANG translocated into the nucleus, enhanced 45S rRNA transcription, promoted cell proliferation and tube formation in human retinal microvascular endothelial cells. In conclusion, the hypoxic conditions regulate the expression of ANG and EMT via an activation of HIF-1α signaling. It provides molecular evidence for potential therapy strategies of treating CNV. PMID:27259982

  6. Differentiation of cones in cultured rabbit retina: effects of retinal pigment epithelial cell-conditioned medium.

    PubMed

    Mack, Andreas F; Uhlmann, Daniela; Germer, Angela; Szél, Agoston; Enzmann, Volker; Reichenbach, Andreas

    2003-04-24

    This study was aimed at investigating the postnatal differentiation of cone photoreceptors in the rabbit retina in an organotypic explant culture system. Both short wavelength (S) and middle wavelength (M) cone opsins were expressed in culture but M cones appeared only in retinal explants from the dorsal half of the eye. Stimulating the explants with retinal pigment epithelial cell (RPE) conditioned medium resulted in a suppression of opsin expression despite of an increase of the number of presumptive peanut agglutinin-labeled cones. These results suggest that at birth the immature cones are largely undetermined in terms of their final cone identity although some positional information ('dorsal' vs. 'ventral' retina) is present. Furthermore, factors from RPE may inhibit as well as stimulate different steps of cone cell differentiation. PMID:12676342

  7. Regulation of aryl hydrocarbon receptor-mediated transcription in human retinal pigmented epithelial cells.

    PubMed

    Jin, Hong Lan; Jeong, Kwang Won

    2016-04-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor with pleiotropic effects in normal physiology or vascular development, xenobiotic metabolism, and cancer. A previous study has reported that BRG1, a component of the SWI/SNF complex, is a coactivator for AHR and is recruited to the promoter region of the CYP1A1 gene in mouse hepatocytes. Recent data suggest that AHR is also expressed in human retinal pigment epithelial cells (ARPE-19), which play a crucial role in retinal physiology and the visual cycle. Multiple studies have shown that the AHR plays an important role in the pathogenesis of retinal diseases including age-related macular degeneration. However, the mechanism of AHR transcriptional activation in retinal pigment cells has not been reported. Here, we demonstrate that the AHR signaling pathway is active in ARPE-19 cells, as in hepatocytes, but with different target gene specificity. We also found that chromatin remodeling by the BRG1-containing SWI/SNF complex is required for the AHR-mediated expression of target genes in ARPE-19 cells. We identified a novel enhancer region (-12 kb) of the CYP1A1 gene in ARPE-19 cells, to which both AHR and BRG1 are recruited in a ligand-dependent manner. BRG1 is associated with the AHR in ARPE-19 cells, and the C-terminal activation domain of the AHR directly interacts with BRG1. Furthermore, depletion of BRG1 caused a reduction in chromatin accessibility at the CYP1A1 enhancer. These results suggest that ARPE-19 cells possess an AHR-mediated transcription pathway with different target gene specificity, and that BRG1 is required for AHR-mediated transcription in ARPE-19 cells. PMID:26966070

  8. Regulation of tyrosinase expression and activity in cultured human retinal pigment epithelial cells.

    PubMed

    Abul-Hassan, K; Walmsley, R; Tombran-Tink, J; Boulton, M

    2000-12-01

    The purpose of this study was to investigate the regulation of tyrosinase gene expression and activity in cultured human retinal pigment epithelial (RPE) cells. The tyrosinase promoter (Ty.prom) region (400 bp) was PCR amplified and cloned into a modified mammalian expression vector (pcDNA3.1) upstream of a firefly luciferase (Luc) cDNA and was designated 'pcDNA3.1-Ty.prom.Luc'. The plasmid was co-transfected into RPE cells with a second mammalian expression plasmid (pRL-TK) containing a herpes simplex virus thymidine kinase promoter region upstream of Renilla Luc in a protocol designated the 'dual luciferase assay' (DLA). After co-transfection, cells were treated with a range of potential melanogenic agents; basic fibroblast growth factor (bFGF), methyl methane sulphonate, alpha-melanocyte stimulating hormone, verapamil, phorbol myristate acetate, cholera toxin (CT), pigment epithelium derived factor (PEDF), and L-tyrosine. The expression of tyrosinase promoter and enzymatic activities were determined 48 hr post-transfection using the DLA and DOPA oxidase assays, respectively. Tyrosinase activity could not be detected in RPE cells with any of the treatments. Tyrosinase promoter activity was significantly up-regulated in RPE cells treated with bFGF, PEDF, verapamil, CT and tyrosine compared with control cells. In conclusion, the tyrosinase gene is not only expressed but can be regulated in response to different chemicals in cultured human RPE cells. However, it appears that RPE cells in culture lack a post-transcriptional and/or translational modification point(s), which are necessary for tyrosinase enzymic activity. PMID:11153695

  9. Pirfenidone inhibits migration, differentiation, and proliferation of human retinal pigment epithelial cells in vitro

    PubMed Central

    Wang, Jing; Yang, Yangfan; Xu, Jiangang; Lin, Xianchai; Wu, Kaili

    2013-01-01

    Purpose To investigate the effects of pirfenidone (PFD) on the migration, differentiation, and proliferation of retinal pigment epithelial (RPE) cells and demonstrate whether the drug induces cytotoxicity. Methods Human RPE cells (line D407) were treated with various concentrations of PFD. Cell migration was measured with scratch assay. The protein levels of fibronectin (FN), connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), transforming growth factor beta (TGFβS), and Smads were assessed with western blot analyses. Levels of mRNA of TGFβS, FN, and Snail1 were analyzed using reverse transcriptase–polymerase chain reaction. Cell apoptosis was detected with flow cytometry using the Annexin V/PI apoptosis kit, and the percentages of cells labeled in different apoptotic stage were compared. A Trypan Blue assay was used to assess cell viability. Results PFD inhibited RPE cell migration. Western blot analyses showed that PFD inhibited the expression of FN, α-SMA, CTGF, TGFβ1, TGFβ2, Smad2/3, and Smad4. Similarly, PFD also downregulated mRNA levels of Snail1, FN, TGFβ1, and TGFβ2. No significant differences in cell apoptosis or viability were observed between the control and PFD-treated groups. Conclusions PFD inhibited RPE cell migration, differentiation, and proliferation in vitro and caused no significant cytotoxicity. PMID:24415895

  10. The effect of retinal pigment epithelial cell patch size on growth factor expression

    SciTech Connect

    Vargis, Elizabeth A.; Peterson, Cristen B.; Morrell-Falvey, Jennifer L.; Retterer, Scott T.; Collier, Charles Patrick

    2014-01-30

    The spatial organization of retinal pigment epithelial (RPE) cells grown in culture was controlled using micropatterning techniques in order to examine the effect of patch size on cell health and differentiation. Understanding this effect is a critical step in the development of multiplexed high throughput fluidic assays and provides a model for replicating disease states associated with the deterioration of retinal tissue during age-related macular degeneration (AMD). Microcontact printing of fibronectin on polystyrene and glass substrates was used to promote cell attachment, forming RPE patches of controlled size and shape. These colonies mimic the effect of atrophy and loss-of-function that occurs in the retina during degenerative diseases such as AMD. After 72 hours of cell growth, levels of vascular endothelial growth factor (VEGF), an important biomarker of AMD, were measured. Cells were counted and morphological indicators of cell viability and tight junction formation were assessed via fluorescence microscopy. As a result, up to a twofold increase of VEGF expression per cell was measured as colony size decreased, suggesting that the local microenvironment of, and connections between, RPE cells influences growth factor expression leading to the initiation and progression of diseases such as AMD.

  11. The effect of retinal pigment epithelial cell patch size on growth factor expression

    DOE PAGESBeta

    Vargis, Elizabeth A.; Peterson, Cristen B.; Morrell-Falvey, Jennifer L.; Retterer, Scott T.; Collier, Charles Patrick

    2014-01-30

    The spatial organization of retinal pigment epithelial (RPE) cells grown in culture was controlled using micropatterning techniques in order to examine the effect of patch size on cell health and differentiation. Understanding this effect is a critical step in the development of multiplexed high throughput fluidic assays and provides a model for replicating disease states associated with the deterioration of retinal tissue during age-related macular degeneration (AMD). Microcontact printing of fibronectin on polystyrene and glass substrates was used to promote cell attachment, forming RPE patches of controlled size and shape. These colonies mimic the effect of atrophy and loss-of-function thatmore » occurs in the retina during degenerative diseases such as AMD. After 72 hours of cell growth, levels of vascular endothelial growth factor (VEGF), an important biomarker of AMD, were measured. Cells were counted and morphological indicators of cell viability and tight junction formation were assessed via fluorescence microscopy. As a result, up to a twofold increase of VEGF expression per cell was measured as colony size decreased, suggesting that the local microenvironment of, and connections between, RPE cells influences growth factor expression leading to the initiation and progression of diseases such as AMD.« less

  12. Controlling Retinal Pigment Epithelial Cell Patch Size Influences Growth Factor Expression

    DOE PAGESBeta

    Vargis, Elizabeth A; Peterson, Cristen B; Morrell-Falvey, Jennifer L; Retterer, Scott T; Collier, Pat

    2014-01-01

    The spatial organization of retinal pigment epithelial (RPE) cells grown in culture was controlled using micropatterning techniques in order to examine the effect of patch size on cell health and differentiation. Understanding this effect is a critical step in the development of multiplexed high throughput fluidic assays and provides a model for replicating disease states associated with the deterioration of retinal tissue during age-related macular degeneration (AMD). Microcontact printing of fibronectin on polystyrene and glass substrates was used to promote cell attachment, forming RPE patches of controlled size and shape. These colonies mimic the effect of atrophy and loss-of-function thatmore » occurs in the retina during degenerative diseases such as AMD. After 72 hours of cell growth, levels of vascular endothelial growth factor (VEGF), an important biomarker of AMD, were measured. Cells were counted and morphological indicators of cell viability and tight junction formation were assessed via fluorescence microscopy. Up to a twofold increase of VEGF expression per cell was measured as colony size decreased, suggesting that the local microenvironment of, and connections between, RPE cells influences growth factor expression leading to the initiation and progression of diseases such as AMD.« less

  13. Role of retinal pigment epithelial cell β-catenin signaling in experimental proliferative vitreoretinopathy.

    PubMed

    Umazume, Kazuhiko; Tsukahara, Rintaro; Liu, Lanhsin; Fernandez de Castro, Juan P; McDonald, Kevin; Kaplan, Henry J; Tamiya, Shigeo

    2014-05-01

    Proliferative vitreoretinopathy is caused by the contraction of fibrotic membranes on the epiretinal surface of the neurosensory retina, resulting in a traction retinal detachment and loss of visual acuity. Retinal pigment epithelial (RPE) cells play an important role in formation of such fibrotic, contractile membranes. We investigated the role of Wnt/β-catenin signaling, a pathway implicated in several fibrotic diseases, in RPE cells in proliferative vitreoretinopathy. In vitro culture of swine RPE sheets resulted in nuclear translocation of β-catenin in dedifferentiated RPE cells. FH535, a specific inhibitor of β-catenin signaling, reduced the outgrowth of cultured RPE sheets and prevented dedifferentiated RPE cell proliferation and migration. It also inhibited formation of contractile membranes by dedifferentiated RPE cells on collagen I matrices. Expression and function of the β-catenin signaling target connexin-43 were down-regulated by FH535, and functional blockade of connexins with carbenoxolone also prevented the in vitro formation of fibrotic, contractile membranes. Intravitreal injection of FH535 in swine also inhibited formation of dense, contractile membranes on the epiretinal surface and prevented development of traction retinal detachment. These findings demonstrate that β-catenin signaling is involved in formation of contractile membranes by dedifferentiated RPE cells and suggest that adjunctive treatment targeting this pathway could be useful in preventing proliferative vitreoretinopathy. PMID:24656918

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

    PubMed Central

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

    2016-01-01

    Background: We evaluated the protective effect of ALS-L1023, an extract of Melissa officinalis L. (Labiatae; lemon balm) against oxidative stress-induced apoptosis in human retinal pigment epithelial cells (ARPE-19 cells). Methods: ARPE-19 cells were incubated with ALS-L1023 for 24 h and then treated with hydrogen peroxide (H2O2). Oxidative stress-induced apoptosis and intracellular generation of reactive oxygen species (ROS) were assessed by flow cytometry. Caspase-3/7 activation and cleaved poly ADP-ribose polymerase (PARP) were measured to investigate the protective role of ALS-L1023 against apoptosis. The protective effect of ALS-L1023 against oxidative stress through activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) was evaluated by Western blot analysis. Results: ALS-L1023 clearly reduced H2O2-induced cell apoptosis and intracellular production of ROS. H2O2-induced oxidative stress increased caspase-3/7 activity and apoptotic PARP cleavage, which were significantly inhibited by ALS-L1023. Activation of the PI3K/Akt pathway was associated with the protective effect of ALS-L1023 on ARPE-19 cells. Conclusions: ALS-L1023 protected human RPE cells against oxidative damage. This suggests that ALS-L1023 has therapeutic potential for the prevention of dry age-related macular degeneration. PMID:26941573

  15. 4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells

    PubMed Central

    Sharma, Abha; Sharma, Rajendra; Chaudhary, Pankaj; Vatsyayan, Rit; Pearce, Virginia; Jeyabal, Prince V.S.; Zimniak, Piotr; Awasthi, Sanjay; Awasthi, Yogesh C.

    2009-01-01

    4-Hydroxynonenal (4-HNE) has been suggested to be involved in stress-induced signaling for apoptosis. In present studies, we have examined the effects of 4-HNE on the intrinsic apoptotic pathway associated with p53 in human retinal pigment epithelial (RPE and ARPE-19) cells. Our results show that 4-HNE causes induction, phosphorylation, and nuclear accumulation of p53 which is accompanied with down regulation of MDM2, activation of the pro-apoptotic p53 target genes viz. p21 and Bax, JNK, caspase3, and onset of apoptosis in treated RPE cells. Reduced expression of p53 by an efficient silencing of the p53 gene resulted in a significant resistance of these cells to 4-HNE-induced cell death. The effects of 4-HNE on the expression and functions of p53 are blocked in GSTA4-4 over expressing cells indicating that 4-HNE-induced, p53-mediated signaling for apoptosis is regulated by GSTs. Our results also show that the induction of p53 in tissues of mGsta4 (-/-) mice correlate with elevated levels of 4-HNE due to its impaired metabolism. Together, these studies suggest that 4-HNE is involved in p53-mediated signaling in in vitro cell cultures as well as in vivo that can be regulated by GSTs. PMID:18930016

  16. Phototoxicity and cytotoxicity of fullerol in human retinal pigment epithelial cells

    SciTech Connect

    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{sub 60}(OH){sub 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{sub 60}(OH){sub 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 muM. Exposure to an 8.5 J.cm{sup -2} dose of visible light in the presence of > 5 muM fullerol induced TBARS formation and early apoptosis, indicating phototoxic damage in the form of lipid peroxidation. Pretreatment with 10 and 20 muM 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 PHI = 0.05 in D{sub 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.

  17. Voltage-activated currents recorded from rabbit pigmented ciliary body epithelial cells in culture.

    PubMed Central

    Fain, G L; Farahbakhsh, N A

    1989-01-01

    1. The whole-cell recording mode of the patch-clamp technique was used to investigate the presence of voltage-activated currents in the isolated pigmented cells from the rabbit ciliary body epithelium grown in culture. 2. In Ringer solution with composition similar to that of the rabbit aqueous humour, depolarizing voltage steps activated a transient inward current and a delayed outward current, while hyperpolarization elicited an inwardly rectified current. 3. The depolarization-activated inward current was mainly carried by Na+ and was blocked by submicromolar concentrations of tetrodotoxin. This current in many cells was sufficiently large to produce a regenerative Na+ spike. 4. The depolarization-activated outward current was carried by K+ and blocked by external TEA and Ba2+. Its activation appeared to be Ca2(+)-independent. 5. The hyperpolarization-activated inward current was almost exclusively carried by K+ and was blocked by Ba2+ and Cs+. For large hyperpolarizations below -120 mV, this current exhibited a biphasic activation with a fast transient peak followed by a slower sag, that appeared to be due to K+ depletion. 6. The voltage-dependent K+ conductances probably act to stabilize the cell membrane resting potential and may also play a role in ion transport. The function of the Na(+)-dependent inward current is unclear, but it may permit the electrically coupled epithelial cells of the ciliary body to conduct propagated action potentials. Images Fig. 2 PMID:2621623

  18. Different death stimuli evoke apoptosis via multiple pathways in retinal pigment epithelial cells.

    PubMed

    Ferrington, Deborah A; Tran, Tina N; Lew, Kathleen L; Van Remmen, Holly; Gregerson, Dale S

    2006-09-01

    Loss of retinal pigment epithelial (RPE) cells via apoptosis plays a prominent role in several retinal degenerative diseases, such as age-related macular degeneration, and with light damage. Strategies for preservation of vision that would interrupt the apoptotic cascade require understanding the molecular events associated with apoptosis. This study investigated the susceptibility of RPE to caspase-dependent and -independent apoptotic pathways when challenged with different stimuli, including oxidants, anti-Fas antibody, and activated cytotoxic T lymphocytes (CTLs). These experiments used novel RPE cell lines developed from wildtype and heterozygous mice with reduced levels of either Mn superoxide dismutatse (SOD) or CuZnSOD. Peroxide and 4-hydroxynonenal induced apoptosis through both caspase-independent and -dependent pathways, respectively. With both oxidants, translocation of apoptosis inducing factor into the nucleus was observed. Cells containing reduced levels of CuZnSOD were the most susceptible to oxidant-induced cell death. Targeted killing by CTLs and activation of the Fas death receptor induced caspase-dependent apoptosis. These results show stimulus-specific activation of either the caspase-dependent or -independent pathway. Since cultured RPE express the protein components required for different apoptotic pathways, they provide a good model system for studying molecular events associated with multiple signals that lead to cell death. PMID:16682026

  19. Photobiomodulation with 670 nm light increased phagocytosis in human retinal pigment epithelial cells

    PubMed Central

    Fuma, Shinichiro; Murase, Hiromi; Kuse, Yoshiki; Tsuruma, Kazuhiro; Shimazawa, Masamitsu

    2015-01-01

    Purpose Photobiomodulation is the treatment with light in the far-red to near-infrared region of the spectrum and has been reported to have beneficial effects in various animal models of disease, including an age-related macular degeneration (AMD) mouse model. Previous reports have suggested that phagocytosis is reduced by age-related increased oxidative stress in AMD. Therefore, we investigated whether photobiomodulation improves phagocytosis caused by oxidative stress in the human retinal pigment epithelial (ARPE-19) cell line. Methods ARPE-19 cells and human primary retinal pigment epithelium (hRPE) cells were incubated and irradiated with near-infrared light (670 nm LED light, 2,500 lx, twice a day, 250 s/per time) for 4 d. Next, hydrogen peroxide (H2O2) and photoreceptor outer segments (POS) labeled using a pH-sensitive fluorescent dye were added to the cell culture, and phagocytosis was evaluated by measuring the fluorescence intensity. Furthermore, cell death was observed by double staining with Hoechst33342 and propidium iodide after photobiomodulation. CM-H2DCFDA, JC-1 dye, and CCK-8 were added to the cell culture to investigate the reactive oxygen species (ROS) production, mitochondrial membrane potential, and cell viability, respectively. We also investigated the expression of phagocytosis-related proteins, such as focal adhesion kinase (FAK) and Mer tyrosine kinase (MerTK). Results Oxidative stress inhibited phagocytosis, and photobiomodulation increased the oxidative stress-induced hypoactivity of phagocytosis in ARPE-19 cells and hRPE cells. Furthermore, H2O2 and photobiomodulation did not affect cell death in this experimental condition. Photobiomodulation reduced ROS production but did not affect cell viability or mitochondrial membrane potential. The expression of phosphorylated MerTK increased, but phosphorylated FAK was not affected by photobiomodulation. Conclusions These findings indicate that near-infrared light photobiomodulation (670 nm) may

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

    PubMed

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

    2013-01-01

    Human visual system is exposed to high levels of natural and artificial lights of different spectra and intensities along lifetime. Light-emitting diodes (LEDs) are the basic lighting components in screens of PCs, phones and TV sets; hence it is so important to know the implications of LED radiations on the human visual system. The aim of this study was to investigate the effect of LEDs radiations on human retinal pigment epithelial cells (HRPEpiC). They were exposed to three light-darkness (12 h/12 h) cycles, using blue-468 nm, green-525 nm, red-616 nm and white light. Cellular viability of HRPEpiC was evaluated by labeling all nuclei with DAPI; Production of reactive oxygen species (ROS) was determined by H2DCFDA staining; mitochondrial membrane potential was quantified by TMRM staining; DNA damage was determined by H2AX histone activation, and apoptosis was evaluated by caspases-3,-7 activation. It is shown that LED radiations decrease 75-99% cellular viability, and increase 66-89% cellular apoptosis. They also increase ROS production and DNA damage. Fluorescence intensity of apoptosis was 3.7% in nonirradiated cells and 88.8%, 86.1%, 83.9% and 65.5% in cells exposed to white, blue, green or red light, respectively. This study indicates three light-darkness (12 h/12 h) cycles of exposure to LED lighting affect in vitro HRPEpiC. PMID:22989198

  1. Biocompatibility of the vital dye Acid Violet-17 on retinal pigment epithelial cells

    PubMed Central

    Tura, Ayşegül; Alt, Aizhan; Lüke, Julia; Grisanti, Salvatore; Haritoglou, Christos; Meyer, Carsten H; Nassar, Khaled; Lüke, Matthias

    2016-01-01

    Purpose To examine the viability and differentiation of retinal pigment epithelial (RPE) cells after exposure to the vital dye Acid Violet-17 (AV-17). Methods Bovine RPE cells were incubated with AV-17 (0.0625–0.5 mg/mL) for 30 seconds or 5 minutes. Viability was determined by live/dead staining, cleaved CASP3 immunostainings, and MTT test. Actin cytoskeleton was visualized by Alexa 488-phalloidin. Immunocytochemistry was performed to determine the levels of ZO-1, CTNNB1, and KRT19. Results Exposure to AV-17 at the concentrations of 0.25–0.5 mg/mL resulted in a dose-dependent decrease in viability, the loss of ZO-1 from tight junctions, translocation of CTNNB1 into the cytoplasm and nucleus, disarrangement of the actin cytoskeleton, and a slight increase in KRT19. Conclusion AV-17 at a concentration <0.125 mg/mL is likely to be well tolerated by the RPE cells, whereas the concentrations from 0.25 mg/mL onward can reduce viability and induce dedifferentiation particularly after long-term exposure. PMID:27536056

  2. Downregulation of p22phox in Retinal Pigment Epithelial Cells Inhibits Choroidal Neovascularization in Mice

    PubMed Central

    Li, Qiuhong; Dinculescu, Astra; Shan, Zhiying; Miller, Rehae; Pang, Jijing; Lewin, Alfred S; Raizada, Mohan K; Hauswirth, William W

    2016-01-01

    Choroidal neovascularization (CNV) occurs in a variety of chorioretinal diseases including age-related macular degeneration (AMD), and is the major cause of severe visual loss in patients with AMD. Oxidative stress has been thought to play an important role in the development of CNV. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is one of the major intracellular sources of reactive oxygen species (ROS) in the vascular system. In this study, we examined the expression of p22phox, an integral subunit in the NADPH oxidase complex, in the mouse eye. We determined that p22phox is expressed in the retinal pigment epithelial (RPE) cells and inner retinal neurons. A small-interfering RNA (siRNA) designed against p22phox efficiently reduced the expression of the protein in the eye when delivered by means of recombinant adeno-associated virus (AAV) vector. Vector treatment inhibited CNV in the mouse when delivered into the subretinal space where RPE cells were transduced. These results suggest that NADPH oxidase–mediated ROS production in RPE cells may play an important role in the pathogenesis of neovascular AMD, and that this pathway may represent a new target for therapeutic intervention in AMD. PMID:18665154

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

    SciTech Connect

    Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. ); Pandey, S. )

    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.

  4. Propofol Decreases Endoplasmic Reticulum Stress–Mediated Apoptosis in Retinal Pigment Epithelial Cells

    PubMed Central

    Xu, Yue; Zhang, Ting; Zhang, Shaochong

    2016-01-01

    Age-related macular degeneration (AMD) is the major cause of loss of sight globally. There is currently no effective treatment available. Retinal pigment epithelial (RPE) cells are an important part of the outer blood-retina barrier and their death is a determinant of AMD. Propofol, a common clinically used intravenous anesthetic agent, has been shown to act as an efficacious neuroprotective agent with antioxidative and anti-inflammatory properties in vivo and in vitro. However, little is known about its effects on RPE cells. The purpose of our research was to investigate whether propofol could protect RPE cells from apoptosis through endoplasmic reticulum (ER) stress–dependent pathways. To this end, prior to stimulation with thapsigargin (TG), ARPE-19 cells were pretreated with varying concentrations of propofol. A protective effect of propofol in TG-treated ARPE-9 was apparent, TUNEL and flow cytometric assays showed decreased apoptosis. We further demonstrated that propofol pretreatment attenuated or inhibited the effects caused by TG, such as upregulation of Bax, BiP, C/EBP homologous protein (CHOP), active caspase 12, and cleaved caspase 3, and downregulation of Bcl2. It also decreased the TG-induced levels of ER stress–related molecules such as p-PERK, p-eIF2α, and ATF4. Furthermore, it downregulated the expression of nuclear factor κB (NF-κB). This study elucidated novel propofol-induced cellular mechanisms for antiapoptotic activities in RPE cells undergoing ER stress and demonstrated the potential value of using propofol in the treatment of AMD. PMID:27311010

  5. Planar Microdevices Enhance Transport of Large Molecular Weight Molecules Across Retinal Pigment Epithelial Cells

    PubMed Central

    Wade, Jennifer S.; Desai, Tejal A.

    2014-01-01

    Large molecular weight drug delivery to the posterior eye is challenging due to cellular barriers that hinder drug transport. Understanding how to enhance transport across the retinal barrier is important for the design of new drug delivery systems. A novel mechanism to enhance drug transport is the use of geometric properties, which has not been extensively explored in the retina. Planar SU-8/ Poly(ethyleneglycol)dimethacrylate microdevices were constructed using photolithography to deliver FITC dextran across an in vitro retinal model. The model consists of retinal pigment epithelial (RPE) cells grown to confluence on transwell inserts, which provides an environment to investigate the influence of geometry on paracellular and transcellular delivery of encapsulated large molecules. Planar microdevices enhanced transport of large molecular weight dextrans across different models of RPE in a size dependent fashion. Increased drug permeation across the RPE was observed with the addition of microdevices as compared to a traditional bolus of FITC dextran. This phenomena was initiated by a non-toxic interaction between the microdevices and the retinal tight junction proteins. Suggesting that increased drug transport occurs via a paracellular pathway. These experiments provide evidence to support the future use of planar unidirectional microdevices for delivery of biologics in ocular applications. PMID:24789225

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

    PubMed

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

    2016-07-18

    4-Hydroxy-7-oxo-5-heptenic acid (HOHA)-lactone is a biologically active oxidative truncation product released (t1/2 = 30 min at 37 °C) by nonenzymatic transesterification/deacylation from docosahexaenoate lipids. We now report that HOHA-lactone readily diffuses into retinal pigmented epithelial (RPE) cells where it is metabolized. A reduced glutathione (GSH) Michael adduct of HOHA-lactone is the most prominent metabolite detected by LC-MS in both the extracellular medium and cell lysates. This molecule appeared inside of ARPE-19 cells within seconds after exposure to HOHA-lactone. The intracellular level reached a maximum concentration at 30 min and then decreased with concomitant increases in its level in the extracellular medium, thus revealing a unidirectional export of the reduced GSH-HOHA-lactone adduct from the cytosol to extracellular medium. This metabolism is likely to modulate the involvement of HOHA-lactone in the pathogenesis of human diseases. HOHA-lactone is biologically active, e.g., low concentrations (0.1-1 μM) induce secretion of vascular endothelial growth factor (VEGF) from ARPE-19 cells. HOHA-lactone is also a precursor of 2-(ω-carboxyethyl)pyrrole (CEP) derivatives of primary amino groups in proteins and ethanolamine phospholipids that have significant pathological and physiological relevance to age-related macular degeneration (AMD), cancer, and wound healing. Both HOHA-lactone and the derived CEP can contribute to the angiogenesis that defines the neovascular "wet" form of AMD and that promotes the growth of tumors. While GSH depletion can increase the lethality of radiotherapy, because it will impair the metabolism of HOHA-lactone, the present study suggests that GSH depletion will also increase levels of HOHA-lactone and CEP that may promote recurrence of tumor growth. PMID:27355557

  7. Upregulation of GADD45α in light-damaged retinal pigment epithelial cells

    PubMed Central

    Gao, M-L; Deng, W-L; Huang, N; Wang, Y-Y; Lei, X-L; Xu, Z-Q; Hu, D-N; Cai, J-Q; Lu, F; Jin, Z-B

    2016-01-01

    To better understand the molecular mechanisms responsible for light-induced damage in retinal pigmented epithelial (RPE) cells, we developed an automated device to recapitulate intense light exposure. When compared with human fibroblasts, ARPE-19 cells that had been exposed to blue-rich light-emitting diode-light of 10 000 Lux at 37 °C for 9 h displayed dramatic cellular apoptosis. Collectively, gene expression profiling and qPCR demonstrated that growth arrest and DNA damage-45α (GADD45α) expression was markedly upregulated. Transient knockdown of GADD45α partially attenuated light-damage-induced apoptosis in ARPE-19 cells, whereas GADD45α overexpression dramatically increased it. These results demonstrate the critical function of GADD45α in light-induced RPE cellular apoptosis. Quantitative reverse transcription-PCR and western blotting revealed that the upregulation of GADD45α was under direct control of p53. Moreover, treatment with Ly294002, an inhibitor of AKT phosphorylation, further promoted GADD45α gene transcription in both non-light and light-damaged ARPE-19 cells. Treatment also exacerbated RPE cellular apoptosis after light exposure, confirming that inhibition of Akt phosphorylation increases GADD45α expression. Collectively, our findings reveal that light irrigation induces human RPE cellular apoptosis through upregulation of GADD45α expression mediated through both the p53 and phosphatidylinositol 3-kinase-AKT signaling pathways. These results provide new insights into human retinal diseases elicited by light damage and open a new avenue for disease prevention and treatment. PMID:27551507

  8. Expression and Functional Roles of Caspase-5 in Inflammatory Responses of Human Retinal Pigment Epithelial Cells

    PubMed Central

    Bian, Zong-Mei; Elner, Susan G.; Khanna, Hemant; Murga-Zamalloa, Carlos A.; Patil, Suresh

    2011-01-01

    Purpose. To investigate the expression, activation, and functional involvement of caspase-5 in human retinal pigment epithelial (hRPE) cells. Methods. Expression and activation of caspase-5 in primary cultured hRPE cells, telomerase-immortalized hTERT-RPE1 cells (hTERT-RPE1), or both, were measured after stimulation with proinflammatory agents IL-1β, TNF-α, lipopolysaccharide (LPS), interferon-γ, monocyte coculture, adenosine triphosphate (ATP), or endoplasmic reticulum (ER) stress inducers. Immunomodulating agents dexamethasone (Dex), IL-10, and triamcinolone acetonide (TA) were used to antagonize proinflammatory stimulation. Cell death ELISA and TUNEL staining assays were used to assess apoptosis. Results. Caspase-5 mRNA expression and protein activation were induced by LPS and monocyte-hRPE coculture. Caspase-5 activation appeared as early as 2 hours after challenge by LPS and consistently increased to 24 hours. Meanwhile, caspase-1 expression and protein activation were induced by LPS. Activation of caspase-5 was blocked or reduced by Dex, IL-10, and TA. Activation of caspase-5 and -1 was also enhanced by ATP and ER stress inducers. Expression and activation of caspase-5 were inhibited by a caspase-1–specific inhibitor. Caspase-5 knockdown reduced caspase-1 protein expression and activation and inhibited TNF-α–induced IL-8 and MCP-1. In contrast to caspase-4, the contribution of caspase-5 to stress-induced apoptosis was moderate. Conclusions. Caspase-5 mRNA synthesis, protein expression, and catalytic activation were highly regulated in response to various proinflammatory stimuli, ATP, and ER stress inducers. Mutual activation between caspase-5 and -1 suggests caspase-5 may work predominantly in concert with caspase-1 in modulating hRPE inflammatory responses. PMID:21969293

  9. Upregulation of GADD45α in light-damaged retinal pigment epithelial cells.

    PubMed

    Gao, M-L; Deng, W-L; Huang, N; Wang, Y-Y; Lei, X-L; Xu, Z-Q; Hu, D-N; Cai, J-Q; Lu, F; Jin, Z-B

    2016-01-01

    To better understand the molecular mechanisms responsible for light-induced damage in retinal pigmented epithelial (RPE) cells, we developed an automated device to recapitulate intense light exposure. When compared with human fibroblasts, ARPE-19 cells that had been exposed to blue-rich light-emitting diode-light of 10 000 Lux at 37 °C for 9 h displayed dramatic cellular apoptosis. Collectively, gene expression profiling and qPCR demonstrated that growth arrest and DNA damage-45α (GADD45α) expression was markedly upregulated. Transient knockdown of GADD45α partially attenuated light-damage-induced apoptosis in ARPE-19 cells, whereas GADD45α overexpression dramatically increased it. These results demonstrate the critical function of GADD45α in light-induced RPE cellular apoptosis. Quantitative reverse transcription-PCR and western blotting revealed that the upregulation of GADD45α was under direct control of p53. Moreover, treatment with Ly294002, an inhibitor of AKT phosphorylation, further promoted GADD45α gene transcription in both non-light and light-damaged ARPE-19 cells. Treatment also exacerbated RPE cellular apoptosis after light exposure, confirming that inhibition of Akt phosphorylation increases GADD45α expression. Collectively, our findings reveal that light irrigation induces human RPE cellular apoptosis through upregulation of GADD45α expression mediated through both the p53 and phosphatidylinositol 3-kinase-AKT signaling pathways. These results provide new insights into human retinal diseases elicited by light damage and open a new avenue for disease prevention and treatment. PMID:27551507

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

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

  11. Purinergic regulation of cation conductances and intracellular Ca2+ in cultured rat retinal pigment epithelial cells

    PubMed Central

    Ryan, Jennifer S; Baldridge, William H; Kelly, Melanie E M

    1999-01-01

    We used whole-cell patch clamp and fluorescent calcium imaging techniques to investigate the effects of adenosine 5′-triphosphate (ATP) on membrane currents and intracellular calcium concentration ([Ca2+]i)in rat retinal pigment epithelial (RPE) cells. In 62 % of RPE cells, application of 100 μM ATP elicited a fast inward current at negative membrane potentials. In 38 % of RPE cells recorded, a biphasic response to ATP was observed in which activation of the fast inward current was followed by activation of a delayed outward current. The ATP-activated inward current was a non-selective cation (NSC) current that showed inward rectification, reversed at −1.5 ± 1 mV and was permeable to monovalent cations. The NSC current was insensitive to the P2 purinoceptor antagonists, suramin or PPADS but was activated by the purinoceptor agonists UTP, ADP and 2MeSATP. The outward current activated by ATP reversed at −68 ± 3 mV (equilibrium potential for potassium (EK) = −84 mV) and was blocked by Ba2+ ions, consistent with the activation of a K+ conductance. The outward K+ conductance was also reduced by the maxi-KCa channel blocker iberiotoxin (IbTX; 10 nM), suggesting that ATP activated an outward Ca2+-activated K+ channel in rat RPE cells. The Ca2+-activated K+ current (IK(Ca)) was also activated by the purinoceptor agonists UTP, ADP and 2MeSATP. In fluo-3 or fluo-4 loaded RPE cells, ATP and the pyrimidine agonist UTP elevated [Ca2+]i. The increase in Ca2+ was not dependent on extracellular Ca2+ influx, but was sensitive to the Ca2+-ATPase inhibitor thapsigargin, confirming the involvement of intracellular Ca2+ stores release. These results suggest that rat RPE cells express both P2X purinoceptors that gate activation of a non-selective cation conductance and G protein-coupled P2Y purinoceptors that mediate Ca2+ release from intracellular stores and activation of a calcium-activated K+ current. PMID:10545141

  12. Purinergic regulation of cation conductances and intracellular Ca2+ in cultured rat retinal pigment epithelial cells.

    PubMed

    Ryan, J S; Baldridge, W H; Kelly, M E

    1999-11-01

    1. We used whole-cell patch clamp and fluorescent calcium imaging techniques to investigate the effects of adenosine 5'-triphosphate (ATP) on membrane currents and intracellular calcium concentration ([Ca2+]i)in rat retinal pigment epithelial (RPE) cells. In 62 % of RPE cells, application of 100 microM ATP elicited a fast inward current at negative membrane potentials. In 38 % of RPE cells recorded, a biphasic response to ATP was observed in which activation of the fast inward current was followed by activation of a delayed outward current. 2. The ATP-activated inward current was a non-selective cation (NSC) current that showed inward rectification, reversed at -1.5 +/- 1 mV and was permeable to monovalent cations. The NSC current was insensitive to the P2 purinoceptor antagonists, suramin or PPADS but was activated by the purinoceptor agonists UTP, ADP and 2MeSATP. 3. The outward current activated by ATP reversed at -68 +/- 3 mV (equilibrium potential for potassium (EK) = -84 mV) and was blocked by Ba2+ ions, consistent with the activation of a K+ conductance. The outward K+ conductance was also reduced by the maxi-KCa channel blocker iberiotoxin (IbTX; 10 nM), suggesting that ATP activated an outward Ca2+-activated K+ channel in rat RPE cells. The Ca2+-activated K+ current (IK(Ca)) was also activated by the purinoceptor agonists UTP, ADP and 2MeSATP. 4. In fluo-3 or fluo-4 loaded RPE cells, ATP and the pyrimidine agonist UTP elevated [Ca2+]i. The increase in Ca2+ was not dependent on extracellular Ca2+ influx, but was sensitive to the Ca2+-ATPase inhibitor thapsigargin, confirming the involvement of intracellular Ca2+ stores release. 5. These results suggest that rat RPE cells express both P2X purinoceptors that gate activation of a non-selective cation conductance and G protein-coupled P2Y purinoceptors that mediate Ca2+ release from intracellular stores and activation of a calcium-activated K+ current. PMID:10545141

  13. Iron prochelator BSIH protects retinal pigment epithelial cells against cell death induced by hydrogen peroxide.

    PubMed

    Charkoudian, Louise K; Dentchev, Tzvete; Lukinova, Nina; Wolkow, Natalie; Dunaief, Joshua L; Franz, Katherine J

    2008-12-01

    Dysregulation of localized iron homeostasis is implicated in several degenerative diseases, including Parkinson's, Alzheimer's, and age-related macular degeneration, wherein iron-mediated oxidative stress is hypothesized to contribute to cell death. Inhibiting toxic iron without altering normal metal-dependent processes presents significant challenges for standard small molecule chelating agents. We previously introduced BSIH (isonicotinic acid [2-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-benzylidene]-hydrazide) prochelators that are converted by hydrogen peroxide into SIH (salicylaldehyde isonicotinoyl hydrazone) chelating agents that inhibit iron-catalyzed hydroxyl radical generation. Here, we show that BSIH protects a cultured cell model for retinal pigment epithelium against cell death induced by hydrogen peroxide. BSIH is more stable than SIH in cell culture medium and is more protective during long-term experiments. Repetitive exposure of cells to BSIH is nontoxic, whereas SIH and desferrioxamine induce cell death after repeated exposure. Combined, our results indicate that cell protection by BSIH involves iron sequestration that occurs only when the cells are stressed by hydrogen peroxide. These findings suggest that prochelators discriminate toxic iron from healthy iron and are promising candidates for neuro- and retinal protection. PMID:18835041

  14. Effects of PACAP on intracellular signaling pathways in human retinal pigment epithelial cells exposed to oxidative stress.

    PubMed

    Fabian, E; Reglodi, D; Mester, L; Szabo, A; Szabadfi, K; Tamas, A; Toth, G; Kovacs, K

    2012-11-01

    The integrity of retinal pigment epithelial cells is critical for photoreceptor survival and vision. Pituitary adenylate cyclase activating polypeptide (PACAP) exerts retinoprotective effects against several types of injuries in vivo, including optic nerve transection, retinal ischemia, excitotoxic injuries, UVA-induced lesion, and diabetic retinopathy. In a recent study, we have proven that PACAP is also protective in oxidative stress-induced injury in human pigment epithelial cells (ARPE-19 cells). The aim of the present study was to investigate the possible mechanisms of this protection. ARPE cells were exposed to a 24-h hydrogen peroxide treatment. Expressions of kinases and apoptotic markers were studied by complex array kits and Western blot. Oxidative stress induced the activation of several apoptotic markers, including Bad, Bax, HIF-1α, several heat shock proteins, TNF-related apoptosis-inducing ligand, and Fas-associated protein with death domain, while PACAP treatment decreased them. The changes in the expression of MAP kinases showed that PACAP activated the protective ERK1/2 and downstream CREB, and decreased the activation of the pro-apoptotic p38MAPK and c-Jun N-terminal kinase, an effect opposite to that observed with only oxidative stress. Furthermore, PACAP increased the activation of the protective Akt pathway. In addition, the effects of oxidative stress on several other signaling molecules were counteracted by PACAP treatment (Chk2, Yes, Lyn, paxillin, p53, PLC, STAT4, RSK). These play a role in cell death, cell cycle, inflammation, adhesion, differentiation and proliferation. In summary, PACAP, acting at several levels, influences the balance between pro- and anti-apoptotic factors in favor of anti-apoptosis, thereby providing protection in oxidative stress-induced injury of human retinal pigment epithelial cells. PMID:22644900

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

    PubMed Central

    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

  16. Surface Modified Biodegradable Electrospun Membranes as a Carrier for Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells.

    PubMed

    Sorkio, Anni; Porter, Patrick J; Juuti-Uusitalo, Kati; Meenan, Brian J; Skottman, Heli; Burke, George A

    2015-09-01

    Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells are currently undergoing clinical trials to treat retinal degenerative diseases. Transplantation of hESC-RPE cells in conjuction with a supportive biomaterial carrier holds great potential as a future treatment for retinal degeneration. However, there has been no such biodegradable material that could support the growth and maturation of hESC-RPE cells so far. The primary aim of this work was to create a thin porous poly (L-lactide-co-caprolactone) (PLCL) membrane that could promote attachment, proliferation, and maturation of the hESC-RPE cells in serum-free culture conditions. The PLCL membranes were modified by atmospheric pressure plasma processing and coated with collagen IV to enhance cell growth and maturation. Permeability of the membranes was analyzed with an Ussing chamber system. Analysis with scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy demonstrated that plasma surface treatment augments the surface properties of the membrane, which enhances the binding and conformation of the protein. Cell proliferation assays, reverse transcription-polymerase chain reaction, indirect immunofluoresence staining, trans-epithelial electrical resistance measurements, and in vitro phagocytosis assay clearly demonstrated that the plasma treated PLCL membranes supported the adherence, proliferation, maturation and functionality of hESC-RPE cells in serum-free culture conditions. Here, we report for the first time, how PLCL membranes can be modified with atmospheric pressure plasma processing to enable the formation of a functional hESC-RPE monolayer on a porous biodegradable substrate, which have a potential as a tissue-engineered construct for regenerative retinal repair applications. PMID:25946229

  17. Anthocyanins Protect Against A2E Photooxidation and Membrane Permeabilization in Retinal Pigment Epithelial Cells

    PubMed Central

    Jang, Young P.; Zhou, Jilin; Nakanishi, Koji; Sparrow, Janet R.

    2005-01-01

    The pyridinium bisretinoid A2E, an autofluorescent pigment that accumulates in retinal pigment epithelial cells with age and in some retinal disorders, can mediate a detergent-like perturbation of cell membranes and light-induced damage to the cell. The photodynamic events initiated by the sensitization of A2E include the generation of singlet oxygen and the oxidation of A2E at carbon–carbon double bonds. To assess the ability of plant-derived anthocyanins to modulate adverse effects of A2E accumulation on retinal pigment epithelium (RPE) cells, these flavylium salts were isolated from extracts of bilberry. Nine anthocyanin fractions reflecting monoglycosides of delphinidin, cyanidin, petunidin and malvidin were obtained and all were shown to suppress the photooxidation of A2E at least in part by quenching singlet oxygen. The anthocyanins tested exhibited antioxidant activity of variable efficiency. The structural characteristics relevant to this variability likely included the ability to form a stable quinonoidal anhydro base at neutral pH, a conjugated diene structure in the C (pyrane) ring, the presence of hydroxyl groups on the B (benzene) ring and the relative hydrophobicity conferred by the arrangement of substituents on the B ring. Cells that had taken up anthocyanins also exhibited a resistance to the membrane permeabilization that occurs as a result of the detergent-like action of A2E. PMID:15745429

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

    PubMed Central

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

    2016-01-01

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

  19. Involvement of protein kinase C in phagocytosis of human retinal pigment epithelial cells and induction of matrix metalloproteinase secretion.

    PubMed

    Irschick, Eveline U; Haas, Gertrud; Troger, Josef; Ueberall, Florian; Huemer, Hartwig P

    2009-10-01

    Protein kinase C (PKC) is involved in cell activation. We investigated PKC-mediated pathways and secretion of matrix metalloproteinases (MMPs) in phagocytosis by human retinal pigment epithelial cells (RPE). We used time-resolved fluorometry for europium-labeled microsphere uptake and gel zymography to assay the influence of PKC modulators. PKC inhibitors blocked phagocytosis by RPE. ARPE-19, a human RPE-cell line, showed reduced secretion of MMP-2, although MMP-9 secretion by PKC activation was conserved in both cell types, namely in the primary RPEs and in the RPE-cell line. Particle uptake by RPE cells requires activation of PKC; the use of PKC inhibitors as new anticancer drugs may possibly cause ocular side-effects. PMID:18641922

  20. UVA-induced oxidative damage in retinal pigment epithelial cells after H2O2 or sparfloxacin exposure.

    PubMed

    Verna, L K; Holman, S A; Lee, V C; Hoh, J

    2000-01-01

    Retinal impairment is one of the leading causes of visual loss in an aging human population. To explore a possible cause for retinal damage in the human population, we have monitored DNA oxidation in human retinal pigment epithelial (RPE) cells after exposure to hydrogen peroxide (H2O2) or the quinolone antibacterial sparfloxacin. When H2O2- or sparfloxacin-exposed cells were further exposed to ultraviolet A (UVA) irradiation, oxidative damage to the DNA of these cells was greatly increased over baseline values. This RPE+pharmaceutical-UVA cell system was developed to mimic in vivo retinal degeneration, seen in mouse studies using quinolone and UVA exposure. DNA damage produced by sparfloxacin and UVA in RPE cells could be remedied by the use of antioxidants, indicating a possible in vivo method for prevention or minimization of retinal damage in humans PMID:11201054

  1. Retinal pigment epithelial change and partial lipodystrophy.

    PubMed Central

    Davis, T. M.; Holdright, D. R.; Schulenberg, W. E.; Turner, R. C.; Joplin, G. F.

    1988-01-01

    Cuticular drusen and retinal pigment epithelial changes were found incidentally in a 27 year old Lebanese woman during assessment of partial lipodystrophy. Her vision was normal despite involvement of both maculae. The patient had hypocomplementaemia, but serum C3 nephritic factor was absent and renal function was normal. She had impaired glucose tolerance and a continuous infusion of glucose with model assessment (CIGMA) test revealed low normal tissue insulin sensitivity and high normal pancreatic beta cell function. Mild fasting hypertriglyceridaemia (2.0 mmol/l) may have been secondary to impaired insulin sensitivity. Endocrine function was otherwise normal apart from a completely absent growth hormone response to adequate hypoglycaemia. The simultaneous occurrence of partial lipodystrophy and retinal pigmentary epithelial and basement membrane changes appears to be a newly recognized syndrome. Images Figure 1 Figure 2 PMID:3255937

  2. A Method for the Isolation and Culture of Adult Rat Retinal Pigment Epithelial (RPE) Cells to Study Retinal Diseases

    PubMed Central

    Heller, Janosch P.; Kwok, Jessica C. F.; Vecino, Elena; Martin, Keith R.; Fawcett, James W.

    2015-01-01

    Diseases such as age-related macular degeneration (AMD) affect the retinal pigment epithelium (RPE) and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 min yielded 4 × 104 viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch’s membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch’s membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases. PMID:26635529

  3. Effect of Storage Temperature on Key Functions of Cultured Retinal Pigment Epithelial Cells

    PubMed Central

    Pasovic, Lara; Eidet, Jon Roger; Brusletto, Berit S.; Lyberg, Torstein; Utheim, Tor P.

    2015-01-01

    Purpose. Replacement of the diseased retinal pigment epithelium (RPE) with cells capable of performing the specialized functions of the RPE is the aim of cell replacement therapy for treatment of macular degenerative diseases. A storage method for RPE is likely to become a prerequisite for the establishment of such treatment. Herein, we analyze the effect of storage temperature on key functions of cultured RPE cells. Methods. Cultured ARPE-19 cells were stored in Minimum Essential Medium at 4°C, 16°C, and 37°C for seven days. Total RNA was isolated and the gene expression profile was determined using DNA microarrays. Comparison of the microarray expression values with qRT-PCR analysis of selected genes validated the results. Results. Expression levels of several key genes involved in phagocytosis, pigment synthesis, the visual cycle, adherens, and tight junctions, and glucose and ion transport were maintained close to control levels in cultures stored at 4°C and 16°C. Cultures stored at 37°C displayed regulational changes in a larger subset of genes related to phagocytosis, adherens, and tight junctions. Conclusion. RPE cultures stored at 4°C and 16°C for one week are capable of maintaining the expression levels of genes important for key RPE functions close to control levels. PMID:26448872

  4. Novel Compstatin Family Peptides Inhibit Complement Activation by Drusen-Like Deposits in Human Retinal Pigmented Epithelial Cell Cultures

    PubMed Central

    Gorham, Ronald D.; Forest, David L.; Tamamis, Phanourios; de Victoria, Aliana López; Kraszni, Márta; Kieslich, Chris A.; Banna, Christopher D.; Bellows-Peterson, Meghan L.; Larive, Cynthia K.; Floudas, Christodoulos A.; Archontis, Georgios; Johnson, Lincoln V.; Morikis, Dimitrios

    2013-01-01

    We have used a novel human retinal pigmented epithelial (RPE) cell-based model that mimics drusen biogenesis and the pathobiology of age-related macular degeneration to evaluate the efficacy of newly designed peptide inhibitors of the complement system. The peptides belong to the compstatin family and, compared to existing compstatin analogs, have been optimized to promote binding to their target, complement protein C3, and to enhance solubility by improving their polarity/hydrophobicity ratios. Based on analysis of molecular dynamics simulation data of peptide-C3 complexes, novel binding features were designed by introducing intermolecular salt bridge-forming arginines at the N-terminus and at position -1 of N-terminal dipeptide extensions. Our study demonstrates that the RPE cell assay has discriminatory capability for measuring the efficacy and potency of inhibitory peptides in a macular disease environment. PMID:23954241

  5. High glucose promotes the migration of retinal pigment epithelial cells through increased oxidative stress and PEDF expression.

    PubMed

    Farnoodian, Mitra; Halbach, Caroline; Slinger, Cassidy; Pattnaik, Bikash R; Sorenson, Christine M; Sheibani, Nader

    2016-09-01

    Defects in the outer blood-retinal barrier have significant impact on the pathogenesis of diabetic retinopathy and macular edema. However, the detailed mechanisms involved remain largely unknown. This is, in part, attributed to the lack of suitable animal and cell culture models, including those of mouse origin. We recently reported a method for the culture of retinal pigment epithelial (RPE) cells from wild-type and transgenic mice. The RPE cells are responsible for maintaining the integrity of the outer blood-retinal barrier whose dysfunction during diabetes has a significant impact on vision. Here we determined the impact of high glucose on the function of RPE cells. We showed that high glucose conditions resulted in enhanced migration and increased the level of oxidative stress in RPE cells, but minimally impacted their rate of proliferation and apoptosis. High glucose also minimally affected the cell-matrix and cell-cell interactions of RPE cells. However, the expression of integrins and extracellular matrix proteins including pigment epithelium-derived factor (PEDF) were altered under high glucose conditions. Incubation of RPE cells with the antioxidant N-acetylcysteine under high glucose conditions restored normal migration and PEDF expression. These cells also exhibited increased nuclear localization of the antioxidant transcription factor Nrf2 and ZO-1, reduced levels of β-catenin and phagocytic activity, and minimal effect on production of vascular endothelial growth factor, inflammatory cytokines, and Akt, MAPK, and Src signaling pathways. Thus high glucose conditions promote RPE cell migration through increased oxidative stress and expression of PEDF without a significant effect on the rate of proliferation and apoptosis. PMID:27440660

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

    PubMed

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

    2016-01-01

    To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV), self-complementary adeno-associated virus 2 (scAAV2) with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con) or constitutively active Rap1a (scAAV2-CARap1a) showed strong GFP at the 5 × 10(8) viral particle/µl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS)-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE)/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/β-catenin complexes, increased transepithelial electrical resistance through an interaction of β-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC) transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes. PMID:27606349

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

    PubMed Central

    Sugimoto, Masahiko; Kondo, Mineo

    2016-01-01

    Aim. We investigated whether lecithin-bound iodine (LBI) can protect the integrity of tight junctions of retinal pigment epithelial cells from hypoxia. Method. Cultured human retinal pigment epithelial (ARPE-19) cells were pretreated with LBI. To mimic hypoxic conditions, cells were incubated with CoCl2. We compared the integrity of the tight junctions (TJs) of control to cells with either LBI alone, CoCl2 alone, or LBI + CoCl2. The levels of cytokines in the conditioned media were also determined. Results. Significant decrease in the zonula occludens-1 (ZO-1) intensity in the CoCl2 group compared to the control (5787.7 ± 4126.4 in CoCl2 group versus 29244.6 ± 2981.2 in control; average ± standard deviation). But the decrease was not significant in the LBI + CoCl2 (27189.0 ± 11231.1). The levels of monocyte chemoattractant protein-1 (MCP-1) and Chemokine (C-C Motif) Ligand 11 (CCL-11) were significantly higher in the CoCl2 than in the control (340.8 ± 43.3 versus 279.7 ± 68.3 pg/mL for MCP-1, and 15.2 ± 12.9 versus 12.5 ± 6.1 pg/mL for CCL-11. With LBI pretreatment, the levels of both cytokines were decreased to 182.6 ± 23.8 (MCP-1) and 5.46 ± 1.9 pg/mL for CCL-11). Blockade of MCP-1 or CCL-11 also shows similar result representing TJ protection from hypoxic stress. Conclusions. LBI results in a protective action from hypoxia. PMID:27340563

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

    PubMed Central

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

    2016-01-01

    To test the hypothesis that increased Rap1a activity specifically in retinal pigment epithelial cells resists choroidal neovascularization (CNV), self-complementary adeno-associated virus 2 (scAAV2) with RPE65-promoter-driven GFP vectors were generated and introduced subretinally into Rap1b-deficient mice. Six-week-old mice that received subretinal control (scAAV2-Con) or constitutively active Rap1a (scAAV2-CARap1a) showed strong GFP at the 5 × 108 viral particle/µl dose 5 weeks later without altering retinal morphology or function. Compared to scAAV2-Con- or phosphate-buffered saline (PBS)-injected, eyes injected with scAAV2-CARap1a had increased Rap1 in retinal pigment epithelial (RPE)/choroidal lysates and a significant reduction in CNV volume 7 days after laser, comparable to eyes that received intravitreal anti-VEGF versus IgG control. scAAV2-CARap1a-, but not anti-VEGF-, injected eyes had increased pan-cadherin in RPE/choroids. In cultured RPE cells, increased active Rap1a inhibited TNFα-induced disassociation of junctional pan-cadherin/β-catenin complexes, increased transepithelial electrical resistance through an interaction of β-catenin with phosphorylated scaffold protein, IQGAP1, and inhibited choroidal endothelial cell (CEC) transmigration of an RPE monolayer. This evidence shows that increased Rap1a activity specifically in RPE cells is sufficient to reduce CEC transmigration and CNV and involves IQGAP1-mediated protection of RPE junctional complexes. PMID:27606349

  9. Apr3 accelerates the senescence of human retinal pigment epithelial cells.

    PubMed

    Han, Song; Lu, Qingjun; Wang, Ningli

    2016-04-01

    Senescence of retinal pigment epithelium (RPE) cells is a major contributor to age‑related macular degeneration (AMD). However, the molecular mechanisms underlying RPE dysfunction are not well understood. Apoptosis related protein 3 (Apr3) was originally cloned from HL‑60 cells induced by all‑trans retinoic acid (ATRA). Preliminary data revealed elevated Apr3 expression in the tissues of aged mice, suggesting that it is involved in the aging process. The present study demonstrated that Apr3 mRNA and protein levels were markedly increased in aged mouse RPE cells. Elevated Apr3 expression was also observed during premature senescence induced by oxidative stress (H2O2 and tert‑BHP) in ARPE‑19 cells. Moreover, Apr3 overexpression promoted cellular senescence in ARPE‑19 cells, as characterized by enhanced senescence‑associated β‑galactosidase activity, reduced cell proliferation and increased expression of the senescence markers p53 and p21. In addition, it was demonstrated that overexpression of Apr3‑N, a truncated counterpart of Apr3, abrogated Apr3‑induced phenotypes. It was concluded that Apr3 expression was induced in replicative and premature senescence of RPE cells and its overexpression accelerated senescence of ARPE‑19 cells, which provides important insights into the function of Apr3 in senescence‑associated diseases. PMID:26934949

  10. Up-Regulation of ENO1 by HIF-1α in Retinal Pigment Epithelial Cells after Hypoxic Challenge Is Not Involved in the Regulation of VEGF Secretion

    PubMed Central

    Zheng, Feihui; Jang, Wai-Chi; Fung, Frederic K. C.; Lo, Amy C. Y.; Wong, Ian Y. H.

    2016-01-01

    Purpose Alpha-enolase (ENO1), a major glycolytic enzyme, is reported to be over-expressed in various cancer tissues. It has been demonstrated to be regulated by the Hypoxia-inducible factor 1-α (HIF-1α), a crucial transcriptional factor implicated in tumor progression and cancer angiogenesis. Choroidal neovascularization (CNV), which is a leading cause of severe vision loss caused by newly formed blood vessels in the choroid, is also engendered by hypoxic stress. In this report, we investigated the expression of ENO1 and the effects of its down-regulation upon cobalt (II) chloride-induced hypoxia in retinal pigment epithelial cells, identified as the primary source of ocular angiogenic factors. Methods HIF-1α-diminished retinal pigment epithelial cells were generated by small interfering RNA (siRNA) technology in ARPE-19 cells, a human retinal pigment epithelial cell line. Both normal and HIF-1α-diminished ARPE-19 cells were then subjected to hypoxic challenge using cobalt (II) chloride (CoCl2) or anaerobic chamber. The relation between ENO1 expression and vascular endothelial growth factor (VEGF) secretion by retinal pigment epithelial cells were examined. Protein levels of HIF-1α and ENO1 were analyzed using Western Blot, while VEGF secretion was essayed by enzyme-linked immunosorbent assay (ELISA). Cytotoxicity after hypoxia was detected by Lactate Dehydrogenase (LDH) Assay. Results Upon 24 hr of CoCl2-induced hypoxia, the expression levels of ENO1 and VEGF were increased along with HIF-1α in ARPE-19 cells, both of which can in turn be down-regulated by HIF-1α siRNA application. However, knockdown of ENO1 alone or together with HIF-1α did not help suppress VEGF secretion in hypoxic ARPE-19 cells. Conclusion ENO1 was demonstrated to be up-regulated by HIF-1α in retinal pigment epithelial cells in response to hypoxia, without influencing VEGF secretion. PMID:26882120

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

    PubMed Central

    2009-01-01

    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 maculopathies. PMID:19413901

  12. Retinal Pigment Epithelial Cells Mitigate the Effects of Complement Attack by Endocytosis of C5b-9

    PubMed Central

    Georgiannakis, Apostolos; Burgoyne, Tom; Lueck, Katharina; Futter, Clare; Greenwood, John

    2015-01-01

    Retinal pigment epithelial (RPE) cell death is a hallmark of age-related macular degeneration. The alternative pathway of complement activation is strongly implicated in RPE cell dysfunction and loss in age-related macular degeneration; therefore, it is critical that RPE cells use molecular strategies to mitigate the potentially harmful effects of complement attack. We show that the terminal complement complex C5b-9 assembles rapidly on the basal surface of cultured primary porcine RPE cells but disappears over 48 h without any discernable adverse effects on the cells. However, in the presence of the dynamin inhibitor dynasore, C5b-9 was almost completely retained at the cell surface, suggesting that, under normal circumstances, it is eliminated via the endocytic pathway. In support of this idea, we observed that C5b-9 colocalizes with the early endosome marker EEA1 and that, in the presence of protease inhibitors, it can be detected in lysosomes. Preventing the endocytosis of C5b-9 by RPE cells led to structural defects in mitochondrial morphology consistent with cell stress. We conclude that RPE cells use the endocytic pathway to prevent the accumulation of C5b-9 on the cell surface and that processing and destruction of C5b-9 by this route are essential for RPE cell survival. PMID:26324770

  13. Nanoparticle-mediated gene transfer specific to retinal pigment epithelial cells.

    PubMed

    Koirala, Adarsha; Makkia, Rasha S; Cooper, Mark J; Naash, Muna I

    2011-12-01

    Previously, we demonstrated that CK30PEG10k-compacted DNA nanoparticles (NPs) efficiently target photoreceptor cells and improve visual function in a retinitis pigmentosa model. Here, we test the ability of these NPs in driving transgene expression in the retinal pigment epithelium (RPE), using an RPE-specific reporter vector (VMD2-eGFP). NPs, uncompacted plasmid, or saline were subretinally delivered to adult BALB/c mice. NP-based expression was specific to RPE cells and caused no deleterious effects on retinal structure and function. eGFP expression levels in NP-injected eyes peaked at post-injection day 2 (PI-2), stabilized at levels ~3-fold higher than in naked DNA-injected eyes, and remained elevated at the latest time-point examined (PI-30). Unlike naked DNA, which only transfected cells at the site of injection, NPs were able to transfect cells throughout the RPE. Subretinal injections of rhodamine labeled NPs and naked DNA showed comparable initial uptake into RPE cells. However, at PI-7 and -30 days significantly more fluorescence was detected inside the RPE of NP-injected eyes compared to naked DNA, suggesting NPs are stable inside the cell which could possibly lead to higher and sustained expression. Overall, our results demonstrate that NPs can efficiently deliver genes to the RPE and hold great potential for the treatment of RPE-associated diseases. PMID:21885113

  14. Efficiency of Membrane Protein Expression Following Infection with Recombinant Adenovirus of Polarized Non-Transformed Human Retinal Pigment Epithelial Cells.

    PubMed

    Müller, Claudia; Blenkinsop, Timothy A; Stern, Jeffrey H; Finnemann, Silvia C

    2016-01-01

    Transient expression of exogenous proteins facilitates studies of molecular mechanisms and utility for transplantation of retinal pigment epithelial (RPE) cells in culture. Here, we compared expression of the membrane protein β5 integrin-GFP (β5-GFP) in two recently established models of differentiated human RPE, adult RPE stem cell-derived RPE and primary fetal RPE, upon infection with recombinant adenovirus or transfection with DNA in liposomes. We varied viral titer and duration of virus incubation and examined β5-GFP and the tight junction marker ZO-1 in manipulated cells by confocal microscopy. Fewer than 5 % of cells expressed β5-GFP after liposome-mediated transfection. The percentage of cells with detectable β5-GFP exceeded 90 % after adenovirus infection for as little as 1 h. Decreasing virus titer two-fold did not alter the fraction of cells expressing β5-GFP but increased variability of β5-GFP level among cells. In cells with low expression levels, β5-GFP localized mostly to the apical plasma membrane like endogenous αvβ5 integrin. In cells with high expression levels, β5-GFP localized to the cytoplasm in addition to the apical surface suggesting accumulation in trafficking compartments. Altogether, adenovirus delivery yields efficient exogenous membrane protein expression of correct polarity in differentiated human RPE cells in culture. PMID:26427482

  15. Photochemistry and photocytotoxicity of alkaloids from Goldenseal (Hydrastis canadensis L.) 3: effect on human lens and retinal pigment epithelial cells.

    PubMed

    Chignell, Colin F; Sik, Robert H; Watson, Mary A; Wielgus, Albert R

    2007-01-01

    The dried root or rhizome of Goldenseal (Hydrastis canadensis L.) contains several alkaloids including berberine, hydrastine, palmatine and lesser amounts of canadine and hydrastinine. Preparations derived from Goldenseal have been used to treat skin and eye ailments. Berberine, the major alkaloid in Goldenseal root powder, has been used in eye drops to treat trachoma, a disease characterized by keratoconjunctivitis. Berberine and palmatine are also present in extracts from Berberis amurensis Ruprecht (Berberidaceae) which are used to treat ocular disorders. We have previously shown that Goldenseal alkaloids are phototoxic to keratinocytes (Chem Res Toxicol. 14, 1529, 2001; ibid 19, 739, 2006) and now report their effect on human lens and retinal pigment epithelial cells. Human lens epithelial cells (HLE-B3) were severely damaged when incubated with berberine (25 microM) and exposed to UVA (5 J cm(-2)). Under the same conditions, palmatine was less phototoxic and hydrastine, canadine and hydrastinine were inactive. Moderate protection against berberine phototoxicity was afforded by the antioxidants ascorbate (2 mM) and N-acetylcysteine (5 mM). When exposed to UVA (5 J cm(-2)) both berberine (10 microM) and palmatine (10 microM) caused mild DNA damage as determined by the alkaline comet assay which measures single strand breaks. Berberine and palmatine are the only Goldenseal alkaloids with appreciable absorption above 400 nm. Because light at wavelengths below 400 nm is cut off by the anterior portion of the adult human eye only berberine and palmatine were tested for phototoxicity to human retinal pigment epithelial (hRPE) cells. Although berberine did damage hRPE cells when irradiated with visible light (lambda > 400 nm) approximately 10 times higher concentrations were required to produce the same amount of damage as seen in lens cells. Palmatine was not phototoxic to hRPE cells. Neither berberine nor palmatine photodamaged DNA in hRPE. Infusions of Goldenseal

  16. Autophagy and mitochondrial alterations in human retinal pigment epithelial cells induced by ethanol: implications of 4-hydroxy-nonenal

    PubMed Central

    Flores-Bellver, M; Bonet-Ponce, L; Barcia, J M; Garcia-Verdugo, J M; Martinez-Gil, N; Saez-Atienzar, S; Sancho-Pelluz, J; Jordan, J; Galindo, M F; Romero, F J

    2014-01-01

    Retinal pigment epithelium has a crucial role in the physiology and pathophysiology of the retina due to its location and metabolism. Oxidative damage has been demonstrated as a pathogenic mechanism in several retinal diseases, and reactive oxygen species are certainly important by-products of ethanol (EtOH) metabolism. Autophagy has been shown to exert a protective effect in different cellular and animal models. Thus, in our model, EtOH treatment increases autophagy flux, in a concentration-dependent manner. Mitochondrial morphology seems to be clearly altered under EtOH exposure, leading to an apparent increase in mitochondrial fission. An increase in 2′,7′-dichlorofluorescein fluorescence and accumulation of lipid peroxidation products, such as 4-hydroxy-nonenal (4-HNE), among others were confirmed. The characterization of these structures confirmed their nature as aggresomes. Hence, autophagy seems to have a cytoprotective role in ARPE-19 cells under EtOH damage, by degrading fragmented mitochondria and 4-HNE aggresomes. Herein, we describe the central implication of autophagy in human retinal pigment epithelial cells upon oxidative stress induced by EtOH, with possible implications for other conditions and diseases. PMID:25032851

  17. Effect of Toxoplasma gondii infection on the junctional complex of retinal pigment epithelial cells.

    PubMed

    Nogueira, Alanderson R; Leve, Fernanda; Morgado-Diaz, José; Tedesco, Roberto Carlos; Pereira, Mirian Claudia S

    2016-04-01

    Ocular toxoplasmosis is the most frequent cause of uveitis, leading to partial or total loss of vision, with the retina the main affected structure. The cells of the retinal pigment epithelium (RPE) play an important role in the physiology of the retina and formation of the blood-retinal barrier. Several pathogens induce barrier dysfunction by altering tight junction (TJ) integrity. Here, we analysed the effect of infection by Toxoplasma gondii on TJ integrity in ARPE-19 cells. Loss of TJ integrity was demonstrated in T. gondii-infected ARPE-19 cells, causing increase in paracellular permeability and disturbance of the barrier function of the RPE. Confocal microscopy also revealed alteration in the TJ protein occludin induced by T. gondii infection. Disruption of junctional complex was also evidenced by scanning and transmission electron microscopy. Cell-cell contact loss was noticed in the early stages of infection by T. gondii with the visualization of small to moderate intercellular spaces. Large gaps were mostly observed with the progression of the infection. Thus, our data suggest that the alterations induced by T. gondii in the structural organization of the RPE may contribute to retinal injury evidenced by ocular toxoplasmosis. PMID:26928468

  18. Concurrent expression of heme oxygenase-1 and p53 in human retinal pigment epithelial cell line

    SciTech Connect

    Lee, Sang Yull; Jo, Hong Jae; Kim, Kang Mi; Song, Ju Dong; Chung, Hun Taeg; Park, Young Chul

    2008-01-25

    Heme oxygenase-1 (HO-1) is a stress-responsive protein that is known to regulate cellular functions such as cell proliferation, inflammation, and apoptosis. Here, we investigated the effects of HO activity on the expression of p53 in the human retinal pigment epithelium (RPE) cell line ARPE-19. Cobalt protoporphyrin (CoPP) induced the expression of both HO-1 and p53 without significant toxicity to the cells. In addition, the blockage of HO activity with the iron chelator DFO or with HO-1 siRNA inhibited the CoPP-induced expression of p53. Similarly, zinc protoporphyrin (ZnPP), an inhibitor of HO, suppressed p53 expression in ARPE-19 cells, although ZnPP increased the level of HO-1 protein while inhibiting HO activity. Also, CoPP-induced p53 expression was not affected by the formation of reactive oxygen species (ROS). Based on these results, we conclude that HO activity is involved in the regulation of p53 expression in a ROS-independent mechanism, and also suggest that the expression of p53 in ARPE-19 cells is associated with heme metabolites such as biliverdin/bilirubin, carbon monoxide, and iron produced by the activity of HO.

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

    SciTech Connect

    Bridges, Christy C. . E-mail: bridges_cc@mercer.edu; Battle, Jamie R.; Zalups, Rudolfs K.

    2007-06-01

    Inorganic mercury (Hg{sup 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{sup 2+} exposure in that it mediates transport of substances to the photoreceptor cells. In order for Hg{sup 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{sup 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{sup 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{sup 2+}, was assessed in ARPE-19 cells exposed to the following S-conjugates of Hg{sup 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{sup +}-dependent and was inhibited by neutral and cationic amino acids. RT-PCR analyses identified systems B{sup 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{sup 0,+} and ASC. These amino acid transporters may play a role in the retinal toxicity observed following exposure to mercury.

  20. Hydroxyl PAMAM dendrimer-based gene vectors for transgene delivery to human retinal pigment epithelial cells

    NASA Astrophysics Data System (ADS)

    Mastorakos, Panagiotis; Kambhampati, Siva P.; Mishra, Manoj K.; Wu, Tony; Song, Eric; Hanes, Justin; Kannan, Rangaramanujam M.

    2015-02-01

    Ocular gene therapy holds promise for the treatment of numerous blinding disorders. Despite the significant progress in the field of viral and non-viral gene delivery to the eye, significant obstacles remain in the way of achieving high-level transgene expression without adverse effects. The retinal pigment epithelium (RPE) is involved in the pathogenesis of retinal diseases and is a key target for a number of gene-based therapeutics. In this study, we addressed the inherent drawbacks of non-viral gene vectors and combined different approaches to design an efficient and safe dendrimer-based gene-delivery platform for delivery to human RPE cells. We used hydroxyl-terminated polyamidoamine (PAMAM) dendrimers functionalized with various amounts of amine groups to achieve effective plasmid compaction. We further used triamcinolone acetonide (TA) as a nuclear localization enhancer for the dendrimer-gene complex and achieved significant improvement in cell uptake and transfection of hard-to-transfect human RPE cells. To improve colloidal stability, we further shielded the gene vector surface through incorporation of PEGylated dendrimer along with dendrimer-TA for DNA complexation. The resultant complexes showed improved stability while minimally affecting transgene delivery, thus improving the translational relevance of this platform.Ocular gene therapy holds promise for the treatment of numerous blinding disorders. Despite the significant progress in the field of viral and non-viral gene delivery to the eye, significant obstacles remain in the way of achieving high-level transgene expression without adverse effects. The retinal pigment epithelium (RPE) is involved in the pathogenesis of retinal diseases and is a key target for a number of gene-based therapeutics. In this study, we addressed the inherent drawbacks of non-viral gene vectors and combined different approaches to design an efficient and safe dendrimer-based gene-delivery platform for delivery to human RPE

  1. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway.

    PubMed

    Eidet, J R; Reppe, S; Pasovic, L; Olstad, O K; Lyberg, T; Khan, A Z; Fostad, I G; Chen, D F; Utheim, T P

    2016-01-01

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin's potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated. PMID:26940175

  2. The Silk-protein Sericin Induces Rapid Melanization of Cultured Primary Human Retinal Pigment Epithelial Cells by Activating the NF-κB Pathway

    PubMed Central

    Eidet, J. R.; Reppe, S.; Pasovic, L.; Olstad, O. K.; Lyberg, T.; Khan, A. Z.; Fostad, I. G.; Chen, D. F.; Utheim, T. P.

    2016-01-01

    Restoration of the retinal pigment epithelial (RPE) cells to prevent further loss of vision in patients with age-related macular degeneration represents a promising novel treatment modality. Development of RPE transplants, however, requires up to 3 months of cell differentiation. We explored whether the silk protein sericin can induce maturation of primary human retinal pigment epithelial (hRPE) cells. Microarray analysis demonstrated that sericin up-regulated RPE-associated transcripts (RPE65 and CRALBP). Upstream analysis identified the NF-κB pathway as one of the top sericin-induced regulators. ELISA confirmed that sericin stimulates the main NF-κB pathway. Increased levels of RPE-associated proteins (RPE65 and the pigment melanin) in the sericin-supplemented cultures were confirmed by western blot, spectrophotometry and transmission electron microscopy. Sericin also increased cell density and reduced cell death following serum starvation in culture. Inclusion of NF-κB agonists and antagonists in the culture medium showed that activation of the NF-κB pathway appears to be necessary, but not sufficient, for sericin-induced RPE pigmentation. We conclude that sericin promotes pigmentation of cultured primary hRPE cells by activating the main NF-κB pathway. Sericin’s potential role in culture protocols for rapid differentiation of hRPE cells derived from embryonic or induced pluripotent stem cells should be investigated. PMID:26940175

  3. Regenerating Retinal Pigment Epithelial Cells to Cure Blindness: A Road Towards Personalized Artificial Tissue

    PubMed Central

    Jha, Balendu Shekhar

    2015-01-01

    Retinal pigment epithelium (RPE) is a polarized monolayer tissue that functions to support the health and integrity of retinal photoreceptors (PRs). RPE atrophy has been linked to pathogenesis of age-related macular degeneration (AMD), a leading cause of blindness in elderly in the USA. RPE atrophy in AMD leads to the PR cell death and vision loss. It is thought that replacing diseased RPE with healthy RPE tissue can prevent PR cell death. Retinal surgical innovations have provided proof-of-principle data that autologous RPE tissue can replace diseased macular RPE and provide visual rescue in AMD patients. Current efforts are focused on developing an in vitro tissue using natural and synthetic scaffolds to generate a polarized functional RPE monolayer. In the future, these tissue-engineering approaches combined with pluripotent stem cell technology will lead to the development of personalized and “off-the-shelf” cell therapies for AMD patients. This review summarizes the historical development and ongoing efforts in surgical and in vitro tissue engineering techniques to develop a three-dimensional therapeutic native RPE tissue substitute. PMID:26146605

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

    PubMed

    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. PMID:26875723

  5. Hypoxia-induced metabolic stress in retinal pigment epithelial cells is sufficient to induce photoreceptor degeneration

    PubMed Central

    Kurihara, Toshihide; Westenskow, Peter D; Gantner, Marin L; Usui, Yoshihiko; Schultz, Andrew; Bravo, Stephen; Aguilar, Edith; Wittgrove, Carli; Friedlander, Mollie SH; Paris, Liliana P; Chew, Emily; Siuzdak, Gary; Friedlander, Martin

    2016-01-01

    Photoreceptors are the most numerous and metabolically demanding cells in the retina. Their primary nutrient source is the choriocapillaris, and both the choriocapillaris and photoreceptors require trophic and functional support from retinal pigment epithelium (RPE) cells. Defects in RPE, photoreceptors, and the choriocapillaris are characteristic of age-related macular degeneration (AMD), a common vision-threatening disease. RPE dysfunction or death is a primary event in AMD, but the combination(s) of cellular stresses that affect the function and survival of RPE are incompletely understood. Here, using mouse models in which hypoxia can be genetically triggered in RPE, we show that hypoxia-induced metabolic stress alone leads to photoreceptor atrophy. Glucose and lipid metabolism are radically altered in hypoxic RPE cells; these changes impact nutrient availability for the sensory retina and promote progressive photoreceptor degeneration. Understanding the molecular pathways that control these responses may provide important clues about AMD pathogenesis and inform future therapies. DOI: http://dx.doi.org/10.7554/eLife.14319.001 PMID:26978795

  6. Intracellular delivery of dendrimer triamcinolone acetonide conjugates into microglial and human retinal pigment epithelial cells.

    PubMed

    Kambhampati, Siva P; Mishra, Manoj K; Mastorakos, Panagiotis; Oh, Yumin; Lutty, Gerard A; Kannan, Rangaramanujam M

    2015-09-01

    Triamcinolone acetonide (TA) is a potent, intermediate-acting, steroid that has anti-inflammatory and anti-angiogenic activity. Intravitreal administration of TA has been used for diabetic macular edema, proliferative diabetic retinopathy and exudative age-related macular degeneration (AMD). However, the hydrophobicity, lack of solubility, and the side effects limit its effectiveness in the treatment of retinal diseases. In this study, we explore a PAMAM dendrimer-TA conjugate (D-TA) as a potential strategy to improve intracellular delivery and efficacy of TA to target cells. The conjugates were prepared with a high drug payload (∼ 21%) and were readily soluble in saline. Compared to free TA, D-TA demonstrated a significantly improved toxicity profile in two important target [microglial and human retinal pigment epithelium (RPE)] cells. The D-TA was ∼ 100-fold more effective than free TA in its anti-inflammatory activity (measured in microglia), and in suppressing VEGF production (in hypoxic RPE cells). Dendrimer-based delivery may improve the efficacy of TA towards both its key targets of inflammation and VEGF production, with significant clinical implications. PMID:25701805

  7. Intracellular delivery of dendrimer triamcinolone acetonide conjugates into microglial and human retinal pigment epithelial cells

    PubMed Central

    Kambhampati, Siva P.; Mishra, Manoj K.; Mastorakos, Panagiotis; Oh, Yumin; Lutty, Gerard A.; Kannan, Rangaramanujam M.

    2016-01-01

    Triamcinolone acetonide (TA) is a potent, intermediate-acting, steroid that has anti-inflammatory and anti-angiogenic activity. Intravitreal administration of TA has been used for diabetic macular edema, proliferative diabetic retinopathy and exudative age-related macular degeneration (AMD). However, the hydrophobicity, lack of solubility, and the side effects limit its effectiveness in the treatment of retinal diseases. In this study, we explore a PAMAM dendrimer-TA conjugate (D-TA) as a potential strategy to improve intracellular delivery and efficacy of TA to target cells. The conjugates were prepared with a high drug payload (~21%) and were readily soluble in saline. Compared to free TA, D-TA demonstrated a significantly improved toxicity profile in two important target [microglial and human retinal pigment epithelium (RPE)] cells. The D-TA was ~100-fold more effective than free TA in its anti-inflammatory activity (measured in microglia), and in suppressing VEGF production (in hypoxic RPE cells). Dendrimer-based delivery may improve the efficacy of TA towards both its key targets of inflammation and VEGF production, with significant clinical implications. PMID:25701805

  8. Cytokine regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) production by human retinal pigment epithelial cells

    PubMed Central

    Crane, I J; Kuppner, M C; Mckillop-Smith, S; Wallace, C A; Forrester, J V

    1999-01-01

    GM-CSF is an important regulator of macrophage, granulocyte and dendritic cell behaviour and function. These cell types have been implicated in the retinal damage characteristic of endogenous posterior uveitis. Dendritic cells in the choroid have access to retinal antigens processed by the retinal pigment epithelial (RPE) cells of the blood–retinal barrier and are thought to be candidates for the presentation of antigen in uveoretinitis. We therefore investigated the production of GM-CSF and its regulation in human RPE cells. IL-1β, tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) all stimulated GM-CSF production by RPE cells and a combination of these cytokines increased GM-CSF production over five-fold compared with that with the individual cytokines alone. Interferon-gamma (IFN-γ) rapidly down-regulated these responses. IFN-γ did not appear to be acting directly on IL-1β or via the synthesis of another protein. GM-CSF mRNA expression showed the same pattern of response to these cytokines, indicating transcriptional or pre-transcriptional regulation, and there was no evidence that IFN-γ was acting by destabilizing GM-CSF mRNA. These results are generally important in understanding the ways in which cytokine regulation differs between different cell types and also more specifically for determining ways in which a cytokine with a significant role in the development of autoimmune uveoretinitis may be manipulated. PMID:9933455

  9. Effect of bevacizumab (Avastin™) on mitochondrial function of in vitro retinal pigment epithelial, neurosensory retinal and microvascular endothelial cells

    PubMed Central

    Luthra, Saurabh; Sharma, Ashish; Dong, Joyce; Neekhra, Aneesh; Gramajo, Ana L; Seigel, Gail M; Kenney, M Cristina; Kuppermann, Baruch D

    2013-01-01

    Purpose: To evaluate the effect of bevacizumab on the mitochondrial function of human retinal pigment epithelial (ARPE-19), rat neurosensory retinal (R28) and human microvascular endothelial (HMVEC) cells in culture. Materials and Methods: ARPE-19 and R28 cells were treated with 0.125, 0.25, 0.50 and 1 mg/ml of bevacizumab. The HMVEC cultures were treated with 0.125, 0.25, 0.50 and 1 mg/ml of bevacizumab or 1 mg/ml of immunoglobulin G (control). Mitochondrial function assessed by mitochondrial dehydrogenase activity (MDA) was determined using the WST-1 assay. Results: Bevacizumab doses of 0.125 to 1 mg/ml for 5 days did not significantly affect the MDA of ARPE-19 cells. Bevacizumab treatment at 0.125 and 0.25 mg/ml (clinical dose) did not significantly affect the MDA of R28 cells; however, 0.50 and 1 mg/ml doses significantly reduced the R28 cell mitochondrial function. All doses of bevacizumab significantly reduced the MDA of proliferating and non-proliferating HMVEC. Conclusion: Bevacizumab exposure for 5 days was safe at clinical doses in both ARPE-19 and R28 retinal neurosensory cells in culture. By contrast, bevacizumab exposure at all doses show a significant dose-dependent decrease in mitochondrial activity in both the proliferating and non-proliferating HMVEC in vitro. This suggests a selective action of bevacizumab on endothelial cells at clinical doses. PMID:24413824

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

    PubMed

    Takahashi, Eri; Fukushima, Ayako; Haga, Akira; Inomata, Yasuya; Ito, Yasuhiro; Fukushima, Mikiko; Tanihara, Hidenobu

    2016-02-12

    In rhegmatogenous retinal detachment (RRD), scattered RPE cells from the basement membrane into the vitreous cavity undergo an epithelial mesenchymal transition (EMT) and form the intraocular fibrous membrane in response to vitreous fluid. We investigated whether exposure to vitreous samples was associated with EMT-associated signals and mesenchymal characters. Human vitreous samples were collected from patients with RRD, epiretinal membrane (ERM), or macular hole (MH). We evaluated the effects of vitreous on ARPE-19 cells in suspension cultures using poly 2-hydroxyethyl methacrylate-coated dishes and three-dimensional (3D) Matrigel cultures. We found that exposure to vitreous samples did not induce morphological changes or accelerate wound closure in monolayers. Several samples showed increased phosphorylation of Smad2 and nuclear translocation of nuclear factor-κB. Mechanical stress triggered an elevation of phosphorylation levels in Smad2. In addition, exposure to vitreous fluid increased the phosphorylation of p38 mitogen-activated protein kinase in cell suspension cultures after mechanical stress. Moreover, ARPE-19 cells showed a stellate invasive phenotype in 3D Matrigel cultures with vitreous samples. In this study, we demonstrated that mechanical stress and vitreous were associated with EMT-associated signals and invasive phenotypes in 3D cultures but not in monolayers. These results have important implications for the role of vitreous humor in the induction of EMT and intraocular fibrosis. PMID:26802464

  11. MicroRNA-29 regulates high-glucose-induced apoptosis in human retinal pigment epithelial cells through PTEN.

    PubMed

    Lin, Xiaohui; Zhou, Xiyuan; Liu, Danning; Yun, Lixia; Zhang, Lina; Chen, Xiaohai; Chai, Qinghe; Li, Langen

    2016-04-01

    Hyperglycemia or high-glucose (HG)-induced apoptosis in human retinal pigment epithelial (RPE) cells is a characteristic process in diabetic retinopathy. In our study, we examined whether microRNA-29 (miR-29) may regulate HG-induced RPE cell apoptosis. Human RPE cell line, ARPE-19 cells, was treated with various high concentration of glucose in vitro. HG-induced RPE cell apoptosis was examined by terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay and miR-29 gene expression by quantitative RT-PCR (qRT-PCR). miR-29 was then downregulated in RPE cells, and its effect on HG-induced apoptosis was examined by TUNEL assay and western blot assay on caspase-7 protein. Association of miR-29 on its downstream target, PTEN, in HG-induced RPE cell apoptosis was evaluated by dual-luciferase assay and qRT-PCR. PTEN was silenced in RPE cells. The effects of PTEN downregulation on miR-29-mediated HG-induced RPE cell apoptosis were also examined by TUNEL and western blot assays. HG induced significant apoptosis in RPE cells in a dose-dependent manner. miR-29 was upregulated by HG in RPE cells. miR-29 downregulation protected HG-induced apoptosis and reduced the production of caspase-7 protein in RPE cells. PTEN was shown to be directly downregulated by HG and then upregulated by miR-29 downregulation in RPE cells. Small interfering RNA (siRNA)-mediated PTEN downregulation reversed the protective effect of miR-29 downregulation on HG-induced RPE cell apoptosis. This study demonstrates that miR-29, through inverse association of PTEN, plays an important role in the process of HG-induced apoptosis in RPE cells. PMID:26822433

  12. Iris pigment epithelial cysts in a newborn

    PubMed Central

    Zargar, Shabnam; Prendiville, Kevin John; Martinez, Eladio

    2016-01-01

    Purpose: We report a case of iris pigment epithelial cysts in a newborn and discuss the importance of an accurate diagnosis for prevention of amblyopia. Methods: We describe a case of an abnormal red reflex seen on a newborn exam. Results: A full-term female born via normal spontaneous vaginal delivery without any complications was seen in the newborn nursery. She was noted to have an abnormal eye exam. Pupils were large with circular dark excrescences of the iris pigment epithelium. She was referred to a pediatric ophthalmologist where she was noted to fixate and follow faces. No afferent pupillary defect was seen. OD red reflex was normal whereas OS red reflex was blocked mostly by dark excrescences. A 2–3 mm dark brown lesion was seen in the OD iris and a 3–5 mm dark brown lesion was seen in the OS iris, consistent with a pupillary iris pigment epithelial cyst. Central visual axis was clear OU. Glaucoma was not present and patching was not performed. Observations and clinical photographs were recommended with follow-up in three months. Conclusion: Iris pigment epithelial cysts are uncommonly seen in children. The primary care provider first seeing a newborn must be aware of lesions obscuring a red reflex with appropriate follow-up. Follow-up in three months with IOP measurements is recommended. Iris pigment epithelial cysts in children may be a cause of amblyopia, thus prompt evaluation is important for prognostic purposes and the prevention of amblyopia. PMID:27625966

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

    SciTech Connect

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

    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

  14. Allicin attenuates H₂O₂-induced cytotoxicity in retinal pigmented epithelial cells by regulating the levels of reactive oxygen species.

    PubMed

    Tu, Gerile; Zhang, Yu-Feng; Wei, Wei; Li, Langen; Zhang, Yanmei; Yang, Jia; Xing, Yiqiao

    2016-03-01

    Retinal pigmented epithelial cell (RPE) oxidative stress is known to have a vital role in the etiology of age‑related macular degeneration (AMD). The present study aimed to investigate whether allicin, a natural product with antioxidant activity, was able to protect RPEs (ARPE‑19) from hydrogen peroxide (H2O2)‑induced damage, and to determine the underlying mechanisms. The 3-(4,5-dimethylthiazol-2-yl)-2,5‑diphenyl tetrazolium bromide assay was used to determine cellular viability, and reactive oxygen species (ROS) were detected using a ROS Assay kit. The results demonstrated that allicin was able to protect ARPE‑19 cells from H2O2‑induced damage in a dose‑dependent manner. In addition, allicin attenuated oxidative stress by reducing the levels of intracellular ROS and malondialdehyde (MDA), and enhancing the glutathione/glutathione disulfide (GSSG) ratio. With regards to the underlying mechanism, allicin was able to markedly modulate the expression levels of ROS‑associated enzymes, including superoxide dismutase, NADPH oxidase 4 and NAD(P)H dehydrogenase quinone 1, and elevate the activity of nuclear factor erythroid 2‑related factor 2 in the H2O2‑stimulated ARPE‑19 cells. These results suggested that allicin may exert protective effects against H2O2‑induced cytotoxicity in RPEs via ROS regulation. PMID:26781848

  15. Effects of Bevacizumab on Bcl-2 Expression and Apoptosis in Retinal Pigment Epithelial Cells under Oxidative Stress

    PubMed Central

    Kim, Sukjin; Kim, Young Jun; Kim, Na Rae

    2015-01-01

    Purpose To evaluate the effects of bevacizumab on expression of B-cell leukemia/lymphoma (Bcl)-2 and apoptosis in retinal pigment epithelial (RPE) cells under oxidative stress conditions. Methods RPE cells were treated with H2O2 (0, 100, 200, 300, and 400 µM) and bevacizumab at or above the doses normally used in clinical practice (0, 0.33, 0.67, 1.33, and 2.67 mg/mL). Cell apoptosis was measured using flow cytometry with annexin V-fluorescein isothiocyanate. The expression of Bcl-2 mRNA was determined using reverse transcription polymerase chain reaction. Results Under low oxidative stress conditions (H2O2 100 µM), cell apoptosis was not significantly different at any concentration of bevacizumab, but Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL). Under moderate oxidative stress conditions (H2O2 200 µM), Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL), but cell apoptosis increased only at 2.67 mg/mL of bevacizumab. Under high oxidative stress (300 µM) conditions, cell apoptosis increased at high concentrations of bevacizumab (1.33 and 2.67 mg/mL), but it did not correlate with Bcl-2 expression. Conclusions Withdrawal of vascular endothelial growth factor can lead to RPE cell apoptosis and influences the expression of anti-apoptotic genes such as Bcl-2 under oxidative stress conditions. Since oxidative stress levels of each patient are unknown, repeated injections of intravitreal bevacizumab, as in eyes with age-related macular degeneration, might influence RPE cell survival. PMID:26635460

  16. Calcium overload is associated with lipofuscin formation in human retinal pigment epithelial cells fed with photoreceptor outer segments

    PubMed Central

    Zhang, L; Hui, Y-N; Wang, Y-S; Ma, J-X; Wang, J-B; Ma, L-N

    2011-01-01

    Purpose To investigate the role of Ca2+ in lipofuscin formation in human retinal pigment epithelial (RPE) cells that phagocytize bovine photoreceptor outer segments (POSs). Methods Cultured human RPE cells fed with 2 × 107 per l bovine POS were treated with flunarizine, an antagonist of Ca2+ channel, or/and centrophenoxine, a lipofuscin scavenger. The Ca2+ changes and lipofuscin formation were measured with fluoresence dye Fluo-3/AM ester, laser scanning confocal microscopy (LSCM) and flow cytometry (FCM). The activity of RPE cells was measured by methyl thiazolyl tetrazolium (MTT) assay and argyrophilic nucleolar organizer regions (AgNORs) assay. Results The Ca2+ fluorescence intensity (CFI) of RPE cells fed with POS was significantly increased compared with the controls (165.36±29.92 U). It reached a peak with 777.33±63.86 U (P<0.01) at 12 h, and then decreased but still maintained a high level of 316.90±36.07 U (P<0.01) for 4 days. Flunarizine and centrophenoxine significantly decreased the Ca2+ overload to 227.18±14.00 U at 12 h and 211.06±20.45 U at 4 days. FCM confirmed these changes. The drugs also showed an inhibitory effect on the lipofuscin formation. The proliferation rate of the cells fed with POS increased significantly. Both drugs had inhibitory effects on the activity of the cultured cells. This tendency was confirmed by AgNORs assay. Conclusions The Ca2+ inflow initiated lipofuscin accumulation in RPE cells fed with POS. Flunarizine and centrophenoxine can decrease Ca2+ overload and lipofuscin formation in RPE cells, accompanied by maintaining cellular vitality. PMID:21311572

  17. LYTAK1, a TAK1 inhibitor, suppresses proliferation and epithelial-mesenchymal transition in retinal pigment epithelium cells

    PubMed Central

    CHEN, ZHEN; MEI, YAN; LEI, HUO; TIAN, RUN; NI, NINGHUA; HAN, FANG; GAN, SHENGWEI; SUN, SHANQUAN

    2016-01-01

    The proliferation of retinal pigment epithelium (RPE) cells following epithelial-mesenchymal transition (EMT) is critical in proliferative vitreoretinopathy (PVR), which results in retinal detachment and the loss of vision. The current study was conducted to examine the importance of transforming growth factor β-1 (TGF-β1)-activated kinase 1 (TAK1) inhibitor (LYTAK1) in regulating EMT and the proliferation of RPE cells. RPE cells were pre-treated with increasing concentrations of LYTAK1 prior to treatment with TGF-β1 for 24 h. The effect of LYTAK1 on RPE cell proliferation was examined using a Cell Counting kit-8 assay. The expression levels of TAK1, smooth muscle actin, fibronectin, p-Smad2, p-Smad3, nuclear factor (NF)-κB p65 and IκB kinase α were detected by western blotting. LYTAK1 suppressed the proliferation and migration of RPE cells. Additionally, LYTAK1 significantly prevented TGF-β1-induced EMT by decreasing the levels of fibronectin and α-smooth muscle actin. It was demonstrated that the effects of LYTAK1 were via the Smad signaling pathway. The present study also determined, that the underlying mechanism of the effects of LYTAK1 on EMT in RPE cells involves downregulation of the NF-κB signaling pathway. In conclusion, TAK1 transcription factor was shown to be important in TGF-β1-induced EMT in human RPE cells. Thus, the results of this study aid in elucidating the pathogenesis of human PVR. In addition, this study suggests that specific inhibition by LYTAK1 may provide a novel approach for the treatment and prevention of PVR. PMID:27175834

  18. Role of ZnS Nanoparticles on Endoplasmic Reticulum Stress-mediated Apoptosis in Retinal Pigment Epithelial Cells.

    PubMed

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

    2016-04-01

    Age-related macular degeneration (AMD) is the leading cause for irreversible visual impairment affecting 30-50 million individuals every year. Oxidative stress and endoplasmic reticulum stress have been identified as crucial factors for the pathogenesis of AMD. Current treatments do not focus on underlying stimuli responsible for the disease like AMD. Zinc is an important trace metal in retina and its deficiency leads to AMD. Recent studies on zinc sulphide nanoparticles (ZnS-NPs) are gaining attention in the field of physical and biological research. In this present study, in investigating the role of ZnS-NPs on hydrogen peroxide and thapsigargin-treated primary mice retinal pigment epithelial (MRPE) cells, we synthesized ZnS-NPs and characterized using atomic force microscope (AFM) and SEM-EDX. The ZnS-NPs abrogate the primary MRPE cell death through inhibition of oxidative stress-induced reactive oxygen species production and cell permeability. Oxidant molecules hydrogen peroxide and thapsigargin alter unfolded protein response such as glucose-regulated protein 78 (GRP78) and C/EBP homology protein (CHOP) expressions, whereas ZnS-NPs-pre-treated primary MRPE cells downregulated the overexpression of such proteins. The expressions of apoptotic proteins caspase 12 and cleaved caspase 9 and caspase 3 were also significantly controlled in ZnS-NPs-treated primary MRPE cells when comparing with thapsigargin- and hydrogen peroxide-treated cells. From these results, ZnS-NPs stabilize reactive oxygen species elevation, when subjected to hydrogen peroxide- and thapsigargin-mediated oxidant injury and helps in maintaining normal homeostasis through regulating endoplasmic reticulum (ER) stress response proteins which is the lead cause for apoptosis-mediated pathogenesis of AMD. PMID:26329999

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

    PubMed Central

    van der Burght, Barbro W.; Hansen, Morten; Olsen, Jørgen; Zhou, Jilin; Wu, Yalin; Nissen, Mogens H.; Sparrow, Janet R.

    2016-01-01

    Purpose Accumulation of bisretinoids as lipofuscin in retinal pigment epithelial (RPE) cells is implicated in the pathogenesis of some blinding diseases including age-related macular degeneration (AMD). To identify genes whose expression may change under conditions of bisretinoid accumulation, we investigated the differential gene expression in RPE cells that had accumulated the lipofuscin fluorophore A2E and were exposed to blue light (430 nm). Methods A2E-laden RPE cells were exposed to blue light (A2E/430 nm) at various time intervals. Cell death was quantified using Dead Red staining, and RNA levels for the entire genome was determined using DNA microarrays (Affymetrix GeneChip Human Genome 2.0 Plus). Array results for selected genes were confirmed by real-time reverse-transcriptase polymerase chain reaction. Results Principal component analysis revealed that the A2E-laden RPE cells irradiated with blue light were clearly distinguishable from the control samples. We found differential regulation of genes belonging to the following functional groups: transcription factors, stress response, apoptosis and immune response. Among the last mentioned were downregulation of four genes that coded for proteins that have an inhibitory effect on the complement cascade: (complement factor H, complement factor H-related 1, complement factor I and vitronectin) and of two belonging to the classical pathway (complement component 1, s subcomponent and complement component 1, r subcomponent). Conclusion This study demonstrates that blue light irradiation of A2E-laden RPE cells can alter the transcription of genes belonging to different functional pathways including stress response, apoptosis and the immune response. We suggest that these molecules may be associated to the pathogenesis of AMD and can potentially serve as future therapeutic targets. PMID:23742627

  20. Fenofibrate inhibits the expression of VEGFC and VEGFR-3 in retinal pigmental epithelial cells exposed to hypoxia

    PubMed Central

    ZHAO, JIANFENG; GENG, YU; HUA, HAIRONG; CUN, BIYUN; CHEN, QIANBO; XI, XIAOTING; YANG, LIUSHU; LI, YAN

    2015-01-01

    The aim of the present study was to examine the mechanisms through which fenofibrate inhibits the ability of human retinal pigment epithelial cells (RPE cells) exposed to hypoxia to stimulate the proliferation and migration of human umbilical vein endothelial cells (HUVECs). For this purpose, RPE cells and HUVECs were divided into the following groups: RPE-normoxia, RPE + fenofibrate, RPE-hypoxia, RPE hypoxia + fenofibrate; HUVECs normal culture and HUVECs + RPE-hypoxia culture supernatant. RPE cell hypoxia was induced by cobalt(II) chloride (CoCl2). A superoxide anion probe was used to measure the production of superoxide anion, which is indicative of hypoxic conditions. Cell proliferation was assessed by MTT assay, and the expression of vascular endothelial growth factor C (VEGFC) and vascular endothelial growth factor receptor-3 (VEGFR-3) in the RPE cell culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA). The migration ability of the HUVECs was determined by scratch-wound assay, and the angiogenic ability of the HUVECs was examined by measuring cell lumen formation. The mRNA and protein expression levels of VEGFC and VEGFR-3 in the RPE cells were measured by RT-qPCR and western blot analysis, respectively. Our results revealed that fenofibrate inhibited the increase in the expression and release of VEGFC and VEGFR-3 into the RPE cell culture supernatant induced by exposure to hypoxia. The culture of HUVECs in medium supernatant of RPE cells epxosed to hypoxia enhanced the viability and migration ability of the HUVECs and promoted lumen formation; these effects were inhibited by fenofibrate. In conclusion, our data demonstrated that the exposure of RPE cells to hypoxia induced the expression and release of VEGFC and VEGFR-3 into the cell culture supernatant. The culture of HUVECs in conditioned medium from RPE cells exposed to hypoxia increased VEGFC and VEGFR-3 expression, and promoted the proliferation and migration of the HUVECs, as

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

    PubMed

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

    2016-08-01

    One risk factor of neovascular age-related macular degeneration is systemic hypertension; hypertension is mainly caused by extracellular hyperosmolarity after consumption of dietary salt. In retinal pigment epithelial (RPE) cells, high extracellular osmolarity induces vascular endothelial growth factor (VEGF)-A (Hollborn et al. in Mol Vis 21:360-377, 2015). The aim of the present study was to determine whether extracellular hyperosmolarity and chemical hypoxia trigger the expression of further VEGF family members including placental growth factor (PlGF) in human RPE cells. Hyperosmotic media were made up by addition of 100 mM NaCl or sucrose. Chemical hypoxia was induced by CoCl2. Gene expression was quantified by real-time RT-PCR, and secretion of PlGF-2 was investigated with ELISA. Nuclear factor of activated T cell 5 (NFAT5) was depleted using siRNA. Extracellular hyperosmolarity triggered expression of VEGF-A, VEGF-D, and PlGF genes, and secretion of PlGF-2. Hypoosmolarity decreased PlGF gene expression. Hypoxia induced expression of VEGF-A, VEGF-B, VEGF-D, and PlGF genes. Extracellular hyperosmolarity and hypoxia produced additive PlGF gene expression. Both hyperosmolarity and hypoxia induced expression of KDR and FLT-4 receptor genes, while hyperosmolarity caused neuropilin-2 and hypoxia neuropilin-1 gene expression. The hyperosmotic, but not the hypoxic, PlGF gene expression was in part mediated by NFAT5. The expression of PlGF in RPE cells depends on the extracellular osmolarity. The data suggest that high consumption of dietary salt may exacerbate the angiogenic response of RPE cells in the hypoxic retina via transcriptional activation of various VEGF family member genes. PMID:27230578

  2. Gene delivery and expression in human retinal pigment epithelial cells: effects of synthetic carriers, serum, extracellular matrix and viral promoters.

    PubMed

    Urtti, A; Polansky, J; Lui, G M; Szoka, F C

    2000-01-01

    Non-viral gene therapy is a potential treatment to many incurable retinal diseases. To fulfill this promise, plasmid DNA must be delivered to the retinal target cells. We evaluated the efficacy of synthetic DNA complexing compounds in transfecting primary human retinal pigment epithelial (RPE) cells in vitro. Fetal human RPE cells were cultured with or without extracellular matrix (ECM), produced using calf corneal endothelial cells. Plasmids encoding nuclear localizing beta galactosidase or luciferase (pRSVLuc, pCLuc4, pSV2Luc) were complexed in water at various +/- charge ratios using cationic lipids (Lipofectin, DOTAP, DOGS), polyethylene imines (25 and 750 kDa), and with degraded 6th generation starburst polyamidoamine dendrimers. Luciferase was quantified using a luminometric assay and beta galactosidase with X-gal staining. Toxicities of transfections were evaluated with the MTT-assay. Using beta galactosidase as the reporter gene naked DNA did not transfect RPE cells at measurable levels whereas 1-5% of the cells expressed histochemically detectable amounts of the gene after transfection with cationic lipid DNA complexes. In RPE cells, Rous sarcoma virus and cytomegalovirus (CMV) were more efficient promoters than SV40 in driving luciferase expression, and CMV was chosen for further experiments. At optimal complex charge ratios, expression levels of luciferase were > 10(9) light units/mg protein after transfection using dendrimers and PEI25, while transfection mediated with the other carriers resulted in luciferase expression levels of 10(7)-10(9) light units/mg protein or less. In general, dendrimers and large molecular weight PEI were less toxic than cationic lipids or PEI25 to RPE cells. Serum and ECM decreased gene expression to the RPE cells with all carriers. Despite low percentage of transfected cells the transgene expression per RPE cell is high, important feature in the retinal tissue with small dimensions, in particular in the case of secreted gene

  3. Subretinal transplantation of putative retinal pigment epithelial cells derived from human embryonic stem cells in rat retinal degeneration model

    PubMed Central

    Park, Un Chul; Cho, Myung Soo; Park, Jung Hyun; Kim, Sang Jin; Ku, Seung-Yup; Choi, Young Min; Moon, Shin Yong

    2011-01-01

    Objective To differentiate the human embryonic stem cells (hESCs) into the retinal pigment epithelium (RPE) in the defined culture condition and determine its therapeutic potential for the treatment of retinal degenerative diseases. Methods The embryoid bodies were formed from hESCs and attached on the matrigel coated culture dishes. The neural structures consisting neural precursors were selected and expanded to form rosette structures. The mechanically isolated neural rosettes were differentiated into pigmented cells in the media comprised of N2 and B27. Expression profiles of markers related to RPE development were analyzed by reverse transcription-polymerase chain reaction and immunostaining. Dissociated putative RPE cells (105 cells/5 µL) were transplanted into the subretinal space of rat retinal degeneration model induced by intravenous sodium iodate injection. Animals were sacrificed at 1, 2, and 4 weeks after transplantation, and immnohistochemistry study was performed to verify the survival of the transplanted cells. Results The putative RPE cells derived from hESC showed characteristics of the human RPE cells morphologically and expressed molecular markers and associated with RPE fate. Grafted RPE cells were found to survive in the subretinal space up to 4 weeks after transplantation, and the expression of RPE markers was confirmed with immunohistochemistry. Conclusion Transplanted RPE cells derived from hESC in the defined culture condition successfully survived and migrated within subretinal space of rat retinal degeneration model. These results support the feasibility of the hESC derived RPE cells for cell-based therapies for retinal degenerative disease. PMID:22384445

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

    PubMed Central

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

    2015-01-01

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

  5. miR-17-3p Exacerbates Oxidative Damage in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Tian, Bo; Maidana, Daniel E.; Dib, Bernard; Miller, John B.; Bouzika, Peggy; Miller, Joan W.; Vavvas, Demetrios G.; Lin, Haijiang

    2016-01-01

    Oxidative stress has been shown to contribute to the development of age-related macular degeneration (AMD). MicroRNAs (miRNA) are small non-coding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression. We showed miR-17-3p to be elevated in macular RPE cells from AMD patients and in ARPE-19 cells under oxidative stress. Transfection of miR-17-3p mimic in ARPE-19 induced cell death and exacerbated oxidative lethality that was alleviated by miR-17-3p inhibitor. The expression of antioxidant enzymes manganese superoxide dismutase (MnSOD) and thioredoxin reductase-2 (TrxR2) were suppressed by miR-17-3p mimic and reversed by miR-17-3p inhibitor. These results suggest miR-17-3p aggravates oxidative damage-induced cell death in human RPE cells, while miR-17-3p inhibitor acts as a potential protector against oxidative stress by regulating the expression of antioxidant enzymes. PMID:27505139

  6. Pinosylvin-mediated protection against oxidative stress in human retinal pigment epithelial cells

    PubMed Central

    Koskela, Ali; Reinisalo, Mika; Hyttinen, Juha M. T.; Kaarniranta, Kai

    2014-01-01

    Purpose In this work, we investigated the ability of pinosylvin (PS), 3,5-dihydroxy-trans-stilbene, to modulate oxidative stress in human RPE cells. PS, a stilbenoid polyphenol, occurs in high concentrations in bark byproducts and therefore represents an attractive bioactive compound for health-promoting applications. Methods First, we evaluated the toxicity range of PS by exposing ARPE-19 cells to 0.1–200 µM concentrations of PS for 24 h followed by the cell viability test. In the next stage, the ARPE-19 cells were preincubated in PS for 24 h followed by hydroquinone (HQ) exposure without PS for another 24 h. The cell viability test was conducted after HQ exposure. To elucidate the potential mechanisms behind PS-mediated protection against oxidative stress, the ARPE-19 cells were treated with 5 µM PS for 6 h, and mRNA was extracted at four time points (2 h, 6 h, 12 h, 24 h) to determine changes in the expression of nuclear factor-erythroid 2-related factor-2 (Nrf2), sequestosome 1 (p62/SQSTM1), heme oxygenase-1 (HO-1), and glutathione S-transferase pi 1 (GSTP1) genes. To clarify the molecular mechanism behind PS-mediated protection further, the ARPE-19 cells were transfected with p62 and Nrf2 siRNAs for 24 h, and the roles of p62, Nrf2, and its target gene HO-1 in conferring protection against oxidative stress were studied with quantitative real-time PCR (qRT-PCR) and the cell viability test. Results PS treatment at concentrations of 5 and 10 µM significantly enhanced cell survival from oxidative stress. The expression levels of an enzyme with antioxidative, anti-inflammatory, and immunomodulatory properties, HO-1, were increased by PS treatment and correlated strongly with cell survival. PS treatment did not elevate the expression levels of Nrf2 or its target genes, p62 or GSTP1, even though it had a clear effect on the expression of HO-1, another gene controlled by Nrf2. RNA interference analysis further confirmed the important role of Nrf2 and HO-1 in PS

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

    PubMed Central

    Plaza Reyes, Alvaro; Petrus-Reurer, Sandra; Antonsson, Liselotte; Stenfelt, Sonya; Bartuma, Hammurabi; Panula, Sarita; Mader, Theresa; Douagi, Iyadh; André, Helder; Hovatta, Outi; Lanner, Fredrik; Kvanta, Anders

    2015-01-01

    Summary 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. PMID:26724907

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

    PubMed

    Plaza Reyes, Alvaro; Petrus-Reurer, Sandra; Antonsson, Liselotte; Stenfelt, Sonya; Bartuma, Hammurabi; Panula, Sarita; Mader, Theresa; Douagi, Iyadh; André, Helder; Hovatta, Outi; Lanner, Fredrik; Kvanta, Anders

    2016-01-12

    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. PMID:26724907

  9. Regulation of reduced-folate transporter-1 (RFT-1) in retinal pigment epithelial cells by folate

    PubMed Central

    Naggar, Hany; VanElls, Tracy K.; Ganapathy, Vadivel; Smith, Sylvia B.

    2013-01-01

    Purpose Reduced-folate transporter-1 (RFT-1), a typical transport protein with twelve membrane-spanning domains, transports reduced-folates, such as N5-methyltetrahydrofolate (MTF), the predominant circulating form of folate. RFT-1 is localized to the RPE apical membrane and transports folate from RPE to photoreceptor cells. We asked whether RFT-1 activity in RPE is altered under high folate conditions. Methods ARPE-19 cells were cultured 24, 48 or 72 h in medium containing either 0.5 nM, 5.0 nM or 2.26 µM MTF and the activity of RFT-1 was assessed by determining the uptake of N5-MTF. Semi-quantitative RT-PCR and western blot analysis were used to study RFT-1 gene and protein expression. Results Cells treated for 72 h with 2.26 µM MTF showed a significant (40%) decrease in MTF uptake compared to cells exposed to 0.5 nM or 5 nM MTF. The effect of high concentrations of folate on RFT-1 activity was specific. Kinetic analysis showed that folate-induced attenuation of RFT-1 activity was associated with a decrease in the maximal velocity of the transporter, but no change in the substrate affinity. Steady-state levels of RFT-1 mRNA and protein decreased significantly in the presence of excess folate. Conclusions Excess folate levels folate downregulate RFT-1 in RPE. This study represents the first molecular analysis of the regulation of RFT-1 by folate in RPE and reveals attenuation of the activity and expression of a folate transport protein under conditions of high levels of folate. PMID:15875363

  10. C5a and toll-like receptor 4 crosstalk in retinal pigment epithelial cells

    PubMed Central

    Zhu, Yi; Dai, Bingling; Li, Yongguo

    2015-01-01

    Purpose To investigate the effect of the complement activation product C5a on toll-like receptor (TLR) 4-induced responses in RPE cells. Methods Confluent cultures of human RPE cells (ARPE-19) were stimulated with C5a, lipopolysaccharide (LPS), or a combination of the two. The expression of TLR4 was determined by real-time PCR and flow cytometry. Cytokine profiles were determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). The phosphorylation of p38, ERK 1/2, and JNK was measured by flow cytometry. Results C5a stimulation enhanced the expression of TLR4 in a dose- and time-dependent manner. C5a was able to stimulate the production of TLR4-induced IL-6 and IL-8 by ARPE-19 cells. Blocking experiments showed that the effect of C5a on cytokine production was mediated via C5aR. ERK1/2, but not JNK or p38, were involved in the production of IL-6 and IL-8. Conclusions The results indicate that C5a can induce the TLR4 expression and enhance the production of TLR4-induced IL-6 and IL-8 by ARPE-19. The effect of C5a on cytokine production was mediated by C5aR and the phosphorylation of ERK1/2. PMID:26487798

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

    SciTech Connect

    Zhang, Wenjie; Zhang, Xiaomei; Lu, Hong; Matsukura, Makoto; Zhao, Jien; Shinohara, Makoto

    2013-05-10

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

  12. Autologous transplantation of genetically modified iris pigment epithelial cells: A promising concept for the treatment of age-related macular degeneration and other disorders of the eye

    NASA Astrophysics Data System (ADS)

    Semkova, Irina; Kreppel, Florian; Welsandt, Gerhard; Luther, Thomas; Kozlowski, Jolanta; Janicki, Hanna; Kochanek, Stefan; Schraermeyer, Ulrich

    2002-10-01

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

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

    PubMed

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

    2015-08-01

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

  14. Biomimetic collagen I and IV double layer Langmuir-Schaefer films as microenvironment for human pluripotent stem cell derived retinal pigment epithelial cells.

    PubMed

    Sorkio, Anni E; Vuorimaa-Laukkanen, Elina P; Hakola, Hanna M; Liang, Huamin; Ujula, Tiina A; Valle-Delgado, Juan José; Österberg, Monika; Yliperttula, Marjo L; Skottman, Heli

    2015-05-01

    The environmental cues received by the cells from synthetic substrates in vitro are very different from those they receive in vivo. In this study, we applied the Langmuir-Schaefer (LS) deposition, a variant of Langmuir-Blodgett technique, to fabricate a biomimetic microenvironment mimicking the structure and organization of native Bruch's membrane for the production of the functional human embryonic stem cell derived retinal pigment epithelial (hESC-RPE) cells. Surface pressure-area isotherms were measured simultaneously with Brewster angle microscopy to investigate the self-assembly of human collagens type I and IV on air-subphase interface. Furthermore, the structure of the prepared collagen LS films was characterized with scanning electron microscopy, atomic force microscopy, surface plasmon resonance measurements and immunofluorescent staining. The integrity of hESC-RPE on double layer LS films was investigated by measuring transepithelial resistance and permeability of small molecular weight substance. Maturation and functionality of hESC-RPE cells on double layer collagen LS films was further assessed by RPE-specific gene and protein expression, growth factor secretion, and phagocytic activity. Here, we demonstrated that the prepared collagen LS films have layered structure with oriented fibers corresponding to architecture of the uppermost layers of Bruch's membrane and result in increased barrier properties and functionality of hESC-RPE cells as compared to the commonly used dip-coated controls. PMID:25771016

  15. MEK/ERK pathway mediates UVB-induced AQP1 downregulation and water permeability impairment in human retinal pigment epithelial cells.

    PubMed

    Jiang, Qin; Cao, Cong; Lu, Shan; Kivlin, Rebecca; Wallin, Brittany; Chu, Wenming; Bi, Zhigang; Wang, Xinru; Wan, Yinsheng

    2009-06-01

    Aquaporins (AQPs) are a family of 13 small ( approximately 30 kDa/monomer), hydrophobic, integral membrane proteins. AQPs are expressed in various epithelial and endothelial cells involved in fluid transport. Here, we demonstrated for the first time that AQP1 is expressed in cultured human retinal pigment epithelial (RPE) cells (ARPE-19 cell line). Ultraviolet radiation (UVB) and H2O2, two major factors causing RPE cell damage, induced AQP1 downregulation which was mediated by MEK/ERK activation. UV and H2O2 as well as AQP1-specific siRNA knockdown impaired water permeability of ARPE-19 cells. Notably, pretreatment with all-trans retinoic acid attenuated UV- and H2O2-induced AQP1 downregulation and water permeability impairment. Considering that water permeability is involved in multiple functions of RPE cells such as cellular junction formation, fluid or protein exchange and barrier formation, our data elucidated a novel mechanism through which UV radiation and oxidative stress induce eye cell damage. Our results further support the notion that all-trans retinoic acid might be useful for protection against UV or oxidative stress-induced eye cell damage. PMID:19424603

  16. LEDGF1-326 Decreases P23H and Wild Type Rhodopsin Aggregates and P23H Rhodopsin Mediated Cell Damage in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Baid, Rinku; Scheinman, Robert I.; Shinohara, Toshimichi; Singh, Dhirendra P.; Kompella, Uday B.

    2011-01-01

    Background P23H rhodopsin, a mutant rhodopsin, is known to aggregate and cause retinal degeneration. However, its effects on retinal pigment epithelial (RPE) cells are unknown. The purpose of this study was to determine the effect of P23H rhodopsin in RPE cells and further assess whether LEDGF1-326, a protein devoid of heat shock elements of LEDGF, a cell survival factor, reduces P23H rhodopsin aggregates and any associated cellular damage. Methods ARPE-19 cells were transiently transfected/cotransfected with pLEDGF1-326 and/or pWT-Rho (wild type)/pP23H-Rho. Rhodopsin mediated cellular damage and rescue by LEDGF1-326 was assessed using cell viability, cell proliferation, and confocal microscopy assays. Rhodopsin monomers, oligomers, and their reduction in the presence of LEDGF1-326 were quantified by western blot analysis. P23H rhodopsin mRNA levels in the presence and absence of LEDGF1-326 was determined by real time quantitative PCR. Principal Findings P23H rhodopsin reduced RPE cell viability and cell proliferation in a dose dependent manner, and disrupted the nuclear material. LEDGF1-326 did not alter P23H rhodopsin mRNA levels, reduced its oligomers, and significantly increased RPE cell viability as well as proliferation, while reducing nuclear damage. WT rhodopsin formed oligomers, although to a smaller extent than P23H rhodopsin. Further, LEDGF1-326 decreased WT rhodopsin aggregates. Conclusions P23H rhodopsin as well as WT rhodopsin form aggregates in RPE cells and LEDGF1-326 decreases these aggregates. Further, LEDGF1-326 reduces the RPE cell damage caused by P23H rhodopsin. LEDGF1-326 might be useful in treating cellular damage associated with protein aggregation diseases such as retinitis pigmentosa. PMID:21915354

  17. The marine n-3 PUFA DHA evokes cytoprotection against oxidative stress and protein misfolding by inducing autophagy and NFE2L2 in human retinal pigment epithelial cells

    PubMed Central

    Johansson, Ida; Monsen, Vivi Talstad; Pettersen, Kristine; Mildenberger, Jennifer; Misund, Kristine; Kaarniranta, Kai; Schønberg, Svanhild; Bjørkøy, Geir

    2015-01-01

    Accumulation and aggregation of misfolded proteins is a hallmark of several diseases collectively known as proteinopathies. Autophagy has a cytoprotective role in diseases associated with protein aggregates. Age-related macular degeneration (AMD) is the most common neurodegenerative eye disease that evokes blindness in elderly. AMD is characterized by degeneration of retinal pigment epithelial (RPE) cells and leads to loss of photoreceptor cells and central vision. The initial phase associates with accumulation of intracellular lipofuscin and extracellular deposits called drusen. Epidemiological studies have suggested an inverse correlation between dietary intake of marine n-3 polyunsaturated fatty acids (PUFAs) and the risk of developing neurodegenerative diseases, including AMD. However, the disease-preventive mechanism(s) mobilized by n-3 PUFAs is not completely understood. In human retinal pigment epithelial cells we find that physiologically relevant doses of the n-3 PUFA docosahexaenoic acid (DHA) induce a transient increase in cellular reactive oxygen species (ROS) levels that activates the oxidative stress response regulator NFE2L2/NRF2 (nuclear factor, erythroid derived 2, like 2). Simultaneously, there is a transient increase in intracellular protein aggregates containing SQSTM1/p62 (sequestosome 1) and an increase in autophagy. Pretreatment with DHA rescues the cells from cell cycle arrest induced by misfolded proteins or oxidative stress. Cells with a downregulated oxidative stress response, or autophagy, respond with reduced cell growth and survival after DHA supplementation. These results suggest that DHA both induces endogenous antioxidants and mobilizes selective autophagy of misfolded proteins. Both mechanisms could be relevant to reduce the risk of developing aggregate-associate diseases such as AMD. PMID:26237736

  18. A novel fluorescence-based assay for measuring A2E removal from human retinal pigment epithelial cells to screen for age-related macular degeneration inhibitors.

    PubMed

    Jin, Hong Lan; Lee, Sung-Chan; Kwon, Yong Sam; Choung, Se-Young; Jeong, Kwang Won

    2016-01-01

    Age-related macular degeneration (AMD) is a common retinal disease that leads to irreversible central vision loss in the elderly population. Recent studies have identified many factors related to the development of dry AMD, such as aging, cigarette smoking, genetic predispositions, and oxidative stress, eventually inducing the accumulation of lipofuscin, which is one of the most critical risk factors. One of the major lipofuscins in retinal pigment epithelial (RPE) cells is N-retinylidene-N-retinylethanolamine (also known as A2E), a pyridinium bis-retinoid. Currently there is a lack of effective therapy to prevent or restore vision loss caused by dry AMD. Recent studies have shown that 430 nm blue light induces the oxidation of A2E and the activation of caspase-3 to subsequently cause the death of RPE cells, suggesting that removal of A2E from retinal pigment cells might be critical for preventing AMD. Here, we developed a fluorescence-labeled A2E analog (A2E-BDP) that functions similar to A2E in RPE cells, but is more sensitive to detection than A2E. A2E-BDP-based tracing of intracellular A2E will be helpful, not only for studying the accumulation and removal of A2E in human RPE cells but also for identifying possible inhibitors of AMD. PMID:26604166

  19. Clearance of autophagy-associated dying retinal pigment epithelial cells - a possible source for inflammation in age-related macular degeneration.

    PubMed

    Szatmári-Tóth, M; Kristóf, E; Veréb, Z; Akhtar, S; Facskó, A; Fésüs, L; Kauppinen, A; Kaarniranta, K; Petrovski, G

    2016-01-01

    Retinal pigment epithelial (RPE) cells can undergo different forms of cell death, including autophagy-associated cell death during age-related macular degeneration (AMD). Failure of macrophages or dendritic cells (DCs) to engulf the different dying cells in the retina may result in the accumulation of debris and progression of AMD. ARPE-19 and primary human RPE cells undergo autophagy-associated cell death upon serum depletion and oxidative stress induced by hydrogen peroxide (H2O2). Autophagy was revealed by elevated light-chain-3 II (LC3-II) expression and electron microscopy, while autophagic flux was confirmed by blocking the autophago-lysosomal fusion using chloroquine (CQ) in these cells. The autophagy-associated dying RPE cells were engulfed by human macrophages, DCs and living RPE cells in an increasing and time-dependent manner. Inhibition of autophagy by 3-methyladenine (3-MA) decreased the engulfment of the autophagy-associated dying cells by macrophages, whereas sorting out the GFP-LC3-positive/autophagic cell population or treatment by the glucocorticoid triamcinolone (TC) enhanced it. Increased amounts of IL-6 and IL-8 were released when autophagy-associated dying RPEs were engulfed by macrophages. Our data suggest that cells undergoing autophagy-associated cell death engage in clearance mechanisms guided by professional and non-professional phagocytes, which is accompanied by inflammation as part of an in vitro modeling of AMD pathogenesis. PMID:27607582

  20. Ca2+ channels in retinal pigment epithelial cells regulate vascular endothelial growth factor secretion rates in health and disease

    PubMed Central

    Rosenthal, Rita; Heimann, Heinrich; Agostini, Hansjürgen; Martin, Gottfried; Hansen, Lutz Lothar

    2007-01-01

    Purpose Choroidal neovascularization (CNV) is the most severe complication in age-related macular degeneration. The major angiogenic factor involved is vascular endothelial growth factor (VEGF) secreted by the retinal pigment epithelium (RPE). Since RPE cells express neuroendocrine L-type Ca2+ channels we investigated their involvement in VEGF secretion in normal RPE cells and RPE cells from patients with CNV. Methods Freshly isolated and cultured RPE cells were studied using the patch-clamp technique and ELISA-based secretion assays. Results Both freshly isolated and cultured cells showed whole-cell Ba2+ currents with properties of L-type Ca2+ currents: high activation threshold, sensitivity to dihydropyridines (10 μM nifedipine) and slow inactivation. VEGF-A secretion was elevated by BayK8644 (10 μM) or basic fibroblast growth factor (bFGF, 10 ng/ml), both of which are able to activate L-type channels. Cells from CNV tissue also showed nifedipine-sensitive Ba2+ currents, which displayed a voltage-dependent activation at more negative potentials, faster inactivation and changed regulation by tyrosine kinase pp60c-src. The CNV RPE cells showed higher VEGF secretion rates which were reduced by nifedipine. Conclusions Thus, L-type Ca2+ channels in normal RPE cells regulate the secretion of VEGF. RPE cells from eyes with CNV maintain a VEGF secretion regulated by nifedipine-sensitve Ca2+ channels which might be of importance for the development of CNV. PMID:17417605

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

    PubMed Central

    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

  2. Formation of lipofuscin in cultured retinal pigment epithelial cells exposed to pre-oxidized photoreceptor outer segments.

    PubMed

    Wihlmark, U; Wrigstad, A; Roberg, K; Brunk, U T; Nilsson, S E

    1996-04-01

    Accumulation of lipofuscin in the retinal pigment epithelium (RPE) with increasing age may affect essential supportive functions for the photoreceptors. Earlier, we described a model system for the study of lipofuscinogenesis in RPE cell cultures and showed that mild oxidative stress enhances lipofuscin formation from phagocytized photoreceptor outer segments (POS). In the present study, bovine POS were photo-oxidized, and turned into a lipofuscin-like material, by irradiation with UV light. Transmission electron microscopy of irradiated POS showed loss of the normal stacks of the disk membranes with conversion into an amorphous osmiophilic electron-dense mass. The formation of thiobarbituric acid reactive substances (TBARS), estimated during the irradiation process, indicated lipid peroxidation. Irradiated POS also showed a strong granular yellow autofluorescence. RPE cell cultures, kept at 21% ambient oxygen, were fed daily for 3, 5 or 7 days with either (i) UV-peroxidized POS, (ii) native POS or (iii) culture medium only. RPE cells fed irradiated POS showed significantly higher levels of lipofuscin-specific autofluorescence compared to cells exposed to native POS after 3 days (p = 0.0056), 5 days (p = 0.0037) and 7 days (p = 0.0020), and to the non-exposed control cells (3 days: p = 0.005, 5 days: p = 0.0037, 7 days: p = 0.0094). The lipofuscin content of cells exposed to irradiated POS increased significantly between days 3 and 7 (p = 0.0335). Ultrastructural studies showed much more numerous and larger lipofuscin-like inclusions in RPE cells fed irradiated POS compared to cells exposed to native POS. In the control cells, lipofuscin-like granules were small and sparse. It appears that exposing RPE cells to previously peroxidized POS, thus artificially converted to lipofuscin and obviously not digestible by the lysosomal enzymes, accelerates the formation of severely lipofuscin-loaded cells. The results will be useful for further studies of possible harmful

  3. Efficient delivery and functional expression of transfected modified mRNA in human embryonic stem cell-derived retinal pigmented epithelial cells.

    PubMed

    Hansson, Magnus L; Albert, Silvia; González Somermeyer, Louisa; Peco, Rubén; Mejía-Ramírez, Eva; Montserrat, Núria; Izpisua Belmonte, Juan Carlos

    2015-02-27

    Gene- and cell-based therapies are promising strategies for the treatment of degenerative retinal diseases such as age-related macular degeneration, Stargardt disease, and retinitis pigmentosa. Cellular engineering before transplantation may allow the delivery of cellular factors that can promote functional improvements, such as increased engraftment or survival of transplanted cells. A current challenge in traditional DNA-based vector transfection is to find a delivery system that is both safe and efficient, but using mRNA as an alternative to DNA can circumvent these major roadblocks. In this study, we show that both unmodified and modified mRNA can be delivered to retinal pigmented epithelial (RPE) cells with a high efficiency compared with conventional plasmid delivery systems. On the other hand, administration of unmodified mRNA induced a strong innate immune response that was almost absent when using modified mRNA. Importantly, transfection of mRNA encoding a key regulator of RPE gene expression, microphthalmia-associated transcription factor (MITF), confirmed the functionality of the delivered mRNA. Immunostaining showed that transfection with either type of mRNA led to the expression of roughly equal levels of MITF, primarily localized in the nucleus. Despite these findings, quantitative RT-PCR analyses showed that the activation of the expression of MITF target genes was higher following transfection with modified mRNA compared with unmodified mRNA. Our findings, therefore, show that modified mRNA transfection can be applied to human embryonic stem cell-derived RPE cells and that the method is safe, efficient, and functional. PMID:25555917

  4. Bisretinoid-mediated Complement Activation on Retinal Pigment Epithelial Cells Is Dependent on Complement Factor H Haplotype*

    PubMed Central

    Radu, Roxana A.; Hu, Jane; Jiang, Zhichun; Bok, Dean

    2014-01-01

    Age-related macular degeneration (AMD) is a common central blinding disease of the elderly. Homozygosity for a sequence variant causing Y402H and I62V substitutions in the gene for complement factor H (CFH) is strongly associated with risk of AMD. CFH, secreted by many cell types, including those of the retinal pigment epithelium (RPE), is a regulatory protein that inhibits complement activation. Recessive Stargardt maculopathy is another central blinding disease caused by mutations in the gene for ABCA4, a transporter in photoreceptor outer segments (OS) that clears retinaldehyde and prevents formation of toxic bisretinoids. Photoreceptors daily shed their distal OS, which are phagocytosed by the RPE cells. Here, we investigated the relationship between the CFH haplotype of human RPE (hRPE) cells, exposure to OS containing bisretinoids, and complement activation. We show that hRPE cells of the AMD-predisposing CFH haplotype (HH402/VV62) are attacked by complement following exposure to bisretinoid-containing Abca4−/− OS. This activation was dependent on factor B, indicating involvement of the alternative pathway. In contrast, hRPE cells of the AMD-protective CFH haplotype (YY402/II62) showed no complement activation following exposure to either Abca4−/− or wild-type OS. The AMD-protective YY402/II62 hRPE cells were more resistant to the membrane attack complex, whereas HH402/VV62 hRPE cells showed significant membrane attack complex deposition following ingestion of Abca4−/− OS. These results suggest that bisretinoid accumulation in hRPE cells stimulates activation and dysregulation of complement. Cells with an intact complement negative regulatory system are protected from complement attack, whereas cells with reduced CFH synthesis because of the Y402H and I62V substitutions are vulnerable to disease. PMID:24550392

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

    SciTech Connect

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

    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.

  6. Hyperplastic neuroretinopathy and disorder of pigment epithelial cells precede accelerated retinal degeneration in the SJL/N mouse.

    PubMed

    Caffé, A R; Szél, A; Juliusson, B; Hawkins, R; van Veen, T

    1993-02-01

    We have found a complex eye disease in the SJL/N mouse. This animal is closely related to the SJL/J mouse, which is homozygous for retinal degeneration (rd) and which also suffers from extraocular reticulum cell sarcomas at around 200 days of age. In the SJL/N animal, a high incidence of subretinal tumor is present at 9 days after birth. Furthermore, we have observed an extensive neuroretinal hyperplasia, a phenomenon that is termed "hyperplastic neuroretinopathy", and that is probably the consequence of elevated levels of cytokines in the animals. In addition to these anomalies, the SJL/N mouse shows progressive dystrophy of the retinal pigment epithelium (RPE) from day 4 onwards, and accelerated photoreceptor cell degeneration is completed by day 16. The early RPE dystrophy appears to be a secondary autoimmune disease, since cells in this structure and in the choroid develop MHC class II antigens, whereas we suspect that the accelerated photoreceptor cell loss is induced by a soluble toxic agent. The F1 progeny derived from cross-breeding the SJL/N and Balb/c +/+ strains also shows a high incidence of subretinal tumor and hyperplastic neuroretinopathy, but neither the RPE dystrophy nor retinal degeneration. PMID:8384083

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

    PubMed

    Tokarz, Paulina; Kaarniranta, Kai; Blasiak, Janusz

    2016-04-01

    Epigenetic modifications influence DNA damage response (DDR). In this study we explored the role of DNA methylation and histone acetylation in DDR in cells challenged with acute or chronic oxidative stress. We used retinal pigment epithelial cells (ARPE-19), which natively are exposed to oxidative stress due to permanent exposure to light and high blood flow. We employed a DNA methyltransferase inhibitor - RG108 (RG), or a histone deacetylase inhibitor - valproic acid (VA). ARPE-19 cells were exposed to tert-butyl hydroperoxide, an acute oxidative stress inducer, or glucose oxidase, which slowly liberates low-doses of hydrogen peroxide in the presence of glucose, creating chronic conditions. VA and RG reduced level of intracellular reactive oxygen species and DNA damage in ARPE-19 cells in normal condition and in oxidative stress. This protective effect of VA and RG was associated with the up-regulated expression of antioxidant enzyme genes: CAT, GPx1, GPx4, SOD1 and SOD2. RG decreased the number of cells in G2/M checkpoint in response to chronic oxidative stress. Neither RG nor VA changed the DNA repair or apoptosis induced by oxidative stress. Therefore, certain epigenetic manipulations may protect ARPE-19 cells from detrimental effects of oxidative stress by modulation of antioxidative enzyme gene expression, which may be further explored in pharmacological studies on oxidative stress-related eye diseases. PMID:26899469

  8. Involvement of TonEBP/NFAT5 in osmoadaptative response of human retinal pigmented epithelial cells to hyperosmolar stress

    PubMed Central

    Libert, Sarah; Willermain, François; Weber, Célia; Bryla, Angélic; Salik, Dany; Gregoire, Françoise; Bolaky, Nargis; Caspers, Laure; Perret, Jason

    2016-01-01

    Purpose: Macular edema, a frequently encountered complication of diabetic retinopathy (DR), results from alterations of the blood retinal barrier (BRB) and leads to modifications of the retinal pigmented epithelium (RPE) functions. Osmolar changes of the surrounding medium could be responsible for modifications of the RPE functions leading to disturbance of retinal homeostasis. The expression, activation and function of the key hyperosmolar response factor Tonicity Enhancer Binding Protein (TonEBP also called nuclear factor of activated T-cell 5 - NFTA5) was investigated in ARPE-19 cells, derived from human RPE, in response to hyperosmolar stimulation. Methods: ARPE-19 cells were exposed to hyperosmolar medium. TonEBP mRNA and protein levels were quantified by qRT-PCR and semi-quantitative Western blot. TonEBP nuclear translocation was investigated by immunofluorescence. TonEBP transactivation activity was measured using a reported plasmid containing TonEBP binding sites. Results: In response to hyperosmolar stimulation of ARPE-19 cells, a dose-dependent increase in TonEBP mRNA and protein levels, as well as TonEBP nuclear translocation were observed. TonEBP transactivation activity was further demonstrated using a reporter plasmid containing TonEBP binding sites. A dominant negative form of TonEBP abolished NaCl-induced increase in TonEBP transactivation activity, and inhibited the increase of the target genes aldose reductase and sodium-dependent taurine transporter mRNA levels. SB203580, an inhibitor of two of the p38 protein kinase’s isoforms (p38α and p38β) inhibited the TonEBP nuclear translocation and transactivation activity in ARPE-19 cells exposed to hyperosmolar stimulation. Conclusions: Our data demonstrates the involvement of TonEBP in the mechanisms responsible for osmoadaptation to hyperosmolar stress in RPE cells. Given the emerging role of TonEBP in different pathological pathways, these data open new perspectives for the analysis of the

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

    PubMed Central

    Kim, Dong-Il; Park, Min-Jung; Choi, Joo-Hee; Kim, In-Seon; Han, Ho-Jae; Yoon, Kyung-Chul; Park, Sang-Woo; Lee, Min-Young; Oh, Ki-Seok; Park, Soo-Hyun

    2015-01-01

    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. PMID:26583059

  10. Synergistic effects of gamma interferon on inflammatory mediators that induce interleukin-6 gene expression and secretion by human retinal pigment epithelial cells.

    PubMed Central

    Nagineni, C N; Detrick, B; Hooks, J J

    1994-01-01

    The retinal pigment epithelial (RPE) cell is a potent regulatory cell within the retina. It helps to maintain normal retinal activity, and following gamma interferon (IFN-gamma) exposure, it may express major histocompatibility complex class II molecules and function as an antigen-presenting cell. Since interleukin-1 (IL-1) and IL-6 are potent cytokines observed in ocular inflammatory processes, we initiated studies to evaluate conditions which enable RPE cells to produce these cytokines. Cultures of human RPE cells from two eye donors were established and characterized, and enzyme immunoassays were employed to screen for IL-1 and IL-6 production. Treatment of RPE cells with lipopolysaccharide (LPS) or recombinant tumor necrosis factor alpha, IL-1, or IFN-gamma resulted in a significant level of secretion of IL-6. In contrast, treatment with recombinant epidermal growth factor, basic fibroblast growth factor, platelet-derived growth factor, or transforming growth factor alpha, or LPS can dramatically augment the secretion of IL-6 by RPE cells. Thus, these inflammatory mediators can act alone or synergistically with IFN-gamma to activate RPE cells and dramatically increase the expression and secretion of IL-6. In contrast, IL-1 was not detected following stimulation with any of the above-mentioned cytokines or LPS. Characterization of IL-6 protein production by RPE cells revealed that 98% of the protein is promptly secreted by the cell, its induction is dependent upon the time and concentration of the stimulant, and the continuous presence of the stimulant is required for IL-6 production. Moreover, Western blot (immunoblot) analysis of secreted proteins revealed that IL-6 was produced in multiple molecular forms.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8556503

  11. Transplantation of human retinal pigment epithelial cells in the nucleus accumbens of cocaine self-administering rats provides protection from seeking.

    PubMed

    Venkiteswaran, Kala; Alexander, Danielle N; Puhl, Matthew D; Rao, Anand; Piquet, Amanda L; Nyland, Jennifer E; Subramanian, Megha P; Iyer, Puja; Boisvert, Matthew M; Handly, Erin; Subramanian, Thyagarajan; Grigson, Patricia Sue

    2016-05-01

    Chronic exposure to drugs and alcohol leads to damage to dopaminergic neurons and their projections in the 'reward pathway' that originate in the ventral tegmental area (VTA) and terminate in the nucleus accumbens (NAc). This damage is thought to contribute to the signature symptom of addiction: chronic relapse. In this study we show that bilateral transplants of human retinal pigment epithelial cells (RPECs), a cell mediated dopaminergic and trophic neuromodulator, into the medial shell of the NAc, rescue rats with a history of high rates of cocaine self-administration from drug-seeking when returned, after 2 weeks of abstinence, to the drug-associated chamber under extinction conditions (i.e., with no drug available). Excellent survival was noted for the transplant of RPECs in the shell and/or the core of the NAc bilaterally in all rats that showed behavioral recovery from cocaine seeking. Design based unbiased stereology of tyrosine hydroxylase (TH) positive cell bodies in the VTA showed better preservation (p<0.035) in transplanted animals compared to control animals. This experiment shows that the RPEC graft provides beneficial effects to prevent drug seeking in drug addiction via its effects directly on the NAc and its neural network with the VTA. PMID:26562520

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

    PubMed

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

    2016-04-30

    In this work, we carried out a comparative study of four different niosome formulations based on the same cationic lipid and non-ionic tensoactive. The niosomes prepared by oil-in-water emulsion technique (o/w) only differed in the helper lipid composition: squalene, cholesterol, squalane or no helper lipid. Niosomes and nioplexes elaborated upon the addition of pCMS-EGFP reporter plasmid were characterized in terms of size, zeta potential and polydispersity index. The capacity of the niosomes to condense, release and protect the DNA against enzymatic degradation was evaluated by agarose gel electrophoresis. In vitro experiments were carried out to evaluate transfection efficiency and cell viability in retinal pigment epithelial cells. Moreover, uptake and intracellular trafficking studies were performed to further understand the role of the helper lipids in the transfection process. Interestingly, among all tested formulations, niosomes elaborated with squalene as helper lipid were the most efficient transfecting cells. Such transfection efficiency could be attributed to their higher cellular uptake and the particular entry pathways used, where macropinocytosis pathway and lysosomal release played an important role. Therefore, these results suggest that helper lipid composition is a crucial step to be considered in the design of niosome formulation for retinal gene delivery applications since clearly modulates the cellular uptake, internalization mechanism and consequently, the final transfection efficiency. PMID:26956159

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

    SciTech Connect

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

    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

  14. A novel polysaccharide compound derived from algae extracts protects retinal pigment epithelial cells from high glucose-induced oxidative damage in vitro.

    PubMed

    Xie, Peiyu; Fujii, Isao; Zhao, Ji'en; Shinohara, Makoto; Matsukura, Makoto

    2012-01-01

    Diabetic retinopathy is a common complication of diabetes mellitus (DM). The oxidative damage inflicted on retinal pigment epithelial (RPE) cells by high glucose closely approximates the molecular basis for the loss of vision associated with this disease. We investigate a novel algae-derived polysaccharide compound for its role in protecting ARPE-19 cells from high glucose-induced oxidative damage. ARPE-19 cells were cultured for 4 d with normal concentration of D-glucose, and exposed to either normal or high concentrations of D-glucose in the presence or absence of the polysaccharide compound at variety of concentrations for another 48 h. Taurine was used as a positive control. Activity of super oxide dismutase (SOD) and concentration of glutathione (GSH) were measured as well as cytotoxicity of high glucose and the polysaccharide compound. To analyse cellular damage by high glucose, activation of Annexin V and p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) were examined. Our results showed that a significant cellular damage on ARPE-19 cells after 48 h treatment with high glucose, accompanied by a decrease in SOD activity and GSH concentration; high glucose also caused ARPE-19 cell apoptosis and activation of p38MAPK and ERK. As the non-toxic polysaccharide compound protected ARPE-19 cells from high glucose-induced cellular damage, the compound recovered SOD activity and concentration of GSH in the cells. The compound also abrogated the cell apoptosis and activation of p38MAPK and ERK. Therefore, the polysaccharide compound derived from algae extracts could be unique candidate for a new class of anti-DM and anti-oxidative damage. PMID:22975494

  15. TNF-α Mediates PKCδ/JNK1/2/c-Jun-Dependent Monocyte Adhesion via ICAM-1 Induction in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Lee, I-Ta; Liu, Shiau-Wen; Chi, Pei-Ling; Lin, Chih-Chung; Hsiao, Li-Der; Yang, Chuen-Mao

    2015-01-01

    Retinal inflammatory diseases induced by cytokines, such as tumor necrosis factor-α (TNF-α) are associated with an up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the retinal pigment epithelial cells (RPECs). Retinal pigment epithelium (RPE) is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). However, the detailed mechanisms of TNF-α-induced ICAM-1 expression are largely unclear in human RPECs. We demonstrated that in RPECs, TNF-α could induce ICAM-1 protein and mRNA expression and promoter activity, and monocyte adhesion. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of PKCs (Ro318220), PKCδ (Rottlerin), MEK1/2 (U0126), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of TNFR1, TRAF2, JNK2, p42, or c-Jun. We showed that TNF-α could stimulate the TNFR1 and TRAF2 complex formation. TNF-α-stimulated JNK1/2 was also reduced by Rottlerin or SP600125. However, Rottlerin had no effect on TNF-α-induced p42/p44 MAPK phosphorylation. We observed that TNF-α induced c-Jun phosphorylation which was inhibited by Rottlerin or SP600125. On the other hand, TNF-α-stimulated ICAM-1 promoter activity was prominently lost in RPECs transfected with the point-mutated AP-1 ICAM-1 promoter plasmid. These results suggest that TNF-α-induced ICAM-1 expression and monocyte adhesion is mediated through a TNFR1/TRAF2/PKCδ/JNK1/2/c-Jun pathway in RPECs. These findings concerning TNF-α-induced ICAM-1 expression in RPECs imply that TNF-α might play an important role in ocular inflammation and diseases. PMID:25675437

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

    PubMed

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

    2016-06-17

    Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein αB-crystallin (αB) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of αB via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where αB expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and αB-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of αB results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for αB in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes. PMID:27129211

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

    SciTech Connect

    Wang, Cheng-hu; Cao, Guo-Fan; Jiang, Qin; Yao, Jin

    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.

  18. Polarized Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cell Monolayers Have Higher Resistance to Oxidative Stress-Induced Cell Death Than Nonpolarized Cultures

    PubMed Central

    Hsiung, Jamie; Zhu, Danhong

    2015-01-01

    Oxidative stress-mediated injury to the retinal pigment epithelium (RPE) is a major factor involved in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. Human embryonic stem cell (hESC)-derived RPE cells are currently being evaluated for their potential for cell therapy in AMD patients through subretinal injection of cells in suspension and subretinal placement as a polarized monolayer. To gain an understanding of how transplanted RPE cells will respond to the highly oxidatively stressed environment of an AMD patient eye, we compared the survival of polarized and nonpolarized RPE cultures following oxidative stress treatment. Polarized, nonpolarized/confluent, nonpolarized/subconfluent hESC-RPE cells were treated with H2O2. Terminal deoxynucleotidyl transferase dUTP nick end labeling stains revealed the highest amount of cell death in subconfluent hESC-RPE cells and little cell death in polarized hESC-RPE cells with H2O2 treatment. There were higher levels of proapoptotic factors (phosphorylated p38, phosphorylated c-Jun NH2-terminal kinase, Bax, and cleaved caspase 3 fragments) in treated nonpolarized RPE—particularly subconfluent cells—relative to polarized cells. On the other hand, polarized RPE cells had constitutively higher levels of cell survival and antiapoptotic signaling factors such as p-Akt and Bcl-2, as well as antioxidants superoxide dismutase 1 and catalase relative to nonpolarized cells, that possibly contributed to polarized cells’ higher tolerance to oxidative stress compared with nonpolarized RPE cells. Subconfluent cells were particularly sensitive to oxidative stress-induced apoptosis. These results suggest that implantation of polarized hESC-RPE monolayers for treating AMD patients with geographic atrophy should have better survival than injections of hESC-RPE cells in suspension. PMID:25411476

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

    PubMed Central

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

    2009-01-01

    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 C2 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). Overexpression 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 C2 ceramide suggests that increased ceramide levels will preferentially affect non-polarized RPE, as are found in late

  20. Retinal pigment epithelial hamartoma--unusual manifestations.

    PubMed Central

    Rosenberg, P. R.; Walsh, J. B.

    1984-01-01

    Hamartoma of the retinal pigment epithelium is an uncommon tumour of young adults. We have seen 2 patients with this clinical diagnosis, both with unusual manifestations. In one patient growth of the tumour was observed over a 5-year period. In the second patient arterial-arterial anastomoses were detected at a site distal to the tumour. Images PMID:6722077

  1. Oxidative stress sensitizes retinal pigmented epithelial (RPE) cells to complement-mediated injury in a natural antibody-, lectin pathway-, and phospholipid epitope-dependent manner.

    PubMed

    Joseph, Kusumam; Kulik, Liudmila; Coughlin, Beth; Kunchithapautham, Kannan; Bandyopadhyay, Mausumi; Thiel, Steffen; Thielens, Nicole M; Holers, V Michael; Rohrer, Bärbel

    2013-05-01

    Uncontrolled activation of the alternative complement pathway (AP) is thought to be associated with age-related macular degeneration. Previously, we have shown that in retinal pigmented epithelial (RPE) monolayers, oxidative stress reduced complement inhibition on the cell surface, resulting in sublytic complement activation and loss of transepithelial resistance (TER), but the potential ligand and pathway involved are unknown. ARPE-19 cells were grown as monolayers on transwell plates, and sublytic complement activation was induced with H2O2 and normal human serum. TER deteriorated rapidly in H2O2-exposed monolayers upon adding normal human serum. Although the effect required AP activation, AP was not sufficient, because elimination of MASP, but not C1q, prevented TER reduction. Reconstitution experiments to unravel essential components of the lectin pathway (LP) showed that both ficolin and mannan-binding lectin can activate the LP through natural IgM antibodies (IgM-C2) that recognize phospholipid cell surface modifications on oxidatively stressed RPE cells. The same epitopes were found on human primary embryonic RPE monolayers. Likewise, mouse laser-induced choroidal neovascularization, an injury that involves LP activation, could be increased in antibody-deficient rag1(-/-) mice using the phospholipid-specific IgM-C2. In summary, using a combination of depletion and reconstitution strategies, we have shown that the LP is required to initiate the complement cascade following natural antibody recognition of neoepitopes, which is then further amplified by the AP. LP activation is triggered by IgM bound to phospholipids. Taken together, we have defined novel mechanisms of complement activation in oxidatively stressed RPE, linking molecular events involved in age-related macular degeneration, including the presence of natural antibodies and neoepitopes. PMID:23493397

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

    SciTech Connect

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

    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.

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

    PubMed

    Bantseev, Vladimir; Youn, Hyun-Yi

    2006-12-01

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

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

    SciTech Connect

    Wang, Jiying; Ohno-Matsui, Kyoko; Morita, Ikuo

    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

  5. Protective effect of autophagy on human retinal pigment epithelial cells against lipofuscin fluorophore A2E: implications for age-related macular degeneration

    PubMed Central

    Zhang, J; Bai, Y; Huang, L; Qi, Y; Zhang, Q; Li, S; Wu, Y; Li, X

    2015-01-01

    Age-related macular degeneration (AMD) is the leading cause of central vision loss in the elderly. Degeneration of retinal pigment epithelial (RPE) cells is a crucial causative factor responsible for the onset and progression of AMD. A2E, a major component of toxic lipofuscin implicated in AMD, is deposited in RPE cells with age. However, the mechanism whereby A2E may contribute to the pathogenesis of AMD remains unclear. We demonstrated that A2E was a danger signal of RPE cells, which induced autophagy and decreased cell viability in a concentration- and time-dependent manner. Within 15 min after the treatment of RPE with 25 μM A2E, the induction of autophagosome was detected by transmission electron microscopy. After continuous incubating RPE cells with A2E, intense punctate staining of LC3 and increased expression of LC3-II and Beclin-1 were identified. Meanwhile, the levels of intercellular adhesion molecule (ICAM), interleukin (IL)1β, IL2, IL-6, IL-8, IL-17A, IL-22, macrophage cationic peptide (MCP)-1, stromal cell-derived factor (SDF)-1, and vascular endothelial growth factor A (VEGFA) were elevated. The autophagic inhibitor 3-methyladenine (3-MA) and activator rapamycin were also used to verify the effect of autophagy on RPE cells against A2E. Our results revealed that 3-MA decreased the autophagosomes and LC3 puncta induced by A2E, increased inflammation-associated protein expression including ICAM, IL1β, IL2, IL-6, IL-8, IL-17A, IL-22, and SDF-1, and upregulated VEGFA expression. Whereas rapamycin augmented the A2E-mediated autophagy, attenuated protein expression of inflammation-associated and angiogenic factors, and blocked the Akt/mTOR pathway. Taken together, A2E induces autophagy in RPE cells at the early stage of incubation, and this autophagic response can be inhibited by 3-MA or augmented by rapamycin via the mTOR pathway. The enhancement of autophagy has a protective role in RPE cells against the adverse effects of A2E by reducing the

  6. Protective effect of autophagy on human retinal pigment epithelial cells against lipofuscin fluorophore A2E: implications for age-related macular degeneration.

    PubMed

    Zhang, J; Bai, Y; Huang, L; Qi, Y; Zhang, Q; Li, S; Wu, Y; Li, X

    2015-01-01

    Age-related macular degeneration (AMD) is the leading cause of central vision loss in the elderly. Degeneration of retinal pigment epithelial (RPE) cells is a crucial causative factor responsible for the onset and progression of AMD. A2E, a major component of toxic lipofuscin implicated in AMD, is deposited in RPE cells with age. However, the mechanism whereby A2E may contribute to the pathogenesis of AMD remains unclear. We demonstrated that A2E was a danger signal of RPE cells, which induced autophagy and decreased cell viability in a concentration- and time-dependent manner. Within 15 min after the treatment of RPE with 25 μM A2E, the induction of autophagosome was detected by transmission electron microscopy. After continuous incubating RPE cells with A2E, intense punctate staining of LC3 and increased expression of LC3-II and Beclin-1 were identified. Meanwhile, the levels of intercellular adhesion molecule (ICAM), interleukin (IL)1β, IL2, IL-6, IL-8, IL-17A, IL-22, macrophage cationic peptide (MCP)-1, stromal cell-derived factor (SDF)-1, and vascular endothelial growth factor A (VEGFA) were elevated. The autophagic inhibitor 3-methyladenine (3-MA) and activator rapamycin were also used to verify the effect of autophagy on RPE cells against A2E. Our results revealed that 3-MA decreased the autophagosomes and LC3 puncta induced by A2E, increased inflammation-associated protein expression including ICAM, IL1β, IL2, IL-6, IL-8, IL-17A, IL-22, and SDF-1, and upregulated VEGFA expression. Whereas rapamycin augmented the A2E-mediated autophagy, attenuated protein expression of inflammation-associated and angiogenic factors, and blocked the Akt/mTOR pathway. Taken together, A2E induces autophagy in RPE cells at the early stage of incubation, and this autophagic response can be inhibited by 3-MA or augmented by rapamycin via the mTOR pathway. The enhancement of autophagy has a protective role in RPE cells against the adverse effects of A2E by reducing the

  7. Lyar Is a New Ligand for Retinal Pigment Epithelial Phagocytosis.

    PubMed

    Guo, Feiye; Ding, Ying; Caberoy, Nora B; Alvarado, Gabriela; Liu, Robert; Shen, Chen; Yu, Jisu; Zhou, Yixiong; Salero, Enrique; LeBlanc, Michelle E; Wang, Weiwen; Li, Wei

    2015-10-01

    Phagocytosis is critical to tissue homeostasis, as highlighted by phagocytosis defect of retinal pigment epithelial (RPE) cells with debris accumulation, photoreceptor degeneration and blindness. Phagocytosis ligands are the key to delineating molecular mechanisms and functional roles of phagocytes, but are traditionally identified in individual cases with technical challenges. We recently developed open reading frame phage display (OPD) for phagocytosis-based functional cloning (PFC) to identify unknown ligands. One of the identified ligands was Ly-1 antibody reactive clone (Lyar) with functions poorly defined. Herein, we characterized Lyar as a new ligand to stimulate RPE phagocytosis. In contrast to its reported nucleolar expression, immunohistochemistry showed that Lyar was highly expressed in photoreceptor outer segments (POSs) of the retina. Cytoplasmic Lyar was released from apoptotic cells, and selectively bound to shed POSs and apoptotic cells, but not healthy cells. POS vesicles engulfed through Lyar-dependent pathway were targeted to phagosomes and colocalized with phagosome marker Rab7. These results suggest that Lyar is a genuine RPE phagocytosis ligand, which in turn supports the validity of OPD/PFC as the only available approach for unbiased identification of phagocytosis ligands with broad applicability to various phagocytes. PMID:25735755

  8. Temsirolimus inhibits proliferation and migration in retinal pigment epithelial and endothelial cells via mTOR inhibition and decreases VEGF and PDGF expression.

    PubMed

    Liegl, Raffael; Koenig, Susanna; Siedlecki, Jakob; Haritoglou, Christos; Kampik, Anselm; Kernt, Marcus

    2014-01-01

    Due to their high prevalence, retinal vascular diseases including age related macular degeneration (AMD), retinal vein occlusions (RVO), diabetic retinopathy (DR) and diabetic macular edema have been major therapeutic targets over the last years. The pathogenesis of these diseases is complex and yet not fully understood. However, increased proliferation, migration and angiogenesis are characteristic cellular features in almost every retinal vascular disease. The introduction of vascular endothelial growth factor (VEGF) binding intravitreal treatment strategies has led to great advances in the therapy of these diseases. While the predominant part of affected patients benefits from the specific binding of VEGF by administering an anti-VEGF antibody into the vitreous cavity, a small number of non-responders exist and alternative or additional therapeutic strategies should therefore be evaluated. The mammalian target of rapamycin (mTOR) is a central signaling pathway that eventually triggers up-regulation of cellular proliferation, migration and survival and has been identified to play a key role in angiogenesis. In the present study we were able to show that both retinal pigment epithelial (RPE) cells as wells as human umbilical vein endothelial cells (HUVEC) are inhibited in proliferating and migrating after treatment with temsirolimus in non-toxic concentrations. Previous studies suggest that the production of VEGF, platelet derived growth factor (PDGF) and other important cytokines is not only triggered by hypoxia but also by mTOR itself. Our results indicate that temsirolimus decreases VEGF and PDGF expression on RNA and protein levels significantly. We therefore believe that the mTOR inhibitor temsirolimus might be a promising drug in the future and it seems worthwhile to evaluate complementary therapeutic effects with anti-VEGF drugs for patients not profiting from mono anti-VEGF therapy alone. PMID:24586308

  9. Osmolarity and spectrophotometric property of brilliant blue green define the degree of toxicity on retinal pigment epithelial cells exposed to surgical endoilluminator

    PubMed Central

    Balaiya, Sankarathi; Sambhav, Kumar; Cook, William B; Chalam, Kakarla V

    2016-01-01

    Objective To evaluate the effect of varying concentrations of brilliant blue green (BBG) and their different biochemical characteristics on retinal pigment epithelial (RPE) cells under xenon light source illumination at varying distances to identify safe parameters for intraoperative use. Methods Human retinal RPE cells (ARPE-19) were exposed to two concentrations (0.25 and 0.50 mg/mL) of BBG and illuminated with a xenon surgical illuminator at varying distances (10 and 25 mm), intensity levels, and time intervals (1, 5, and 15 minutes). Additionally, the effect of osmolarity was examined by diluting BBG in different concentrations of glucose. Cytotoxicity of BBG and osmolarity effects on cell viability were evaluated using a WST-1 assay. Light absorption and emission characteristic of BBG in different solvents were measured using a plate reader at different wavelengths. Lastly, the activity of caspase-3 was also studied. Results Cell viability of ARPE-19 cells was 77.4%±12.7%, 78.7%±17.0%, and 65.0%±19.7% at 1, 5, and 15 minutes to exposure of high illumination xenon light at 10 mm (P<0.05) compared to controls. At both distances of illumination (10 and 25 mm), similar cell viabilities were seen between 1 and 5 minutes of exposure. However, there was a decline in viability when the illumination was carried out to 15 minutes in all groups (P<0.05). There was no significant reduction in cell viability in presence or absence of xenon light in different osmolar solutions concentrations of glucose (P>0.05). Maximal light absorption of BBG was noted between 540 and 680 nm. Activated caspase-3 level was not significant in both the concentrations of BBG (P>0.05). Conclusion Our findings suggest that BBG at 0.25 mg/mL during vitreoretinal surgery is safe and not toxic to RPE cells up to 5 minutes under focal high illumination (10 mm) and up to 15 minutes under medium diffuse illumination (25 mm). BBG was safe to be mixed with isotonic glucose solution at the

  10. Chloroquine and Hydroxychloroquine Increase Retinal Pigment Epithelial Layer Permeability.

    PubMed

    Korthagen, Nicoline M; Bastiaans, Jeroen; van Meurs, Jan C; van Bilsen, Kiki; van Hagen, P Martin; Dik, Willem A

    2015-07-01

    Antimalarials chloroquine (CQ) and hydroxychloroquine (HCQ) are widely used as antiinflammatory drugs, but side effects include retinopathy and vision loss. The objective of this study was to examine the effect of CQ and HCQ on the barrier integrity of retinal pigment epithelial (RPE) cell monolayers in vitro. Permeability of ARPE-19 cell monolayers was determined using Fluorescein isothiocyanate (FITC)-labeled dextran. The influence of CQ and HCQ on cell death and the expression tight junction molecules was examined. CQ and HCQ significantly increased ARPE-19 monolayer permeability after 3 and 18 h, respectively, and enhanced mRNA levels for claudin-1 and occludin. Cytotoxicity was only observed after 18 h exposure. Thus, CQ and HCQ rapidly enhance RPE barrier permeability in vitro, independent of cytotoxicity or loss of zonula occludens-1, claudin-1, and occludin expression. Our findings suggest that CQ/HCQ-induced permeability of the RPE layer may contribute to blood-retinal barrier breakdown in case of CQ/HCQ-induced retinopathy. PMID:25752684

  11. Lutein and zeaxanthin supplementation reduces photo-oxidative damage and modulates the expression of inflammation-related genes in retinal pigment epithelial cells

    PubMed Central

    Bian, Qingning; Gao, Shasha; Zhou, Jilin; Qin, Jian; Taylor, Allen; Johnson, Elizabeth J.; Tang, Guangwen; Sparrow, Janet R.; Gierhart, Dennis; Shang, Fu

    2012-01-01

    Oxidative damage and inflammation are related to the pathogenesis of age-related macular degeneration (AMD). Epidemiologic studies suggest that insufficient dietary lutein and zeaxanthin intake or lower serum zeaxanthin levels are associated with increased risk for AMD. The objective of this work is to test the protective effects of lutein and zeaxanthin against photo-oxidative damage to retinal pigment epithelial cells (RPE) and oxidation-induced changes in expression of inflammation-related genes. To mimic lipofuscin-mediated photo-oxidation in vivo, we used ARPE-19 cells that accumulated A2E, a lipofuscin fluorophore and photosensitizer, as a model system to investigate the effects of lutein and zeaxanthin supplementation. The data show that supplementation with lutein or zeaxanthin in the medium resulted in accumulation of lutein or zeaxanthin in the RPE cells. The concentrations of lutein and zeaxanthin in the cells were 2–14-fold of that detected in the medium, indicating that ARPE-19 cells actively take up lutein or zeaxanthin. As compared with untreated cells, exposure of A2E-containing RPE to blue light resulted in a 40–60% decrease in proteasome activity, a 50–80% decrease in expression of CFH and MCP-1, and an ~ 20-fold increase in expression of IL-8. The photo-oxidation-induced changes in expression of MCP-1, IL-8 and CFH were similar to those caused by chemical inhibition of the proteasome, suggesting that inactivation of the proteasome is involved in the photo-oxidation-induced alteration in expression of these inflammation-related genes. Incubation of the A2E-containing RPE with lutein or zeaxanthin prior to blue light exposure significantly attenuated the photo-oxidation-induced inactivation of the proteasome and photo-oxidation induced changes in expression of MCP-1, IL-8, and CFH. Together, these data indicate that lutein or zeaxanthin modulates inflammatory responses in cultured RPE in response to photo-oxidation. Protecting the proteasome

  12. Efficient delivery of NF-κB siRNA to human retinal pigment epithelial cells with hyperbranched cationic polysaccharide derivative-based nanoparticles

    PubMed Central

    Liu, Zhenzhen; Gong, Haijun; Zeng, Rui; Liang, Xuan; Zhang, Li-Ming; Yang, Liqun; Lan, Yuqing

    2015-01-01

    A hyperbranched cationic polysaccharide derivative-mediated small interfering (si)RNA interference strategy was proposed to inhibit nuclear transcription factor-kappa B (NF-κB) activation in human retinal pigment epithelial (hRPE) cells for the gene therapy of diabetic retinopathy. Two hyperbranched cationic polysaccharide derivatives containing the same amount of cationic residues, but with different branching structures and molecular weights, including 3-(dimethylamino)-1-propylamine-conjugated glycogen (DMAPA-Glyp) and amylopectin (DMAPA-Amp) derivatives, were developed for the efficient delivery of NF-κB siRNA into hRPE cells. The DMAPA-Glyp derivative showed lower toxicity against hRPE cells. Furthermore, the DMAPA-Glyp derivative more readily condensed siRNA and then formed the nanoparticles attributed to its higher branching architecture when compared to the DMAPA-Amp derivative. Both DMAPA-Glyp/siRNA and DMAPA-Amp/siRNA nanoparticles were able to protect siRNA from degradation by nuclease in 25% fetal bovine serum. The particle sizes of the DMAPA-Glyp/siRNA nanoparticles (70–120 nm) were smaller than those of the DMAPA-Amp/siRNA nanoparticles (130–180 nm) due to the higher branching architecture and lower molecular weight of the DMAPA-Glyp derivative. In addition, the zeta potentials of the DMAPA-Glyp/siRNA nanoparticles were higher than those of the DMAPA-Glyp/siRNA nanoparticles. As a result, siRNA was much more efficiently transferred into hRPE cells using the DMAPA-Glyp/siRNA nanoparticles rather than the DMAPA-Amp/siRNA nanoparticles. This led to significantly high levels of suppression on the expression levels of NF-κB p65 messenger RNA and protein in the cells transfected with DMAPA-Glyp/siRNA nanoparticles. This work provides a potential approach to promote hyperbranched polysaccharide derivatives as nonviral siRNA vectors for the inhibition of NF-κB activation in hRPE cells. PMID:25897219

  13. Inflammasome priming increases retinal pigment epithelial cell susceptibility to lipofuscin phototoxicity by changing the cell death mechanism from apoptosis to pyroptosis.

    PubMed

    Brandstetter, Carolina; Patt, Joshua; Holz, Frank G; Krohne, Tim U

    2016-08-01

    Progressive death of retinal pigment epithelium (RPE) cells is a hallmark of age-related macular degeneration (AMD), the leading cause of blindness in all developed countries. Photooxidative damage and activation of the NLRP3 inflammasome have been suggested as contributing factors to this process. We investigated the effects of inflammasome activation on oxidative damage-induced RPE cell death. In primary human RPE cells and ARPE-19 cells, lipofuscin accumulated following incubation with oxidatively modified photoreceptor outer segments. Oxidative stress was induced by blue light irradiation (dominant wavelength: 448nm, irradiance: 0.8mW/cm(2), duration: 3 to 6h) of lipofuscin-loaded cells and resulted in cell death by apoptosis. Prior inflammasome priming by IL-1α or complement activation product C5a altered the cell death mechanism to pyroptosis and resulted in a significant increase of the phototoxic effect. Following IL-1α priming, viability 24h after irradiation was reduced in primary RPE cells and ARPE-19 cells from 65.3% and 56.7% to 22.6% (p=0.003) and 5.1% (p=0.0002), respectively. Inflammasome-mediated IL-1β release occurred only in association with pyroptotic cell lysis. Inflammasome priming by conditioned media of pyroptotic cells likewise increased cell death. Suppression of inflammasome activation by inhibition of caspase-1 or cathepsins B and L significantly reduced cell death in primed cells. In summary, inflammasome priming by IL-1α, C5a, or conditioned media of pyroptotic cells increases RPE cell susceptibility to photooxidative damage-mediated cell death and changes the mechanism of induced cell death from apoptosis to pyroptosis. This process may contribute to RPE degeneration in AMD and provide new targets for intervention. PMID:27240191

  14. Effect of high mobility group box 1 on the human retinal pigment epithelial cell in high-glucose condition

    PubMed Central

    Fu, Desheng; Tian, Xiaofeng

    2015-01-01

    Diabetic retinopathy (DR) remains a prevalent complication of diabetes and one of the leading causes of blindness among working-age adults. However, the detailed molecular mechanism of the development of DR was still unclear by now. HMGB1 is a non-histone DNA-binding protein and serves as a structural component to facilitate the assembly of nucleoprotein complexes in the nucleus. In the present study, we examined the serum level of HMGB1 and VEGFA in the DR patients. Besides, we also detect the association between HMGB1 and VEGFA level. In the advanced in-vitro study, we detect the protective effect of HMGB1 on the RPE cells in high glucose condition. In this study, we demonstrated that HMGB1 and VEGFA expressions were upregulated in serum samples of DR patients. Advanced analysis showed that HMGB1 and VEGFA level was positively associated. In the in-vitro study, it was found that up-regulation of HMGB1 inhibited the RPE cell viability and induce the apoptosis. Besides, HMGB1 treatment would up-regulate the expression of VEGFA in the RPE cells in high glucose condition. In conclusion, we have demonstrated that HMGB1 and VEGFA are key players in the ability to suppress cell viability and induce apoptosis. The result of this current experiments shed light into the mechanism by which HMGB1 works. Besides, we also present the data of case control study data, our results showed that HMGB1 might be used as biomarkers of DR. PMID:26770371

  15. Defined culture of human embryonic stem cells and xeno-free derivation of retinal pigmented epithelial cells on a novel, synthetic substrate.

    PubMed

    Pennington, Britney O; Clegg, Dennis O; Melkoumian, Zara K; Hikita, Sherry T

    2015-02-01

    Age-related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno-free) are preferred for clinical translation. This avoids exposing AMD patients to animal-derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II-SC, which is a novel, synthetic animal-derived component-free, RGD peptide-containing copolymer compliant with good manufacturing practices designed for xeno-free stem cell culture. Cells on Synthemax II-SC were compared with cultures grown with xenogeneic and xeno-free control substrates. This report demonstrates that Synthemax II-SC supports long-term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE-specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium-derived factor. Both hESCs and hESC-RPE maintained normal karyotypes after long-term culture on Synthemax II-SC. Furthermore, RPE generated on Synthemax II-SC are functional when seeded onto parylene-C scaffolds designed for clinical use. These experiments suggest that Synthemax II-SC is a suitable, defined substrate for hESC culture and the xeno-free derivation of RPE for cellular therapies. PMID:25593208

  16. Multiple pigmented basal cell carcinomas.

    PubMed

    Shoji, T; Lee, J; Hong, S H; Oh, C H; Kim, W K; Bhawan, J

    1998-04-01

    Basal cell carcinoma is the most common of all skin cancers and the most prevalent one among Caucasians. Rarely, these tumors are seen in other races. We report a 77-year-old Korean woman who presented with multiple darkly pigmented enlarging nodules on her scalp, face, trunk, and extremities. The patient had first noted a 6-mm pigmented lesion on her left eyebrow 10 years previously. Since then, other lesions had appeared in many locations on her body. She had been otherwise healthy and without a history of exposure to arsenic or radiation. There was no family history of skin cancer, xeroderma pigmentosum, or basal cell nevus syndrome. On physical examination, multiple darkly pigmented dome-shaped papules and nodules were present on her scalp, face, right forearm, lower abdomen, and inguinal areas. They ranged in size from 0.5 mm to 2 cm. The larger ones showed central ulceration. Multiple biopsy specimens from different sites showed pigmented basal cell carcinomas. Clinically, there was no evidence of nevus sebaceus, xeroderma pigmentosum, basal cell nevus syndrome, or immunodeficiency. Clinical workup including chest radiography, abdominal ultrasound, bone scan, and brain computerized axial tomography scan did not demonstrate primary or secondary tumors. The results of serologic and hematologic tests were also within normal limits. This is an unusual case report of multiple pigmented basal cell carcinomas in an Asian woman without any predisposing risk factors. PMID:9557792

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    SciTech Connect

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

    2014-03-28

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

  20. 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

    PubMed Central

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

    2014-01-01

    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. PMID:25136316

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

    PubMed

    Liu, Xiaobin; Ward, Keith; Xavier, Christy; Jann, Jamieson; Clark, Abbot F; Pang, Iok-Hou; Wu, Hongli

    2016-08-01

    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 200μM H2O2 for 6h. 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 100nM) 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. PMID:26773873

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

    PubMed Central

    Liu, Xiaobin; Ward, Keith; Xavier, Christy; Jann, Jamieson; Clark, Abbot F.; Pang, Iok-Hou; Wu, Hongli

    2015-01-01

    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. PMID:26773873

  3. Epithelial Cell Adhesion Molecule

    PubMed Central

    Trzpis, Monika; McLaughlin, Pamela M.J.; de Leij, Lou M.F.H.; Harmsen, Martin C.

    2007-01-01

    The epithelial cell adhesion molecule (EpCAM, CD326) is a glycoprotein of ∼40 kd that was originally identified as a marker for carcinoma, attributable to its high expression on rapidly proliferating tumors of epithelial origin. Normal epithelia express EpCAM at a variable but generally lower level than carcinoma cells. In early studies, EpCAM was proposed to be a cell-cell adhesion molecule. However, recent insights revealed a more versatile role for EpCAM that is not limited only to cell adhesion but includes diverse processes such as signaling, cell migration, proliferation, and differentiation. Cell surface expression of EpCAM may actually prevent cell-cell adhesion. Here, we provide a comprehensive review of the current knowledge on EpCAM biology in relation to other cell adhesion molecules. We discuss the implications of the newly identified functions of EpCAM in view of its prognostic relevance in carcinoma, inflammatory pathophysiology, and tissue development and regeneration as well as its role in normal epithelial homeostasis. PMID:17600130

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

    PubMed

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

    2016-04-01

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

  5. Inhibition of APE1/Ref-1 redox activity rescues human retinal pigment epithelial cells from oxidative stress and reduces choroidal neovascularization

    PubMed Central

    Li, Y.; Liu, X.; Zhou, T.; Kelley, M.R.; Edwards, P.; Gao, H.; Qiao, X.

    2014-01-01

    The effectiveness of current treatment for age related macular degeneration (AMD) by targeting one molecule is limited due to its multifactorial nature and heterogeneous pathologies. Treatment strategy to target multiple signaling pathways or pathological components in AMD pathogenesis is under investigation for better clinical outcome. Inhibition of the redox function of apurinic endonuclease 1/redox factor-1 (APE1) was found to suppress endothelial angiogenesis and promote neuronal cell recovery, thereby may serve as a potential treatment for AMD. In the current study, we for the first time have found that a specific inhibitor of APE1 redox function by a small molecule compound E3330 regulates retinal pigment epithelium (RPEs) cell response to oxidative stress. E3330 significantly blocked sub-lethal doses of oxidized low density lipoprotein (oxLDL) induced proliferation decline and senescence advancement of RPEs. At the same time, E3330 remarkably decreased the accumulation of intracellular reactive oxygen species (ROS) and down-regulated the productions of monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF), as well as attenuated the level of nuclear factor-κB (NF-κB) p65 in RPEs. A panel of stress and toxicity responsive transcription factors that were significantly upregulated by oxLDL was restored by E3330, including Nrf2/Nrf1, p53, NF-κB, HIF1, CBF/NF-Y/YY1, and MTF-1. Further, a single intravitreal injection of E3330 effectively reduced the progression of laser-induced choroidal neovascularization (CNV) in mouse eyes. These data revealed that E3330 effectively rescued RPEs from oxidative stress induced senescence and dysfunctions in multiple aspects in vitro, and attenuated laser-induced damages to RPE–Bruch׳s membrane complex in vivo. Together with its previously established anti-angiogenic and neuroprotection benefits, E3330 is implicated for potential use for AMD treatment. PMID:24624338

  6. Epithelial stem cells.

    PubMed

    Draheim, Kyle M; Lyle, Stephen

    2011-01-01

    It is likely that adult epithelial stem cells will be useful in the treatment of diseases, such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa, and alopecias. Additionally, other skin problems such as burn wounds, chronic wounds, and ulcers will benefit from stem cell-related therapies. However, there are many questions that need to be answered before this goal can be realized. The most important of these questions is what regulates the adhesion of stem cells to the niche versus migration to the site of injury. We have started to identify the mechanisms involved in this decision-making process. PMID:21618097

  7. Resveratrol inhibits epithelial-mesenchymal transition of retinal pigment epithelium and development of proliferative vitreoretinopathy

    PubMed Central

    Ishikawa, Keijiro; He, Shikun; Terasaki, Hiroto; Nazari, Hossein; Zhang, Huiming; Spee, Christine; Kannan, Ram; Hinton, David R

    2015-01-01

    Proliferative vitreoretinopathy (PVR) is a serious complication of retinal detachment and ocular trauma, and its recurrence may lead to irreversible vision loss. Epithelial to mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is a critical step in the pathogenesis of PVR, which is characterized by fibrotic membrane formation and traction retinal detachment. In this study, we investigated the potential impact of resveratrol (RESV) on EMT and the fibrotic process in cultured RPE cells and further examined the preventive effect of RESV on PVR development using a rabbit model of PVR. We found that RESV induces mesenchymal to epithelial transition (MET) and inhibits transforming growth factor-β2(TGF-β2)-induced EMT of RPE cells by deacetylating SMAD4. The effect of RESV on MET was dependent on sirtuin1 activation. RESV suppressed proliferation, migration and fibronectin synthesis induced by platelet-derived growth factor-BB or TGF-β2. In vivo, RESV inhibited the progression of experimental PVR in rabbit eyes. Histological findings showed that RESV reduced fibrotic membrane formation and decreased α-SMA expression in the epiretinal membranes. These results suggest the potential use of RESV as a therapeutic agent to prevent the development of PVR by targeting EMT of RPE. PMID:26552368

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

    PubMed

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

    2015-05-01

    Retinitis pigmentosa (RP) is a group of visual impairments characterized by progressive rod photoreceptor cell loss due to a genetic background. Pigment epithelium-derived factor (PEDF) predominantly secreted by the retinal pigmented epithelium (RPE) has been reported to protect photoreceptors in retinal degeneration models, including rd1. In addition, clinical trials are currently underway outside Japan using human mesenchymal stromal cells and human neural stem cells to protect photoreceptors in RP and dry age-related macular degeneration, respectively. Thus, this study aimed to investigate the rescue effects of induced pluripotent stem (iPS)-RPE cells in comparison with those types of cells used in clinical trials on photoreceptor degeneration in rd1 mice. Cells were injected into the subretinal space of immune-suppressed 2-week-old rd1 mice. The results demonstrated that human iPS-RPE cells significantly attenuated photoreceptor degeneration on postoperative days (PODs) 14 and 21 and survived longer up to at least 12 weeks after operation than the other two types of graft cells with less immune responses and apoptosis. The mean PEDF concentration in the intraocular fluid in RPE-transplanted eyes was more than 1 µg/ml at PODs 14 and 21, and this may have contributed to the protective effect of RPE transplantation. Our findings suggest that iPS-RPE cells serve as a competent source to delay photoreceptor degeneration through stable survival in degenerating ocular environment and by releasing neuroprotective factors such as PEDF. PMID:25728228

  9. Synergistic protective effects of escin and low‑dose glucocorticoids against vascular endothelial growth factor‑induced blood‑retinal barrier breakdown in retinal pigment epithelial and umbilical vein endothelial cells.

    PubMed

    Zhang, Fenglan; Man, Xuejing; Yu, Huajun; Liu, Limei; Li, Yuanbin

    2015-02-01

    Previous studies have shown that escin possesses glucocorticoid (GC)‑like anti‑edematous and anti‑inflammatory effects. The present study was designed to investigate whether escin exhibits synergistic protective effects against blood‑retinal barrier (BRB) breakdown when combined with GC in an in vitro monolayer BRB model, based on retinal pigment epithelial (RPE) cells and human umbilical vein endothelial cells (HUVECs). The results showed that low concentrations of escin and triamcinolone acetonide (TA) administered separately did not affect BRB trans‑endothelial (epithelium) resistance (TEER). However, when administered together, escin and TA significantly inhibited reduced BRB TEER following treatment with vascular endothelial growth factor (VEGF). Furthermore, low‑concentrations of escin and TA administered together significantly increased the expression levels of occludin and ZO‑1. This demonstrates that escin and GC have synergistic protective effects against BRB breakdown, and the molecular mechanisms may be related to the upregulation of occludin and ZO‑1 expression. The combination of escin with GC indicates a potential beneficial strategy for the treatment of breakdown of the BRB. PMID:25370688

  10. Caffeic acid phenethyl ester reduces the secretion of vascular endothelial growth factor through the inhibition of the ROS, PI3K and HIF-1α signaling pathways in human retinal pigment epithelial cells under hypoxic conditions.

    PubMed

    Paeng, Sung Hwa; Jung, Won-Kyo; Park, Won Sun; Lee, Dae-Sung; Kim, Gi-Young; Choi, Yung Hyun; Seo, Su-Kil; Jang, Won Hee; Choi, Jung Sik; Lee, Young-Min; Park, Saegwang; Choi, Il-Whan

    2015-05-01

    Choroidal neovascularization (CNV) can lead to progressive and severe visual loss. Vascular endothelial growth factor (VEGF) promotes the development of CNV. Caffeic acid phenethyl ester (CAPE), a biologically active component of the honeybee (Apis mellifera) propolis, has been demonstrated to have several interesting biological regulatory properties. The objective of this study was to determine whether treatment with CAPE results in the inhibition of the production of vascular endothelial growth factor (VEGF) in retinal pigment epithelial cells (RPE cells) under hypoxic conditions and to explore the possible underlying mechanisms. An in vitro experimental model of hypoxia was used to mimic an ischemic microenvironment for the RPE cells. Human RPE cells (ARPE-19) were exposed to hypoxia with or without CAPE pre-treatment. ARPE-19 cells were used to investigate the pathway involved in the regulation of VEGF production under hypoxic conditions, based on western blot analysis, enzyme-linked immunosorbent assay (ELISA) and electrophoretic mobility shift assay (EMSA). The amount of VEGF released from the hypoxia-exposed cells was significantly higher than that of the normoxic controls. Pre-treatment with CAPE suppressed the hypoxia-induced production of VEGF in the ARPE-19 cells, and this effect was inhibited through the attenuation of reactive oxygen species (ROS) production, and the inhibition of phosphoinositide 3-kinase (PI3K)/AKT and hypoxia-inducible factor-1α (HIF-1α) expression. These in vitro findings suggest that CAPE may prove to be a novel anti-angiogenic agent for the treatment of diseases associated with CNV. PMID:25738890

  11. Inhibitory Effect of Bone Morphogenetic Protein 4 in Retinal Pigment Epithelial-Mesenchymal Transition

    PubMed Central

    Yao, Haipei; Li, Hui; Yang, Shuai; Li, Min; Zhao, Chun; Zhang, Jingfa; Xu, Guotong; Wang, Fang

    2016-01-01

    Proliferative vitreoretinopathy (PVR), a serious vision-threatening complication of retinal detachment (RD), is characterized by the formation of contractile fibrotic membranes, in which epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a major event. Recent studies suggest an important role of bone morphogenetic protein 4 (BMP4) in the suppression of fibrosis. In this study, we aimed to investigate the role of BMP4 in the pathological process of PVR, particularly in the EMT of RPE cells. We found that BMP4 and its receptors were co-labelled with cytokeratin and α-SMA positive cells within the PVR membrane. Moreover, the mRNA and protein expression levels of BMP4 were decreased whereas BMP4 receptors ALK2, ALK3 and ALK6 were increased during TGF-β-induced EMT in primary RPE cells. Exogenous BMP4 inhibited TGF-β-induced epithelial marker down-regulation, as well as mesenchymal marker up-regulation at both the mRNA and protein levels in RPE cells. In addition, BMP4 treatment attenuated the TGF-β-induced gel contraction, cell migration and Smad2/3 phosphorylation. However, knockdown of endogenous BMP4 stimulated changes in EMT markers. Our results confirm the hypothesis that BMP4 might inhibit TGF-β-mediated EMT in RPE cells via the Smad2/3 pathway and suppress contraction. This might represent a potential treatment for PVR. PMID:27586653

  12. Inhibitory Effect of Bone Morphogenetic Protein 4 in Retinal Pigment Epithelial-Mesenchymal Transition.

    PubMed

    Yao, Haipei; Li, Hui; Yang, Shuai; Li, Min; Zhao, Chun; Zhang, Jingfa; Xu, Guotong; Wang, Fang

    2016-01-01

    Proliferative vitreoretinopathy (PVR), a serious vision-threatening complication of retinal detachment (RD), is characterized by the formation of contractile fibrotic membranes, in which epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a major event. Recent studies suggest an important role of bone morphogenetic protein 4 (BMP4) in the suppression of fibrosis. In this study, we aimed to investigate the role of BMP4 in the pathological process of PVR, particularly in the EMT of RPE cells. We found that BMP4 and its receptors were co-labelled with cytokeratin and α-SMA positive cells within the PVR membrane. Moreover, the mRNA and protein expression levels of BMP4 were decreased whereas BMP4 receptors ALK2, ALK3 and ALK6 were increased during TGF-β-induced EMT in primary RPE cells. Exogenous BMP4 inhibited TGF-β-induced epithelial marker down-regulation, as well as mesenchymal marker up-regulation at both the mRNA and protein levels in RPE cells. In addition, BMP4 treatment attenuated the TGF-β-induced gel contraction, cell migration and Smad2/3 phosphorylation. However, knockdown of endogenous BMP4 stimulated changes in EMT markers. Our results confirm the hypothesis that BMP4 might inhibit TGF-β-mediated EMT in RPE cells via the Smad2/3 pathway and suppress contraction. This might represent a potential treatment for PVR. PMID:27586653

  13. The expression and function of vascular endothelial growth factor in retinal pigment epithelial (RPE) cells is regulated by 4-hydroxynonenal (HNE) and glutathione S-transferaseA4-4

    SciTech Connect

    Vatsyayan, Rit; Lelsani, Poorna Chandra Rao; Chaudhary, Pankaj; Kumar, Sushil; Awasthi, Sanjay; Awasthi, Yogesh C.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Low concentration of HNE (0.1-1.0 {mu}M) induced secretion of VEGF in RPE cells. Black-Right-Pointing-Pointer VEGF secreted medium of RPE cells promoted proliferation of endothelial cells. Black-Right-Pointing-Pointer VEGFR2 expression was attenuated with increasing concentrations of HNE. Black-Right-Pointing-Pointer These effects of HNE could be blocked by the over expression of GSTA4-4 in cells. -- Abstract: It is well established that 4-hydroxynonenal (HNE) plays a major role in oxidative stress-induced signaling and the toxicity of oxidants. Surprisingly our recent studies also demonstrate that low levels of HNE generated during oxidative stress promote cell survival mechanisms and proliferation. Since the expression and secretion of VEGF is known to be affected by Oxidative stress, during present studies, we have examined dose dependent effect of HNE on VEGF expression and secretion in a model of retinal pigment epithelial (RPE) cells in culture. Results of these studies showed that while inclusion of 0.1 {mu}M HNE in the medium caused increased secretion of VEGF, its secretion and expression was significantly suppressed in the presence of >5 {mu}M HNE in the media. These concentration dependent hormetic effects of HNE on VEGF secretion could be blocked by the over expression of GSTA4-4 indicating that these effects were specifically attributed to HNE and regulated by GSTA4-4. VEGF secreted into the media showed angiogenic properties as indicated by increased migration and tube formation of HUVEC in matrigel when grown in media from RPE cells treated with 1 {mu}M HNE. The corresponding media from GSTA4-4 over expressing RPE cells had no effect on migration and tube formation of HUVEC in matrigel. These results are consistent with earlier studies showing that at low concentrations, HNE promotes proliferative mechanisms and suggest that HNE induces VEGF secretion from RPE cells that acts in a paracrine fashion to induce

  14. Substance P promotes the recovery of oxidative stress-damaged retinal pigmented epithelial cells by modulating Akt/GSK-3β signaling

    PubMed Central

    Baek, Sang-Min; Yu, Seung-Young; Son, Youngsook

    2016-01-01

    Purpose Senescence of the retina causes an accumulation of reactive oxygen species (ROS). Oxidative stress associated with ROS can damage RPE cells, leading to neovascularization and severe ocular disorders, including age-related macular degeneration (AMD). Thus, the early treatment of the damage caused by oxidative stress is critical for preventing the development of ocular diseases such as AMD. In this study, we examined the role of substance P (SP) in the recovery of RPE cells damaged by oxidative stress. Methods To induce oxidative stress, RPE cells were treated with H2O2 at various doses. Recovery from oxidative stress was studied following treatment with SP by analyzing cell viability, cell proliferation, cell apoptosis, and Akt/glycogen synthase kinase (GSK)-3β activation in RPE cells in vitro. Results H2O2 treatment reduced cellular viability in a dose-dependent manner. SP inhibited the reduction of cell viability due to H2O2 and caused increased cell proliferation and decreased cell apoptosis. Cell survival under oxidative stress requires the activation of Akt signaling that enables cells to resist oxidative stress-induced damage. SP treatment activated Akt/GSK-3β signaling in RPE cells, which were damaged due to oxidative stress, and the inhibition of Akt signaling in SP-treated RPE cells prevented SP-induced recovery. Pretreatment with the neurokinin 1 receptor (NK1R) antagonist reduced the recovery effect of SP on damaged RPE cells. Conclusions SP can protect RPE cells from oxidant-induced cell death by activating Akt/GSK-3β signaling via NK1R. This study suggests the possibility of SP as a treatment for oxidative stress-related diseases. PMID:27582624

  15. Exposing human retinal pigmented epithelial cells to red light in vitro elicits an adaptive response to a subsequent 2-μm laser challenge

    NASA Astrophysics Data System (ADS)

    Schuster, K. J.; Estlack, L. E.; Wigle, J. C.

    2013-03-01

    The objective of this study was to elucidate cellular mechanisms of protection against laser-induced thermal killing utilizing an in vitro retina model. When exposed to a 1-sec pulse of 2-μm laser radiation 24 hr after illuminating hTERT-RPE cells with red light (preconditioning), the cells are more resistant to thermal challenge than unilluminated controls (adaptive response). Results of efforts to understand the physiology of this effect led us to two genes: Vascular Endothelial Growth Factor C (VEGF-C) and Micro RNA 146a (miR-146a). Transfecting wild type (WT) cells with siRNA for VEGF-C and miR-146a mRNA resulted in knockdown strains (VEGF-C(KD) and miR- 146a(-)) with 10% and 30% (respectively) of the constitutive levels expressed in the WT cells. To induce gene expression, WT or KD cells were preconditioned with 1.44 to 5.40 J/cm2, using irradiances between 0.40 and 1.60 mW/cm2 of either 671-nm (diode) or 637-nm (laser) radiation. Probit analysis was used to calculate threshold damage irradiance, expressed as ED50, between 10 and 100 W/cm2 for the 2-μm laser pulse. In the WT cells there is a significant increase in ED50 (p 0.05) with the maximum response occurring at 2.88 J/cm2 in the preconditioned cells. Neither KD cell strain showed a significant increase in the ED50, although some data suggest the response may just be decreased in the knockdown cells instead of absent. So far the response does not appear to be dependent upon either wavelength (637 vs. 671 nm) or coherence (laser vs. LED), but there is an irradiance dependence.

  16. Erp29 Attenuates Cigarette Smoke Extract–Induced Endoplasmic Reticulum Stress and Mitigates Tight Junction Damage in Retinal Pigment Epithelial Cells

    PubMed Central

    Huang, Chuangxin; Wang, Joshua J.; Jing, Guangjun; Li, Junhua; Jin, Chenjin; Yu, Qiang; Falkowski, Marek W.; Zhang, Sarah X.

    2015-01-01

    Purpose Endoplasmic reticulum protein 29 (ERp29) is a novel chaperone that was recently found decreased in human retinas with AMD. Herein, we examined the effect of ERp29 on cigarette smoke–induced RPE apoptosis and tight junction disruption. Methods Cultured human RPE (HRPE) cells (ARPE-19) or mouse RPE eyecup explants were exposed to cigarette smoke extract (CSE) for short (up to 24 hours) or long (up to 3 weeks) periods. Expression of ERp29 was up- and downregulated by adenovirus and siRNA, respectively. Endoplasmic reticulum stress markers, apoptosis, and cell death, the expression and distribution of tight junction protein ZO-1, transepithelial electrical resistance (TEER), and F-actin expression were examined. Results Endoplasmic reticulum protein 29 was significantly increased by short-term exposure to CSE in ARPE-19 cells or eyecup explants but was reduced after 3-week exposure. Overexpression of ERp29 increased the levels of GRP78, p58IPK, and Nrf-2, while reducing p-eIF2α and C/EBP homologous protein (CHOP), and protected RPE cells from CSE-induced apoptosis. In contrast, knockdown of ERp29 decreased the levels of p58IPK and Nrf2, but increased p-eIF2α and CHOP and exacerbated CSE-triggered cell death. In addition, overexpression of ERp29 attenuated CSE-induced reduction in ZO-1 and enhanced the RPE barrier function, as measured by TEER. Knockdown of ERp29 decreased the level of ZO-1 protein. These effects were associated with changes in the expression of cytoskeleton F-actin. Conclusions Endoplasmic reticulum protein 29 attenuates CSE-induced ER stress and enhances cell viability and barrier integrity of RPE cells, and therefore may act as a protective mechanism for RPE survival and activity. PMID:26431474

  17. Oxidative stress-induced premature senescence dysregulates VEGF and CFH expression in retinal pigment epithelial cells: Implications for Age-related Macular Degeneration

    PubMed Central

    Marazita, Mariela C.; Dugour, Andrea; Marquioni-Ramella, Melisa D.; Figueroa, Juan M.; Suburo, Angela M.

    2015-01-01

    Oxidative stress has a critical role in the pathogenesis of Age-related Macular Degeneration (AMD), a multifactorial disease that includes age, gene variants of complement regulatory proteins and smoking as the main risk factors. Stress-induced premature cellular senescence (SIPS) is postulated to contribute to this condition. In this study, we hypothesized that oxidative damage, promoted by endogenous or exogenous sources, could elicit a senescence response in RPE cells, which would in turn dysregulate the expression of major players in AMD pathogenic mechanisms. We showed that exposure of a human RPE cell line (ARPE-19) to a cigarette smoke concentrate (CSC), not only enhanced Reactive Oxygen Species (ROS) levels, but also induced 8-Hydroxydeoxyguanosine-immunoreactive (8-OHdG) DNA lesions and phosphorylated-Histone 2AX-immunoreactive (p-H2AX) nuclear foci. CSC-nuclear damage was followed by premature senescence as shown by positive senescence associated-β-galactosidase (SA-β-Gal) staining, and p16INK4a and p21Waf-Cip1 protein upregulation. N-acetylcysteine (NAC) treatment, a ROS scavenger, decreased senescence markers, thus supporting the role of oxidative damage in CSC-induced senescence activation. ARPE-19 senescent cultures were also established by exposure to hydrogen peroxide (H2O2), which is an endogenous stress source produced in the retina under photo-oxidation conditions. Senescent cells upregulated the proinflammatory cytokines IL-6 and IL-8, the main markers of the senescence-associated secretory phenotype (SASP). Most important, we show for the first time that senescent ARPE-19 cells upregulated vascular endothelial growth factor (VEGF) and simultaneously downregulated complement factor H (CFH) expression. Since both phenomena are involved in AMD pathogenesis, our results support the hypothesis that SIPS could be a principal player in the induction and progression of AMD. Moreover, they would also explain the striking association of this disease

  18. The response of human retinal pigmented epithelial cells in vitro to changes in nitric oxide concentration stimulated by low levels of red light

    NASA Astrophysics Data System (ADS)

    Lavey, Brent J.; Estlack, Larry E.; Schuster, Kurt J.; Rockwell, Benjamin A.; Wigle, Jeffrey C.

    2013-03-01

    The goal of this project is to explore the role of nitric oxide (NO) in regulating the response of hTERT-RPE to low-level exposures to red light. Exposure to low-level red light has been shown to positively affect wound healing, reduce pain, and encourage cell proliferation. The current explanation for this effect is described as an interaction between the photons and cytochrome c oxidase (Cco), which plays a role in regulation of intracellular NO levels in addition to being the mitochondrial protein complex where reduction of oxygen occurs in the process of oxidative phosphorylation. Exposure to 2.88 J/cm2 of 671-nm and 637-nm light shows a two-fold increase in NO immediately after exposure, and a 56% increase in ATP measured at ~5 h post exposure. Levels of NF-κB mRNA and protein were measured at six and 24 h, respectively, and found to increase six fold, correlating with increases in NO levels. Light-stimulated increased levels of NO also correlated with an 11-fold increase in Bcl-2 and a 70% decrease in Bax mRNA levels, relative to controls. NF-κB promotes cell growth and Bcl-2 is an apoptosis suppressor protein. Bax is a positive apoptotic effector protein. These results support the hypothesis that light-induced changes in the intracellular levels of NO play a role in the beneficial effects of low-level light photobiomodulation

  19. Eph and Ephrin function in dispersal and epithelial insertion of pigmented immunocytes in sea urchin embryos.

    PubMed

    Krupke, Oliver A; Zysk, Ivona; Mellott, Dan O; Burke, Robert D

    2016-01-01

    The mechanisms that underlie directional cell migration are incompletely understood. Eph receptors usually guide migrations of cells by exclusion from regions expressing Ephrin. In sea urchin embryos, pigmented immunocytes are specified in vegetal epithelium, transition to mesenchyme, migrate, and re-enter ectoderm, distributing in dorsal ectoderm and ciliary band, but not ventral ectoderm. Immunocytes express Sp-Eph and Sp-Efn is expressed throughout dorsal and ciliary band ectoderm. Interfering with expression or function of Sp-Eph results in rounded immunocytes entering ectoderm but not adopting a dendritic form. Expressing Sp-Efn throughout embryos permits immunocyte insertion in ventral ectoderm. In mosaic embryos, immunocytes insert preferentially in ectoderm expressing Sp-Efn. We conclude that Sp-Eph signaling is necessary and sufficient for epithelial insertion. As well, we propose that immunocytes disperse when Sp-Eph enhances adhesion, causing haptotactic movement to regions of higher ligand abundance. This is a distinctive example of Eph/Ephrin signaling acting positively to pattern migrating cells. PMID:27474796

  20. Eph and Ephrin function in dispersal and epithelial insertion of pigmented immunocytes in sea urchin embryos

    PubMed Central

    Krupke, Oliver A; Zysk, Ivona; Mellott, Dan O; Burke, Robert D

    2016-01-01

    The mechanisms that underlie directional cell migration are incompletely understood. Eph receptors usually guide migrations of cells by exclusion from regions expressing Ephrin. In sea urchin embryos, pigmented immunocytes are specified in vegetal epithelium, transition to mesenchyme, migrate, and re-enter ectoderm, distributing in dorsal ectoderm and ciliary band, but not ventral ectoderm. Immunocytes express Sp-Eph and Sp-Efn is expressed throughout dorsal and ciliary band ectoderm. Interfering with expression or function of Sp-Eph results in rounded immunocytes entering ectoderm but not adopting a dendritic form. Expressing Sp-Efn throughout embryos permits immunocyte insertion in ventral ectoderm. In mosaic embryos, immunocytes insert preferentially in ectoderm expressing Sp-Efn. We conclude that Sp-Eph signaling is necessary and sufficient for epithelial insertion. As well, we propose that immunocytes disperse when Sp-Eph enhances adhesion, causing haptotactic movement to regions of higher ligand abundance. This is a distinctive example of Eph/Ephrin signaling acting positively to pattern migrating cells. DOI: http://dx.doi.org/10.7554/eLife.16000.001 PMID:27474796

  1. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

    ABSTRACT Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell–matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical–basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity. For further reading, please see related articles: ‘ERM proteins at a glance’ by Andrea McClatchey (J. Cell Sci. 127, 3199–3204). ‘Establishment of epithelial polarity – GEF who's minding the GAP?’ by Siu Ngok et al. (J. Cell Sci. 127, 3205–3215). PMID:24994933

  2. Localized choroidal thickness variation and pigment epithelial detachment in dome-shaped macula with subretinal fluid.

    PubMed

    Deobhakta, Avnish; Ross, Adam H; Helal, John; Maia, André; Freund, K Bailey

    2015-03-01

    The objective of this report is to demonstrate that individuals with dome-shaped macula can develop persistent subretinal fluid due to abrupt changes in the thickness of the choroid, making it unlikely to be reported. Additionally, these patients often have pigment epithelial detachments, suggestive of possible choroidal neo-vascularization. These two qualities can often lead to persistent subretinal fluid that is refractory to treatment. PMID:25835309

  3. Free-Floating Iris Pigmented Epithelial Cyst in the Anterior Chamber

    PubMed Central

    Rotsos, Tryfon; Bagikos, Georgios; Christou, Spyridon; Symeonidis, Chrysanthos; Papadaki, Thekla; Papaeuthimiou, Ioannis; Miltsakakis, Dimitrios

    2016-01-01

    An unusual case of a free-floating peripheral pigmented cyst in the anterior chamber is presented. A 30-year-old Caucasian male presented reporting a visual defect on his right eye in prone position over the past year. Slit-lamp examination revealed a small pigmented free-floating peripheral iris cyst at the 6 o'clock position in the anterior chamber. Ultrasound biomicroscopy revealed an unfixed epithelial pigmented cyst with an extremely thin wall and no internal reflectivity. Due to the lack of severity of visual disturbance of the patient, no surgical treatment was indicated. The patient is to be followed up annually and advised to return immediately in case of pain or any visual symptoms. Free-floating iris cysts in the anterior chamber are uncommon and remain stable in the majority of cases. Management includes only regular observation until any complications arise. PMID:26904334

  4. Free-Floating Iris Pigmented Epithelial Cyst in the Anterior Chamber.

    PubMed

    Rotsos, Tryfon; Bagikos, Georgios; Christou, Spyridon; Symeonidis, Chrysanthos; Papadaki, Thekla; Papaeuthimiou, Ioannis; Miltsakakis, Dimitrios

    2016-01-01

    An unusual case of a free-floating peripheral pigmented cyst in the anterior chamber is presented. A 30-year-old Caucasian male presented reporting a visual defect on his right eye in prone position over the past year. Slit-lamp examination revealed a small pigmented free-floating peripheral iris cyst at the 6 o'clock position in the anterior chamber. Ultrasound biomicroscopy revealed an unfixed epithelial pigmented cyst with an extremely thin wall and no internal reflectivity. Due to the lack of severity of visual disturbance of the patient, no surgical treatment was indicated. The patient is to be followed up annually and advised to return immediately in case of pain or any visual symptoms. Free-floating iris cysts in the anterior chamber are uncommon and remain stable in the majority of cases. Management includes only regular observation until any complications arise. PMID:26904334

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

    PubMed Central

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

    2016-01-01

    The disruption of retinal pigment epithelial (RPE) function and the degeneration of photoreceptors are cardinal features of age related macular degeneration (AMD); however there are still gaps in our understanding of underlying biological processes. Excess homocysteine (Hcy) has been reported to be elevated in plasma of patients with AMD. This study aimed to evaluate the direct effect of hyperhomocysteinemia (HHcy) on structure and function of RPE. Initial studies in a mouse model of HHcy, in which cystathionine-β-synthase (cbs) was deficient, revealed abnormal RPE cell morphology with features similar to that of AMD upon optical coherence tomography (OCT), fluorescein angiography (FA), histological, and electron microscopic examinations. These features include atrophy, vacuolization, hypopigmentation, thickened basal laminar membrane, hyporeflective lucency, choroidal neovascularization (CNV), and disturbed RPE–photoreceptor relationship. Furthermore, intravitreal injection of Hcy per se in normal wild type (WT) mice resulted in diffuse hyper-fluorescence, albumin leakage, and CNV in the area of RPE. In vitro experiments on ARPE-19 showed that Hcy dose-dependently reduced tight junction protein expression, increased FITC dextran leakage, decreased transcellular electrical resistance, and impaired phagocytic activity. Collectively, our results demonstrated unreported effects of excess Hcy levels on RPE structure and function that lead to the development of AMD-like features. PMID:26885895

  6. Ion Channels in Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Palmer, Lawrence G.

    Ion channels in epithelial cells serve to move ions, and in some cases fluid, between compartments of the body. This function of the transfer of material is fundamentally different from that of the transfer of information, which is the main job of most channels in excitable cells. Nevertheless the basic construction of the channels is similar in many respects in the two tissue types. This chapter reviews the nature of channels in epithelia and discusses how their functions have evolved to accomplish the basic tasks for which they are responsible. I will focus on three channel types: epithelial Na+ channels, inward-rectifier K+ channels, and CFTR Cl- channels.

  7. Bilateral Refractive Changes in Vascularized Pigment Epithelial Detachment Treated by Anti-VEGF Therapy.

    PubMed

    Hanhart, Joel; Chowers, Itay

    2015-01-01

    We report the case of a patient bilaterally treated with anti-VEGF compounds for bilateral massive vascularized retinal pigment epithelial detachment (PED). During the years prior to treatment, PED growth was accompanied by gradual hypermetropization. After right intraocular injection of bevacizumab followed by three bilateral aflibercept injections, the PED flattened resulting in a rapid relative myopization. This case illustrates ocular refractive properties associated with PED and its response to treatment. This case also highlights the importance of assessing refraction in age-related macular degeneration patients experiencing substantial PED amplitude changes. PMID:26955349

  8. Epithelial Sodium Channels in Pulmonary Epithelial Progenitor and Stem Cells

    PubMed Central

    Liu, Yang; Jiang, Bi-Jie; Zhao, Run-Zhen; Ji, Hong-Long

    2016-01-01

    Regeneration of the epithelium of mammalian lungs is essential for restoring normal function following injury, and various cells and mechanisms contribute to this regeneration and repair. Club cells, bronchioalveolar stem cells (BASCs), and alveolar type II epithelial cells (ATII) are dominant stem/progenitor cells for maintaining epithelial turnover and repair. Epithelial Na+ channels (ENaC), a critical pathway for transapical salt and fluid transport, are expressed in lung epithelial progenitors, including club and ATII cells. Since ENaC activity and expression are development- and differentiation-dependent, apically located ENaC activity has therefore been used as a functional biomarker of lung injury repair. ENaC activity may be involved in the migration and differentiation of local and circulating stem/progenitor cells with diverse functions, eventually benefiting stem cells spreading to re-epithelialize injured lungs. This review summarizes the potential roles of ENaC expressed in native progenitor and stem cells in the development and regeneration of the respiratory epithelium. PMID:27570489

  9. Epithelial cells and Von Gierke's disease.

    PubMed

    Negishi, H; Benke, P J

    1977-08-01

    Epithelial cells and not fibroblasts from human liver and amniotic fluid contain inducible glucose-6-phosphatase (G-6-Pase) activity. The diagnosis of Von Gierke's disease has been made in a patient with hepatomegaly utilizing cultured epithelial cells grown from a liver biopsy. G-6-Pase activity in epithelial cells from this patient could not be induced by dibutyryl cyclic AMP and theophylline. This is the first use of epithelial cells for diagnosis of a metabolic disease. G-6-Pase activity in cloned epithelial cells from amniotic fluid increases 2- to 3-fold after 24-hr exposure to dibutyryl cyclic AMP and theophylline. The prenatal diagnosis of Von Gierke's disease may be possible in a laboratory experienced with these techniques if epithelial cell growth is obtained from amniotic fluid. PMID:196249

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

    PubMed Central

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

    2016-01-01

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

  11. In Vivo Imaging of the Human Retinal Pigment Epithelial Mosaic Using Adaptive Optics Enhanced Indocyanine Green Ophthalmoscopy

    PubMed Central

    Tam, Johnny; Liu, Jianfei; Dubra, Alfredo; Fariss, Robert

    2016-01-01

    Purpose The purpose of this study was to establish that retinal pigment epithelial (RPE) cells take up indocyanine green (ICG) dye following systemic injection and that adaptive optics enhanced indocyanine green ophthalmoscopy (AO-ICG) enables direct visualization of the RPE mosaic in the living human eye. Methods A customized adaptive optics scanning light ophthalmoscope (AOSLO) was used to acquire high-resolution retinal fluorescence images of residual ICG dye in human subjects after intravenous injection at the standard clinical dose. Simultaneously, multimodal AOSLO images were also acquired, which included confocal reflectance, nonconfocal split detection, and darkfield. Imaging was performed in 6 eyes of three healthy subjects with no history of ocular or systemic diseases. In addition, histologic studies in mice were carried out. Results The AO-ICG channel successfully resolved individual RPE cells in human subjects at various time points, including 20 minutes and 2 hours after dye administration. Adaptive optics-ICG images of RPE revealed detail which could be correlated with AO dark-field images of the same cells. Interestingly, there was a marked heterogeneity in the fluorescence of individual RPE cells. Confirmatory histologic studies in mice corroborated the specific uptake of ICG by the RPE layer at a late time point after systemic ICG injection. Conclusions Adaptive optics-enhanced imaging of ICG dye provides a novel way to visualize and assess the RPE mosaic in the living human eye alongside images of the overlying photoreceptors and other cells. PMID:27564519

  12. Origins of adult pigmentation: diversity in pigment stem cell lineages and implications for pattern evolution

    PubMed Central

    Spiewak, Jessica E.

    2014-01-01

    Summary Teleosts comprise about half of all vertebrate species and exhibit an extraordinary diversity of adult pigment patterns that function in shoaling, camouflage and mate choice and have played important roles in speciation. Here, we review recent studies that have identified several distinct neural crest lineages, with distinct genetic requirements, that give rise to adult pigment cells in fishes. These lineages include post-embryonic, peripheral nerve associated stem cells that generate black melanophores and iridescent iridophores, cells derived directly from embryonic neural crest cells that generate yellow-orange xanthophores, and bipotent stem cells that generate both melanophores and xanthophores. This complexity in adult chromatophore lineages has implications for our understanding of adult traits, melanoma, and the evolutionary diversification of pigment cell lineages and patterns. PMID:25421288

  13. Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase-MTORC1 signaling

    PubMed Central

    Valapala, Mallika; Wilson, Christine; Hose, Stacey; Bhutto, Imran A; Grebe, Rhonda; Dong, Aling; Greenbaum, Seth; Gu, Limin; Sengupta, Samhita; Cano, Marisol; Hackett, Sean; Xu, Guotong; Lutty, Gerard A; Dong, Lijin; Sergeev, Yuri; Handa, James T; Campochiaro, Peter; Wawrousek, Eric; Zigler, Jr, J Samuel; Sinha, Debasish

    2014-01-01

    In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V0-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the βA3- and βA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis. PMID:24468901

  14. Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase-MTORC1 signaling.

    PubMed

    Valapala, Mallika; Wilson, Christine; Hose, Stacey; Bhutto, Imran A; Grebe, Rhonda; Dong, Aling; Greenbaum, Seth; Gu, Limin; Sengupta, Samhita; Cano, Marisol; Hackett, Sean; Xu, Guotong; Lutty, Gerard A; Dong, Lijin; Sergeev, Yuri; Handa, James T; Campochiaro, Peter; Wawrousek, Eric; Zigler, J Samuel; Sinha, Debasish

    2014-03-01

    In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V0-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the βA3- and βA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis. PMID:24468901

  15. Internal pigment cells respond to external UV radiation in frogs.

    PubMed

    Franco-Belussi, Lilian; Nilsson Sköld, Helen; de Oliveira, Classius

    2016-05-01

    Fish and amphibians have pigment cells that generate colorful skins important for signaling, camouflage, thermoregulation and protection against ultraviolet radiation (UVR). However, many animals also have pigment cells inside their bodies, on their internal organs and membranes. In contrast to external pigmentation, internal pigmentation is remarkably little studied and its function is not well known. Here, we tested genotoxic effects of UVR and its effects on internal pigmentation in a neotropical frog, Physalaemus nattereri We found increases in body darkness and internal melanin pigmentation in testes and heart surfaces and in the mesenterium and lumbar region after just a few hours of UVR exposure. The melanin dispersion in melanomacrophages in the liver and melanocytes in testes increased after UV exposure. In addition, the amount of melanin inside melanomacrophages cells also increased. Although mast cells were quickly activated by UVR, only longer UVR exposure resulted in genotoxic effects inside frogs, by increasing the frequency of micronuclei in red blood cells. This is the first study to describe systemic responses of external UVR on internal melanin pigmentation, melanomacrophages and melanocytes in frogs and thus provides a functional explanation to the presence of internal pigmentation. PMID:26944494

  16. Epithelial organization, cell polarity and tumorigenesis.

    PubMed

    McCaffrey, Luke Martin; Macara, Ian G

    2011-12-01

    Epithelial cells comprise the foundation for the majority of organs in the mammalian body, and are the source of approximately 90% of all human cancers. Characteristically, epithelial cells form intercellular adhesions, exhibit apical/basal polarity, and orient their mitotic spindles in the plane of the epithelial sheet. Defects in these attributes result in the tissue disorganization associated with cancer. Epithelia undergo self-renewal from stem cells, which might in some cases be the cell of origin for cancers. The PAR polarity proteins are master regulators of epithelial organization, and are closely linked to signaling pathways such as Hippo, which orchestrate proliferation and apoptosis to control organ size. 3D ex vivo culture systems can now faithfully recapitulate epithelial organ morphogenesis, providing a powerful approach to study both normal development and the initiating events in carcinogenesis. PMID:21782440

  17. Epithelial TRPV1 signaling accelerates gingival epithelial cell proliferation.

    PubMed

    Takahashi, N; Matsuda, Y; Yamada, H; Tabeta, K; Nakajima, T; Murakami, S; Yamazaki, K

    2014-11-01

    Transient receptor potential cation channel subfamily V member 1 (TRPV1), a member of the calcium-permeable thermosensitive transient receptor potential superfamily, is a sensor of thermal and chemical stimuli. TRPV1 is activated by noxious heat (> 43°C), acidic conditions (pH < 6.6), capsaicin, and endovanilloids. This pain receptor was discovered on nociceptive fibers in the peripheral nervous system. TRPV1 was recently found to be expressed by non-neuronal cells, such as epithelial cells. The oral gingival epithelium is exposed to multiple noxious stimuli, including heat and acids derived from endogenous and exogenous substances; however, whether gingival epithelial cells (GECs) express TRPV1 is unknown. We show that both TRPV1 mRNA and protein are expressed by GECs. Capsaicin, a TRPV1 agonist, elevated intracellular Ca(2+) levels in the gingival epithelial cell line, epi 4. Moreover, TRPV1 activation in epi 4 cells accelerated proliferation. These responses to capsaicin were inhibited by a specific TRPV1 antagonist, SB-366791. We also observed GEC proliferation in capsaicin-treated mice in vivo. No effects were observed on GEC apoptosis by epithelial TRPV1 signaling. To examine the molecular mechanisms underlying this proliferative effect, we performed complementary (c)DNA microarray analysis of capsaicin-stimulated epi 4 cells. Compared with control conditions, 227 genes were up-regulated and 232 genes were down-regulated following capsaicin stimulation. Several proliferation-related genes were validated by independent experiments. Among them, fibroblast growth factor-17 and neuregulin 2 were significantly up-regulated in capsaicin-treated epi 4 cells. Our results suggest that functional TRPV1 is expressed by GECs and contributes to the regulation of cell proliferation. PMID:25266715

  18. Symmetry breaking mechanism for epithelial cell polarization

    NASA Astrophysics Data System (ADS)

    Veglio, A.; Gamba, A.; Nicodemi, M.; Bussolino, F.; Serini, G.

    2009-09-01

    In multicellular organisms, epithelial cells form layers separating compartments responsible for different physiological functions. At the early stage of epithelial layer formation, each cell of an aggregate defines an inner and an outer side by breaking the symmetry of its initial state, in a process known as epithelial polarization. By integrating recent biochemical and biophysical data with stochastic simulations of the relevant reaction-diffusion system, we provide evidence that epithelial cell polarization is a chemical phase-separation process induced by a local bistability in the signaling network at the level of the cell membrane. The early symmetry breaking event triggering phase separation is induced by adhesion-dependent mechanical forces localized in the point of convergence of cell surfaces when a threshold number of confluent cells is reached. The generality of the emerging phase-separation scenario is likely common to many processes of cell polarity formation.

  19. Airway epithelial cell responses to ozone injury

    SciTech Connect

    Leikauf, G.D.; Simpson, L.G.; Zhao, Qiyu

    1995-03-01

    The airway epithelial cell is an important target in ozone injury. Once activated, the airway epithelium responds in three phases. The initial, or immediate phase, involves activation of constitutive cells, often through direct covalent interactions including the formation of secondary ozonolysis products-hydroxyhydroperoxides, aldehydes, and hydrogen peroxide. Recently, we found hydroxyhydroperoxides to be potent agonists; of bioactive eicosanoid formation by human airway epithelial cells in culture. Other probable immediate events include activation and inactivation of enzymes present on the epithelial surface (e.g., neutral endopeptidase). During the next 2 to 24 hr, or early phase, epithelial cells respond by synthesis and release of chemotactic factors, including chemokines-macrophage inflammatory protein-2, RANTES, and interleukin-8. Infiltrating leukocytes during this period also release elastase, an important agonist of epithelial cell mucus secretion and additional chemokine formation. The third (late) phase of ozone injury is characterized by eosinophil or monocyte infiltration. Cytokine expression leads to alteration of structural protein synthesis, with increases in fibronectin evident by in situ hybridization. Synthesis of epithelial antiproteases, e.g., secretary leukocyte protease inhibitor, may also increase locally 24 to 48 hr after elastase concentrations become excessive. Thus, the epithelium is not merely a passive barrier to ozone injury but has a dynamic role in directing the migration, activating, and then counteracting inflammatory cells. Through these complex interactions, epithelial cells can be viewed as the initiators (alpha) and the receptors (omega) of ozone-induced airway disease. 51 refs., 2 figs., 3 tabs.

  20. Tetraspanin 3c requirement for pigment cell interactions and boundary formation in zebrafish adult pigment stripes

    PubMed Central

    Inoue, Shinya; Kondo, Shigeru; Parichy, David M.; Watanabe, Masakatsu

    2014-01-01

    Summary Skin pigment pattern formation in zebrafish requires pigment-cell autonomous interactions between melanophores and xanthophores, yet the molecular bases for these interactions remain largely unknown. Here, we examined the dali mutant that exhibits stripes in which melanophores are intermingled abnormally with xanthophores. By in vitro cell culture, we found that melanophores of dali mutants have a defect in motility and that interactions between melanophores and xanthophores are defective as well. Positional cloning and rescue identified dali as tetraspanin 3c (tspan3c), encoding a transmembrane scaffolding protein expressed by melanophores and xanthophores. We further showed that dali mutant Tspan3c expressed in HeLa cell exhibits a defect in N-glycosylation and is retained inappropriately in the endoplasmic reticulum. Our results are the first to identify roles for a tetraspanin superfamily protein in skin pigment pattern formation and suggest new mechanisms for the establishment and maintenance of zebrafish stripe boundaries. PMID:24734316

  1. Cell Division Drives Epithelial Cell Rearrangements during Gastrulation in Chick.

    PubMed

    Firmino, Joao; Rocancourt, Didier; Saadaoui, Mehdi; Moreau, Chloe; Gros, Jerome

    2016-02-01

    During early embryonic development, cells are organized as cohesive epithelial sheets that are continuously growing and remodeled without losing their integrity, giving rise to a wide array of tissue shapes. Here, using live imaging in chick embryo, we investigate how epithelial cells rearrange during gastrulation. We find that cell division is a major rearrangement driver that powers dramatic epithelial cell intercalation events. We show that these cell division-mediated intercalations, which represent the majority of epithelial rearrangements within the early embryo, are absolutely necessary for the spatial patterning of gastrulation movements. Furthermore, we demonstrate that these intercalation events result from overall low cortical actomyosin accumulation within the epithelial cells of the embryo, which enables dividing cells to remodel junctions in their vicinity. These findings uncover a role for cell division as coordinator of epithelial growth and remodeling that might underlie various developmental, homeostatic, or pathological processes in amniotes. PMID:26859350

  2. High Dose Intravitreal Bevacizumab for Refractory Pigment Epithelial Detachment in Age-related Macular Degeneration

    PubMed Central

    Lee, Dong Kyu; Kim, Soon Hyun; You, Yong Sung

    2016-01-01

    Purpose Intravitreal anti-vascular endothelial growth factor (anti-VEGF) is the first choice of treatment for age-related macular degeneration. However, quite a few eyes treated using conventional dose anti-VEGF (CDAV) have persistent pigment epithelial detachment (PED) on optical coherence tomography. This study investigated the efficacy and safety of high dose anti-VEGF (HDAV) for refractory PED. Methods In this retrospective study, 31 eyes of neovascular age-related macular degeneration patients with persistent PED findings despite six or more intravitreal injections of CDAV (bevacizumab 1.25 mg or ranibizumab 2.5 mg) were analyzed. Changes in visual outcome, central foveal thickness, and PED height were compared before and after HDAV (bevacizumab 5.0 mg) for these refractory PED cases. Results The mean age of patients was 67.7 years. The number of CDAV injections was 12.1. The number of HDAV injections was 3.39. Best-corrected visual acuity in logarithm of the minimum angle of resolution before and after HDAV was 0.49 and 0.41 (p < 0.001), respectively. Central foveal thickness before and after HDAV was 330.06 and 311.10 µm (p = 0.125), respectively. PED height before and after HDAV was 230.28 and 204.07 µm (p = 0.014), respectively. There were no serious adverse reactions in all the eyes. Conclusions Increasing the dose of bevacizumab in refractory PED may be a possible treatment option. PMID:27478353

  3. Retinal pigment epithelial detachments and tears, and progressive retinal degeneration in light chain deposition disease

    PubMed Central

    Spielberg, Leigh H; Heckenlively, John R; Leys, Anita M

    2013-01-01

    Background/purpose Light-chain deposition disease (LCDD) is a rare condition characterised by deposition of monoclonal immunoglobulin light chains (LCs) in tissues, resulting in varying degrees of organ dysfunction. This study reports the characteristic clinical ocular findings seen in advanced LCDD upon development of ocular fundus changes. This is the first report to describe this entity in vivo in a series of patients. Methods A case series of ocular fundus changes in three patients with kidney biopsy-proven LCDD. All patients underwent best corrected visual acuity (BCVA) exam, perimetry, colour fundus photography and fluorescein angiography; two patients underwent indocyanine green angiography, optical coherence tomography, ultrasound and electroretinography; and one patient underwent fundus autofluorescence. Results Three patients, 53–60 years old at initial presentation, were studied. All three presented with night blindness, poor dark adaptation, metamorphopsia and visual loss. Examination revealed serous and serohaemorrhagic detachments, multiple retinal pigment epithelial (RPE) tears, diffuse RPE degeneration and progressive fibrotic changes. Neither choroidal neovascularisation nor other vascular abnormalities were present. Final best corrected visual acuity (BCVA) ranged from 20/40 to 20/300. Conclusions Progressive LC deposition in the fundus seems to damage RPE pump function with flow disturbance between choroid and retina. This pathogenesis can explain the evolution to RPE detachments and subsequent rips and progressive retinal malfunction. PMID:23385633

  4. High Concentration of Zinc in Sub-retinal Pigment Epithelial Deposits

    SciTech Connect

    Lengyel,I.; Flinn, J.; Peto, T.; Linkous, D.; Cano, K.; Bird, A.; Lanzirotti, A.; Frederickson, C.; van Kuijk, F.

    2007-01-01

    One of the hallmarks of age-related macular degeneration (AMD), the leading cause of blindness in the elderly in Western societies, is the accumulation of sub-retinal pigment epithelial deposits (sub-RPE deposits), including drusen and basal laminar deposits, in Bruch's membrane (BM). The nature and the underlying mechanisms of this deposit formation are not fully understood. Because we know that zinc contributes to deposit formation in neurodegenerative diseases, we tested the hypothesis that zinc might be involved in deposit formation in AMD. Using zinc specific fluorescent probes and microprobe synchrotron X-ray fluorescence we showed that sub-RPE deposits in post-mortem human tissues contain unexpectedly high concentrations of zinc, including abundant bio-available (ionic and/or loosely protein bound) ions. Zinc accumulation was especially high in the maculae of eyes with AMD. Internal deposit structures are especially enriched in bio-available zinc. Based on the evidence provided here we suggest that zinc plays a role in sub-RPE deposit formation in the aging human eye and possibly also in the development and/or progression of AMD.

  5. Nucleus Morphometry in Cultured Epithelial Cells Correlates with Phenotype.

    PubMed

    Khan, Ayyad Z; Utheim, Tor P; Jackson, Catherine J; Reppe, Sjur; Lyberg, Torstein; Eidet, Jon R

    2016-06-01

    Phenotype of cultured ocular epithelial transplants has been shown to affect clinical success rates following transplantation to the cornea. The purpose of this study was to evaluate the relationship between cell nucleus morphometry and phenotype in three types of cultured epithelial cells. This study provides knowledge for the development of a non-invasive method of determining the phenotype of cultured epithelium before transplantation. Cultured human conjunctival epithelial cells (HCjE), human epidermal keratinocytes (HEK), and human retinal pigment epithelial cells (HRPE) were analyzed by quantitative immunofluorescence. Assessments of nucleus morphometry and nucleus-to-cytoplasm ratio (N/C ratio) were performed using ImageJ. Spearman's correlation coefficient was employed for statistical analysis. Levels of the proliferation marker PCNA in HCjE, HEK, and HRPE correlated positively with nuclear area. Nuclear area correlated significantly with levels of the undifferentiated cell marker ABCG2 in HCjE. Bmi1 levels, but not p63α levels, correlated significantly with nuclear area in HEK. The N/C ratio did not correlate significantly with any of the immunomarkers in HCjE (ABCG2, CK7, and PCNA) and HRPE (PCNA). In HEK, however, the N/C ratio was negatively correlated with levels of the undifferentiated cell marker CK14 and positively correlated with Bmi1 expression. The size of the nuclear area correlated positively with proliferation markers in all three epithelia. Morphometric indicators of phenotype in cultured epithelia can be identified using ImageJ. Conversely, the N/C ratio did not show a uniform relationship with phenotype in HCjE, HEK, or HRPE. N/C ratio therefore, may not be a useful morphometric marker for in vitro assessment of phenotype in these three epithelia. PMID:27329312

  6. Odontogenic epithelial stem cells: hidden sources.

    PubMed

    Padma Priya, Sivan; Higuchi, Akon; Abu Fanas, Salem; Pooi Ling, Mok; Kumari Neela, Vasantha; Sunil, P M; Saraswathi, T R; Murugan, Kadarkarai; Alarfaj, Abdullah A; Munusamy, Murugan A; Kumar, Suresh

    2015-12-01

    The ultimate goal of dental stem cell research is to construct a bioengineered tooth. Tooth formation occurs based on the well-organized reciprocal interaction of epithelial and mesenchymal cells. The dental mesenchymal stem cells are the best explored, but because the human odontogenic epithelium is lost after the completion of enamel formation, studies on these cells are scarce. The successful creation of a bioengineered tooth is achievable only when the odontogenic epithelium is reconstructed to produce a replica of natural enamel. This article discusses the untapped sources of odontogenic epithelial stem cells in humans, such as those present in the active dental lamina in postnatal life, in remnants of dental lamina (the gubernaculum cord), in the epithelial cell rests of Malassez, and in reduced enamel epithelium. The possible uses of these stem cells in regenerative medicine, not just for enamel formation, are discussed. PMID:26367485

  7. Dermoscopic features of small size pigmented basal cell carcinomas.

    PubMed

    Takahashi, Asuka; Hara, Hiroyuki; Aikawa, Miwa; Ochiai, Toyoko

    2016-05-01

    Dermoscopic images of histologically proven pigmented basal cell carcinomas (BCC) were retrospectively assessed to compare the dermoscopic features of BCC of 3 mm or less in diameter (n = 6) with BCC of 4-6 mm in diameter (n = 11). All lesions lacked the presence of a pigment network. BCC with a diameter of 3 mm or less had fewer positive dermoscopic features compared with the 4-6 mm in diameter BCC. Multiple blue-gray globules and large blue-gray ovoid nests were frequently present. Dermoscopy is a useful tool for early diagnosis of pigmented BCC, even when they are small. PMID:26458728

  8. Endothelial Cells Promote Pigmentation through Endothelin Receptor B Activation.

    PubMed

    Regazzetti, Claire; De Donatis, Gian Marco; Ghorbel, Houda Hammami; Cardot-Leccia, Nathalie; Ambrosetti, Damien; Bahadoran, Philippe; Chignon-Sicard, Bérengère; Lacour, Jean-Philippe; Ballotti, Robert; Mahns, Andre; Passeron, Thierry

    2015-12-01

    Findings of increased vascularization in melasma lesions and hyperpigmentation in acquired bilateral telangiectatic macules suggested a link between pigmentation and vascularization. Using high-magnification digital epiluminescence dermatoscopy, laser confocal microscopy, and histological examination, we showed that benign vascular lesions of the skin have restricted but significant hyperpigmentation compared with the surrounding skin. We then studied the role of microvascular endothelial cells in regulating skin pigmentation using an in vitro co-culture model using endothelial cells and melanocytes. These experiments showed that endothelin 1 released by microvascular endothelial cells induces increased melanogenesis signaling, characterized by microphthalmia-associated transcription factor phosphorylation, and increased tyrosinase and dopachrome tautomerase levels. Immunostaining for endothelin 1 in vascular lesions confirmed the increased expression on the basal layer of the epidermis above small vessels compared with perilesional skin. Endothelin acts through the activation of endothelin receptor B and the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK)1/2, and p38, to induce melanogenesis. Finally, culturing of reconstructed skin with microvascular endothelial cells led to increased skin pigmentation that could be prevented by inhibiting EDNRB. Taken together these results demonstrated the role of underlying microvascularization in skin pigmentation, a finding that could open new fields of research for regulating physiological pigmentation and for treating pigmentation disorders such as melasma. PMID:26308584

  9. Immunohistochemical characterisation of epithelial cells of rodent harderian glands in primary culture

    PubMed Central

    DJERIDANE, YASMINA; SIMONNEAUX, VALERIE; KLOSEN, PAUL; VIVIEN-ROELS, BERTHE; PEVET, PAUL

    1999-01-01

    The aims of the current investigation were (1) to establish an efficient procedure for the isolation of rodent harderian gland cells and to define conditions for maintenance of viable differentiated cells; (2) to compare the in vitro growth pattern of cultured epithelial cells; and (3) to characterise the cultured epithelial cells from 3 rodent species: Wistar rats, Syrian hamsters and Djungarian hamsters. We have established primary culture conditions that permit the maintenance of viable and differentiated secretory cells from adult rodent harderian gland. This study demonstrates that the cell growth pattern is faster in hamsters than in rats and despite morphological changes, epithelial cells reestablish their distinctive (biochemical/metabolic) phenotype as indicated by lipid-containing vacuoles, porphyrin pigment and serotonin and tryptophan hydroxylase labelling. PMID:10634691

  10. Energy metabolism in intestinal epithelial cells during maturation along the crypt-villus axis.

    PubMed

    Yang, Huansheng; Wang, Xiaocheng; Xiong, Xia; Yin, Yulong

    2016-01-01

    Intestinal epithelial cells continuously migrate and mature along crypt-villus axis (CVA), while the changes in energy metabolism during maturation are unclear in neonates. The present study was conducted to test the hypothesis that the energy metabolism in intestinal epithelial cells would be changed during maturation along CVA in neonates. Eight 21-day-old suckling piglets were used. Intestinal epithelial cells were isolated sequentially along CVA, and proteomics was used to analyze the changes in proteins expression in epithelial cells along CVA. The identified differentially expressed proteins were mainly involved in cellular process, metabolic process, biological regulation, pigmentation, multicellular organizational process and so on. The energy metabolism in intestinal epithelial cells of piglets was increased from the bottom of crypt to the top of villi. Moreover, the expression of proteins related to the metabolism of glucose, most of amino acids, and fatty acids was increased in intestinal epithelial cells during maturation along CVA, while the expression of proteins related to glutamine metabolism was decreased from crypt to villus tip. The expression of proteins involved in citrate cycle was also increased intestinal epithelial cells during maturation along CVA. Moreover, dietary supplementation with different energy sources had different effects on intestinal structure of weaned piglets. PMID:27558220

  11. Energy metabolism in intestinal epithelial cells during maturation along the crypt-villus axis

    PubMed Central

    Yang, Huansheng; Wang, Xiaocheng; Xiong, Xia; Yin, Yulong

    2016-01-01

    Intestinal epithelial cells continuously migrate and mature along crypt-villus axis (CVA), while the changes in energy metabolism during maturation are unclear in neonates. The present study was conducted to test the hypothesis that the energy metabolism in intestinal epithelial cells would be changed during maturation along CVA in neonates. Eight 21-day-old suckling piglets were used. Intestinal epithelial cells were isolated sequentially along CVA, and proteomics was used to analyze the changes in proteins expression in epithelial cells along CVA. The identified differentially expressed proteins were mainly involved in cellular process, metabolic process, biological regulation, pigmentation, multicellular organizational process and so on. The energy metabolism in intestinal epithelial cells of piglets was increased from the bottom of crypt to the top of villi. Moreover, the expression of proteins related to the metabolism of glucose, most of amino acids, and fatty acids was increased in intestinal epithelial cells during maturation along CVA, while the expression of proteins related to glutamine metabolism was decreased from crypt to villus tip. The expression of proteins involved in citrate cycle was also increased intestinal epithelial cells during maturation along CVA. Moreover, dietary supplementation with different energy sources had different effects on intestinal structure of weaned piglets. PMID:27558220

  12. Induced pluripotency of human prostatic epithelial cells.

    PubMed

    Zhao, Hongjuan; Sun, Ning; Young, Sarah R; Nolley, Rosalie; Santos, Jennifer; Wu, Joseph C; Peehl, Donna M

    2013-01-01

    Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that

  13. Progression of Retinal Pigment Epithelial Atrophy in Antiangiogenic Therapy of Neovascular Age-Related Macular Degeneration

    PubMed Central

    Schütze, Christopher; Wedl, Manuela; Baumann, Bernhard; Pircher, Michael; Hitzenberger, Christoph K.; Schmidt-Erfurth, Ursula

    2015-01-01

    Purpose To monitor retinal pigment epithelial (RPE) atrophy progression during antiangiogenic therapy of neovascular age-related macular degeneration (AMD) over 2 years using polarization-sensitive optical coherence tomography (OCT). Design Prospective interventional case series. Methods setting: Clinical practice. study population: Thirty patients (31 eyes) with treatment-naïve neovascular AMD. observation procedures: Standard intravitreal therapy (0.5 mg ranibizumab) was administered monthly during the first year and pro re nata (PRN; as-needed) during the second year. Spectral-domain (SD) OCT and polarization-sensitive OCT (selectively imaging the RPE) examinations were performed at baseline and at 1, 3, 6, 12, and 24 months using a standardized protocol. RPE-related changes were evaluated using a semi-automated polarization-sensitive OCT segmentation algorithm and correlated with SD OCT and fundus autofluorescence (FAF) findings. main outcome measures: RPE response, geographic atrophy (GA) progression. Results Atrophic RPE changes included RPE thinning, RPE porosity, focal RPE atrophy, and development of GA. Early RPE loss (ie, RPE porosity, focal atrophy) increased progressively during initial monthly treatment and remained stable during subsequent PRN-based therapy. GA developed in 61% of eyes at month 24. Mean GA area increased from 0.77 mm2 at 12 months to 1.10 mm2 (standard deviation = 1.09 mm2) at 24 months. Reactive accumulation of RPE-related material at the lesion borders increased until month 3 and subsequently decreased. Conclusions Progressive RPE atrophy and GA developed in the majority of eyes. RPE migration signifies certain RPE plasticity. Polarization-sensitive OCT specifically images RPE-related changes in neovascular AMD, contrary to conventional imaging methods. Polarization-sensitive OCT allows for precisely monitoring the sequence of RPE-related morphologic changes. PMID:25769245

  14. Epithelial Cell Shedding and Barrier Function

    PubMed Central

    Williams, J. M.; Duckworth, C. A.; Burkitt, M. D.; Watson, A. J. M.; Campbell, B. J.

    2015-01-01

    The intestinal epithelium is a critical component of the gut barrier. Composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, this delicate structure prevents the transfer of harmful microorganisms, antigens, and toxins from the gut lumen into the circulation. The equilibrium between the rate of apoptosis and shedding of senescent epithelial cells at the villus tip, and the generation of new cells in the crypt, is key to maintaining tissue homeostasis. However, in both localized and systemic inflammation, this balance may be disturbed as a result of pathological IEC shedding. Shedding of IECs from the epithelial monolayer may cause transient gaps or microerosions in the epithelial barrier, resulting in increased intestinal permeability. Although pathological IEC shedding has been observed in mouse models of inflammation and human intestinal conditions such as inflammatory bowel disease, understanding of the underlying mechanisms remains limited. This process may also be an important contributor to systemic and intestinal inflammatory diseases and gut barrier dysfunction in domestic animal species. This review aims to summarize current knowledge about intestinal epithelial cell shedding, its significance in gut barrier dysfunction and host-microbial interactions, and where research in this field is directed. PMID:25428410

  15. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    EPA Science Inventory

    The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

  16. Epithelial Cell Regulation of Allergic Diseases.

    PubMed

    Gour, Naina; Lajoie, Stephane

    2016-09-01

    Allergic diseases, which have escalated in prevalence in recent years, arise as a result of maladaptive immune responses to ubiquitous environmental stimuli. Why only certain individuals mount inappropriate type 2 immune responses to these otherwise harmless allergens has remained an unanswered question. Mounting evidence suggests that the epithelium, by sensing its environment, is the central regulator of allergic diseases. Once considered to be a passive barrier to allergens, epithelial cells at mucosal surfaces are now considered to be the cornerstone of the allergic diathesis. Beyond their function as maintaining barrier at mucosal surfaces, mucosal epithelial cells through the secretion of mediators like IL-25, IL-33, and TSLP control the fate of downstream allergic immune responses. In this review, we will discuss the advances in recent years regarding the process of allergen recognition and secretion of soluble mediators by epithelial cells that shape the development of the allergic response. PMID:27534656

  17. Mechanisms of protein delivery to melanosomes in pigment cells

    PubMed Central

    Sitaram, Anand; Marks, Michael S.

    2012-01-01

    SUMMARY Vertebrate pigment cells in the eye and skin are useful models for cell types that use specialized endosomal trafficking pathways to partition cargo proteins to unique lysosome-related organelles such as melanosomes. This review describes current models of protein trafficking required for melanosome biogenesis in mammalian melanocytes. PMID:22505665

  18. Hydroxyl PAMAM dendrimer-based gene vectors for transgene delivery to human retinal pigment epithelial cells†

    PubMed Central

    Mastorakos, Panagiotis; Kambhampati, Siva P.; Mishra, Manoj K.; Wu, Tony; Song, Eric; Hanes, Justin

    2016-01-01

    Ocular gene therapy holds promise for the treatment of numerous blinding disorders. Despite the significant progress in the field of viral and non-viral gene delivery to the eye, significant obstacles remain in the way of achieving high-level transgene expression without adverse effects. The retinal pigment epithelium (RPE) is involved in the pathogenesis of retinal diseases and is a key target for a number of gene-based therapeutics. In this study, we addressed the inherent drawbacks of non-viral gene vectors and combined different approaches to design an efficient and safe dendrimer-based gene-delivery platform for delivery to human RPE cells. We used hydroxyl-terminated polyamidoamine (PAMAM) dendrimers functionalized with various amounts of amine groups to achieve effective plasmid compaction. We further used triamcinolone acetonide (TA) as a nuclear localization enhancer for the dendrimer-gene complex and achieved significant improvement in cell uptake and transfection of hard-to-transfect human RPE cells. To improve colloidal stability, we further shielded the gene vector surface through incorporation of PEGylated dendrimer along with dendrimer-TA for DNA complexation. The resultant complexes showed improved stability while minimally affecting transgene delivery, thus improving the translational relevance of this platform. PMID:25213606

  19. Innate lymphoid cells regulate intestinal epithelial cell glycosylation.

    PubMed

    Goto, Yoshiyuki; Obata, Takashi; Kunisawa, Jun; Sato, Shintaro; Ivanov, Ivaylo I; Lamichhane, Aayam; Takeyama, Natsumi; Kamioka, Mariko; Sakamoto, Mitsuo; Matsuki, Takahiro; Setoyama, Hiromi; Imaoka, Akemi; Uematsu, Satoshi; Akira, Shizuo; Domino, Steven E; Kulig, Paulina; Becher, Burkhard; Renauld, Jean-Christophe; Sasakawa, Chihiro; Umesaki, Yoshinori; Benno, Yoshimi; Kiyono, Hiroshi

    2014-09-12

    Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host-microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria-dependent and -independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation. PMID:25214634

  20. Pigment Cell Progenitors in Zebrafish Remain Multipotent through Metamorphosis.

    PubMed

    Singh, Ajeet Pratap; Dinwiddie, April; Mahalwar, Prateek; Schach, Ursula; Linker, Claudia; Irion, Uwe; Nüsslein-Volhard, Christiane

    2016-08-01

    The neural crest is a transient, multipotent embryonic cell population in vertebrates giving rise to diverse cell types in adults via intermediate progenitors. The in vivo cell-fate potential and lineage segregation of these postembryonic progenitors is poorly understood, and it is unknown if and when the progenitors become fate restricted. We investigate the fate restriction in the neural crest-derived stem cells and intermediate progenitors in zebrafish, which give rise to three distinct adult pigment cell types: melanophores, iridophores, and xanthophores. By inducing clones in sox10-expressing cells, we trace and quantitatively compare the pigment cell progenitors at four stages, from embryogenesis to metamorphosis. At all stages, a large fraction of the progenitors are multipotent. These multipotent progenitors have a high proliferation ability, which diminishes with fate restriction. We suggest that multipotency of the nerve-associated progenitors lasting into metamorphosis may have facilitated the evolution of adult-specific traits in vertebrates. PMID:27453500

  1. Protons Sensitize Epithelial Cells to Mesenchymal Transition

    PubMed Central

    Wang, Minli; Hada, Megumi; Saha, Janapriya; Sridharan, Deepa M.; Pluth, Janice M.; Cucinotta, Francis A.

    2012-01-01

    Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1. PMID:22844446

  2. Pigment cell localizations in anuran ventral skin at climactic metamorphosis.

    PubMed

    Denèfle, J P; Lechaire, J P

    1991-09-01

    In anuran amphibians, the specific color pattern of the skin is expressed after metamorphosis, and its formation involves pigment cell migrations. Pigment cells are differently distributed in the tadpole, larval, and froglet skin. To learn more about their fate during metamorphic climax and in the young froglet, we focused our attention on the different localizations of larval melanophores and iridophores in the ventral skin of Rana esculenta before and during skin homing. Localizations of melanophores and iridophores can be elucidated at the developmental stages suggested by Taylor and Kollros (TK stages). At TK stage II (during early premetamorphosis), large melanophores beneath the larval skin are detected. At TK stage X, dispersed melanophores lie under bundles of muscular striated fibrils near the larval skin; they are also observed at the vascular level. At TK stage XVII (prometamorphosis), melanophores are extended on the inner side of the basement lamellar collagen. At the end of prometamorphosis, iridophores are located with melanophores in the separating space between attached basement collagen and derived basement collagen. At TK stage XX (earlier climax), melanophores and iridophores are detected inside the upper extremities of fractures opened in the derived basement collagen. At TK stage XXIV (later climax), both types of larval pigment cells are observed in the inner extremities of breaks derived from the fractures. During climax, these pigment cells occupy the well-formed breaks. At TK stage XXV in young froglet, the pigment cells remain alone in the breaks formed in the derived basement collagen. Briefly, breaks in the basement lamellar collagen are opened by invading cell processes of mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1750384

  3. Respiratory epithelial cells orchestrate pulmonary innate immunity.

    PubMed

    Whitsett, Jeffrey A; Alenghat, Theresa

    2015-01-01

    The epithelial surfaces of the lungs are in direct contact with the environment and are subjected to dynamic physical forces as airway tubes and alveoli are stretched and compressed during ventilation. Mucociliary clearance in conducting airways, reduction of surface tension in the alveoli, and maintenance of near sterility have been accommodated by the evolution of a multi-tiered innate host-defense system. The biophysical nature of pulmonary host defenses are integrated with the ability of respiratory epithelial cells to respond to and 'instruct' the professional immune system to protect the lungs from infection and injury. PMID:25521682

  4. Photoaging of retinal pigment epithelial melanosomes: The effect of photobleaching on morphology and reactivity of the pigment granules.

    PubMed

    Zadlo, Andrzej; Szewczyk, Grzegorz; Sarna, Michal; Kozinska, Anna; Pilat, Anna; Kaczara, Patrycja; Sarna, Tadeusz

    2016-08-01

    To elucidate the mechanism of age-related changes in antioxidant and photoprotective properties of human retinal pigment epithelium (RPE) melanosomes, the effect of in vitro photoaging of bovine RPE melanosomes was examined employing an array of complementary spectroscopic and analytical methods. Electron paramagnetic resonance (EPR) spectroscopy, saturation recovery EPR, atomic force microscopy (AFM) and dynamic light scattering (DLS) were used to determine melanin content of control and photobleached melanosomes, and to monitor changes in their morphology. Methylene blue (MB), TEMPO choline, dysprosium(III) ions and singlet oxygen were employed as molecular probes to characterize the efficiency of control and photobleached melanosomes to interact with different reagents. EPR oximetry, UV-vis absorption spectroscopy, iodometric assay of lipid hydroperoxides and time-resolved singlet oxygen phosphorescence were used to analyze the efficiency of photobleached and untreated melanosomes to inhibit MB-photosensitized oxidation of liposomal lipids. The obtained results revealed that, compared to untreated melanosomes, moderately photobleached melanosomes protected unsaturated lipids less efficiently against photosensitized peroxidiation, while weakly photobleached melanosomes were actually better antioxidant and photoprotective agents. The observed changes could be attributed to two effects - modification of the melanosome morphology and oxidative degradation of the melanin functional groups induced by different degree of photobleaching. While the former increases the accessibility of melanin nanoaggregates to reagents, the latter reduces the efficiency of melanin to interact with chemical and physical agents. PMID:27338854

  5. Vortex or whorl formation of cultured human corneal epithelial cells induced by magnetic fields.

    PubMed

    Dua, H S; Singh, A; Gomes, J A; Laibson, P R; Donoso, L A; Tyagi, S

    1996-01-01

    The terms 'vortex keratopathy' and 'hurricane keratopathy' describe two similar conditions affecting the corneal surface. In the former, a vortex or whorl pattern is seen on the corneal surface and is due to the deposition of substances such as pigment, iron or drugs in the epithelial cells. In the latter, a similar pattern is presented by migrating epithelial cells but, unlike the former, the pattern is rendered more visible by fluorescein staining. Both represent the migratory pattern of normal epithelial cells which is otherwise not visible due to the slow rate of epithelial turnover and migration. The whorl pattern has a clockwise predisposition in the majority of cases and is hypothesised to be due to the influence of ocular electro-magnetic fields on the migrating epithelial cells. In this study we tested in vitro the effect of static magnetic fields on corneal epithelial cells. We were able to reproduce dramatic vortex or whorl patterns in response to magnetic fields, but without preferential migration towards the North or South Pole. PMID:8944095

  6. Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition

    PubMed Central

    Muir, Amanda B.; Dods, Kara; Noah, Yuli; Toltzis, Sarit; Chandramouleeswaran, Prasanna Modayur; Lee, Anna; Benitez, Alain; Bedenbaugh, Adam; Falk, Gary W.; Wells, Rebecca G.; Nakagawa, Hiroshi; Wang, Mei-Lun

    2015-01-01

    Background and Aims Eosinophilic esophagitis (EoE) is an allergic inflammatory disease that leads to esophageal fibrosis and stricture. We have recently shown that in EoE, esophageal epithelial cells undergo an epithelial to mesenchymal transition (EMT), characterized by gain of mesenchymal markers and loss of epithelial gene expression. Whether epithelial cells exposed to profibrotic cytokines can also acquire the functional characteristics of activated myofibroblasts, including migration, contraction, and extracellular matrix deposition, is relevant to our understanding and treatment of EoE-associated fibrogenesis. In the current study, we characterize cell migration, contraction, and collagen production by esophageal epithelial cells that have undergone cytokine-induced EMT in vitro. Methods and Results Stimulation of human non-transformed immortalized esophageal epithelial cells (EPC2-hTERT) with profibrotic cytokines TNFα, TGFβ, and IL1β for three weeks led to acquisition of mesenchymal αSMA and vimentin, and loss of epithelial E-cadherin expression. Upon removal of the profibrotic stimulus, epithelial characteristics were partially rescued. TGFβ stimulation had a robust effect upon epithelial collagen production. Surprisingly, TNFα stimulation had the most potent effect upon cell migration and contraction, exceeding the effects of the prototypical profibrotic cytokine TGFβ. IL1β stimulation alone had minimal effect upon esophageal epithelial migration, contraction, and collagen production. Conclusions Esophageal epithelial cells that have undergone EMT acquire functional characteristics of activated myofibroblasts in vitro. Profibrotic cytokines exert differential effects upon esophageal epithelial cells, underscoring complexities of fibrogenesis in EoE, and implicating esophageal epithelial cells as effector cells in EoE-associated fibrogenesis. PMID:25183431

  7. EDAC: Epithelial defence against cancer-cell competition between normal and transformed epithelial cells in mammals.

    PubMed

    Kajita, Mihoko; Fujita, Yasuyuki

    2015-07-01

    During embryonic development or under certain pathological conditions, viable but suboptimal cells are often eliminated from the cellular society through a process termed cell competition. Cell competition was originally identified in Drosophila where cells with different properties compete for survival; 'loser' cells are eliminated from tissues and consequently 'winner' cells become dominant. Recent studies have shown that cell competition also occurs in mammals. While apoptotic cell death is the major fate for losers in Drosophila, outcompeted cells show more variable phenotypes in mammals, such as cell death-independent apical extrusion and cellular senescence. Molecular mechanisms underlying these processes have been recently revealed. Especially, in epithelial tissues, normal cells sense and actively eliminate the neighbouring transformed cells via cytoskeletal proteins by the process named epithelial defence against cancer (EDAC). Here, we introduce this newly emerging research field: cell competition in mammals. PMID:25991731

  8. Altered melanocyte differentiation and retinal pigmented epithelium transdifferentiation induced by Mash1 expression in pigment cell precursors.

    PubMed

    Lanning, Jessica L; Wallace, Jaclyn S; Zhang, Deming; Diwakar, Ganesh; Jiao, Zhongxian; Hornyak, Thomas J

    2005-10-01

    Transcription factor genes governing pigment cell development that are associated with spotting mutations in mice include members of several structural transcription factor classes but not members of the basic helix-loop-helix (bHLH) class, important for neurogenesis and myogenesis. To determine the effects of bHLH factor expression on pigment cell development, the neurogenic bHLH factor Mash1 was expressed early in pigment cell development in transgenic mice from the dopachrome tautomerase (Dct) promoter. Dct:Mash1 transgenic founders exhibit variable microphthalmia and patchy coat color hypopigmentation. Transgenic F1 mice exhibit microphthalmia with complete coat color dilution. Marker analysis demonstrates that Mash1 expression in the retinal pigmented epithelium (RPE) initiates neurogenesis in this cell layer, whereas expression in remaining neural crest-derived melanocytes alters their differentiation, in part by profoundly downregulating expression of the p (pink-eyed dilution) gene, while maintaining their cell fate. The effects of transcriptional perturbation of pigment cell precursors by Mash1 further highlight differences between pigment cells of distinct developmental origins, and suggest a mechanism for the alteration of melanogenesis to result in marked coat color dilution. PMID:16185282

  9. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    SciTech Connect

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  10. Pigment cell differentiation in sea urchin blastula-derived primary cell cultures.

    PubMed

    Ageenko, Natalya V; Kiselev, Konstantin V; Dmitrenok, Pavel S; Odintsova, Nelly A

    2014-07-01

    The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchins than in those intact. The possible roles of the specific components of the coelomic fluids in the pigment differentiation process and the quantitative measurement of the production of naphthoquinone pigments during cultivation were examined by MALDI and electrospray ionization mass spectrometry. Echinochrome A and spinochrome E were produced by the cultivated cells of the sand dollar Scaphechinus mirabilis in all tested media, while only spinochromes were found in the cultivated cells of another sea urchin, Strongylocentrotus intermedius. The expression of genes associated with the induction of pigment differentiation was increased in cells cultivated in the presence of shikimic acid, a precursor of naphthoquinone pigments. Our results should contribute to the development of new techniques in marine biotechnology, including the generation of cell cultures producing complex bioactive compounds with therapeutic potential. PMID:24979272

  11. Pigment Cell Differentiation in Sea Urchin Blastula-Derived Primary Cell Cultures

    PubMed Central

    Ageenko, Natalya V.; Kiselev, Konstantin V.; Dmitrenok, Pavel S.; Odintsova, Nelly A.

    2014-01-01

    The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchins than in those intact. The possible roles of the specific components of the coelomic fluids in the pigment differentiation process and the quantitative measurement of the production of naphthoquinone pigments during cultivation were examined by MALDI and electrospray ionization mass spectrometry. Echinochrome A and spinochrome E were produced by the cultivated cells of the sand dollar Scaphechinus mirabilis in all tested media, while only spinochromes were found in the cultivated cells of another sea urchin, Strongylocentrotus intermedius. The expression of genes associated with the induction of pigment differentiation was increased in cells cultivated in the presence of shikimic acid, a precursor of naphthoquinone pigments. Our results should contribute to the development of new techniques in marine biotechnology, including the generation of cell cultures producing complex bioactive compounds with therapeutic potential. PMID:24979272

  12. Differentiation of Lens-Like Structures from Newt Iris Epithelial Cells In Vitro

    PubMed Central

    Eguchi, Goro; Abe, Shin-Ichi; Watanabe, Kenji

    1974-01-01

    Dissociated cells of pigmented iris epithelium from adult newts grew intensively in monolayer cultures after a lag of two to three weeks. During the lag period, depigmentation occurred in many cells. When cultures became confluent five to six weeks after seeding, many tiny lens-like structures (30-70 per plate) differentiated from dense foci of amelanotic epithelial cells. These lens-like structures appeared in all cultures originated from cells of ventral as well as dorsal iris. The identification of these structures as lens was established by both immunological and ultrastructural techniques. Images PMID:4216028

  13. DNA typing of epithelial cells after strangulation.

    PubMed

    Wiegand, P; Kleiber, M

    1997-01-01

    DNA typing was carried out on epithelial cells which were transferred from the hands of the suspect onto the neck of the victim. In an experimental study 16 suspect-victim combinations were investigated for estimating the typing success. Alternatively to an attack against the neck, the upper arm was used for "strangulation". PCR typing was carried out using the short tandem repeat systems (STRs) HumCD4, HumVWF31A (VWA) and Hum-FIBRA (FGA) and the success rate was > 70% for all 3 systems. In most of the cases mixed patterns containing the phenotype of the suspect and the victim were obtained. In a case where strangulation was the cause of death, epithelial cells could be removed from the neck of the victim. The DNA pattern of the suspect could be successfully amplified using four STRs, demonstrating the applicability of this approach for practical casework. PMID:9274940

  14. Pigment developed to protect spacecraft/solar cells from Sun's harmful rays.

    NASA Technical Reports Server (NTRS)

    1995-01-01

    A pigment (phthalocyanine) is studied at the Marshall Materials and Processes Lab. The pigment has the ability to protect spacecraft against the harmful effects of the Sun's ultraviolet rays, and to increase the efficiency and life of solar cells.

  15. FULLEROL CYTOTOXICITY AND PHOTOTOXICITY IN HUMAN RETINAL PIGMENT EPITHELIAL CELLS.

    EPA Science Inventory

    Purpose:

    Water-soluble fullerenes [nano-C60 ,fullerol] have recently been shown to exhibit antibacterial and antiviral activity including inhibition of HIV protease. In addition to their potential to be used as drugs, they are also being tested as carriers to deliver drugs...

  16. Control of local immunity by airway epithelial cells.

    PubMed

    Weitnauer, M; Mijošek, V; Dalpke, A H

    2016-03-01

    The lung is ventilated by thousand liters of air per day. Inevitably, the respiratory system comes into contact with airborne microbial compounds, most of them harmless contaminants. Airway epithelial cells are known to have innate sensor functions, thus being able to detect microbial danger. To avoid chronic inflammation, the pulmonary system has developed specific means to control local immune responses. Even though airway epithelial cells can act as proinflammatory promoters, we propose that under homeostatic conditions airway epithelial cells are important modulators of immune responses in the lung. In this review, we discuss epithelial cell regulatory functions that control reactivity of professional immune cells within the microenvironment of the airways and how these mechanisms are altered in pulmonary diseases. Regulation by epithelial cells can be divided into two mechanisms: (1) mediators regulate epithelial cells' innate sensitivity in cis and (2) factors are produced that limit reactivity of immune cells in trans. PMID:26627458

  17. Generation of Mouse Lung Epithelial Cells

    PubMed Central

    Kasinski, Andrea L.; Slack, Frank J.

    2016-01-01

    Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One approach involves utilizing cells derived from the model under evaluation. The approach to generate such cells varies based on the cell of origin and often the genetics of the cell. Here we describe the steps involved in generating epithelial cells from the lungs of KrasLSL-G12D/+; p53LSL-R172/+ mice (Kasinski and Slack, 2012). These mice develop aggressive lung adenocarcinoma following cre-recombinase dependent removal of a stop cassette in the transgenes and subsequent expression of Kra-G12D and p53R172. While this protocol may be useful for the generation of epithelial lines from other genetic backgrounds, it should be noted that the Kras; p53 cell line generated here is capable of proliferating in culture without any additional genetic manipulation that is often needed for less aggressive backgrounds.

  18. Transcriptional Landscape of Glomerular Parietal Epithelial Cells

    PubMed Central

    Gharib, Sina A.; Pippin, Jeffrey W.; Ohse, Takamoto; Pickering, Scott G.; Krofft, Ronald D.; Shankland, Stuart J.

    2014-01-01

    Very little is known about the function of glomerular parietal epithelial cells (PECs). In this study, we performed genome-wide expression analysis on PEC-enriched capsulated vs. PEC-deprived decapsulated rat glomeruli to determine the transcriptional state of PECs under normal conditions. We identified hundreds of differentially expressed genes that mapped to distinct biologic modules including development, tight junction, ion transport, and metabolic processes. Since developmental programs were highly enriched in PECs, we characterized several of their candidate members at the protein level. Collectively, our findings confirm that PECs are multifaceted cells and help define their diverse functional repertoire. PMID:25127402

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

    PubMed

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

    2016-01-01

    Age-related macular degeneration (AMD) is a medical condition usually affecting older adults and resulting in a loss of vision in the macula, the center of the visual field. The dry form of this disease presents with atrophy of the retinal pigment epithelium, resulting in the detachment of the retina and loss of photoreceptors. Cigarette smoke is one main risk factor for dry AMD and increases the risk of developing the disease by three times. In order to understand the influence of cigarette smoke on retinal pigment epithelial cells, cultured human ARPE-19 cells were treated with cigarette smoke extract for 24 h. Using quantitative mass spectrometry more than 3000 proteins were identified and their respective abundances were compared between cigarette smoke-treated and untreated cells. Altogether 1932 proteins were quantified with at least two unique peptides, with 686 proteins found to be significantly differentially abundant with p > 0.05. Of these proteins the abundance of 64 proteins was at least 2-fold down-regulated after cigarette smoke treatment while 120 proteins were 2-fold up-regulated. The analysis of associated biological processes revealed an alteration of proteins involved in RNA processing and transport as well as extracellular matrix remodelling in response to cigarette smoke treatment. PMID:26427490

  20. Formation of a Neurosensory Organ by Epithelial Cell Slithering.

    PubMed

    Kuo, Christin S; Krasnow, Mark A

    2015-10-01

    Epithelial cells are normally stably anchored, maintaining their relative positions and association with the basement membrane. Developmental rearrangements occur through cell intercalation, and cells can delaminate during epithelial-mesenchymal transitions and metastasis. We mapped the formation of lung neuroepithelial bodies (NEBs), innervated clusters of neuroendocrine/neurosensory cells within the bronchial epithelium, revealing a targeted mode of cell migration that we named "slithering," in which cells transiently lose epithelial character but remain associated with the membrane while traversing neighboring epithelial cells to reach cluster sites. Immunostaining, lineage tracing, clonal analysis, and live imaging showed that NEB progenitors, initially distributed randomly, downregulate adhesion and polarity proteins, crawling over and between neighboring cells to converge at diametrically opposed positions at bronchial branchpoints, where they reestablish epithelial structure and express neuroendocrine genes. There is little accompanying progenitor proliferation or apoptosis. Activation of the slithering program may explain why lung cancers arising from neuroendocrine cells are highly metastatic. PMID:26435104

  1. Ouabain modulates ciliogenesis in epithelial cells

    PubMed Central

    Larre, Isabel; Castillo, Aida; Flores-Maldonado, Catalina; Contreras, Ruben G.; Galvan, Ivan; Muñoz-Estrada, Jesus; Cereijido, Marcelino

    2011-01-01

    The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na+ and H2O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na+,K+-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life. PMID:22143774

  2. Is the inflammasome relevant for epithelial cell function?

    PubMed

    Santana, Patricia T; Martel, Jan; Lai, Hsin-Chih; Perfettini, Jean-Luc; Kanellopoulos, Jean M; Young, John D; Coutinho-Silva, Robson; Ojcius, David M

    2016-02-01

    Inflammasomes are intracellular protein complexes that sense microbial components and damage of infected cells. Following activation by molecules released by pathogens or injured cells, inflammasomes activate caspase-1, allowing secretion of the pro-inflammatory cytokines IL-1β and IL-18 from innate immune cells. Inflammasomes are also expressed in epithelial cells, where their function has attracted less attention. Nonetheless, depending on the tissue, epithelial inflammasomes can mediate inflammation, wound healing, and pain sensitivity. We review here recent findings on inflammasomes found in epithelial tissues, highlighting the importance of these protein complexes in the response of epithelial tissues to microbial infections. PMID:26546965

  3. Coevolution of neoplastic epithelial cells and multilineage stroma via polyploid giant cells during immortalization and transformation of mullerian epithelial cells

    PubMed Central

    Zhang, Shiwu; Mercado-Uribe, Imelda; Sood, Anil; Bast, Robert C.; Liu, Jinsong

    2016-01-01

    Stromal cells are generally considered to be derived primarily from the host's normal mesenchymal stromal cells or bone marrow. However, the origins of stromal cells have been quite controversial. To determine the role of polyploidy in tumor development, we examined the fate of normal mullerian epithelial cells during the immortalization and transformation process by tracing the expression of SV40 large T antigen. Here we show that immortalized or HRAS-transformed mullerian epithelial cells contain a subpopulation of polyploid giant cells that grow as multicellular spheroids expressing hematopoietic markers in response to treatment with CoCl2. The immortalized or transformed epithelial cells can transdifferentiate into stromal cells when transplanted into nude mice. Immunofluorescent staining revealed expression of stem cell factors OCT4, Nanog, and SOX-2 in spheroid, whereas expression of embryonic stem cell marker SSEA1 was increased in HRAS-transformed cells compared with their immortalized isogenic counterparts. These results suggest that normal mullerian epithelial cells are intrinsically highly plastic, via the formation of polyploid giant cells and activation of embryonic stem-like program, which work together to promote the coevolution of neoplastic epithelial cells and multiple lineage stromal cells. PMID:27382431

  4. Establishment of Hertwig's epithelial root sheath/epithelial rests of Malassez cell line from human periodontium.

    PubMed

    Nam, Hyun; Kim, Ji-Hye; Kim, Jae-Won; Seo, Byoung-Moo; Park, Joo-Cheol; Kim, Jung-Wook; Lee, Gene

    2014-07-01

    Human Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-β1. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth. PMID:25081036

  5. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    SciTech Connect

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  6. Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation

    PubMed Central

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2016-01-01

    Summary Cellular senescence suppresses cancer by arresting cells at risk of malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation and branching morphogenesis. Furthermore, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts – the ability to alter epithelial differentiation – that might also explain the loss of tissue function and organization that is a hallmark of aging. PMID:15657080

  7. Henipavirus pathogenesis in human respiratory epithelial cells.

    PubMed

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz; Rockx, Barry

    2013-03-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  8. Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

    PubMed Central

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J. Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz

    2013-01-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  9. Nuclear microscopy of rat colon epithelial cells

    NASA Astrophysics Data System (ADS)

    Ren, M.; Rajendran, Reshmi; Ng, Mary; Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank; Jenner, Andrew Michael

    2011-10-01

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  10. Regulation of intestinal epithelial cells transcriptome by enteric glial cells: impact on intestinal epithelial barrier functions

    PubMed Central

    2009-01-01

    Background Emerging evidences suggest that enteric glial cells (EGC), a major constituent of the enteric nervous system (ENS), are key regulators of intestinal epithelial barrier (IEB) functions. Indeed EGC inhibit intestinal epithelial cells (IEC) proliferation and increase IEB paracellular permeability. However, the role of EGC on other important barrier functions and the signalling pathways involved in their effects are currently unknown. To achieve this goal, we aimed at identifying the impact of EGC upon IEC transcriptome by performing microarray studies. Results EGC induced significant changes in gene expression profiling of proliferating IEC after 24 hours of co-culture. 116 genes were identified as differentially expressed (70 up-regulated and 46 down-regulated) in IEC cultured with EGC compared to IEC cultured alone. By performing functional analysis of the 116 identified genes using Ingenuity Pathway Analysis, we showed that EGC induced a significant regulation of genes favoring both cell-to-cell and cell-to-matrix adhesion as well as cell differentiation. Consistently, functional studies showed that EGC induced a significant increase in cell adhesion. EGC also regulated genes involved in cell motility towards an enhancement of cell motility. In addition, EGC profoundly modulated expression of genes involved in cell proliferation and cell survival, although no clear functional trend could be identified. Finally, important genes involved in lipid and protein metabolism of epithelial cells were shown to be differentially regulated by EGC. Conclusion This study reinforces the emerging concept that EGC have major protective effects upon the IEB. EGC have a profound impact upon IEC transcriptome and induce a shift in IEC phenotype towards increased cell adhesion and cell differentiation. This concept needs to be further validated under both physiological and pathophysiological conditions. PMID:19883504