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

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Richard Rosen ◽  
Tommaso Vagaggini ◽  
Yueqin Chen ◽  
Dan-Ning Hu
Keyword(s):  
Culture Medium ◽  
Retinal Pigment ◽  
Retinal Diseases ◽  
Hypoxia Inducible Factors ◽  
Rpe Cells ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Cell Hypoxia ◽  
Important Stimulus

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

The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo

1988 ◽  
Vol 91 (2) ◽  
pp. 303-312
Author(s):  
N.M. McKechnie ◽  
M. Boulton ◽  
H.L. Robey ◽  
F.J. Savage ◽  
I. Grierson
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cytokeratin 18 ◽  
Rpe Cells ◽  
Human Retinal Pigment Epithelium ◽  
Cytoskeletal Elements

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

scholarly journals Pathogenic Effects of Mineralocorticoid Pathway Activation in Retinal Pigment Epithelium

2021 ◽  
Vol 22 (17) ◽  
pp. 9618
Author(s):  
Jérémie Canonica ◽  
Min Zhao ◽  
Tatiana Favez ◽  
Emmanuelle Gelizé ◽  
Laurent Jonet ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Retinal Pigment Epithelium ◽  
Barrier Function ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Diseases ◽  
Mesenchymal Transition ◽  
Rpe Cells ◽  
Pathway Activation ◽  
And Migration

Glucocorticoids are amongst the most used drugs to treat retinal diseases of various origins. Yet, the transcriptional regulations induced by glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation in retinal pigment epithelium cells (RPE) that form the outer blood–retina barrier are unknown. Levels of endogenous corticoids, ligands for MR and GR, were measured in human ocular media. Human RPE cells derived from induced pluripotent stem cells (iRPE) were used to analyze the pan-transcriptional regulations induced by aldosterone—an MR-specific agonist, or cortisol or cortisol + RU486—a GR antagonist. The retinal phenotype of transgenic mice that overexpress the human MR (P1.hMR) was analyzed. In the human eye, the main ligand for GR and MR is cortisol. The iRPE cells express functional GR and MR. The subset of genes regulated by aldosterone and by cortisol + RU-486, and not by cortisol alone, mimics an imbalance toward MR activation. They are involved in extracellular matrix remodeling (CNN1, MGP, AMTN), epithelial–mesenchymal transition, RPE cell proliferation and migration (ITGB3, PLAUR and FOSL1) and immune balance (TNFSF18 and PTX3). The P1.hMR mice showed choroidal vasodilation, focal alteration of the RPE/choroid interface and migration of RPE cells together with RPE barrier function alteration, similar to human retinal diseases within the pachychoroid spectrum. RPE is a corticosteroid-sensitive epithelium. MR pathway activation in the RPE regulates genes involved in barrier function, extracellular matrix, neural regulation and epithelial differentiation, which could contribute to retinal pathology.

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

2017 ◽  
Vol 117 (04) ◽  
pp. 750-757
Author(s):  
Xin Jia ◽  
Chen Zhao ◽  
Qishan Chen ◽  
Yuxiang Du ◽  
Lijuan Huang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Retinal Pigment Epithelium ◽  
Cell Survival ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Pigment Epithelium Cell ◽  
Photoreceptor Cells ◽  
Rpe Cells

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

scholarly journals A Higher Proportion of Eicosapentaenoic Acid (EPA) When Combined with Docosahexaenoic Acid (DHA) in Omega-3 Dietary Supplements Provides Higher Antioxidant Effects in Human Retinal Cells

Antioxidants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 828 ◽  
Author(s):  
Manuel Saenz de Viteri ◽  
María Hernandez ◽  
Valentina Bilbao-Malavé ◽  
Patricia Fernandez-Robredo ◽  
Jorge González-Zamora ◽  
...  
Keyword(s):  
Docosahexaenoic Acid ◽  
Eicosapentaenoic Acid ◽  
Cell Viability ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Ethyl Esters ◽  
Omega 3 ◽  
Rpe Cells ◽  
Antioxidant Effects

Retinal pigment epithelium (RPE) is a key regulator of retinal function and is directly related to the transport, delivery, and metabolism of long-chain n-3 polyunsaturated fatty acids (n3-PUFA), in the retina. Due to their functions and location, RPE cells are constantly exposed to oxidative stress. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have shown to have antioxidant effects by different mechanisms. For this reason, we designed an in vitro study to compare 10 formulations of DHA and EPA supplements from different origins and combined in different proportions, evaluating their effect on cell viability, cell proliferation, reactive oxygen species production, and cell migration using ARPE-19 cells. Furthermore, we assessed their ability to rescue RPE cells from the oxidative conditions seen in diabetic retinopathy. Our results showed that the different formulations of n3-PUFAs have a beneficial effect on cell viability and proliferation and are able to restore oxidative induced RPE damage. We observed that the n3-PUFA provided different results alone or combined in the same supplement. When combined, the best results were obtained in formulations that included a higher proportion of EPA than DHA. Moreover, n3-PUFA in the form of ethyl-esters had a worse performance when compared with triglycerides or phospholipid based formulations.

scholarly journals Nanoscopic Approach to Study the Early Stages of Epithelial to Mesenchymal Transition (EMT) of Human Retinal Pigment Epithelial (RPE) Cells In Vitro

Life ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 128
Author(s):  
Lilia A. Chtcheglova ◽  
Andreas Ohlmann ◽  
Danila Boytsov ◽  
Peter Hinterdorfer ◽  
Siegfried G. Priglinger ◽  
...  
Keyword(s):  
Structural Changes ◽  
Epithelial To Mesenchymal Transition ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cell Phenotype ◽  
Cytoskeletal Reorganization ◽  
Mesenchymal Transition ◽  
Rpe Cells ◽  
Early Stages

The maintenance of visual function is supported by the proper functioning of the retinal pigment epithelium (RPE), representing a mosaic of polarized cuboidal postmitotic cells. Damage factors such as inflammation, aging, or injury can initiate the migration and proliferation of RPE cells, whereas they undergo a pseudo-metastatic transformation or an epithelial to mesenchymal transition (EMT) from cuboidal epithelioid into fibroblast-like or macrophage-like cells. This process is recognized as a key feature in several severe ocular pathologies, and is mimicked by placing RPE cells in culture, which provides a reasonable and well-characterized in vitro model for a type 2 EMT. The most obvious characteristic of EMT is the cell phenotype switching, accompanied by the cytoskeletal reorganization with changes in size, shape, and geometry. Atomic force microscopy (AFM) has the salient ability to label-free explore these characteristics. Based on our AFM results supported by the genetic analysis of specific RPE differentiation markers, we elucidate a scheme for gradual transformation from the cobblestone to fibroblast-like phenotype. Structural changes in the actin cytoskeletal reorganization at the early stages of EMT lead to the development of characteristic geodomes, a finding that may reflect an increased propensity of RPE cells to undergo further EMT and thus become of diagnostic significance.

scholarly journals Identification of cell surface markers and establishment of monolayer differentiation to retinal pigment epithelial cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Alvaro Plaza Reyes ◽  
Sandra Petrus-Reurer ◽  
Sara Padrell Sánchez ◽  
Pankaj Kumar ◽  
Iyadh Douagi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Retinal Pigment Epithelial ◽  
Retinal Pigment ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
In Vitro Differentiation ◽  
Rpe Cells ◽  
Pigment Epithelial ◽  
Differentiation Efficiency

AbstractIn vitro differentiation of human pluripotent stem cells into functional retinal pigment epithelial (RPE) cells provides a potentially unlimited source for cell based reparative therapy of age-related macular degeneration. Although the inherent pigmentation of the RPE cells have been useful to grossly evaluate differentiation efficiency and allowed manual isolation of pigmented structures, accurate quantification and automated isolation has been challenging. To address this issue, here we perform a comprehensive antibody screening and identify cell surface markers for RPE cells. We show that these markers can be used to isolate RPE cells during in vitro differentiation and to track, quantify and improve differentiation efficiency. Finally, these surface markers aided to develop a robust, direct and scalable monolayer differentiation protocol on human recombinant laminin-111 and −521 without the need for manual isolation.

scholarly journals Negative Impact of Hypoxia on Tryptophan 2,3-Dioxygenase Function

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Frank Elbers ◽  
Claudia Woite ◽  
Valentina Antoni ◽  
Sara Stein ◽  
Hiroshi Funakoshi ◽  
...  
Keyword(s):  
Essential Amino Acid ◽  
Negative Impact ◽  
Ex Vivo ◽  
T Cell Proliferation ◽  
Effector Functions ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Oxygen Conditions

Tryptophan is an essential amino acid for hosts and pathogens. The liver enzyme tryptophan 2,3-dioxygenase (TDO) provokes, by its ability to degrade tryptophan to N-formylkynurenine, the precursor of the immune-relevant kynurenines, direct and indirect antimicrobial and immunoregulatory states. Up to now these TDO-mediated broad-spectrum effector functions have never been observed under hypoxiain vitro, although physiologic oxygen concentrations in liver tissue are low, especially in case of infection. Here we analysed recombinant expressed human TDO andex vivomurine TDO functions under different oxygen conditions and show that TDO-induced restrictions of clinically relevant pathogens (bacteria, parasites) and of T cell proliferation are abrogated under hypoxic conditions. We pinpointed the loss of TDO efficiency to the reduction of TDO activity, since cell survival and TDO protein levels were unaffected. In conclusion, the potent antimicrobial as well as immunoregulatory effects of TDO were substantially impaired under hypoxic conditions that pathophysiologically occurin vivo. This might be detrimental for the appropriate host immune response towards relevant pathogens.

scholarly journals Reduction of lipid accumulation rescues Bietti’s crystalline dystrophy phenotypes

2018 ◽  
Vol 115 (15) ◽  
pp. 3936-3941 ◽  
Author(s):  
Masayuki Hata ◽  
Hanako O. Ikeda ◽  
Sachiko Iwai ◽  
Yuto Iida ◽  
Norimoto Gotoh ◽  
...  
Keyword(s):  
Free Cholesterol ◽  
Cell Damage ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Patient Specific ◽  
Rpe Cells ◽  
Underlying Mechanisms ◽  
Induced Pluripotent ◽  
Bietti's Crystalline Dystrophy

Bietti’s crystalline dystrophy (BCD) is an intractable and progressive chorioretinal degenerative disease caused by mutations in the CYP4V2 gene, resulting in blindness in most patients. Although we and others have shown that retinal pigment epithelium (RPE) cells are primarily impaired in patients with BCD, the underlying mechanisms of RPE cell damage are still unclear because we lack access to appropriate disease models and to lesion-affected cells from patients with BCD. Here, we generated human RPE cells from induced pluripotent stem cells (iPSCs) derived from patients with BCD carrying a CYP4V2 mutation and successfully established an in vitro model of BCD, i.e., BCD patient-specific iPSC-RPE cells. In this model, RPE cells showed degenerative changes of vacuolated cytoplasm similar to those in postmortem specimens from patients with BCD. BCD iPSC-RPE cells exhibited lysosomal dysfunction and impairment of autophagy flux, followed by cell death. Lipidomic analyses revealed the accumulation of glucosylceramide and free cholesterol in BCD-affected cells. Notably, we found that reducing free cholesterol by cyclodextrins or δ-tocopherol in RPE cells rescued BCD phenotypes, whereas glucosylceramide reduction did not affect the BCD phenotype. Our data provide evidence that reducing intracellular free cholesterol may have therapeutic efficacy in patients with BCD.

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