scholarly journals Functional and morphological analysis of the subretinal injection of retinal pigment epithelium cells

2012 ◽  
Vol 29 (2) ◽  
pp. 83-93 ◽  
Author(s):  
MAREN ENGELHARDT ◽  
CHINATSU TOSHA ◽  
VANDA S. LOPES ◽  
BRYAN CHEN ◽  
LISA NGUYEN ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Host Cell ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Primary Cultures ◽  
Host Cells ◽  
Apical Surface ◽  
Noninvasive Methods ◽  
Rpe Cells ◽  
Subretinal Injection

AbstractReplacement of retinal pigment epithelium (RPE) cells by transplantation is a potential treatment for some retinal degenerations. Here, we used a combination of invasive and noninvasive methods to characterize the structural and functional consequences of subretinal injection of RPE cells. Pigmented cells from primary cultures were injected into albino mice. Recovery was monitored over 8 weeks by fundus imaging, spectral domain optical coherence tomography (sdOCT), histology, and electroretinography (ERG). sdOCT showed that retinal reattachment was nearly complete by 1 week. ERG response amplitudes were reduced after injection, with cone-mediated function then recovering better than rod function. Photoreceptor cell loss was evident by sdOCT and histology, near the site of injection, and is likely to have been the main cause of incomplete recovery. With microscopy, injected cells were identified by the presence of apical melanosomes. They either established contact with Bruch’s membrane, and thus became part of the RPE monolayer, or were located on the apical surface of the host’s cells, resulting in apposition of the basal surface of the injected cell with the apical surface of the host cell and the formation of a series of desmosomal junctions. RPE cell density was not increased, indicating that the incorporation of an injected cell into the RPE monolayer was concomitant with the loss of a host cell. The transplanted and remaining host cells contained large vacuoles of ingested debris as well as lipofuscin-like granules, suggesting that they had scavenged the excess injected and host cells, and were stressed by the high digestive load. Therefore, although significant functional and structural recovery was observed, the consequences of this digestive stress may be a concern for longer-term health, especially where RPE cell transplantation is used to treat diseases that include lipofuscin accumulation as part of their pathology.

scholarly journals Regulation of phagolysosomal activity by miR-204 critically influences structure and function of retinal pigment epithelium/retina

2019 ◽  
Vol 28 (20) ◽  
pp. 3355-3368 ◽  
Author(s):  
Congxiao Zhang ◽  
Kiyoharu J Miyagishima ◽  
Lijin Dong ◽  
Aaron Rising ◽  
Malika Nimmagadda ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Primary Cultures ◽  
Structure And Function ◽  
Apical Surface ◽  
Altered Expression ◽  
And Function ◽  
The Impact

Abstract MicroRNA-204 (miR-204) is expressed in pulmonary, renal, mammary and eye tissue, and its reduction can result in multiple diseases including cancer. We first generated miR-204−/− mice to study the impact of miR-204 loss on retinal and retinal pigment epithelium (RPE) structure and function. The RPE is fundamentally important for maintaining the health and integrity of the retinal photoreceptors. miR-204−/− eyes evidenced areas of hyper-autofluorescence and defective photoreceptor digestion, along with increased microglia migration to the RPE. Migratory Iba1+ microglial cells were localized to the RPE apical surface where they participated in the phagocytosis of photoreceptor outer segments (POSs) and contributed to a persistent build-up of rhodopsin. These structural, molecular and cellular outcomes were accompanied by decreased light-evoked electrical responses from the retina and RPE. In parallel experiments, we suppressed miR-204 expression in primary cultures of human RPE using anti-miR-204. In vitro suppression of miR-204 in human RPE similarly showed abnormal POS clearance and altered expression of autophagy-related proteins and Rab22a, a regulator of endosome maturation. Together, these in vitro and in vivo experiments suggest that the normally high levels of miR-204 in RPE can mitigate disease onset by preventing generation of oxidative stress and inflammation originating from intracellular accumulation of undigested photoreactive POS lipids. More generally, these results implicate RPE miR-204-mediated regulation of autophagy and endolysosomal interaction as a critical determinant of normal RPE/retina structure and function.

scholarly journals Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton.

1991 ◽  
Vol 112 (5) ◽  
pp. 863-872 ◽  
Author(s):  
D Gundersen ◽  
J Orlowski ◽  
E Rodriguez-Boulan
Keyword(s):  
Retinal Pigment Epithelium ◽  
Epithelial Cells ◽  
Current Knowledge ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Opposite Polarity ◽  
Apical Surface ◽  
Rpe Cells ◽  
Membrane Cytoskeleton

In striking contrast to most other transporting epithelia (e.g., urinary or digestive systems), where Na,K-ATPase is expressed basolaterally, the retinal pigment epithelium (RPE) cells display Na,K-ATPase pumps on the apical membrane. We report here studies aimed to identify the mechanisms underlying this polarity "reversal" of the RPE Na,K-ATPase. By immunofluorescence on thin frozen sections, both alpha and beta subunits were localized on the apical surface of both freshly isolated rat RPE monolayers and RPE monolayers grown in culture. The polarity of the RPE cell is not completely reversed, however, since aminopeptidase, an apically located protein in kidney epithelia, was also found on the apical surface of RPE cells. We used subunit- and isoform-specific cDNA probes to determine that RPE Na,K-ATPase has the same isoform (alpha 1) as the one found in kidney. Ankyrin and fodrin, proteins of the basolateral membrane cytoskeleton of kidney epithelial cells known to be associated with the Na,K-ATPase (Nelson, W. J., and R. W. Hammerton. 1989. J. Cell Biol. 110:349-357) also displayed a reversed apical localization in RPE and were intimately associated to Na,K-ATPase, as revealed by cross-linking experiments. These results indicate that an entire membrane-cytoskeleton complex is assembled with opposite polarity in RPE cells. We discuss our observations in the context of current knowledge on protein sorting mechanisms in epithelial cells.

Apical sorting of influenza hemagglutinin by transcytosis in retinal pigment epithelium

1997 ◽  
Vol 110 (15) ◽  
pp. 1717-1727 ◽  
Author(s):  
V.L. Bonilha ◽  
A.D. Marmorstein ◽  
L. Cohen-Gould ◽  
E. Rodriguez-Boulan
Keyword(s):  
Plasma Membrane ◽  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Subretinal Space ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Apical Surface ◽  
Rpe Cells

The retinal pigment epithelium is endowed with a unique distribution of certain plasma membrane proteins. Na+,K+-ATPase, for instance, is polarized to the apical surface of RPE, rather than to the basolateral surface as in most other epithelia. To study the sorting pathways of RPE cells, we used temperature sensitive mutants of influenza and vesicular stomatitis virus (VSV) to synchronize the transport of hemagglutinin (HA) and VSV G protein (VSV G) along the biosynthetic pathway of the RPE cell line RPE-J. After HA and VSV G accumulated in the trans-Golgi network of RPE-J cells kept at 20 degrees C, transfer to the permissive temperature (32 degrees C) resulted in the transport of both HA and VSV G to the basolateral plasma membrane. Later, while VSV G remained basolateral, HA progressively reversed its polarity, eventually becoming apical. Further analysis demonstrated that the reversal of HA polarity was due to transcytosis of HA from the basolateral to the apical surface of RPE-J cells. To determine whether HA followed a transcytotic route in RPE in vivo, influenza and VSV were injected into the subretinal space of rat eyes. Again, both HA and VSV G were initially observed at the basolateral surface of RPE cells. However, whereas VSV G remained there, HA progressively redistributed to the apical surface. These findings demonstrated that RPE cells use a transcytotic pathway for the targeting of at least some apical proteins to their destination.

The polarity of the plasma membrane protein RET-PE2 in retinal pigment epithelium is developmentally regulated

1996 ◽  
Vol 109 (13) ◽  
pp. 3025-3034 ◽  
Author(s):  
A.D. Marmorstein ◽  
V.L. Bonilha ◽  
S. Chiflet ◽  
J.M. Neill ◽  
E. Rodriguez-Boulan
Keyword(s):  
Retinal Pigment Epithelium ◽  
Laser Scanning ◽  
Biochemical Characterization ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Primary Cultures ◽  
Laser Scanning Confocal Microscopy ◽  
Apical Surface ◽  
Adult Rat ◽  
Interphotoreceptor Matrix

The retinal pigment epithelium (RPE) differs from other epithelia in that the apical surface is not free; instead, it interacts with both photoreceptors and a specialized extracellular material, the interphotoreceptor matrix. Biochemical characterization of the apical and basolateral surfaces of RPE in adult rat eye cups, using a novel in situ biotinylation assay, revealed very different protein compositions and identified a major surface antigen, RET-PE2, with a predominantly apical distribution (approximately 74%). The apical polarity of RET-PE2 was confirmed by immunofluorescence and laser scanning confocal microscopy. In striking contrast, RET-PE2 antigen was preferentially basolateral in primary cultures derived from adult rat RPE and in an immortalized RPE cell line (RPE-J). Under all conditions, RET-PE2 was highly soluble in Triton X-100 (> 81% at 4 degrees C), suggesting that its redistribution was not dependent on changes in cytoskeletal interactions. Analysis of the localization of RET-PE2 in normal rats at postnatal (PN) days 1, 7, and 14 indicated that RET-PE2 redistributes from predominantly basolateral to predominantly apical during that time. Since photoreceptors develop during the first two weeks after birth in the rat, our results suggest that the apical redistribution of RET-PE2 is dependent on the establishment of adult interactions between the RPE and the neural retina and/or the interphotoreceptor matrix, either via direct contacts or through alterations in the intracellular sorting patterns of RPE cells.

Immortalization of polarized rat retinal pigment epithelium

1993 ◽  
Vol 104 (1) ◽  
pp. 37-49 ◽  
Author(s):  
I.R. Nabi ◽  
A.P. Mathews ◽  
L. Cohen-Gould ◽  
D. Gundersen ◽  
E. Rodriguez-Boulan
Keyword(s):  
Retinal Pigment Epithelium ◽  
Tight Junction ◽  
Cell Line ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Primary Cultures ◽  
Neural Retina ◽  
Permissive Temperature ◽  
Apical Surface ◽  
Junction Protein

Rat retinal pigment epithelial (RPE) cells were immortalized by infection with a temperature-sensitive tsA SV40 virus and following cloning and selection for epithelial properties the polarized RPE-J cell line was obtained. At the permissive temperature of 33 degrees C, RPE-J cells behave as an immortalized cell line. When RPE-J cells are grown on nitrocellulose filters coated with a thin layer of Matrigel in the presence of 10(−8) M retinoic acid for 6 days at 33 degrees C and then switched for 33–36 hours to the non-permissive temperature of 40 degrees C, they acquire a differentiated polarized RPE phenotype. Under these growth conditions, RPE-J cells exhibit circumferential staining for the tight-junction protein ZO-1 and acquire a transepithelial resistance of 350 ohms cm2. Morphologically, RPE-J cells exhibit a characteristic RPE morphology with extensive apical microvilli as well as numerous dense bodies including premelanosomes and varied multilamellar structures. Ruthenium red labeling revealed the frequent basal localization of the tight junction. The cells were identified to be of rat RPE origin by their expression of the rat RPE marker RET-PE2 and their ability to phagocytose latex beads. While RPE-J cells are capable of sorting influenza and vesicular stomatitis virus to the apical and basal surfaces, respectively, the Na,K-ATPase is not polarized and the neural cell adhesion molecule, N-CAM, is localized exclusively to the lateral surface. In vivo the apical surface of RPE interacts with the adjacent neural retina and the Na,K-ATPase and N-CAM are both apical; the altered polarity of these two proteins in RPE-J cells may be a consequence of the absence of apical interaction with the neural retina in culture. Previous studies of RPE have been restricted to the use of primary cultures and the RPE-J cell line should prove an excellent model system for the study of the mechanisms determining the characteristic polarity and functions of the retinal pigment epithelium.

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.

Microfilament organization and wound repair in retinal pigment epithelium

1995 ◽  
Vol 73 (9-10) ◽  
pp. 709-722 ◽  
Author(s):  
Vitauts. I. Kalnins ◽  
Martin Sandig ◽  
Greg J. Hergott ◽  
Haruhiko Nagai
Keyword(s):  
Cell Proliferation ◽  
Cell Migration ◽  
Retinal Pigment Epithelium ◽  
Wound Repair ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Wound Edge ◽  
Rpe Cells ◽  
Oriented Parallel ◽  
Experimentally Induced

Several systems of microfilaments (MF) associated with adherens-type junctions between adjacent retinal pigment epithelial (RPE) cells and between these cells and the substratum play an important role in maintaining the integrity and organization of the RPE. They include prominent, contractile circumferential MF bundles that are associated with the zonula adherens (ZA) junctions. In chick RPE, these junctions are assembled from smaller subunits thus giving greater structural flexibility to the junctional region. Because the separation of the junctions requires trypsin and low calcium, both calcium-dependent and -independent mechanisms are involved in keeping adjacent RPE cells attached to one another. Another system of MF bundles that crosses the cell at the level of ZA junctions can be induced to form by stretching the epithelium. The MF bundles forming this system are oriented in the direction in which the RPE is stretched, thereby preventing the overextension of the cell in any one direction. The system may be useful as an indicator of the direction in which tension is experienced by RPE during development of the eye, in animal models of disease and during repair of experimentally induced wounds. Numerous single-cell wounds resulting from death of RPE cells by apoptosis at various stages of repair are normally present in developing chick and adult mammalian RPE. These wounds are repaired by the spreading of adjacent RPE cells and by the contraction of MF bundles oriented parallel to the wound edge, which develop during this time. As a result of the spreading in the absence of cell proliferation, the RPE cells increase in diameter with age. Experimentally induced wounds made by removing 5–10 RPE cells are repaired by a similar mechanism within 24 h. In repair of larger wounds, over 125 μm in width, the MF bundles oriented parallel to the wound edge characteristic of spreading cells are later replaced by stress fibers (SFs) that run perpendicularly to the wound edge and interact with the substratum at focal contacts (FCs) as RPE cells start to migrate. Cell proliferation is induced in cells along the wound edge only when the wounds are wide enough to require cell migration. In the presence of antibodies to beta-1-integrins, a component of FCs, cell spreading is not prevented but both cell migration and cell proliferation are inhibited. Thus, only the organization of the cytoskeleton characteristic of migrating RPE cells that have SFs that interact with the substratum at FCs, is associated with the induction of cell proliferation.Key words: retinal pigment epithelium, microfilaments, wound repair.

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.

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