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.

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.

scholarly journals Epithelial-to-Mesenchymal Transition of RPE Cells In Vitro Confers Increased β1,6-N-Glycosylation and Increased Susceptibility to Galectin-3 Binding

PLoS ONE ◽  
2016 ◽  
Vol 11 (1) ◽  
pp. e0146887 ◽  
Author(s):  
Claudia S. Priglinger ◽  
Jara Obermann ◽  
Christoph M. Szober ◽  
Juliane Merl-Pham ◽  
Uli Ohmayer ◽  
...  
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Mesenchymal Transition ◽  
Rpe Cells ◽  
Galectin 3 ◽  
Increased Susceptibility

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 Transcriptome-Wide Analysis of CXCR5 Deficient Retinal Pigment Epithelial (RPE) Cells Reveals Molecular Signatures of RPE Homeostasis

Biomedicines ◽  
2020 ◽  
Vol 8 (6) ◽  
pp. 147 ◽  
Author(s):  
Madhu Sudhana Saddala ◽  
Anton Lennikov ◽  
Anthony Mukwaya ◽  
Hu Huang
Keyword(s):  
Epithelial Mesenchymal Transition ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Enrichment Analysis ◽  
Age Related Macular Degeneration ◽  
Pathway Enrichment Analysis ◽  
Molecular Signatures ◽  
Mesenchymal Transition ◽  
Rpe Cells ◽  
Age Related

Age-related macular degeneration (AMD) is the most common cause of irreversible blindness in the elderly population. In our previous studies, we found that deficiency of CXCR5 causes AMD-like pathological phenotypes in mice, characterized by abnormalities and dysfunction of the retinal pigment epithelium (RPE) cells. The abnormalities included abnormal cellular shape and impaired barrier function. In the present study, primary RPE cells were derived separately from CXCR5 knockout (KO) mice and from C57BL6 wild type (WT). The isolated primary cells were cultured for several days, and then total RNA was isolated and used for library preparation, sequencing, and the resultant raw data analyzed. Relative to the WT, a total of 1392 differentially expressed genes (DEG) were identified. Gene ontology analysis showed various biological processes, cellular components, and molecular functions were enriched. Pathway enrichment analysis revealed several pathways, including the PI3K-Akt signaling, mTOR signaling, FoxO, focal adhesion, endocytosis, ubiquitin-mediated proteolysis, TNFα-NF-kB Signaling, adipogenesis genes, p53 signaling, Ras, autophagy, epithelial–mesenchymal transition (EMT), and mitochondrial pathway. This study explores molecular signatures associated with deficiency of CXCR5 in RPE cells. Many of these signatures are important for homeostasis of this tissue. The identified pathways and genes require further evaluation to better understand the pathophysiology of AMD.

scholarly journals A Context-Dependent Role for MiR-124-3p on Cell Phenotype, Viability and Chemosensitivity in Neuroblastoma in vitro

2020 ◽  
Vol 8 ◽  
Author(s):  
John C. Nolan ◽  
Manuela Salvucci ◽  
Steven Carberry ◽  
Ana Barat ◽  
Miguel F. Segura ◽  
...  
Keyword(s):  
Cell Lines ◽  
Epithelial To Mesenchymal Transition ◽  
Neuroblastoma Cell ◽  
Chemotherapy Resistance ◽  
Cellular Transformation ◽  
Cell Phenotype ◽  
Drug Resistant ◽  
Mesenchymal Transition ◽  
Neuroblastoma Cell Lines

Neuroblastoma (NB) is a neural crest-derived tumor, which develops before birth or in early childhood, with metastatic dissemination typically preceding diagnosis. Tumors are characterized by a highly heterogeneous combination of cellular phenotypes demonstrating varying degrees of differentiation along different lineage pathways, and possessing distinct super-enhancers and core regulatory circuits, thereby leading to highly varied malignant potential and divergent clinical outcomes. Cytoskeletal reorganization is fundamental to cellular transformations, including the processes of cellular differentiation and epithelial to mesenchymal transition (EMT), previously reported by our lab and others to coincide with chemotherapy resistance and enhanced metastatic ability of tumor cells. This study set out to investigate the ability of the neuronal miR-124-3p to reverse the cellular transformation associated with drug resistance development and assess the anti-oncogenic role of this miRNA in in vitro models of drug-resistant adrenergic (ADRN) and mesenchymal (MES) neuroblastoma cell lines. Low expression of miR-124-3p in a cohort of neuroblastomas was significantly associated with poor overall and progression-free patient survival. Over-expression of miR-124-3p in vitro inhibited cell viability through the promotion of cell cycle arrest and induction of apoptosis in addition to sensitizing drug-resistant cells to chemotherapeutics in a panel of morphologically distinct neuroblastoma cell lines. Finally, we describe miR-124-3p direct targeting and repression of key up-regulated cytoskeletal genes including MYH9, ACTN4 and PLEC and the reversal of the resistance-associated EMT and enhanced invasive capacity previously reported in our in vitro model (SK-N-ASCis24).

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.

scholarly journals Transcriptomic Profiling of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium Over Time

2019 ◽  
Author(s):  
Grace E. Lidgerwood ◽  
Anne Senabouth ◽  
Casey J.A. Smith-Anttila ◽  
Vikkitharan Gnanasambandapillai ◽  
Dominik C. Kaczorowski ◽  
...  
Keyword(s):  
Stem Cell ◽  
Retinal Pigment Epithelium ◽  
Pluripotent Stem Cell ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Human Pluripotent Stem Cell ◽  
Mesenchymal Transition ◽  
Rpe Cells

AbstractHuman pluripotent stem cell (hPSC)-derived progenies are immature versions of cells, presenting a potential limitation to the accurate modelling of disease associated with maturity or age. Hence, it is important to characterise how closely cells used in culture resemble their native counterparts. In order to select appropriate points in time for RPE cultures to reflect native counterparts, we characterised the transcriptomic profiles of hPSC-derived retinal pigment epithelium (RPE) cells from 1- and 12-month cultures. We differentiated the human embryonic stem cell line H9 into RPE cells, performed single cell RNA-sequencing of a total of 16,576 cells, and analysed the resulting data to assess the molecular changes of RPE cells across these two culture time points. Our results indicate the stability of the RPE transcriptomic signature, with no evidence of an epithelial – mesenchymal transition, and with maturing populations of RPE observed with time in culture. Assessment of gene ontology pathways revealed that as cultures age, RPE cells upregulate expression of genes involved in metal binding and antioxidant functions. This might reflect an increased ability to handle oxidative stress as cells mature. Comparison with native human RPE data confirmed a maturing transcriptional profile of RPE cells in culture. These results suggest that in vitro long-term culture of RPE cells allow the modelling of specific phenotypes observed in native mature tissue. Our work highlights the transcriptional landscape of hPSC-derived RPE as they age in culture, which provides a reference for native and patient-samples to be benchmarked against.

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