AMD-Like Substrate Causes Epithelial Mesenchymal Transition in iPSC-Derived Retinal Pigment Epithelial Cells Wild Type but Not C3-Knockout

The Bruch’s membrane (BrM) is a five-layered extracellular matrix (ECM) that supports the retinal pigment epithelium (RPE). Normal age-related changes in the BrM may lead to RPE cell damage and ultimately to the onset and progression of age-related macular degeneration (AMD), which is the most common cause of visual loss among the elderly. A role for the complement system in AMD pathology has been established, but the disease mechanisms are poorly understood, which hampers the design of efficient therapies to treat millions of patients. In an effort to identify the mechanisms that lead from normal aging to pathology, we have developed a cell-based model using complement deficient human induced pluripotent stem cell (iPSC)-derived RPE cells cultured on an AMD-like ECM that mimics BrM. The data present evidence that changes in the ECM result in loss of differentiation and promote epithelial mesenchymal transition (EMT) of healthy RPE cells. This pathological process is mediated by complement activation and involves the formation of a randomly oriented collagen meshwork that drives the dedifferentiation of the RPE monolayer. Genetic ablation of complement component 3 has a protective effect against EMT but does not prevent the abnormal deposition of collagens. These findings offer new insights into the sequence of events that initiate AMD and may guide the design of efficient therapies to treat this disease with unmet medical needs.

Download Full-text

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

Biomedicines ◽

10.3390/biomedicines8060147 ◽

2020 ◽

Vol 8 (6) ◽

pp. 147 ◽

Cited By ~ 3

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.

Download Full-text

Polarity and epithelial-mesenchymal transition of retinal pigment epithelial cells in proliferative vitreoretinopathy

PeerJ ◽

10.7717/peerj.10136 ◽

2020 ◽

Vol 8 ◽

pp. e10136

Author(s):

Hui Zou ◽

Chenli Shan ◽

Linlin Ma ◽

Jia Liu ◽

Ning Yang ◽

...

Keyword(s):

Proliferative Vitreoretinopathy ◽

Molecular Mechanisms ◽

Epithelial Mesenchymal Transition ◽

Retinal Pigment ◽

Notch Pathway ◽

Retinal Pigment Epithelial Cells ◽

Cell Contact ◽

Mesenchymal Transition ◽

Rpe Cells ◽

Cell Cell

Under physiological conditions, retinal pigment epithelium (RPE) is a cellular monolayer composed of mitotically quiescent cells. Tight junctions and adherens junctions maintain the polarity of RPE cells, and are required for cellular functions. In proliferative vitreoretinopathy (PVR), upon retinal tear, RPE cells lose cell-cell contact, undergo epithelial-mesenchymal transition (EMT), and ultimately transform into myofibroblasts, leading to the formation of fibrocellular membranes on both surfaces of the detached retina and on the posterior hyaloids, which causes tractional retinal detachment. In PVR, RPE cells are crucial contributors, and multiple signaling pathways, including the SMAD-dependent pathway, Rho pathway, MAPK pathways, Jagged/Notch pathway, and the Wnt/β-catenin pathway are activated. These pathways mediate the EMT of RPE cells, which play a key role in the pathogenesis of PVR. This review summarizes the current body of knowledge on the polarized phenotype of RPE, the role of cell-cell contact, and the molecular mechanisms underlying the RPE EMT in PVR, emphasizing key insights into potential approaches to prevent PVR.

Download Full-text

Deletion of TSPO Resulted in Change of Metabolomic Profile in Retinal Pigment Epithelial Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20061387 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1387 ◽

Cited By ~ 3

Author(s):

Abdulwahab Alamri ◽

Lincoln Biswas ◽

David Watson ◽

Xinhua Shu

Keyword(s):

Retinal Pigment Epithelial ◽

Retinal Pigment ◽

Nucleotide Metabolism ◽

Retinal Pigment Epithelial Cells ◽

Age Related Macular Degeneration ◽

Clinical Feature ◽

Rpe Cells ◽

Pigment Epithelial ◽

Age Related ◽

The Impact

Age-related macular degeneration is the main cause of vision loss in the aged population worldwide. Drusen, extracellular lesions formed underneath the retinal pigment epithelial (RPE) cells, are a clinical feature of AMD and associated with AMD progression. RPE cells support photoreceptor function by providing nutrition, phagocytosing outer segments and removing metabolic waste. Dysfunction and death of RPE cells are early features of AMD. The translocator protein, TSPO, plays an important role in RPE cholesterol efflux and loss of TSPO results in increased intracellular lipid accumulation and reactive oxygen species (ROS) production. This study aimed to investigate the impact of TSPO knockout on RPE cellular metabolism by identifying the metabolic differences between wildtype and knockout RPE cells, with or without treatment with oxidized low density lipoprotein (oxLDL). Using liquid chromatography mass spectrometry (LC/MS), we differentiated several metabolic pathways among wildtype and knockout cells. Lipids amongst other intracellular metabolites were the most influenced by loss of TSPO and/or oxLDL treatment. Glucose, amino acid and nucleotide metabolism was also affected. TSPO deletion led to up-regulation of fatty acids and glycerophospholipids, which in turn possibly affected the cell membrane fluidity and stability. Higher levels of glutathione disulphide (GSSG) were found in TSPO knockout RPE cells, suggesting TSPO regulates mitochondrial-mediated oxidative stress. These data provide biochemical insights into TSPO-associated function in RPE cells and may shed light on disease mechanisms in AMD.

Download Full-text

Protective Effects of Curcumin Ester Prodrug, Curcumin Diethyl Disuccinate against H2O2-Induced Oxidative Stress in Human Retinal Pigment Epithelial Cells: Potential Therapeutic Avenues for Age-Related Macular Degeneration

International Journal of Molecular Sciences ◽

10.3390/ijms20133367 ◽

2019 ◽

Vol 20 (13) ◽

pp. 3367 ◽

Cited By ~ 7

Author(s):

Chawanphat Muangnoi ◽

Umar Sharif ◽

Pahweenvaj Ratnatilaka Na Bhuket ◽

Pornchai Rojsitthisak ◽

Luminita Paraoan

Keyword(s):

Oxidative Stress ◽

Macular Degeneration ◽

Retinal Pigment ◽

Parent Drug ◽

Retinal Pigment Epithelial Cells ◽

Age Related Macular Degeneration ◽

Protective Effects ◽

Rpe Cells ◽

Age Related ◽

Potent Drug

Oxidative stress-induced damage to the retinal pigmented epithelium (RPE), a specialised post-mitotic monolayer that maintains retinal homeostasis, contributes to the development of age-related macular degeneration (AMD). Curcumin (Cur), a naturally occurring antioxidant, was previously shown to have the ability to protect RPE cells from oxidative stress. However, poor solubility and bioavailability makes Cur a poor therapeutic agent. As prodrug approaches can mitigate these limitations, we compared the protective properties of the Cur prodrug curcumin diethyl disuccinate (CurDD) against Cur in relation to oxidative stress induced in human ARPE-19 cells. Both CurDD and Cur significantly decreased H2O2-induced reactive oxygen species (ROS) production and protected RPE cells from oxidative stress-induced death. Both drugs exerted their protective effects through the modulation of p44/42 (ERK) and the involvement of downstream molecules Bax and Bcl-2. Additionally, the expression of antioxidant enzymes HO-1 and NQO1 was also enhanced in cells treated with CurDD and Cur. In all cases, CurDD was more effective than its parent drug against oxidative stress-induced damage to ARPE-19 cells. These findings highlight CurDD as a more potent drug compared to Cur against oxidative stress and indicate that its protective effects are exerted through modulation of key apoptotic and antioxidant molecular pathways.

Download Full-text

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

eLife ◽

10.7554/elife.14319 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 80

Author(s):

Toshihide Kurihara ◽

Peter D Westenskow ◽

Marin L Gantner ◽

Yoshihiko Usui ◽

Andrew Schultz ◽

...

Keyword(s):

Pigment Epithelium ◽

Retinal Pigment ◽

Metabolic Stress ◽

Retinal Pigment Epithelial Cells ◽

Age Related Macular Degeneration ◽

Photoreceptor Degeneration ◽

Rpe Cells ◽

Glucose And Lipid Metabolism ◽

Age Related ◽

Cellular Stresses

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.

Download Full-text

Mitophagy in the Retinal Pigment Epithelium of Dry Age-Related Macular Degeneration Investigated in the NFE2L2/PGC-1α-/- Mouse Model

International Journal of Molecular Sciences ◽

10.3390/ijms21061976 ◽

2020 ◽

Vol 21 (6) ◽

pp. 1976 ◽

Cited By ~ 6

Author(s):

Iswariyaraja Sridevi Gurubaran ◽

Johanna Viiri ◽

Ali Koskela ◽

Juha M.T. Hyttinen ◽

Jussi J. Paterno ◽

...

Keyword(s):

Oxidative Stress ◽

Mouse Model ◽

Macular Degeneration ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Pigment Epithelial Cells ◽

Age Related Macular Degeneration ◽

Double Knockout ◽

Rpe Cells ◽

Age Related

Increased oxidative stress and mitochondrial damage are observed in protein aggregation diseases, such as age-related macular degeneration (AMD). We have recently reported elevated levels of oxidative stress markers, damaged mitochondria, accumulating lysosomal lipofuscin and extracellular drusen-like structures in the retinal pigment epithelial cells (RPE) of the dry AMD-resembling NFE2L2/PGC1α double knockout (dKO) mouse model. Here, we provide evidence of a disturbance in the autolysosomal machinery handling mitochondrial clearance in the RPE cells of one-year-old NFE2L2/PGC1α-deficient mice. Confocal immunohistochemical analysis revealed an upregulation of autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) as well as numerous mitophagy markers, such as PTE-induced putative kinase 1 (PINK1) and E3 ubiquitin ligase (PARKIN) together with damaged mitochondria. However, we detected no evidence of increased autolysosome formation in transmission electron micrographs or of colocalization of lysosomal marker LAMP2 (lysosome-associated membrane protein 2) and the mitochondrial marker ATP synthase β in confocal micrographs. Interestingly, we observed an upregulation of late autolysosomal fusion Ras-related protein (Rab7) in the perinuclear space of RPE cells together with autofluorescence aggregates. Our results reveal that there is at least a relative decrease of mitophagy in the RPE cells of NFE2L2/PGC1α dKO mice. This further supports the hypothesis that mitophagy is a putative therapy target in AMD-like pathology.

Download Full-text

Superior Properties of N-Acetylcysteine Ethyl Ester over N-Acetyl Cysteine to Prevent Retinal Pigment Epithelial Cells Oxidative Damage

International Journal of Molecular Sciences ◽

10.3390/ijms22020600 ◽

2021 ◽

Vol 22 (2) ◽

pp. 600

Author(s):

Gian Marco Tosi ◽

Daniela Giustarini ◽

Lorenzo Franci ◽

Alberto Minetti ◽

Francesco Imperatore ◽

...

Keyword(s):

Oxidative Stress ◽

Oxidative Damage ◽

Ethyl Ester ◽

Retinal Pigment ◽

Developed Countries ◽

Retinal Pigment Epithelial Cells ◽

Age Related Macular Degeneration ◽

Retinal Diseases ◽

Rpe Cells ◽

Age Related

Oxidative stress plays a key role in the pathophysiology of retinal diseases, including age-related macular degeneration (AMD) and diabetic retinopathy, which are the major causes of irreversible blindness in developed countries. An excess of reactive oxygen species (ROS) can directly cause functional and morphological impairments in retinal pigment epithelium (RPE), endothelial cells, and retinal ganglion cells. Antioxidants may represent a preventive/therapeutic strategy and reduce the risk of progression of AMD. Among antioxidants, N-acetyl-L-cysteine (NAC) is widely studied and has been proposed to have therapeutic benefit in treating AMD by mitigating oxidative damage in RPE. Here, we demonstrate that N-acetyl-L-cysteine ethyl ester (NACET), a lipophilic cell-permeable cysteine derivative, increases the viability in oxidative stressed RPE cells more efficiently than NAC by reacting directly and more rapidly with oxidizing agents, and that NACET, but not NAC, pretreatment predisposes RPE cells to oxidative stress resistance and increases the intracellular reduced glutathione (GSH) pool available to act as natural antioxidant defense. Moreover, we demonstrate the ability of NACET to increase GSH levels in rats’ eyes after oral administration. In conclusion, even if experiments in AMD animal models are still needed, our data suggest that NACET may play an important role in preventing and treating retinal diseases associated with oxidative stress, and may represent a valid and more efficient alternative to NAC in therapeutic protocols in which NAC has already shown promising results.

Download Full-text

The CD36 Ligand-Promoted Autophagy Protects Retinal Pigment Epithelial Cells from Oxidative Stress

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/6691402 ◽

2021 ◽

Vol 2021 ◽

pp. 1-14

Author(s):

Marie-France Dorion ◽

Mukandila Mulumba ◽

Shuya Kasai ◽

Ken Itoh ◽

William D. Lubell ◽

...

Keyword(s):

Oxidative Stress ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Pigment Epithelial Cells ◽

Age Related Macular Degeneration ◽

Autophagic Flux ◽

Cytoprotective Effect ◽

Rpe Cells ◽

Age Related ◽

Cluster Of Differentiation

The retinal pigment epithelium (RPE) performs many functions that maintain photoreceptor health. Oxidative damage to the RPE is a critical component in the pathogenesis of eye diseases such as age-related macular degeneration (AMD). Ligands of the cluster of differentiation 36 (CD36) have previously preserved photoreceptor integrity in mouse models of AMD. The cytoprotective effect of the CD36 ligand MPE-001 on RPE cells has now been elucidated employing a model of oxidative stress. Sodium iodate (NaIO3) induced formation of reactive oxygen species and apoptosis in human RPE cells, which were decreased by MPE-001 without affecting antioxidant enzyme transcription. Immunoblotting and immunostaining assays showed a restorative effect of MPE-001 on the autophagic flux disrupted by NaIO3, which was associated with an increase in syntaxin 17-positive mature autophagosomes. The cytoprotective effect of MPE-001 was completely abolished by the autophagy inhibitors wortmannin and bafilomycin A1. In conclusion, we report for the first time an autophagy-dependent protection of RPE cells from oxidative stress by a CD36 ligand.

Download Full-text

mTOR activity is essential for retinal pigment epithelium regeneration in zebrafish

10.1101/2021.06.01.446531 ◽

2021 ◽

Author(s):

Jeff Gross ◽

Fangfang Lu ◽

Lyndsay Leach

Keyword(s):

Retinal Pigment Epithelium ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Mtor Pathway ◽

Age Related Macular Degeneration ◽

Model Systems ◽

Rpe Cells ◽

Genetic Ablation ◽

Age Related ◽

Mtor Activity

The retinal pigment epithelium (RPE) plays numerous critical roles in maintaining vision and this is underscored by the prevalence of degenerative blinding diseases like age-related macular degeneration (AMD), in which visual impairment is caused by progressive loss of RPE cells. In contrast to mammals, zebrafish possess the ability to intrinsically regenerate a functional RPE layer after severe injury. The molecular underpinnings of this regenerative process remain largely unknown yet hold tremendous potential for developing treatment strategies to stimulate endogenous regeneration in the human eye. In this study, we demonstrate that the mTOR pathway is activated in RPE cells post-genetic ablation. Pharmacological and genetic inhibition of mTOR activity impaired RPE regeneration, while mTOR activation enhanced RPE recovery post-injury, demonstrating that mTOR activity is necessary and sufficient for RPE regeneration in zebrafish. RNA-seq of RPE isolated from mTOR-inhibited larvae identified a number of genes and pathways dependent on mTOR activity at early and late stages of regeneration; amongst these were components of the immune system, which is emerging as a key regulator of regenerative responses across various tissue and model systems. Our results identify crosstalk between macrophages/microglia and the RPE, wherein mTOR activity in the RPE is required for recruitment of macrophages/microglia to the injury site. In turn, these macrophages/microglia reinforce mTOR activity in regenerating RPE cells. Interestingly, the function of macrophages/microglia in maintaining mTOR activity in the RPE appeared to be inflammation-independent. Taken together, these data identify mTOR activity as a key regulator of RPE regeneration and link the mTOR pathway to immune responses in facilitating RPE regeneration.

Download Full-text

Madecassoside protects retinal pigment epithelial cells against hydrogen peroxide-induced oxidative stress and apoptosis through the activation of Nrf2/HO-1 pathway

Bioscience Reports ◽

10.1042/bsr20194347 ◽

2020 ◽

Vol 40 (10) ◽

Author(s):

Jinzi Zhou ◽

Fenghua Chen ◽

Aimin Yan ◽

Xiaobo Xia

Keyword(s):

Oxidative Stress ◽

Hydrogen Peroxide ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Pigment Epithelial Cells ◽

Age Related Macular Degeneration ◽

Triterpenoid Saponin ◽

Sod Activity ◽

Rpe Cells ◽

Age Related

Abstract Age-related macular degeneration (AMD) is a progressive and degenerative ocular disease associated with oxidative stress. Madecassoside (MADE) is a major bioactive triterpenoid saponin that possesses antioxidative activity. However, the role of MADE in AMD has never been investigated. In the current study, we aimed to evaluate the protective effect of MADE on retinal pigment epithelium (RPE) cells under oxidative stress condition. We used hydrogen peroxide (H2O2) to induce oxidative damage in human RPE cells (ARPE-19 cells). Our results showed that H2O2-caused significant decrease in cell viability and increase in lactate dehydrogenase (LDH) release were dose-dependently attenuated by MADE. MADE treatment also attenuated H2O2-induced reactive oxygen species (ROS) and malondialdehyde (MDA) production in RPE cells. The reduced glutathione (GSH) level and superoxide dismutase (SOD) activity in H2O2-induced ARPE-19 cells were elevated after MADE treatment. MADE also suppressed caspase-3 activity and bax expression, as well as increased bcl-2 expression. Furthermore, H2O2-induced increase in expression levels of HO-1 and nuclear Nrf2 were enhanced by MADE treatment. Finally, knockdown of Nrf2 reversed the protective effects of MADE on H2O2-induced ARPE-19 cells. In conclusion, these findings demonstrated that MADE protected ARPE-19 cells from H2O2-induced oxidative stress and apoptosis by inducing the activation of Nrf2/HO-1 signaling pathway.

Download Full-text