scholarly journals Hydroquinone Induces NLRP3-Independent IL-18 Release from ARPE-19 Cells

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1405
Author(s):  
Niina Bhattarai ◽  
Eveliina Korhonen ◽  
Yashavanthi Mysore ◽  
Kai Kaarniranta ◽  
Anu Kauppinen
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Retinal Disease ◽  
Oxidase Inhibitor ◽  
Age Related Macular Degeneration ◽  
Rpe Cells ◽  
Pyrin Domain ◽  
Age Related ◽  
Domain 3 ◽  
Human Rpe Cells

Age-related macular degeneration (AMD) is a retinal disease leading to impaired vision. Cigarette smoke increases the risk for developing AMD by causing increased reactive oxygen species (ROS) production and damage in the retinal pigment epithelium (RPE). We have previously shown that the cigarette tar component hydroquinone causes oxidative stress in human RPE cells. In the present study, we investigated the propensity of hydroquinone to induce the secretion of interleukin (IL)-1β and IL-18. The activation of these cytokines is usually regulated by the Nucleotide-binding domain, Leucine-rich repeat, and Pyrin domain 3 (NLRP3) inflammasome. ARPE-19 cells were exposed to hydroquinone, and cell viability was monitored using the lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt (MTT) assays. Enzyme-linked immunosorbent assays (ELISAs) were used to measure the levels of proinflammatory cytokines IL-1β and IL-18 as well as NLRP3, caspase-1, and poly (ADP-ribose) polymerase (PARP). Hydroquinone did not change IL-1β release but significantly increased the secretion of IL-18. Cytoplasmic NLRP3 levels increased after the hydroquinone treatment of IL-1α-primed RPE cells, but IL-18 was equally released from primed and nonprimed cells. Hydroquinone reduced the intracellular levels of PARP, which were restored by treatment with the ROS scavenger N-acetyl-cysteine (NAC). NAC concurrently reduced the NLRP3 levels but had no effect on IL-18 release. In contrast, the NADPH oxidase inhibitor ammonium pyrrolidinedithiocarbamate (APDC) reduced the release of IL-18 but had no effect on the NLRP3 levels. Collectively, hydroquinone caused DNA damage seen as reduced intracellular PARP levels and induced NLRP3-independent IL-18 secretion in human RPE cells.

scholarly journals Protective Effect of Melatonin against Oxidative Stress-Induced Apoptosis and Enhanced Autophagy in Human Retinal Pigment Epithelium Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Chih-Chao Chang ◽  
Tien-Yi Huang ◽  
Hsin-Yuan Chen ◽  
Tsui-Chin Huang ◽  
Li-Chun Lin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Oxidative Damage ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Critical Role ◽  
Age Related Macular Degeneration ◽  
Cell Degeneration ◽  
Rpe Cells ◽  
Age Related ◽  
Human Rpe Cells

Age-related macular degeneration (AMD) affects the retinal macula and results in loss of vision, and AMD is the primary cause of blindness and severe visual impairment among elderly people worldwide. AMD is characterized by the accumulation of drusen in the Bruch’s membrane and dysfunction of retinal pigment epithelial (RPE) cells and photoreceptors. The pathogenesis of AMD remains unclear, and no effective treatment exists. Accumulating evidence indicates that oxidative stress plays a critical role in RPE cell degeneration and AMD. Melatonin is an antioxidant that scavenges free radicals, and it has anti-inflammatory, antitumor, and antiangiogenic effects. This study investigated the antioxidative, antiapoptotic, and autophagic effects of melatonin on oxidative damage to RPE cells. We used hydrogen peroxide (H2O2) to stimulate reactive oxygen species production to cause cell apoptosis in ARPE-19 cell lines. Our findings revealed that treatment with melatonin significantly inhibited H2O2-induced RPE cell damage, decreased the apoptotic rate, increased the mitochondrial membrane potential, and increased the autophagy effect. Furthermore, melatonin reduced the Bax/Bcl-2 ratio and the expression levels of the apoptosis-associated proteins cytochrome c and caspase 7. Additionally, melatonin upregulated the expression of the autophagy-related proteins LC3-II and Beclin-1 and downregulated the expression of p62. Thus, melatonin’s effects on autophagy and apoptosis can protect against H2O2-induced oxidative damage in human RPE cells. Melatonin may have multiple protective effects on human RPE cells against H2O2-induced oxidative damage.

scholarly journals Acadesine suppresses TNF-α induced complement component 3 (C3), in retinal pigment epithelial (RPE) cells

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244307
Author(s):  
Nikolaos E. Efstathiou ◽  
Giannis A. Moustafa ◽  
Daniel E. Maidana ◽  
Eleni K. Konstantinou ◽  
Shoji Notomi ◽  
...  
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Complement Component ◽  
Adenosine Kinase ◽  
Age Related Macular Degeneration ◽  
Dependent Manner ◽  
Rpe Cells ◽  
Age Related ◽  
Tnf Α ◽  
Complement Component 3

Rationale Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness in the developed world. Aging, inflammation and complement dysregulation affecting the retinal pigment epithelium (RPE), are considered significant contributors in its pathogenesis and several evidences have linked tumor necrosis factor alpha (TNF-α) and complement component 3 (C3) with AMD. Acadesine, an analog of AMP and an AMP-activated protein kinase (AMPK) activator, has been shown to have cytoprotective effects in human clinical trials as well as having anti-inflammatory and anti-vascular exudative effects in animals. The purpose of this study was to evaluate if acadesine is able to suppress TNF-α induced C3 in RPE cells. Methods ARPE-19 and human primary RPE cells were cultured and allowed to grow to confluence. TNF-α was used for C3 induction in the presence or absence of acadesine. Small molecule inhibitors and siRNA were used to determine if acadesine exerts its effect via the extracellular or intracellular pathway and to evaluate the importance of AMPK for these effects. The expression level of C3 was determined by immunoblot analysis. Results Acadesine suppresses TNF-α induced C3 in a dose dependent manner. When we utilized the adenosine receptor inhibitor dipyridamole (DPY) along with acadesine, acadesine’s effects were abolished, indicating the necessity of acadesine to enter the cell in order to exert it’s action. However, pretreatment with 5-iodotubericidin (5-Iodo), an adenosine kinase (AK) inhibitor, didn’t prevent acadesine from decreasing TNF-α induced C3 expression suggesting that acadesine does not exert its effect through AMP conversion and subsequent activation of AMPK. Consistent with this, knockdown of AMPK α catalytic subunit did not affect the inhibitory effect of acadesine on TNF-α upregulation of C3. Conclusions Our results suggest that acadesine suppresses TNF-α induced C3, likely through an AMPK-independent pathway, and could have potential use in complement over activation diseases.

scholarly journals PGC-1α Protects RPE Cells of the Aging Retina against Oxidative Stress-Induced Degeneration through the Regulation of Senescence and Mitochondrial Quality Control. The Significance for AMD Pathogenesis

2018 ◽  
Vol 19 (8) ◽  
pp. 2317 ◽  
Author(s):  
Kai Kaarniranta ◽  
Jakub Kajdanek ◽  
Jan Morawiec ◽  
Elzbieta Pawlowska ◽  
Janusz Blasiak
Keyword(s):  
Oxidative Stress ◽  
Cellular Senescence ◽  
Mitochondrial Biogenesis ◽  
Vision Loss ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Sirtuin 1 ◽  
Age Related Macular Degeneration ◽  
Rpe Cells ◽  
Age Related

PGC-1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha) is a transcriptional coactivator of many genes involved in energy management and mitochondrial biogenesis. PGC-1α expression is associated with cellular senescence, organismal aging, and many age-related diseases, including AMD (age-related macular degeneration), an important global issue concerning vision loss. We and others have developed a model of AMD pathogenesis, in which stress-induced senescence of retinal pigment epithelium (RPE) cells leads to AMD-related pathological changes. PGC-1α can decrease oxidative stress, a key factor of AMD pathogenesis related to senescence, through upregulation of antioxidant enzymes and DNA damage response. PGC-1α is an important regulator of VEGF (vascular endothelial growth factor), which is targeted in the therapy of wet AMD, the most devastating form of AMD. Dysfunction of mitochondria induces cellular senescence associated with AMD pathogenesis. PGC-1α can improve mitochondrial biogenesis and negatively regulate senescence, although this function of PGC-1α in AMD needs further studies. Post-translational modifications of PGC-1α by AMPK (AMP kinase) and SIRT1 (sirtuin 1) are crucial for its activation and important in AMD pathogenesis.

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 Oxidative stress-mediated TXNIP loss causes RPE dysfunction

2019 ◽  
Vol 51 (10) ◽  
pp. 1-13 ◽  
Author(s):  
Min Ji Cho ◽  
Sung-Jin Yoon ◽  
Wooil Kim ◽  
Jongjin Park ◽  
Jangwook Lee ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Function ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Common Factor ◽  
Age Related Macular Degeneration ◽  
Autophagic Flux ◽  
Blood Retinal Barrier ◽  
Rpe Cells ◽  
Age Related

Abstract The disruption of the retinal pigment epithelium (RPE), for example, through oxidative damage, is a common factor underlying age-related macular degeneration (AMD). Aberrant autophagy also contributes to AMD pathology, as autophagy maintains RPE homeostasis to ensure blood–retinal barrier (BRB) integrity and protect photoreceptors. Thioredoxin-interacting protein (TXNIP) promotes cellular oxidative stress by inhibiting thioredoxin reducing capacity and is in turn inversely regulated by reactive oxygen species levels; however, its role in oxidative stress-induced RPE cell dysfunction and the mechanistic link between TXNIP and autophagy are largely unknown. Here, we observed that TXNIP expression was rapidly downregulated in RPE cells under oxidative stress and that RPE cell proliferation was decreased. TXNIP knockdown demonstrated that the suppression of proliferation resulted from TXNIP depletion-induced autophagic flux, causing increased p53 activation via nuclear localization, which in turn enhanced AMPK phosphorylation and activation. Moreover, TXNIP downregulation further negatively impacted BRB integrity by disrupting RPE cell tight junctions and enhancing cell motility by phosphorylating, and thereby activating, Src kinase. Finally, we also revealed that TXNIP knockdown upregulated HIF-1α, leading to the enhanced secretion of VEGF from RPE cells and the stimulation of angiogenesis in cocultured human retinal microvascular endothelial cells. This suggests that the exposure of RPE cells to sustained oxidative stress may promote choroidal neovascularization, another AMD pathology. Together, these findings reveal three distinct mechanisms by which TXNIP downregulation disrupts RPE cell function and thereby exacerbates AMD pathogenesis. Accordingly, reinforcing or restoring BRB integrity by targeting TXNIP may serve as an effective therapeutic strategy for preventing or attenuating photoreceptor damage in AMD.

scholarly journals Protective Effect of Metformin against Hydrogen Peroxide-Induced Oxidative Damage in Human Retinal Pigment Epithelial (RPE) Cells by Enhancing Autophagy through Activation of AMPK Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Xia Zhao ◽  
Linlin Liu ◽  
Yizhou Jiang ◽  
Marta Silva ◽  
Xuechu Zhen ◽  
...  
Keyword(s):  
Oxidative Damage ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Protective Effects ◽  
Rpe Cells ◽  
Mitochondria Membrane Potential ◽  
Ampk Pathway ◽  
Age Related ◽  
Diabetes Drug

Age-related macular degeneration (AMD) is a leading cause of blindness with limited effective treatment. Although the pathogenesis of this disease is complex and not fully understood, the oxidative damage caused by excessive reactive oxygen species (ROS) in retinal pigment epithelium (RPE) has been considered as a major cause. Autophagy is essential for the degradation of cellular components damaged by ROS, and its dysregulation has been implicated in AMD pathogenesis. Therefore, strategies aiming to boost autophagy could be effective in protecting RPE cells from oxidative damage. Metformin is the first-line anti-type 2 diabetes drug and has been reported to stimulate autophagy in many tissues. We therefore hypothesized that metformin may be able to protect RPE cells against H2O2-induced oxidative damage by autophagy activation. In the present study, we found that metformin attenuated H2O2-induced cell viability loss, apoptosis, elevated ROS levels, and the collapse of the mitochondria membrane potential in D407 cells. Autophagy was stimulated by metformin, and inhibition of autophagy by 3-methyladenine (3-MA) and chloroquine (CQ) or knockdown of Beclin1 and LC3B blocked the protective effects of metformin. In addition, we showed that metformin could activate the AMPK pathway, whereas both pharmacological and genetic inhibitions of AMPK blocked the autophagy-stimulating and protective effects of metformin. Metformin conferred a similar protection against H2O2-induced oxidative damage in primary cultured human RPE cells. Taken together, these results demonstrate that metformin could protect RPE cells from H2O2-induced oxidative damage by stimulating autophagy via the activation of the AMPK pathway, supporting its potential use in the prevention and treatment of AMD.

scholarly journals Suppressor of Cytokine Signaling 2 Regulates Retinal Pigment Epithelium Metabolism by Enhancing Autophagy

2021 ◽  
Vol 15 ◽  
Author(s):  
Xi-Yuan Liu ◽  
Rui Lu ◽  
Jing Chen ◽  
Jie Wang ◽  
Hong-Mei Qian ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Metabolic Regulation ◽  
Glycogen Synthase ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Age Related Macular Degeneration ◽  
Cytokine Signaling ◽  
Suppressor Of Cytokine Signaling ◽  
Rpe Cells ◽  
Age Related

Retinal pigment epithelium (RPE) serves critical functions in maintaining retinal homeostasis. An important function of RPE is to degrade the photoreceptor outer segment fragments daily to maintain photoreceptor function and longevity throughout life. An impairment of RPE functions such as metabolic regulation leads to the development of age-related macular degeneration (AMD) and inherited retinal degenerative diseases. As substrate recognition subunit of a ubiquitin ligase complex, suppressor of cytokine signaling 2 (SOCS2) specifically binds to the substrates for ubiquitination and negatively regulates growth hormone signaling. Herein, we explore the role of SOCS2 in the metabolic regulation of autophagy in the RPE cells. SOCS2 knockout mice exhibited the irregular morphological deposits between the RPE and Bruch’s membrane. Both in vivo and in vitro experiments showed that RPE cells lacking SOCS2 displayed impaired autophagy, which could be recovered by re-expressing SOCS2. SOCS2 recognizes the ubiquitylated proteins and participates in the formation of autolysosome by binding with autophagy receptors and lysosome-associated membrane protein2 (LAMP-2), thereby regulating the phosphorylation of glycogen synthase kinase 3β (GSK3β) and mammalian target of rapamycin (mTOR) during the autophagy process. Our results imply that SOCS2 participates in ubiquitin-autophagy-lysosomal pathway and enhances autophagy by regulating GSK3β and mTOR. This study provides a potential therapeutic target for AMD.

scholarly journals A convenient protocol for establishing a human cell culture model of the outer retina.

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 1107 ◽  
Author(s):  
Savannah A. Lynn ◽  
Eloise Keeling ◽  
Jennifer M. Dewing ◽  
David A. Johnston ◽  
Anton Page ◽  
...  
Keyword(s):  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Experimental Manipulation ◽  
Age Related Macular Degeneration ◽  
Attractive Alternative ◽  
Outer Retina ◽  
Rpe Cells ◽  
Age Related ◽  
Culture Model

The retinal pigment epithelium (RPE) plays a key role in the pathogenesis of several blinding retinopathies. Alterations to RPE structure and function are reported in Age-related Macular Degeneration, Stargardt and Best disease as well as pattern dystrophies. However, the precise role of RPE cells in disease aetiology remains incompletely understood. Many studies into RPE pathobiology have utilised animal models, which only recapitulate limited disease features. Some studies are also difficult to carry out in animals as the ocular space remains largely inaccessible to powerful microscopes. In contrast, in-vitro models provide an attractive alternative to investigating pathogenic RPE changes associated with age and disease. In this article we describe the step-by-step approach required to establish an experimentally versatile in-vitro culture model of the outer retina incorporating the RPE monolayer and supportive Bruch’s membrane (BrM). We show that confluent monolayers of the spontaneously arisen human ARPE-19 cell-line cultured under optimal conditions reproduce key features of native RPE. These models can be used to study dynamic, intracellular and extracellular pathogenic changes using the latest developments in microscopy and imaging technology. We also discuss how RPE cells from human foetal and stem-cell derived sources can be incorporated alongside sophisticated BrM substitutes to replicate the aged/diseased outer retina in a dish. The work presented here will enable users to rapidly establish a realistic in-vitro model of the outer retina that is amenable to a high degree of experimental manipulation which will also serve as an attractive alternative to using animals. This in-vitro model therefore has the benefit of achieving the 3Rs objective of reducing and replacing the use of animals in research. As well as recapitulating salient structural and physiological features of native RPE, other advantages of this model include its simplicity, rapid set-up time and unlimited scope for detailed single-cell resolution and matrix studies.

scholarly journals Genetic LAMP2 deficiency accelerates the age-associated formation of basal laminar deposits in the retina

2019 ◽  
Vol 116 (47) ◽  
pp. 23724-23734 ◽  
Author(s):  
Shoji Notomi ◽  
Kenji Ishihara ◽  
Nikolaos E. Efstathiou ◽  
Jong-Jer Lee ◽  
Toshio Hisatomi ◽  
...  
Keyword(s):  
Basal Lamina ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Critical Role ◽  
Age Related Macular Degeneration ◽  
Bruch's Membrane ◽  
Bruch’S Membrane ◽  
Rpe Cells ◽  
Age Related ◽  
Deficient Mice

The early stages of age-related macular degeneration (AMD) are characterized by the accumulation of basal laminar deposits (BLamDs). The mechanism for BLamDs accumulating between the retinal pigment epithelium (RPE) and its basal lamina remains elusive. Here we examined the role in AMD of lysosome-associated membrane protein-2 (LAMP2), a glycoprotein that plays a critical role in lysosomal biogenesis and maturation of autophagosomes/phagosomes. LAMP2 was preferentially expressed by RPE cells, and its expression declined with age. Deletion of the Lamp2 gene in mice resulted in age-dependent autofluorescence abnormalities of the fundus, thickening of Bruch’s membrane, and the formation of BLamDs, resembling histopathological changes occurring in AMD. Moreover, LAMP2-deficient mice developed molecular signatures similar to those found in human AMD—namely, the accumulation of APOE, APOA1, clusterin, and vitronectin—adjacent to BLamDs. In contrast, collagen 4, laminin, and fibronectin, which are extracellular matrix proteins constituting RPE basal lamina and Bruch’s membrane were reduced in Lamp2 knockout (KO) mice. Mechanistically, retarded phagocytic degradation of photoreceptor outer segments compromised lysosomal degradation and increased exocytosis in LAMP2-deficient RPE cells. The accumulation of BLamDs observed in LAMP2-deficient mice was eventually followed by loss of the RPE and photoreceptors. Finally, we observed loss of LAMP2 expression along with ultramicroscopic features of abnormal phagocytosis and exocytosis in eyes from AMD patients but not from control individuals. Taken together, these results indicate an important role for LAMP2 in RPE function in health and disease, suggesting that LAMP2 reduction may contribute to the formation of BLamDs in AMD.

scholarly journals NLRP3 Inflammasome: Activation and Regulation in Age-Related Macular Degeneration

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Jiangyuan Gao ◽  
Ruozhou Tom Liu ◽  
Sijia Cao ◽  
Jing Z. Cui ◽  
Aikun Wang ◽  
...  
Keyword(s):  
Macular Degeneration ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
The Elderly ◽  
Geographic Atrophy ◽  
Age Related Macular Degeneration ◽  
Inflammasome Activation ◽  
Related Factors ◽  
Rpe Cells ◽  
Age Related

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly in industrialized countries. AMD is a multifactorial disease influenced by both genetic and environmental risk factors. Progression of AMD is characterized by an increase in the number and size of drusen, extracellular deposits, which accumulate between the retinal pigment epithelium (RPE) and Bruch’s membrane (BM) in outer retina. The major pathways associated with its pathogenesis include oxidative stress and inflammation in the early stages of AMD. Little is known about the interactions among these mechanisms that drive the transition from early to late stages of AMD, such as geographic atrophy (GA) or choroidal neovascularization (CNV). As part of the innate immune system, inflammasome activation has been identified in RPE cells and proposed to be a causal factor for RPE dysfunction and degeneration. Here, we will first review the classic model of inflammasome activation, then discuss the potentials of AMD-related factors to activate the inflammasome in both nonocular immune cells and RPE cells, and finally introduce several novel mechanisms for regulating the inflammasome activity.

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