Constancy of ERp29 Expression in Cultured Retinal Pigment Epithelial Cells in the Ccl2/Cx3cr1 Deficient Mouse Model of Age-Related Macular Degeneration (original) (raw)
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The impact of oxidative stress and inflammation on RPE degeneration in non-neovascular AMD
Progress in retinal and eye research, 2017
The retinal pigment epithelium (RPE) is a highly specialized, unique epithelial cell that interacts with photoreceptors on its apical side and with Bruch's membrane and the choriocapillaris on its basal side. Due to vital functions that keep photoreceptors healthy, the RPE is essential for maintaining vision. With aging and the accumulated effects of environmental stresses, the RPE can become dysfunctional and die. This degeneration plays a central role in age-related macular degeneration (AMD) pathobiology, the leading cause of blindness among the elderly in western societies. Oxidative stress and inflammation have both physiological and potentially pathological roles in RPE degeneration. Given the central role of the RPE, this review will focus on the impact of oxidative stress and inflammation on the RPE with AMD pathobiology. Physiological sources of oxidative stress as well as unique sources from photo-oxidative stress, the phagocytosis of photoreceptor outer segments, and ...
Conditional Induction of Oxidative Stress in RPE: A Mouse Model of Progressive Retinal Degeneration
Advances in Experimental Medicine and Biology, 2015
An appropriate animal model is essential to screening drugs or designing a treatment strategy for geographic atrophy. Since oxidative stress contributes to the pathological changes of the retinal pigment epithelium (RPE), we are reporting a new mouse AMD model of retinal degeneration by inducing mitochondrial oxidative stress in RPE. Sod2 the gene for manganese superoxide dismutase (MnSOD) was deleted in RPE layer using conditional knockout strategy. Fundus microscopy, SD-OCT and electroretinography were used to monitor retinal structure and function in living animals and microscopy was used to assess pathology post mortem. Tissue specific deletion of Sod2 caused elevated signs of oxidative stress, RPE dysfunction and showed some key features of AMD. Due to induction of oxidative stress, the conditional knockout mice show progressive reduction in ERG responses and thinning of outer nuclear layer (ONL) compared to non-induced littermates.
New Biomarkers in the Retina and RPE Under Oxidative Stress
Ocular Diseases, 2012
Early Biosignature of Retina Degeneration 123 are available, but most of these models do not represent the full spectrum of pathological changes observed in human AMD. Animal models that mimic the complex and progressive characteristics of AMD are extremely valuable for studying the pathogenesis of AMD and testing different treatment modalities. Phosphoproteins have been studied using chemical and affinity-based methods [Zhou et al., 2008; Tao et al., 2005]. However, the rapid and dynamic nature of the underlying changes and the low abundance of phosphoproteins reflecting their substoichiometry present challenges to the quantitative study of the phosphoproteome. Thus, the isolation of the phosphoproteome or phosphopeptidome represents a potentially advanced step in this analysis. We introduced a system-wide, unbiased, and high-throughput approach to investigate global phosphoproteome of oxidative stress-induced or aged RPE and retinal cells using phosphoproteome enrichment and labeling method. Phosphoprotein-enriched extracts from human RPE cells under stress were separated by two-dimensional (2D) electrophoresis. Serine, threonine, and tyrosine phosphorylation were visualized by 2D phospho-Western blotting and specific phosphorylation sites were analyzed by tandem mass spectrometry. We examined phosphoproteome changes under oxidative stress in vitro and aging-induced phosphoproteome in vivo. Our results suggest a positive correlation between early biomarkers of phosphoproteome under oxidative stress and RPE proteins from AMD patients. The outcome of the current work is the initial delineation of the underlying physiology of oxidative stress-mediated phosphorylation signaling in RPE apoptosis. In addition, our study suggests a stimulus for understanding oxidative stress-induced cytoskeletal changes and the aggregate formation mechanism by phosphorylations. As a consequence, an effective therapeutic approach and animal model based on the modulation of phosphorylations are expected to result.
Free Radical Biology and Medicine, 2018
Non-exudative age-related macular degeneration (NE-AMD) represents the leading cause of blindness in the elderly. The macular retinal pigment epithelium (RPE) lies in a high oxidative environment because its high metabolic demand, mitochondria concentration, reactive oxygen species levels, and macular blood flow. It has been suggested that oxidative stress-induced damage to the RPE plays a key role in NE-AMD pathogenesis. The fact that the disease limits to the macular region raises the question as to why this area is particularly susceptible. We have developed a NE-AMD model induced by superior cervical ganglionectomy (SCGx) in C57BL/6J mice, which reproduces the disease hallmarks exclusively circumscribed to the temporal region of the RPE/outer retina. The aim of this work was analyzing RPE regional differences that could explain AMD localized susceptibility. Lower melanin content, thicker basal infoldings, higher mitochondrial mass, and higher levels of antioxidant enzymes, were found in the temporal RPE compared with the nasal region. Moreover, SCGx induced a decrease in the antioxidant system, and in mitochondria mass, as well as an increase in mitochondria superoxide, lipid peroxidation products, nuclear Nrf2 and heme oxygenase-1 levels, and in the occurrence of damaged mitochondria exclusively at the temporal RPE. These findings suggest that despite the wellknown differences between the human and mouse retina, it might not be NE-AMD pathophysiology which conditions the localization of the disease, but the macular RPE histologic and metabolic specific attributes that make it more susceptible to choroid alterations leading initially to a localized RPE dysfunction/damage, and secondarily to macular degeneration.
Regulation Of Retinal Pigment Epithelial Cell Death Induced By Oxidative Stress
Investigative Ophthalmology & Visual Science, 2006
The retinoic acid derivative fenretinide (FR) is capable of transdifferentiating cultured retinal pigment epithelial (RPE) cells towards a neuronal-like phenotype, but the underlying mechanisms are not understood. To identify genes involved in this process we performed a microarray analysis of RPE cells pre-and post-FR treatment, and observed a marked down-regulation of AnnexinA8 (AnxA8) in transdifferentiated cells. To determine whether AnxA8 plays a role in maintaining RPE cell phenotype we directly manipulated AnxA8 expression in cultured and primary RPE cells using siRNA-mediated gene suppression, and over-expression of AnxA8-GFP in conjunction with exposure to FR. Treatment of RPE cells with AnxA8 siRNA recapitulated exposure to FR, with cell cycle arrest, neuronal transdifferentiation, and concomitant up-regulation of the neuronal markers calretinin and calbindin, as assessed by real-time PCR and immunofluorescence. In contrast, AnxA8 transient over-expression in ARPE-19 cells prevented FR-induced differentiation. Ectopic expression of AnxA8 in AnxA8-depleted cells led to decreased neuronal marker staining, and normal cell growth as judged by phosphohistone H3 staining, cell counting and cleaved caspase-3 levels. These data show that down-regulation of AnxA8 is both necessary and sufficient for neuronal transdifferentiation of RPE cells and reveal an essential role for AnxA8 as a key regulator of RPE phenotype. Retinal pigment epithelial (RPE) cells and the retina are developmentally derived from the same tissue; the optic vesicle neuroectoderm, and throughout life RPE cells perform a variety of functions to support and protect the retina. These include phagocytosis of photoreceptor outer segments 1 , adsorption of free radicals by pigment granules 2 and maintenance of ocular immune privilege by forming the outer blood-retina barrier 3. Another striking feature of RPE cells, in some species, is their capacity to transdifferentiate into precursor cells and regenerate neuronal tissue. Accordingly, in urodele amphibians such as newts, complete retinal regeneration occurs via RPE transdifferentiation following ocular neuronal injury regardless of life stage 4, 5. In mammals, however, the ability of RPE cells to transdifferentiate in vivo is lost during early embryonic development. Therefore, neuronal cell injury, of the type that occurs in neurodegenerative diseases such as retinitis pigmentosa or age-related macular degeneration, usually results in irreversible vision loss 6, 7. However, there is evidence that despite being largely post-mitotic, some mature RPE cells continue to divide 8, 9 mostly in the peripheral retina 10 , as well as during pathological complications following retinal detachment that lead to proliferative vitreoretinopathy 11. In contrast, when cultured ex vivo, RPE cells can be highly proliferative, though this is usually accompanied by substantial de-differentiation manifested as loss of pigment granules, cell polarity and expression of key RPE cell genes such as RPE65 and the Mer tyrosine kinase. Various studies have shown that under suitable conditions, a more authentic RPE cell phenotype can be restored, demonstrating the phenotypic plasticity of these cells in culture 12. Conversely, RPE transdifferentiation can be induced in vitro by basic fibroblast growth factor (bFGF) or retinoic acid (RA) 13-15 , factors known to play a key role in RPE reprogramming during development and retinal regeneration in urodeles 16. In this study, the RA derivative Fenretinide (FR) was used to induce transdifferentiation of RPE cells towards a neuronal-like phenotype as described previously 15, 17. FR exerts its properties in a similar manner to RA; upon
2021
Age-related Macular degeneration (AMD) is a degenerative disease of the macula affecting the elderly population. Treatment options are limited, partly due to the lack of understanding of AMD pathology and the sparse availability of research models, that replicate the complexity of the human macula and the intricate interplay of the genetic, aging and life-style risk factors contributing to AMD. One of the main genetic risks associated with AMD is located on Complement Factor H (CFH) gene, leading to an amino acid substitution in the FH protein (Y402H). However, the mechanism of how this FH variant promotes the onset of AMD remains unclear. Previously, we have shown that FH deprivation in RPE cells, via CFH silencing, leads to increased inflammation, metabolic impairment and vulnerability towards oxidative stress. In this study, we established a novel co-culture model comprised of CFH silenced RPE cells and porcine retinal explants derived from the visual streak of the porcine eyes, ...
Cleavage of the retinal pigment epithelium-specific protein RPE65 under oxidative stress
International Journal of Biological Macromolecules, 2010
The regeneration of the 11-cis-retinyl imine chromophore of rhodopsin during the visual cycle and mechanisms that control this process are central questions in the field of vision research. The retinal pigment epithelium (RPE)-specific protein RPE65 is centrally involved in the isomerization and hydrolysis of alltrans-retinyl esters. In this study, we investigated RPE65 cleavage and potential regulatory mechanisms under oxidative stress conditions. The D407 RPE cell cultures were exposed to H 2 O 2 (100-1000 M). Changes in the levels of RPE65 and proteins related to apoptosis were investigated using gel electrophoresis and western blotting. Mass spectrometry was used to confirm the identity of RPE65. C57BL/6J (M450) and C3HeB/FeJ (L450) mice were used for in vivo experiments. We found that a novel 45 kDa truncated fragment of the RPE65 protein, designated RPE45, appears in RPE cells upon light exposure or oxidative stress. RPE45 is generated in vitro by recombinant caspases via an ubiquitination-dependent mechanism. Collectively, our results indicate that oxidative stress during the visual cycle results in cleavage of RPE65.
Journal of Biological Chemistry, 2013
Background: Age-related macular degeneration (AMD) involves complement activation; however, initiating ligands and essential arms of the complement cascade are unknown. Results: Phospholipid neoepitopes recognized by natural IgM antibodies triggered lectin pathway in RPE injury models. Conclusion: A role for neoepitopes in triggering AMD pathology was identified. Significance: Identifying macular neoepitopes and components of the complement cascade essential for pathology will aid in developing new therapeutic approaches. Uncontrolled activation of the alternative complement pathway (AP) is thought to be associated with age-related macular degeneration. Previously, we have shown that in retinal pigmented epithelial (RPE) monolayers, oxidative stress reduced complement inhibition on the cell surface, resulting in sublytic complement activation and loss of transepithelial resistance (TER), but the potential ligand and pathway involved are unknown. ARPE-19 cells were grown as monolayers on transwell plates, and sublytic complement activation was induced with H 2 O 2 and normal human serum. TER deteriorated rapidly in H 2 O 2-exposed monolayers upon adding normal human serum. Although the effect required AP activation, AP was not sufficient, because elimination of MASP, but not C1q, prevented TER reduction. Reconstitution experiments to unravel essential components of the lectin pathway (LP) showed that both ficolin and mannan-binding lectin can activate the LP through natural IgM antibodies (IgM-C2) that recognize phospholipid cell surface modifications on oxidatively stressed RPE cells. The same epitopes were found on human primary embryonic RPE monolayers. Likewise, mouse laser-induced choroidal neovascularization, an injury that involves LP activation, could be increased in antibody-deficient rag1 ؊/؊ mice using the phospholipid-specific IgM-C2. In summary, using a combination of depletion and reconstitution strategies, we have shown that the LP is required to initiate the complement cascade following natural antibody recognition of neoepitopes, which is then further amplified by the AP. LP activation is triggered by IgM bound to phospholipids. Taken together, we have defined novel mechanisms of complement activation in oxidatively stressed RPE, linking molecular events involved in age-related macular degeneration, including the presence of natural antibodies and neoepitopes. Age-related macular degeneration (AMD), 2 which is characterized by progressive loss of central vision resulting from damage to the photoreceptor cells in the central area of the retina, the macula, is the leading cause of vision loss in the elderly of industrialized nations (1). Although AMD occurs in two forms, neovascular (wet) and atrophic (dry), both are associated with pathological lesions at the retinal pigmented epithelium (RPE)/ choroid interface in the macular region (2). Early AMD is characterized by a thickening of Bruch's membrane, which includes basal linear deposits and drusen (3). Additionally, changes in RPE morphology and pigmentation, and deterioration of its function as blood-retina barrier have been reported (4). Advanced AMD is characterized by additional subtype-specific morphological features exacerbating the early pathological damage (5). Dry AMD, or geographic atrophy, results from the loss of RPE followed by the loss of photoreceptors, whereas wet