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Biochemical and Biophysical Research... Aug 2022Thioredoxin (Trx) family proteins are key players in redox signaling. Here, we have analyzed glutaredoxin (Grx) 1 and Grx2 in age-related macular degeneration (AMD) and...
Thioredoxin (Trx) family proteins are key players in redox signaling. Here, we have analyzed glutaredoxin (Grx) 1 and Grx2 in age-related macular degeneration (AMD) and in retinal pigment epithelial (ARPE-19) cells. We hypothesized that these redoxins regulate cellular functions and signaling circuits such as cell proliferation, Wnt signaling and VEGF release that have been correlated to the pathophysiology of AMD. ARPE-19 cells were transfected with specific siRNAs to silence the expression of Grx1 and Grx2 and were analyzed for proliferation/viability, migration capacity, β-catenin activation, and VEGF release. An active site-mutated C-X-X-S Grx1 was utilized to trap interacting proteins present in ARPE-19 cell extracts. In both, AMD retinas and in ARPE-19 cells incubated under hypoxia/reoxygenation conditions, Grx1 showed an increased nuclear localization. Grx1-silenced ARPE-19 cells showed a significantly reduced proliferation and migration rate. Our trapping approach showed that Grx1 interacts with β-catenin in a dithiol-disulfide exchange reaction. Knock-down of Grx1 led to a reduction in both total and active β-catenin levels. These findings add redox control to the regulatory mechanisms of β-catenin signaling in the retinal pigment epithelium and open the door to novel therapeutic approaches in AMD that is currently treated with VEGF-inhibitors.
Topics: Cell Proliferation; Epithelial Cells; Glutaredoxins; Humans; Macular Degeneration; Retinal Pigment Epithelium; Retinal Pigments; Signal Transduction; Vascular Endothelial Growth Factor A; beta Catenin
PubMed: 35714567
DOI: 10.1016/j.bbrc.2022.06.030 -
Cells May 2023Mutations in the gene cause inherited retinal disease; however, the pathogenic mechanisms associated with RCBTB1 deficiency remain poorly understood. Here, we...
Mutations in the gene cause inherited retinal disease; however, the pathogenic mechanisms associated with RCBTB1 deficiency remain poorly understood. Here, we investigated the effect of RCBTB1 deficiency on mitochondria and oxidative stress responses in induced pluripotent stem cell (iPSC)-derived retinal pigment epithelial (RPE) cells from control subjects and a patient with -associated retinopathy. Oxidative stress was induced with tert-butyl hydroperoxide (tBHP). RPE cells were characterized by immunostaining, transmission electron microscopy (TEM), CellROX assay, MitoTracker assay, quantitative PCR and immunoprecipitation assay. Patient-derived RPE cells displayed abnormal mitochondrial ultrastructure and reduced MitoTracker fluorescence compared with controls. Patient RPE cells displayed increased levels of reactive oxygen species (ROS) and were more sensitive to tBHP-induced ROS generation than control RPE. Control RPE upregulated and expression in response to tBHP treatment; however, this response was highly attenuated in patient RPE. RCBTB1 was co-immunoprecipitated from control RPE protein lysates by antibodies for either UBE2E3 or CUL3. Together, these results demonstrate that deficiency in patient-derived RPE cells is associated with mitochondrial damage, increased oxidative stress and an attenuated oxidative stress response.
Topics: Humans; Reactive Oxygen Species; Antioxidants; Retinal Diseases; Epithelial Cells; Mitochondria; Retinal Pigments; Guanine Nucleotide Exchange Factors
PubMed: 37408192
DOI: 10.3390/cells12101358 -
Journal of Visualized Experiments : JoVE Apr 2023The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed...
The daily phagocytosis of photoreceptor outer segments by the retinal pigment epithelium (RPE) contributes to the accumulation of an intracellular aging pigment termed lipofuscin. The toxicity of lipofuscin is well established in Stargardt's disease, the most common inherited retinal degeneration, but is more controversial in age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. Determining lipofuscin toxicity in humans has been difficult, and animal models of Stargardt's have limited toxicity. Thus, in vitro models that mimic human RPE in vivo are needed to better understand lipofuscin generation, clearance, and toxicity. The majority of cell culture lipofuscin models to date have been in cell lines or have involved feeding RPE a single component of the complex lipofuscin mixture rather than fragments/tips of the entire photoreceptor outer segment, which generates a more complete and physiologic lipofuscin model. Described here is a method to induce the accumulation of lipofuscin-like material (termed undigestible autofluorescence material, or UAM) in highly differentiated primary human pre-natal RPE (hfRPE) and induced pluripotent stem cell (iPSC) derived RPE. UAM accumulated in cultures by repeated feedings of ultraviolet light-treated OS fragments taken up by the RPE via phagocytosis. The key ways that UAM approximates and differs from lipofuscin in vivo are also discussed. Accompanying this model of lipofuscin-like accumulation, imaging methods to distinguish the broad autofluorescence spectrum of UAM granules from concurrent antibody staining are introduced. Finally, to assess the impact of UAM on RPE phagocytosis capacity, a new method for quantifying outer segment fragment/tips uptake and breakdown has been introduced. Termed "Total Consumptive Capacity", this method overcomes potential misinterpretations of RPE phagocytosis capacity inherent in classic outer segment "pulse-chase" assays. The models and techniques introduced here can be used to study lipofuscin generation and clearance pathways and putative toxicity.
Topics: Animals; Humans; Lipofuscin; Retinal Pigments; Phagocytosis; Retinal Pigment Epithelium; Cell Line; Cells, Cultured
PubMed: 37125790
DOI: 10.3791/65242 -
Redox Biology Oct 2020The mitochondrial-derived peptides (MDPs) are a new class of small open reading frame encoded polypeptides with pleiotropic properties. The prominent members are Humanin... (Review)
Review
Mechanisms of protection of retinal pigment epithelial cells from oxidant injury by humanin and other mitochondrial-derived peptides: Implications for age-related macular degeneration.
The mitochondrial-derived peptides (MDPs) are a new class of small open reading frame encoded polypeptides with pleiotropic properties. The prominent members are Humanin (HN) and small HN-like peptide (SHLP) 2, which encode 16S rRNA, while mitochondrial open reading frame of the twelve S c (MOTS-c) encodes 12S rRNA of the mitochondrial genome. While the multifunctional properties of HN and its analog 14-HNG have been well documented, their protective role in the retinal pigment epithelium (RPE)/retina has been investigated only recently. In this review, we have summarized the multiple effects of HN and its analogs, SHLP2 and MOTS-c in oxidatively stressed human RPE and the regulatory pathways of signaling, mitochondrial function, senescence, and inter-organelle crosstalk. Emphasis is given to the mitochondrial functions such as biogenesis, bioenergetics, and autophagy in RPE undergoing oxidative stress. Further, the potential use of HN and its analogs in the prevention of age-related macular degeneration (AMD) are also presented. In addition, the role of novel, long-acting HN elastin-like polypeptides in nanotherapy of AMD and other ocular diseases stemming from oxidative damage is discussed. It is expected MDPs will become a promising group of mitochondrial peptides with valuable therapeutic applications in the treatment of retinal diseases.
Topics: Animals; Caenorhabditis elegans; Endothelial Cells; HEK293 Cells; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Macular Degeneration; Metal Nanoparticles; Mice; Mice, Inbred NOD; Mitochondria; Neurons; Oxidants; Peptides; RNA, Ribosomal, 16S; Retinal Pigments; Silver
PubMed: 32768357
DOI: 10.1016/j.redox.2020.101663 -
Investigative Ophthalmology & Visual... Aug 2023To model the in vivo effects of chloroquine on the retinal pigment epithelium in experimentally tractable cell culture systems and determine the effects of mild...
PURPOSE
To model the in vivo effects of chloroquine on the retinal pigment epithelium in experimentally tractable cell culture systems and determine the effects of mild chloroquine treatment on lysosome function and turnover.
METHODS
Effects of low-dose chloroquine treatment on lysosomal function and accessibility to newly endocytosed cargo were investigated in primary and embryonic stem cell-derived RPE cells and ARPE19 cells using fluorescence and electron microscopy of fluorescent and gold-labeled probes. Lysosomal protein expression and accumulation were measured by quantitative PCR and Western blotting.
RESULTS
Initial chloroquine-induced lysosome neutralization was followed by partial recovery, lysosomal expansion, and accumulation of undegraded endocytic, phagocytic, and autophagic cargo and inhibition of cathepsin D processing. Accumulation of enlarged lysosomes was accompanied by a gradual loss of accessibility of these structures to the endocytic pathway, implying impaired lysosome reformation. Chloroquine-induced accumulation of pro-cathepsin D, as well as the lysosomal membrane protein, LAMP1, was reproduced by treatment with protease inhibitors and preceded changes in lysosomal gene expression.
CONCLUSIONS
Low-dose chloroquine treatment inhibits lysosome reformation, causing a gradual depletion of lysosomes able to interact with cargo-carrying vacuoles and degrade their content. The resulting accumulation of newly synthesized pro-cathepsin D and LAMP1 reflects inhibition of normal turnover of lysosomal constituents and possibly lysosomes themselves. A better understanding of the mechanisms underlying lysosome reformation may reveal new targets for the treatment of chloroquine-induced retinopathy.
Topics: Humans; Chloroquine; Lysosomes; Phagocytosis; Autophagy; Retinal Diseases; Epithelial Cells; Retinal Pigments
PubMed: 37548963
DOI: 10.1167/iovs.64.11.10 -
Viruses Jan 2023Zika virus (ZIKV) causes microcephaly and congenital eye disease. The cellular and molecular basis of congenital ZIKV infection are not well understood. Here, we...
Zika virus (ZIKV) causes microcephaly and congenital eye disease. The cellular and molecular basis of congenital ZIKV infection are not well understood. Here, we utilized a biologically relevant cell-based system of human fetal retinal pigment epithelial cells (FRPEs), hiPSC-derived retinal stem cells (iRSCs), and retinal organoids to investigate ZIKV-mediated ocular cell injury processes. Our data show that FRPEs were highly susceptible to ZIKV infection exhibiting increased apoptosis, whereas iRSCs showed reduced susceptibility. Detailed transcriptomics and proteomics analyses of infected FRPEs were performed. Nucleoside analogue drug treatment inhibited ZIKV replication. Retinal organoids were susceptible to ZIKV infection. The Asian genotype ZIKV exhibited higher infectivity, induced profound inflammatory response, and dysregulated transcription factors involved in retinal organoid differentiation. Collectively, our study shows that ZIKV affects ocular cells at different developmental stages resulting in cellular injury and death, further providing molecular insight into the pathogenesis of congenital eye disease.
Topics: Humans; Zika Virus Infection; Zika Virus; Induced Pluripotent Stem Cells; Retina; Virus Replication; Eye Diseases; Organoids; Epithelial Cells; Retinal Pigments
PubMed: 36680182
DOI: 10.3390/v15010142 -
Immunity, Inflammation and Disease Dec 2022Diosgenin is a natural steroidal compound with reported antidiabetic and many other protective properties. This study aimed to explore the protective effect of diosgenin...
INTRODUCTION
Diosgenin is a natural steroidal compound with reported antidiabetic and many other protective properties. This study aimed to explore the protective effect of diosgenin on high-glucose (HG)-induced retinal pigment epithelial cells.
METHODS
HG-induced ARPE-19 cells were considered as a cell model of diabetic retinopathy (DR). The viability and apoptosis of ARPE-19 cells induced by HG treated with either diosgenin or Compound C (CC; dorsomorphin) were detected by Cell Counting Kit-8 assay and flow cytometric analysis. The expression of apoptosis-related proteins, inflammation-related proteins, and AMPK/Nrf2/HO-1 pathway-related proteins was detected by western blotting. The levels of inflammatory cytokines and detection of oxidative stress indexes were performed using the appropriate assay kits. The messenger RNA expression of inflammatory cytokines was detected by real-time quantitative polymerase chain reaction.
RESULTS
There was no obvious effect of diosgenin on the viability of ARPE-19 cells and the viability of ARPE-19 cells was significantly reduced after HG induction. However, diosgenin increased the viability, inhibited the apoptosis, and reduced the inflammatory response and oxidative stress of ARPE-19 cells induced by HG. In addition, diosgenin could activate the AMPK/Nrf2/HO-1 pathway. CC, an AMPK inhibitor, could reverse the above changes caused by diosgenin treatment in ARPE-19 cells induced by HG.
CONCLUSIONS
Diosgenin could protect ARPE-19 cells from inflammatory damage and oxidative stress induced by HG, by activating the AMPK/Nrf2/HO-1 pathway.
Topics: NF-E2-Related Factor 2; Diosgenin; AMP-Activated Protein Kinases; Oxidative Stress; Cytokines; Epithelial Cells; Retinal Pigments; Glucose
PubMed: 36444632
DOI: 10.1002/iid3.698 -
Frontiers in Immunology 2023Age related macular degeneration (AMD) is the most common cause of blindness in the elderly. Oxidative stress contributes to retinal pigment epithelium (RPE) dysfunction...
Age related macular degeneration (AMD) is the most common cause of blindness in the elderly. Oxidative stress contributes to retinal pigment epithelium (RPE) dysfunction and cell death thereby leading to AMD. Using improved RPE cell model systems, such as human telomerase transcriptase-overexpressing (hTERT) RPE cells (hTERT-RPE), pathophysiological changes in RPE during oxidative stress can be better understood. Using this model system, we identified changes in the expression of proteins involved in the cellular antioxidant responses after induction of oxidative stress. Some antioxidants such as vitamin E (tocopherols and tocotrienols) are powerful antioxidants that can reduce oxidative damage in cells. Alpha-tocopherol (α-Toc or αT) and gamma-tocopherol (γ-Toc or γT) are well-studied tocopherols, but signaling mechanisms underlying their respective cytoprotective properties may be distinct. Here, we determined what effect oxidative stress, induced by extracellularly applied tBHP in the presence and absence of αT and/or γT, has on the expression of antioxidant proteins and related signaling networks. Using proteomics approaches, we identified differential protein expression in cellular antioxidant response pathways during oxidative stress and after tocopherol treatment. We identified three groups of proteins based on biochemical function: glutathione metabolism/transfer, peroxidases and redox-sensitive proteins involved in cytoprotective signaling. We found that oxidative stress and tocopherol treatment resulted in unique changes in these three groups of antioxidant proteins indicate that αT and γT independently and by themselves can induce the expression of antioxidant proteins in RPE cells. These results provide novel rationales for potential therapeutic strategies to protect RPE cells from oxidative stress.
Topics: Humans; Aged; Antioxidants; Proteome; Oxidative Stress; Tocopherols; Macular Degeneration; Epithelial Cells; Retinal Pigments
PubMed: 37153596
DOI: 10.3389/fimmu.2023.1138519 -
Experimental Eye Research Feb 2024Human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) therapies are promising alternatives for the...
Human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) therapies are promising alternatives for the treatment of retinal degenerative diseases caused by RPE degeneration. The generation of autologous RPE cells from human adult donors, which has the advantage of avoiding immune rejection and teratoma formation, is an alternative cell resource to gain mechanistic insight into and test potential therapies for RPE degenerative diseases. Here, we found that limbal stem cells (LSCs) from hESCs and adult primary human limbus have the potential to produce RPE cells and corneal stromal stem cells (CSSCs). We showed that hESC-LSC-derived RPE cells (LSC-RPE) expressed RPE markers, had a phagocytic function, and synthesized tropical factors. Furthermore, during differentiation from LSCs to RPE cells, cells became pigmented, accompanied by a decrease in the level of LSC marker KRT15 and an increase in the level of RPE marker MITF. The Wnt signaling pathway plays a role in LSC-RPE fate transition, promotes MITF expression in the nucleus, and encourages RPE fate transition. In addition, we also showed that primary LSCs (pLSCs) from adult human limbus similar to hESC-LSC could generate RPE cells, which was supported by the co-expression of LSC and RPE cell markers (KRT15/OTX2, KRT15/MITF), suggesting the transition from pLSC to RPE cells, and typical polygonal morphology, melanization, RPE cell marker genes expression (TYR, RPE65), tight junction formation by ZO-1 expression, and the most crucial phagocytotic function. On the other hand, both hESC-LSCs and pLSCs also differentiated into CSSCs (LSC-CSSCs) that expressed stem cell markers (PAX6, NESTIN), presented MSC features, including surface marker expression and trilineage differentiation capability, like those in human CSSCs. Furthermore, the capability of pLSC-CSSC to differentiate into cells expressing keratocyte marker genes (ALDH3A1, PTGDS, PDK4) indicated the potential to induce keratocytes. These results suggest that the adult pLSC is an alternative cell resource, and its application provides a novel potential therapeutic avenue for preventing RPE dysfunction-related retinal degenerative diseases and corneal scarring.
Topics: Humans; Limbal Stem Cells; Induced Pluripotent Stem Cells; Retinal Pigment Epithelium; Cell Differentiation; Epithelial Cells; Retinal Pigments
PubMed: 38171475
DOI: 10.1016/j.exer.2023.109778 -
Discovery Medicine 2022Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell... (Review)
Review
Transepithelial/transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell culture models of endothelial and epithelial monolayers. The value of TEER reflects the physical structure and characteristics of epithelial/endothelial cells. TEER value is a preferred physiological indicator reflecting transport rate of ions and macromolecules through the paracellular pathway, which is used to evaluate permeability of paracellular pathway. TEER value has a high specificity for the permeability of reactive tightly connected complex. TEER value is an effective indicator to evaluate the integrity of cell barrier. The cell barrier not only controls the diffusion penetration of various substances in adjacent intercellular spaces, but also regulates the transport of ions and macromolecules across. On one hand, the cell barrier protects the body from harmful substances; on the other hand, it restricts the entry of therapeutic drugs. Therefore, with the increase of permeability in paraepithelial pathway, the TEER value decreased, otherwise, it increased. In this review article, we compared the advantages and disadvantages of the existing methods for measuring TEER and summarized the factors affecting TEER accuracy, as well as the roles of TEER in mechanisms of retinal pigment epithelial barrier and retinal disorders such as proliferative vitreoretinopathy (PVR), age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa.
Topics: Humans; Electric Impedance; Retinal Pigments; Endothelial Cells; Tight Junctions; Permeability; Epithelial Cells; Retinal Diseases
PubMed: 36274257
DOI: No ID Found