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International Journal of Molecular... Jun 2023Diabetic retinopathy (DR) is the leading cause of vision loss and a critical complication of diabetes with a very complex etiology. The build-up of reactive oxygen...
Diabetic retinopathy (DR) is the leading cause of vision loss and a critical complication of diabetes with a very complex etiology. The build-up of reactive oxygen species (ROS) due to hyperglycemia is recognized as a primary risk factor for DR. Although spermidine, a naturally occurring polyamine, has been reported to have antioxidant effects, its effectiveness in DR has not yet been examined. Therefore, in this study, we investigated whether spermidine could inhibit high glucose (HG)-promoted oxidative stress in human retinal pigment epithelial (RPE) cells. The results demonstrated that spermidine notably attenuated cytotoxicity and apoptosis in HG-treated RPE ARPE-19 cells, which was related to the inhibition of mitochondrial ROS production. Under HG conditions, interleukin (IL)-1β and IL-18's release levels were markedly increased, coupled with nuclear factor kappa B (NF-κB) signaling activation. However, spermidine counteracted the HG-induced effects. Moreover, the expression of nucleotide-binding oligomerization domain-like receptor (NLR) protein 3 (NLRP3) inflammasome multiprotein complex molecules, including TXNIP, NLRP3, ASC, and caspase-1, increased in hyperglycemic ARPE-19 cells, but spermidine reversed these molecular changes. Collectively, our findings demonstrate that spermidine can protect RPE cells from HG-caused injury by reducing ROS and NF-κB/NLRP3 inflammasome pathway activation, indicating that spermidine could be a potential therapeutic compound for DR treatment.
Topics: Humans; Inflammasomes; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Reactive Oxygen Species; Spermidine; Oxidative Stress; Diabetic Retinopathy; Glucose; Epithelial Cells; Retinal Pigments
PubMed: 37445726
DOI: 10.3390/ijms241310550 -
Aging Apr 2023High glucose promotes retinal pigment epithelial cell (RPEC) migration. However, the underlying molecular mechanisms explaining how high fatty acid levels affect RPEC...
High glucose promotes retinal pigment epithelial cell (RPEC) migration. However, the underlying molecular mechanisms explaining how high fatty acid levels affect RPEC migration remain largely unknown. We investigated whether and how palmitic acid (PA) impacts the migration of human RPEC cell line ARPE-19. ARPE-19 cells were treated with varying doses of palmitic acid, and the RPEC migration was evaluated by scratch and transwell migration assays. Cell viability was determined by the CCK-8 method. The levels of epithelial-mesenchymal transition (EMT)-associated proteins, including E-cadherin, vimentin, MMP2, and MMP3, were evaluated by western blot. The microRNAs and mRNAs levels were assessed by quantitative PCR. miRNA targets were predicted with online tools and validated with the luciferase reporter assay. miRNA mimics, inhibitors, and siRNA oligos were used to perform gain-of-function and loss-of-function studies. We found that PA increased viability of ARPE-19 cells, promoted their migration and EMT. PA decreased E-cadherin protein expression, and increased vimentin, MMP2, and MMP3 protein levels. Additionally, PA increased miR-222 expression in ARPE-19 cells, and functionally blocking miR-222 suppressed the PA-induced RPEC migration and EMT. NUMB was identified as a downstream target of miR-222, and NUMB knockdown abolished the effects of PA on promoting the migration and EMT of ARPE-19 cells. Therefore, PA promotes human RPEC migration by upregulating miR-222 expression and downregulating NUMB. This study unravels a novel PA-miR-222-NUMB axis that can be potentially targeted for therapy of high fat acid-related ocular diseases.
Topics: Humans; Cadherins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Epithelial Cells; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Matrix Metalloproteinase 2; Matrix Metalloproteinase 3; MicroRNAs; Palmitic Acid; Retinal Pigments; Vimentin
PubMed: 37566749
DOI: 10.18632/aging.204647 -
Journal of Vision Jul 2021Recently, we reported measurements of heterochromatic flicker photometry (HFP) in 22 young observers, with stimuli that (nominally) modulated only L- and M-cones and...
Recently, we reported measurements of heterochromatic flicker photometry (HFP) in 22 young observers, with stimuli that (nominally) modulated only L- and M-cones and were kept at (approximately) a constant multiple of detection threshold. These equiluminance settings were represented as the angle in the (L, M) cone contrast plane, with the greenish peak of the flicker in quadrant II and the reddish peak in quadrant IV; equiluminance settings were reported as the greenish angle. The mean equiluminance angle was 116.3° (an M:L cone contrast ratio of -2 at equiluminance), but individual differences in the settings were substantial, with the variation across individuals almost five times larger than the within-subject precision in the settings. In the present study we sought to determine the degree to which we could account for our observers' HFP settings by plausible variations in the macular pigment optical density (MPOD), the lens pigment optical density (LPOD), the cone photopigment optical densities (PPOD), and serine/alanine polymorphism in L-cone opsin (λmax shift). Most of the range of our measured equiluminance angles could be accounted for by these factors, although the largest two angles (smallest |ΔM/M: ΔL/L| ratio at equiluminance) could not. Individual differences in HFP have sometimes been taken to indicate variations in the ratio of L:M cone number; our results suggest that most of the individual differences in HFP might be equally well ascribed to physiological factors other than cone number. Simple linear models allow predictions of equiluminance angle, cone adapting level, and artifactual S-cone contrast from the values of the four factors considered here.
Topics: Cone Opsins; Humans; Lens, Crystalline; Photometry; Retinal Cone Photoreceptor Cells
PubMed: 34313713
DOI: 10.1167/jov.21.7.15 -
BMC Ophthalmology Aug 2023Epiretinal membranes in patients with proliferative vitreoretinopathy (PVR) consist of extracellular matrix and a number of cell types including retinal pigment...
BACKGROUND
Epiretinal membranes in patients with proliferative vitreoretinopathy (PVR) consist of extracellular matrix and a number of cell types including retinal pigment epithelial (RPE) cells and fibroblasts, whose contraction causes retinal detachment. In RPE cells depletion of platelet-derived growth factor (PDGF) receptor (PDGFR)β suppresses vitreous-induced Akt activation, whereas in fibroblasts Akt activation through indirect activation of PDGFRα by growth factors outside the PDGF family (non-PDGFs) plays an essential role in experimental PVR. Whether non-PDGFs in the vitreous, however, were also able to activate PDGFRβ in RPE cells remained elusive.
METHODS
The CRISPR/Cas9 technology was utilized to edit a genomic PDGFRB locus in RPE cells derived from an epiretinal membrane (RPEM) from a patient with PVR, and a retroviral vector was used to express a truncated PDGFRβ short of a PDGF-binding domain in the RPEM cells lacking PDGFRβ. Western blot was employed to analyze expression of PDGFRβ and α-smooth muscle actin, and signaling events (p-PDGFRβ and p-Akt). Cellular assays (proliferation, migration and contraction) were also applied in this study.
RESULTS
Expression of a truncated PDGFRβ lacking a PDGF-binding domain in the RPEM cells whose PDGFRB gene has been silent using the CRISPR/Cas9 technology restores vitreous-induced Akt activation as well as cell proliferation, epithelial-mesenchymal transition, migration and contraction. In addition, we show that scavenging reactive oxygen species (ROS) with N-acetyl-cysteine and inhibiting Src family kinases (SFKs) with their specific inhibitor SU6656 blunt the vitreous-induced activation of the truncated PDGFRβ and Akt as well as the cellular events related to the PVR pathogenesis. These discoveries suggest that in RPE cells PDGFRβ can be activated indirectly by non-PDGFs in the vitreous via an intracellular pathway of ROS/SFKs to facilitate the development of PVR, thereby providing novel opportunities for PVR therapeutics.
CONCLUSION
The data shown here will improve our understanding of the mechanism by which PDGFRβ can be activated by non-PDGFs in the vitreous via an intracellular route of ROS/SFKs and provide a conceptual foundation for preventing PVR by inhibiting PDGFRβ transactivation (ligand-independent activation).
Topics: Humans; Receptor, Platelet-Derived Growth Factor beta; Retinal Pigment Epithelium; Proto-Oncogene Proteins c-akt; Ligands; Reactive Oxygen Species; Vitreoretinopathy, Proliferative; Platelet-Derived Growth Factor; Epithelial Cells; Retinal Pigments; Cell Movement
PubMed: 37537538
DOI: 10.1186/s12886-023-03089-8 -
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 -
Developmental Biology Jan 2023Many animals depend on the sense of vision for survival. In eumetazoans, vision requires specialized, light-sensitive cells called photoreceptors. Light reaches the... (Review)
Review
Many animals depend on the sense of vision for survival. In eumetazoans, vision requires specialized, light-sensitive cells called photoreceptors. Light reaches the photoreceptors and triggers the excitation of light-detecting proteins called opsins. Here, we describe the story of visual opsin evolution from the ancestral bilaterian to the extant vertebrate lineages. We explain the mechanisms determining color vision of extant vertebrates, focusing on opsin gene losses, duplications, and the expression regulation of vertebrate opsins. We describe the sequence variation both within and between species that has tweaked the sensitivities of opsin proteins towards different wavelengths of light. We provide an extensive resource of wavelength sensitivities and mutations that have diverged light sensitivity in many vertebrate species and predict how these mutations were accumulated in each lineage based on parsimony. We suggest possible natural and sexual selection mechanisms underlying these spectral differences. Understanding how molecular changes allow for functional adaptation of animals to different environments is a major goal in the field, and therefore identifying mutations affecting vision and their relationship to photic selection pressures is imperative. The goal of this review is to provide a comprehensive overview of our current understanding of opsin evolution in vertebrates.
Topics: Animals; Opsins; Evolution, Molecular; Phylogeny; Vertebrates; Rod Opsins
PubMed: 36370769
DOI: 10.1016/j.ydbio.2022.10.014 -
Immunologic Research Oct 2022In addition to hypoxia, inflammation is capable of inducing vascular endothelial growth factor (VEGF) expression in human retinal pigment epithelial (RPE) cells....
In addition to hypoxia, inflammation is capable of inducing vascular endothelial growth factor (VEGF) expression in human retinal pigment epithelial (RPE) cells. Excessive levels of VEGF promote choroidal neovascularization and thereby contribute to the pathogenesis of wet age-related macular degeneration (AMD). Intravitreal anti-VEGF injections ameliorate pathological vessel neoformation in wet AMD but excessive dampening of VEGF can result in a degeneration of the RPE. In the present study, we induced VEGF production by exposing human ARPE-19 cells to the pro-inflammatory IL-1α and subsequently to hydroquinone, a component of tobacco smoke that is a major environmental risk factor for AMD. Effects were monitored by measuring the levels of VEGF and anti-angiogenic pigment epithelium-derived factor (PEDF) using an enzyme-linked immunosorbent assay (ELISA) technique. In addition, we measured the production of reactive oxygen species (ROS) using the 2',7'-dichlorofluorescin diacetate (H2DCFDA) probe and studied the effects of two anti-oxidants, ammonium pyrrolidinedithiocarbamate (APDC) and N-acetyl-cysteine (NAC), on VEGF production. Cellular and secreted VEGF as well as secreted PEDF levels were reduced at all tested hydroquinone concentrations (10, 50, or 200 µM); these effects were evident prior to any reduction of cell viability evoked by hydroquinone. Cell viability was carefully explored in our previous study and verified by microscoping in the present study. APDC further reduced the VEGF levels, whereas NAC increased them. The 50 μM concentration of hydroquinone increased ROS production in ARPE-19 cells primed with IL-1α. Hydroquinone disturbs the regulatory balance of VEGF and PEDF in inflammatory conditions. These data support the idea that hydroquinone mediates RPE degeneration by reducing VEGF levels and may predispose to dry AMD since VEGF is as well important for retinal integrity.
Topics: Ammonium Compounds; Antioxidants; Cells, Cultured; Cysteine; Humans; Hydroquinones; Reactive Oxygen Species; Retinal Pigment Epithelium; Retinal Pigments; Tobacco Smoke Pollution; Vascular Endothelial Growth Factor A
PubMed: 35661979
DOI: 10.1007/s12026-022-09300-0 -
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 -
Molecules (Basel, Switzerland) Feb 2023Inflammation and elevated expression of high temperature requirement A serine peptidase 1 (HTRA1) are known high risk factors for age-related macular degeneration (AMD)....
Inflammation and elevated expression of high temperature requirement A serine peptidase 1 (HTRA1) are known high risk factors for age-related macular degeneration (AMD). However, the specific mechanism that HTRA1 causes AMD and the relationship between HTRA1 and inflammation remains unclear. We found that lipopolysaccharide (LPS) induced inflammation enhanced the expression of HTRA1, NF-κB, and p-p65 in ARPE-19 cells. Overexpression of HTRA1 up-regulated NF-κB expression, and on the other hand knockdown of HTRA1 down-regulated the expression of NF-κB. Moreover, NF-κB siRNA has no significant effect on the expression of HTRA1, suggesting HTRA1 works upstream of NF-κB. These results demonstrated that HTRA1 plays a pivotal role in inflammation, explaining possible mechanism of overexpressed HTRA1-induced AMD. Celastrol, a very common anti-inflammatory and antioxidant drug, was found to suppress inflammation by inhibiting phosphorylation of p65 protein efficaciously in RPE cells, which may be applied to the therapy of age-related macular degeneration.
Topics: Humans; NF-kappa B; High-Temperature Requirement A Serine Peptidase 1; Lipopolysaccharides; Retinal Pigment Epithelium; Macular Degeneration; Inflammation; Epithelial Cells; Retinal Pigments
PubMed: 36903482
DOI: 10.3390/molecules28052236 -
Frontiers in Immunology 2022Age-related macular degeneration (AMD), the leading cause of irreversible blindness in elderly Caucasian populations, includes destruction of the blood-retina barrier...
Dabigatran and Wet AMD, Results From Retinal Pigment Epithelial Cell Monolayers, the Mouse Model of Choroidal Neovascularization, and Patients From the Medicare Data Base.
BACKGROUND
Age-related macular degeneration (AMD), the leading cause of irreversible blindness in elderly Caucasian populations, includes destruction of the blood-retina barrier (BRB) generated by the retinal pigment epithelium-Bruch's membrane complex (RPE/BrM), and complement activation. Thrombin is likely to get access to those structures upon BRB integrity loss. Here we investigate the potential role of thrombin in AMD by analyzing effects of the thrombin inhibitor dabigatran.
MATERIAL AND METHODS
MarketScan data for patients aged ≥65 years on Medicare was used to identify association between AMD and dabigatran use. ARPE-19 cells grown as mature monolayers were analyzed for thrombin effects on barrier function (transepithelial resistance; TER) and downstream signaling (complement activation, expression of connective tissue growth factor (CTGF), and secretion of vascular endothelial growth factor (VEGF)). Laser-induced choroidal neovascularization (CNV) in mouse is used to test the identified downstream signaling.
RESULTS
Risk of new wet AMD diagnosis was reduced in dabigatran users. In RPE monolayers, thrombin reduced TER, generated unique complement C3 and C5 cleavage products, led to C3d/MAC deposition on cell surfaces, and increased CTGF expression PAR1-receptor activation and VEGF secretion. CNV lesion repair was accelerated by dabigatran, and molecular readouts suggest that downstream effects of thrombin include CTGF and VEGF, but not the complement system.
CONCLUSIONS
This study provides evidence of association between dabigatran use and reduced exudative AMD diagnosis. Based on the cell- and animal-based studies, we suggest that thrombin modulates wound healing and CTGF and VEGF expression, making dabigatran a potential novel treatment option in AMD.
Topics: Animals; Choroidal Neovascularization; Dabigatran; Disease Models, Animal; Epithelial Cells; Medicare; Mice; Retinal Pigments; Thrombin; United States; Vascular Endothelial Growth Factor A; Wet Macular Degeneration
PubMed: 35784301
DOI: 10.3389/fimmu.2022.896274