ABSTRACT
Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) is an age-related macular degeneration-like (AMD-like) retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production in retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus that enabled simple, sensitive, and high-throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix, reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium-derived factor). In vivo, treatment of 8-month-old R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is an important demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of retinal degenerative diseases, including potentially AMD itself.
Subject(s)
Extracellular Matrix Proteins , Extracellular Matrix , Macular Degeneration , Retinal Pigment Epithelium , Animals , Mice , Retinal Pigment Epithelium/pathology , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/drug effects , Macular Degeneration/pathology , Macular Degeneration/genetics , Macular Degeneration/drug therapy , Macular Degeneration/metabolism , Humans , Extracellular Matrix Proteins/metabolism , Extracellular Matrix Proteins/genetics , Extracellular Matrix/metabolism , Extracellular Matrix/drug effects , Pyridines/pharmacology , Pyrimidines/pharmacology , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/genetics , Disease Models, Animal , Retinal Dystrophies/metabolism , Retinal Dystrophies/pathology , Retinal Dystrophies/genetics , Optic Disk Drusen/congenitalABSTRACT
Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are common retinal diseases responsible for most blindness in working-age and elderly populations. Oxidative stress and mitochondrial dysfunction play roles in these pathogenesis, and new therapies counteracting these contributors could be of great interest. Some molecules, like coenzyme Q10 (CoQ10), are considered beneficial to maintain mitochondrial homeostasis and contribute to the prevention of cellular apoptosis. We investigated the impact of adding CoQ10 (Q) to a nutritional antioxidant complex (Nutrof Total®; N) on the mitochondrial status and apoptosis in an in vitro hydrogen peroxide (H2O2)-induced oxidative stress model in human retinal pigment epithelium (RPE) cells. H2O2 significantly increased 8-OHdG levels (p < 0.05), caspase-3 (p < 0.0001) and TUNEL intensity (p < 0.01), and RANTES (p < 0.05), caspase-1 (p < 0.05), superoxide (p < 0.05), and DRP-1 (p < 0.05) levels, and also decreased IL1ß, SOD2, and CAT gene expression (p < 0.05) vs. control. Remarkably, Q showed a significant recovery in IL1ß gene expression, TUNEL, TNFα, caspase-1, and JC-1 (p < 0.05) vs. H2O2, and NQ showed a synergist effect in caspase-3 (p < 0.01), TUNEL (p < 0.0001), mtDNA, and DRP-1 (p < 0.05). Our results showed that CoQ10 supplementation is effective in restoring/preventing apoptosis and mitochondrial stress-related damage, suggesting that it could be a valid strategy in degenerative processes such as AMD or DR.
Subject(s)
Apoptosis , Hydrogen Peroxide , Oxidative Stress , Retinal Pigment Epithelium , Ubiquinone , Humans , Ubiquinone/analogs & derivatives , Ubiquinone/pharmacology , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/drug effects , Oxidative Stress/drug effects , Apoptosis/drug effects , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Mitochondria/metabolism , Mitochondria/drug effects , Antioxidants/pharmacology , Epithelial Cells/metabolism , Epithelial Cells/drug effects , Cell Line , Dietary SupplementsABSTRACT
Purpose: Retinal neovascularization is a significant feature of advanced age-related macular degeneration (AMD) and a major cause of blindness in patients with AMD. However, the underlying mechanism of this pathological neovascularization remains unknown. Iron metabolism has been implicated in various biological processes. This study was conducted to investigate the effects of iron metabolism on retinal neovascularization in neovascular AMD (nAMD). Methods: C57BL/6J and very low-density lipoprotein receptor (VLDLR) knockout (Vldlr-/-) mice, a murine model of nAMD, were used in this study. Bulk-RNA sequencing was used to identify differentially expressed genes. Western blot analysis was performed to test the expression of proteins. Iron chelator deferiprone (DFP) was administrated to the mice by oral gavage. Fundus fluorescein angiography was used to evaluate retinal vascular leakage. Immunofluorescence staining was used to detect macrophages and iron-related proteins. Results: RNA sequencing (RNA-seq) results showed altered transferrin expression in the retina and RPE of Vldlr-/- mice. Disrupted iron homeostasis was observed in the retina and RPE of Vldlr-/- mice. DFP mitigated iron overload and significantly reduced retinal neovascularization and vascular leakage. In addition, DFP suppressed the inflammation in Vldlr-/- retinas. The reduced signals of macrophages were observed at sites of neovascularization in the retina and RPE of Vldlr-/- mice after DFP treatment. Further, the IL-6/JAK2/STAT3 signaling pathway was activated in the retina and RPE of Vldlr-/- mice and reversed by DFP treatment. Conclusions: Disrupted iron metabolism may contribute to retinal neovascularization in nAMD. Restoring iron homeostasis by DFP could be a potential therapeutic approach for nAMD.
Subject(s)
Deferiprone , Disease Models, Animal , Homeostasis , Iron Chelating Agents , Iron , Mice, Inbred C57BL , Mice, Knockout , Retinal Neovascularization , Animals , Deferiprone/pharmacology , Deferiprone/therapeutic use , Iron Chelating Agents/pharmacology , Iron Chelating Agents/therapeutic use , Mice , Iron/metabolism , Retinal Neovascularization/metabolism , Retinal Neovascularization/drug therapy , Retinal Neovascularization/etiology , Retinal Neovascularization/pathology , Fluorescein Angiography , Receptors, LDL/genetics , Receptors, LDL/metabolism , Blotting, Western , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/pathology , Wet Macular Degeneration/drug therapy , Wet Macular Degeneration/metabolism , STAT3 Transcription Factor/metabolism , MaleABSTRACT
BACKGROUND: Age-related macular degeneration (AMD) is a prevalent ocular pathology affecting mostly the elderly population. AMD is characterized by a progressive retinal pigment epithelial (RPE) cell degeneration, mainly caused by an impaired antioxidative defense. One of the AMD therapeutic procedures involves injecting healthy RPE cells into the subretinal space, necessitating pure, healthy RPE cell suspensions. This study aims to electrically characterize RPE cells to demonstrate a possibility using simulations to separate healthy RPE cells from a mixture of healthy/oxidized cells by dielectrophoresis. METHODS: BPEI-1 rat RPE cells were exposed to hydrogen peroxide to create an in-vitro AMD cellular model. Cell viability was evaluated using various methods, including microscopic imaging, impedance-based real-time cell analysis, and the MTS assay. Healthy and oxidized cells were characterized by recording their dielectrophoretic spectra, and electric cell parameters (crossover frequency, membrane conductivity and permittivity, and cytoplasm conductivity) were computed. A COMSOL simulation was performed on a theoretical microfluidic-based dielectrophoretic separation chip using these parameters. RESULTS: Increasing the hydrogen peroxide concentration shifted the first crossover frequency toward lower values, and the cell membrane permittivity progressively increased. These changes were attributed to progressive membrane peroxidation, as they were diminished when measured on cells treated with the antioxidant N-acetylcysteine. The changes in the crossover frequency were sufficient for the efficient separation of healthy cells, as demonstrated by simulations. CONCLUSIONS: The study demonstrates that dielectrophoresis can be used to separate healthy RPE cells from oxidized ones based on their electrical properties. This method could be a viable approach for obtaining pure, healthy RPE cell suspensions for AMD therapeutic procedures.
Subject(s)
Cell Survival , Hydrogen Peroxide , Macular Degeneration , Retinal Pigment Epithelium , Retinal Pigment Epithelium/pathology , Retinal Pigment Epithelium/drug effects , Animals , Rats , Hydrogen Peroxide/toxicity , Hydrogen Peroxide/pharmacology , Electrophoresis/methods , Oxidative Stress , Cells, CulturedABSTRACT
BACKGROUND: Oxidative stress-induced retinal pigment epithelium (RPE) cell damage is a major factor in age-related macular degeneration (AMD). Vitamin D3 (VD3) is a powerful antioxidant and it has been suggested to have anti-aging properties and potential for treating AMD. This study aimed to investigate the effect of VD3 on RPE cell oxidative apoptosis of RPE cells in order to provide experimental evidence for the treatment of AMD. METHODS: Human retinal pigment epithelial cell 19 (ARPE-19) cells were divided into four groups: blank group (untreated), model group (incubated in medium with 400 µmol/L H2O2 for 1 h), VD3 group (incubated in medium with 100 µmol/L VD3 for 24 h), and treatment group (incubated in medium with 400 µmol/L H2O2 for 1 h and 100 µmol/L VD3 for 24 h). Cell viability, cell senescence, ROS content, expression levels of vitamin D specific receptors, Akt, Sirt1, NAMPT, and JNK mRNA expression levels, SOD activity, and MDA, GSH, and GPX levels were measured. RESULTS: We first established an ARPE-19 cell stress model with H2O2. Our control experiment showed that VD3 treatment had no significant effect on ARPE-19 cell viability within 6-48 h. Treating the stressed ARPE-19 cells with VD3 showed mixed results; caspase-3 expression was decreased, Bcl-2 expression was increased, MDA level of ARPE-19 cells was decreased, GSH-PX, GPX and SOD levels were increased, the relative mRNA expression levels of Akt, Sirt1, NAMPT were increased (P < 0.05), and the relative mRNA expression level of JNK was decreased (P < 0.05). CONCLUSION: VD3 can potentially slow the development of AMD.
Subject(s)
Apoptosis , Cell Survival , Oxidative Stress , Retinal Pigment Epithelium , Humans , Oxidative Stress/drug effects , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathology , Cell Survival/drug effects , Apoptosis/drug effects , Macular Degeneration/metabolism , Vitamins/pharmacology , Vitamin D/pharmacology , Antioxidants/pharmacology , Reactive Oxygen Species/metabolism , Cells, Cultured , Sirtuin 1/metabolism , Sirtuin 1/genetics , Cellular Senescence/drug effects , Cell Line , Hydrogen Peroxide/pharmacology , Hydrogen Peroxide/toxicityABSTRACT
PURPOSE: Bietti crystalline dystrophy (BCD) is an inherited retinal degeneration disease caused by mutations in the CYP4V2 gene. Currently, there is no clinical therapy approach available for BCD patients. Previous research has suggested that polyunsaturated fatty acids (PUFAs) may play a significant role in the development of BCD, implicating the involvement of ferroptosis in disease pathogenesis. In this work, we aimed to investigate the interplay between ferroptosis and BCD and to detect potential therapeutic strategies for the disease. METHODS: Genetic-edited RPE cell line was first established in this study by CRISPR-Cas9 technology. Cyp4v3 (the homologous gene of human CYP4V2) knock out (KO) mice have also been used. Lipid profiling and transcriptome analysis of retinal pigment epithelium (RPE) cells from Cyp4v3 KO mice have been conducted. Ferroptosis phenotypes have been first investigated in BCD models in vitro and in vivo, including lipid peroxidation, mitochondrial changes, elevated levels of reactive oxygen species (ROS), and altered gene expression. Additionally, an iron chelator, deferiprone (DFP), has been tested in vitro and in vivo to determine its efficacy in suppressing ferroptosis and restoring the BCD phenotype. RESULTS: Cyp4v3 KO mice exhibited progressive retinal degeneration and lipid accumulation, similar to the BCD phenotype, which was exacerbated by a high-fat diet (HFD). Increased levels of PUFAs, such as EPA (C22:5) and AA (C20:4), were observed in the RPE of Cyp4v3 KO mice. Transcriptome analysis of RPE in Cyp4v3 KO mice revealed changes in genes involved in iron homeostasis, particularly an upregulation of NCOA4, which was confirmed by immunofluorescence. Ferroptosis-related characteristics, including mitochondrial defects, lipid peroxidation, ROS accumulation, and upregulation of related genes, were detected in the RPE both in vitro and in vivo. Abnormal accumulation of ferrous iron was also detected. DFP, an iron chelator administration suppressed ferroptosis phenotype in CYP4V2 mutated RPE. Oral administration of DFP also restored the retinal function and morphology in Cyp4v3 KO mice. CONCLUSION: This study represented the first evidence of the substantial role of ferroptosis in the development of BCD. PUFAs resulting from CYP4V2 mutation may serve as substrates for ferroptosis, potentially working in conjunction with NCOA4-regulated iron accumulation, ultimately leading to RPE degeneration. DFP administration, which chelates iron, has demonstrated its ability to reverse BCD phenotype both in vitro and in vivo, suggesting a promising therapeutic approach in the future.
Subject(s)
Corneal Dystrophies, Hereditary , Ferroptosis , Mice, Knockout , Retinal Pigment Epithelium , Animals , Ferroptosis/genetics , Ferroptosis/drug effects , Corneal Dystrophies, Hereditary/genetics , Corneal Dystrophies, Hereditary/pathology , Corneal Dystrophies, Hereditary/metabolism , Corneal Dystrophies, Hereditary/drug therapy , Humans , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathology , Retinal Pigment Epithelium/drug effects , Mice , Reactive Oxygen Species/metabolism , Retinal Diseases/genetics , Retinal Diseases/pathology , Retinal Diseases/metabolism , Retinal Diseases/drug therapy , Cytochrome P450 Family 4/genetics , Mice, Inbred C57BL , Cell Line , Lipid Peroxidation/drug effectsABSTRACT
Age-related ocular diseases such as age-related macular degeneration, glaucoma, and diabetic retinopathy are major causes of irreversible vision impairment in the elderly. Conventional treatments focus on symptom relief and disease slowdown, often involving surgery, but fall short of providing a cure, leading to substantial vision loss. Regenerative medicine, particularly mesenchymal stem cells (MSCs), holds promise for ocular disease treatment. This study investigates the synergistic potential of combining placenta-derived MSCs (PD-MSCs) with Achyranthis radix extract (ARE) from Achyranthes japonica to enhance therapeutic outcomes. In a 24-h treatment, ARE significantly increased the proliferative capacity of PD-MSCs and delayed their senescence (* p < 0.05). ARE also enhanced antioxidant capabilities and increased the expression of regeneration-associated genes in an in vitro injured model using chemical damages on human retinal pigment epithelial cell line (ARPE-19) (* p < 0.05). These results suggest that ARE-primed PD-MSC have the capability to enhance the activation of genes associated with regeneration in the injured eye via increasing antioxidant properties. Taken together, these findings support the conclusion that ARE-primed PD-MSC may serve as an enhanced source for stem cell-based therapy in ocular diseases.
Subject(s)
Antioxidants , Mesenchymal Stem Cells , Placenta , Plant Extracts , Humans , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Antioxidants/pharmacology , Plant Extracts/pharmacology , Female , Placenta/metabolism , Placenta/drug effects , Pregnancy , Achyranthes/chemistry , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/cytology , Cell Proliferation/drug effects , Cell LineABSTRACT
Purpose: We sought to evaluate the efficacy of growth differentiation factor (GDF)-15 treatment for suppressing epithelial-mesenchymal transition (EMT) and alleviating transforming growth factor ß2 (TGFß2)-induced lens opacity. Methods: To test whether GDF-15 is a molecule that prevents EMT, we pretreated the culture with GDF-15 in neural progenitor cells, retinal pigment epithelial cells, and lens epithelial cells and then treated with factors that promote EMT, GDF-11, and TGFß2, respectively. To further investigate the efficacy of GDF-15 on alleviating lens opacity, we used mouse lens explant culture to mimic secondary cataracts. We pretreated the lens culture with GDF-15 and then added TGFß2 to develop lens opacity (n = 3 for each group). Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to measure EMT protein and gene expression, respectively. Results: In cell culture, GDF-15 pretreatment significantly attenuated EMT marker expression in cultured cells induced by treatment with GDF-11 or TGFß2. In the lens explant culture, GDF-15 pretreatment also reduced mouse lens opacity induced by exposure to TGFß2. Conclusions: Our results indicate that GDF-15 could alleviate TGFß2-induced EMT and is a potential therapeutic agent to slow or prevent posterior capsular opacification (PCO) progression after cataract surgery. Translational Relevance: Cataracts are the leading cause of blindness worldwide, with the only current treatment involving surgical removal of the lens and replacement with an artificial lens. However, PCO, also known as secondary cataract, is a common complication after cataract surgery. The development of an adjuvant that slows the progression of PCO will be beneficial to the field of anterior complications.
Subject(s)
Cataract , Epithelial-Mesenchymal Transition , Growth Differentiation Factor 15 , Lens, Crystalline , Transforming Growth Factor beta2 , Animals , Epithelial-Mesenchymal Transition/drug effects , Transforming Growth Factor beta2/metabolism , Transforming Growth Factor beta2/pharmacology , Growth Differentiation Factor 15/metabolism , Growth Differentiation Factor 15/genetics , Cataract/pathology , Cataract/metabolism , Cataract/prevention & control , Mice , Lens, Crystalline/metabolism , Lens, Crystalline/pathology , Lens, Crystalline/drug effects , Mice, Inbred C57BL , Cells, Cultured , Disease Models, Animal , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Blotting, Western , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/pathology , Retinal Pigment Epithelium/metabolismABSTRACT
Bisphenol A (BPA) may adversely affect human health by inducing oxidative stress and irreversible damage to cells. Bioactive compounds found in some functional foods, individually or in combination, can attenuate the negative effects of BPA exposure; an example is the multi-supplement containing guarana (Gua), selenium (Se), and L-carnitine (LC) -GSC- which has already demonstrated antioxidant, genoprotective, and immunomodulatory activities. This study aimed to determine the effect of GSC and its constituents on oxidative and genotoxic alterations triggered by BPA exposure in the retinal epithelial cell line. The cells exposed to BPA (0.001, 0.01, 0.1, 1, 3, and 10 µM) to determine the lowest concentration required to induce cyto-genotoxicity. ARPE-19 cells were then concomitantly exposed to the selected BPA concentration, GSC, and its components (Gua, 1.07 mg/mL; Se, 0.178 µg/mL; and LC, 1.43 mg/mL). Flow cytometry, biochemical assays, qRT-PCR, genotoxicity, apoptosis, and cellular proliferation. Based on our results, 10 µM of BPA could induce cyto-genotoxic and oxidative alterations. BPA did not alter the Bcl-2/BAX expression ratio but induced Casp3 and Casp8 overexpression, suggesting that apoptosis was induced mainly via the extrinsic pathway. GSC partially reversed the alterations triggered by BPA in ARPE-19 cells. However, Se had unexpected negative effects on ARPE-19 cells. The multi-supplement GSC may attenuate changes in oxidative and genotoxic markers related to exposure of ARPE-19 cells to BPA. our results revealed that the antioxidant, anti-apoptotic, and genoprotective properties of GSC were not universally shared by its individual, once Se did not exhibit any positive impact.
Subject(s)
Apoptosis , Benzhydryl Compounds , Carnitine , Oxidative Stress , Phenols , Retinal Pigment Epithelium , Selenium , Phenols/toxicity , Benzhydryl Compounds/toxicity , Humans , Selenium/pharmacology , Carnitine/pharmacology , Retinal Pigment Epithelium/drug effects , Oxidative Stress/drug effects , Apoptosis/drug effects , Cell Line , Paullinia/chemistry , DNA Damage/drug effects , Antioxidants/pharmacology , Epithelial Cells/drug effects , Flow Cytometry , Dietary SupplementsABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps cicadae (C.cicadae), named "Chan Hua", an anamorph of Isaria cicadae Miquel, is an entomogenous complex formed by fungi parasitizing on the larvae of cicadas and belongs to the Claviciptaceae family and the genus Codyceps, which traditionally holds a significant place in Chinese ethnopharmacology, specifically for eye clarity and as a remedy for age-related ocular conditions. The underlying mechanisms contributing to its eyesight enhancement and potential effectiveness against Age-related macular degeneration (AMD) remain unexplored. AIM OF THE STUDY: This study aims to elucidate the protective role of C.cicadae and its active ingredient, Myriocin (Myr), against AMD. MATERIALS AND METHODS: A chemical inducer was employed to make retinal pigment epithelium (RPE) damage in vitro and in vivo. The key ingredients of C.cicadae and their related mechanisms for anti-AMD were studied through bioinformatic analysis and molecular biological approaches. RESULTS: Myr was identified through high-performance liquid chromatography (HPLC) as an active ingredient in C.cicadae, and demonstrated a protective effect on RPE cells, reducing the structural damage and cell death induced by sodium iodate (SI). Further, Myr reduced eyelid secretions in AMD mice and restored their retinal structure and function. The differentially expressed genes (DEGs) in Myr treatment are primarily associated with TNF and Necroptosis signaling pathways. Molecular docking indicated a strong affinity between TNF and Myr. Myr inhibited the TNF signaling pathway thereby reducing the expression of inflammatory factors in ARPE-19 cells. Additionally, Myr had consistent action with the necroptosis inhibitor Necrostatin-1 (Nec-1), inhibited the RIPK1/RIPK3/MLKL pathway thereby protecting ARPE-19 cells. CONCLUSION: The findings present Myr, as a potent protector against SI-induced AMD, predominantly through modulation of the TNF-RIPK1/RIPK3/MLKL signaling pathway, offering the insights of therapeutic C.cicadae as viable candidates for AMD treatment.
Subject(s)
Cordyceps , Iodates , Macular Degeneration , Retinal Pigment Epithelium , Tumor Necrosis Factor-alpha , Animals , Macular Degeneration/drug therapy , Cordyceps/chemistry , Mice , Tumor Necrosis Factor-alpha/metabolism , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Signal Transduction/drug effects , Humans , Cell Line , Mice, Inbred C57BL , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Male , Necroptosis/drug effects , Fatty Acids, MonounsaturatedABSTRACT
Age-related macular degeneration (AMD) is one of the leading causes of blindness. AMD is currently incurable; the best solution is to prevent its occurrence. To develop drugs for AMD, it is crucial to have a model system that mimics the symptoms and mechanisms in patients. It is most important to develop safer and more effective anti-AMD drug. In this study, the dose of A2E and the intensity of blue light were evaluated to establish an appropriate atrophic in vitro model of AMD and anti-AMD effect and therapeutic mechanism of Codonopsis lanceolata. The experimental groups included a control group an AMD group treated with A2E and blue light, a lutein group treated with 25 µM lutein after AMD induction, and three groups treated with different doses of C. lanceolata (10, 20, and 50 µg/mL) after AMD induction. Intrinsic apoptotic pathway (Bcl-2 family), anti-oxidative system (Keap1/Nrf2/HO-1 antioxidant response element), and anti-carbonyl effect (4-hydroxynonenal [4-HNE]) were evaluated using immunofluorescence, MTT, TUNEL, FACS, and western blotting analyses. A2E accumulation in the cytoplasm of ARPE-19 cells depending on the dose of A2E. Cell viability of ARPE-19 cells according to the dose of A2E and/or blue light intensity. The population of apoptotic or necrotic cells increased based on the A2E dose and blue light intensity. Codonopsis lanceolata dose-dependently prevented cell death which was induced by A2E and blue light. The antiapoptotic effect of that was caused by activating Keap1/Nrf2/HO-1 pathway, suppressing 4-HNE, and modulating Bcl-2 family proteins like increase of antiapoptotic proteins such as Bcl-2 and Bcl-XL and decrease of proapoptotic protein such as Bim. Based on these findings, 30 µM A2E and 20 mW/cm2 blue light on adult retinal pigment epithelium-19 cells was an appropriate condition for AMD model and C. lanceolata shows promise as an anti-AMD agent.
Subject(s)
Apoptosis , Codonopsis , Macular Degeneration , NF-E2-Related Factor 2 , Oxidative Stress , Codonopsis/chemistry , Humans , Macular Degeneration/drug therapy , Macular Degeneration/metabolism , Macular Degeneration/pathology , Oxidative Stress/drug effects , Apoptosis/drug effects , NF-E2-Related Factor 2/metabolism , Plant Extracts/pharmacology , Antioxidants/pharmacology , Kelch-Like ECH-Associated Protein 1/metabolism , Cell Line , Aldehydes/pharmacology , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/pathology , Light/adverse effects , Proto-Oncogene Proteins c-bcl-2/metabolismABSTRACT
BACKGROUND: Retinal pigment epithelial (RPE) cells have a pivotal function in preserving the equilibrium of the retina and moderating the immunological interaction between the choroid and the retina. This study primarily focuses on delineating the protective effect offered by Kaempferol (Kae) against RPE cell damage. METHODS: Bioinformatics analysis was performed on the GSE30719 dataset to identify hub genes associated with RPE. Subsequently, we analyzed the impact of Kae on RPE apoptosis, cell viability, and inflammatory response through cell experiments, and explored the interaction between hub genes and Kae. RESULTS: Based on the GSE30719 dataset, nine hub genes (ISG15, IFIT1, IFIT3, STAT1, OASL, RSAD2, IRF7, MX2, and MX1) were identified, all of which were highly expressed in the GSE30719 case group. Kae could boost the proliferative activity of RPE cells caused by lipopolysaccharide (LPS), as well as reduce apoptosis and the generation of inflammatory factors (tumor necrosis factor receptor (TNFR), interleukin-1beta (IL-1ß)) and cytokines (IL-1, IL-6, IL-12). STAT1 was shown to inhibit cell proliferation, promote apoptosis, and secrete IL-1/IL-6/IL-12 in LPS-induced RPE cells. Moreover, IRF7 was found to interact with STAT1 in LPS-induced RPE cells, and STAT1 could maintain IRF7 levels through deubiquitination. In addition, we also found that the protective effect of Kae on LPS-induced RPE cell injury was mediated through STAT1/IRF7 axis. CONCLUSION: This study provided evidence that Kae protects RPE cells via regulating the STAT1/IRF7 signaling pathways, indicating its potential therapeutic relevance in the diagnosis and management of retinal disorders linked with RPE cell damage.
Subject(s)
Apoptosis , Interferon Regulatory Factor-7 , Kaempferols , Retinal Pigment Epithelium , STAT1 Transcription Factor , Ubiquitination , Humans , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/drug effects , STAT1 Transcription Factor/metabolism , Interferon Regulatory Factor-7/metabolism , Interferon Regulatory Factor-7/genetics , Ubiquitination/drug effects , Apoptosis/drug effects , Kaempferols/pharmacology , Cell Line , Cell Proliferation/drug effects , Epithelial Cells/metabolism , Epithelial Cells/drug effects , Cell Survival/drug effects , Lipopolysaccharides , Proteolysis/drug effects , Signal Transduction/drug effects , Cytokines/metabolism , Cytokines/geneticsABSTRACT
Sulfated marine polysaccharides, so-called fucoidans, have been shown to exhibit anti-inflammatory and immunomodulatory activities in retinal pigment epithelium (RPE). In this study, we tested the effects of different fucoidans (and of fucoidan-treated RPE cells) on retinal microglia to investigate whether its anti-inflammatory effect can be extrapolated to the innate immune cells of the retina. In addition, we tested whether fucoidan treatment influenced the anti-inflammatory effect of RPE cells on retinal microglia. Three fucoidans were tested (FVs from Fucus vesiculosus, Fuc1 and FucBB04 from Laminaria hyperborea) as well as the supernatant of primary porcine RPE treated with fucoidans for their effects on inflammatory activated (using lipopolysaccharide, LPS) microglia cell line SIM-A9 and primary porcine retinal microglia. Cell viability was detected with a tetrazolium assay (MTT), and morphology by Coomassie staining. Secretion of tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL1ß) and interleukin 8 (IL8) was detected with ELISA, gene expression (NOS2 (Nitric oxide synthase 2), and CXCL8 (IL8)) with qPCR. Phagocytosis was detected with a fluorescence assay. FucBB04 and FVs slightly reduced the viability of SIM-A9 and primary microglia, respectively. Treatment with RPE supernatants increased the viability of LPS-treated primary microglia. FVs and FucBB04 reduced the size of LPS-activated primary microglia, indicating an anti-inflammatory phenotype. RPE supernatant reduced the size of LPS-activated SIM-A9 cells. Proinflammatory cytokine secretion and gene expression in SIM-A9, as well as primary microglia, were not significantly affected by fucoidans, but RPE supernatants reduced the secretion of LPS-induced proinflammatory cytokine secretion in SIM-A9 and primary microglia. The phagocytosis ability of primary microglia was reduced by FucBB04. In conclusion, fucoidans exhibited only modest effects on inflammatorily activated microglia by maintaining their cell size under stimulation, while the anti-inflammatory effect of RPE cells on microglia irrespective of fucoidan treatment could be confirmed, stressing the role of RPE in regulating innate immunity in the retina.
Subject(s)
Cell Survival , Microglia , Polysaccharides , Retinal Pigment Epithelium , Microglia/drug effects , Microglia/metabolism , Animals , Polysaccharides/pharmacology , Swine , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/cytology , Cell Survival/drug effects , Phagocytosis/drug effects , Retina/drug effects , Retina/metabolism , Retina/cytology , Cell Line , Lipopolysaccharides/pharmacology , Anti-Inflammatory Agents/pharmacology , Cytokines/metabolismABSTRACT
Uveitis comprises a cluster of intraocular inflammatory disorders characterized by uncontrolled autoimmune responses and excessive oxidative stress leading to vision loss worldwide. In the present study, curcumin (CUR) was conjugated with polyvinylpyrrolidone (PVP) to form PVP-CUR nanoparticles with significantly elevated solubility and outstanding multiple radical scavenging abilities. In vitro studies revealed that PVP-CUR nanoparticles markedly mitigated oxidative stress and reduced apoptosis in a H2O2-induced human retinal pigment epithelial cell line (ARPE-19) and promoted phenotypic polarization from M1 to M2 in an LPS-induced human microglial cell line (HMC3). Further in vivo studies demonstrated the prominent therapeutic effects of PVP-CUR nanoparticles on experimental autoimmune uveitis (EAU), which relieved clinical and pathological progression, improved perfusion and tomographic manifestations of retinal vessels, and reduced blood-retinal barrier (BRB) leakage; these effects may be mediated by mitigating oxidative stress and attenuating macrophage/microglia-elicited inflammation. Notably, treatment with PVP-CUR nanoparticles was shown to regulate metabolite alterations in EAU rats, providing novel insights into the underlying mechanisms involved. Additionally, the PVP-CUR nanoparticles showed great biocompatibility in vivo. In summary, our study revealed that PVP-CUR nanoparticles may serve as effective and safe nanodrugs for treating uveitis and other oxidative stress- and inflammation-related diseases.
Subject(s)
Autoimmune Diseases , Curcumin , Nanoparticles , Oxidative Stress , Povidone , Uveitis , Animals , Curcumin/administration & dosage , Curcumin/pharmacology , Curcumin/chemistry , Curcumin/therapeutic use , Uveitis/drug therapy , Uveitis/immunology , Uveitis/metabolism , Povidone/chemistry , Povidone/administration & dosage , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Humans , Autoimmune Diseases/drug therapy , Cell Line , Oxidative Stress/drug effects , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Rats , Female , Rats, Inbred Lew , Blood-Retinal Barrier/drug effects , Blood-Retinal Barrier/metabolism , MaleABSTRACT
SCOPE: Xanthophylls, vital for ocular defense against blue light and reactive oxygen species, are prone to oxidative degradation; however, they may be regenerated antioxidant-rich plant phenols. Despite certain in vitro evidence, clinical studies show inconsistent findings and this may be due to varying phenolic reduction potentials. Therefore, the current study aims to investigate the ocular protective effect of various plant phenols combined with xanthophyll. METHODS AND RESULTS: Human retinal pigment epithelial cells (ARPE-19) are subjected to oxidative stress induced by hydrogen peroxide (H2O2) after xanthophyll and phenol pretreatment. Assessments include xanthophyll uptake, total antioxidant capacity, cell viability, intracellular reactive oxygen species levels, apoptosis, phagocytosis, and vascular endothelial growth factor formation. The study finds that while the combination of lutein with phenols does not show significant protective effects compared to lutein-only, zeaxanthin combined with phenols exhibits enhanced protection compared to both the zeaxanthin-only and control groups. CONCLUSION: The research reveals the complex relationship between xanthophylls and phenols, suggesting that the advantageous effects of their combination might vary among different xanthophylls. Caution is necessary when applying molecular theories to ocular health, and this necessitates further research, serving as a basis for proposing clinical trials to evaluate the efficacy of specific xanthophyll and phenol combinations.
Subject(s)
Antioxidants , Apoptosis , Cell Survival , Hydrogen Peroxide , Lutein , Oxidative Stress , Retinal Pigment Epithelium , Xanthophylls , Humans , Oxidative Stress/drug effects , Hydrogen Peroxide/pharmacology , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Cell Line , Cell Survival/drug effects , Apoptosis/drug effects , Xanthophylls/pharmacology , Lutein/pharmacology , Antioxidants/pharmacology , Phenols/pharmacology , Reactive Oxygen Species/metabolism , Vascular Endothelial Growth Factor A/metabolism , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Zeaxanthins/pharmacology , Phagocytosis/drug effectsABSTRACT
Age-related macular degeneration (AMD) is one of the major causes of blindness in the elderly worldwide. Anti-vascular endothelial growth factor (VEGF) drugs have been widely used to treat the neovascular type of AMD (nAMD). However, VEGF acts not only as a pro-angiogenic factor but also as an anti-apoptotic factor in the eyes. In this study, we found that anti-VEGF drugs, including bevacizumab (Bev), ranibizumab (Ran), and aflibercept (Afl), induced epithelial-mesenchymal transition (EMT) in ARPE-19 cells in vitro, accompanied by the induction of CCN2, a potent pro-fibrotic factor. Similarly, intravitreal injection of Afl into mouse eyes resulted in EMT in the retinal pigmented epithelium (RPE). Co-treatment with CCN5, an anti-fibrotic factor that down-regulates CCN2 expression, significantly attenuated the adverse effects of the anti-VEGF drugs both in vitro and in vivo. Inhibition of the VEGF signaling pathway with antagonists of VEGF receptors, SU5416 and ZM323881, induced EMT and up-regulated CCN2 in ARPE-19 cells. Additionally, knock-down of CCN2 with siRNA abolished the adverse effects of the anti-VEGF drugs in ARPE-19 cells. Collectively, these results suggest that anti-VEGF drugs induce EMT in RPE through the induction of CCN2 and that co-treatment with CCN5 attenuates the adverse effects of anti-VEGF drugs in mouse eyes.
Subject(s)
Epithelial-Mesenchymal Transition , Retinal Pigment Epithelium , Vascular Endothelial Growth Factor A , Epithelial-Mesenchymal Transition/drug effects , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/pathology , Animals , Humans , Mice , Vascular Endothelial Growth Factor A/metabolism , Macular Degeneration/metabolism , Macular Degeneration/pathology , Macular Degeneration/drug therapy , Macular Degeneration/chemically induced , Cell Line , Bevacizumab/pharmacology , CCN Intercellular Signaling Proteins/metabolism , CCN Intercellular Signaling Proteins/genetics , Angiogenesis Inhibitors/pharmacology , Ranibizumab/pharmacology , Recombinant Fusion Proteins/pharmacology , Signal Transduction/drug effects , Repressor Proteins , Receptors, Vascular Endothelial Growth FactorABSTRACT
Excessive hydrogen peroxide (H2O2) generated during retinal cell metabolic activity could lead to oxidative degeneration of retinal pigment epithelium (RPE) tissue, a specific pathological process implicated in various retinal diseases resulting in blindness, which can be mitigated by taking dietary antioxidants to prevent inflammation and impaired cellular dysfunction. This study tested the hypothesis that damages induced by oxidative stresses can be mitigated by lutein in a H2O2-challenged model, which was based on an ARPE-19 cell monolayer cultured on three-dimensional (3D)-printed fibrous scaffolds. Pretreating these models with lutein (0.5 µM) for 24 h can significantly lower the oxidative stress and maintain phagocytosis and barrier function. Moreover, lutein can modulate the NLRP3 inflammasome, leading to a â¼40% decrease in the pro-inflammatory cytokine (IL-1ß and IL-18) levels. Collectively, this study suggests that the 3D RPE model is an effective tool to examine the capability of lutein to modulate cellular functionalities and regulate NLRP3 inflammation.
Subject(s)
Hydrogen Peroxide , Inflammasomes , Lutein , NLR Family, Pyrin Domain-Containing 3 Protein , Oxidative Stress , Retinal Pigment Epithelium , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/cytology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Humans , Inflammasomes/metabolism , Inflammasomes/drug effects , Hydrogen Peroxide/metabolism , Lutein/pharmacology , Oxidative Stress/drug effects , Cell Line , Interleukin-1beta/metabolism , Interleukin-1beta/genetics , Interleukin-18/metabolism , Models, BiologicalSubject(s)
Apoptosis , Heme Oxygenase-1 , Hydrogen Peroxide , NF-E2-Related Factor 2 , Oxidative Stress , Retinal Pigment Epithelium , Signal Transduction , NF-E2-Related Factor 2/metabolism , Oxidative Stress/drug effects , Hydrogen Peroxide/toxicity , Hydrogen Peroxide/pharmacology , Apoptosis/drug effects , Humans , Heme Oxygenase-1/metabolism , Heme Oxygenase-1/genetics , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/drug effects , Signal Transduction/drug effects , Triterpenes/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Cell Line , Membrane Proteins/metabolism , Membrane Proteins/genetics , AnimalsABSTRACT
Here, we test whether early visual and OCT rod energy-linked biomarkers indicating pathophysiology in nicotinamide nucleotide transhydrogenase (Nnt)-null 5xFAD mice also occur in Nnt-intact 5xFAD mice and whether these biomarkers can be pharmacologically treated. Four-month-old wild-type or 5xFAD C57BL/6 substrains with either a null (B6J) Nnt or intact Nnt gene (B6NTac) and 5xFAD B6J mice treated for one month with either R-carvedilol + vehicle or only vehicle (0.01% DMSO) were studied. The contrast sensitivity (CS), external limiting membrane-retinal pigment epithelium (ELM-RPE) thickness (a proxy for low pH-triggered water removal), profile shape of the hyperreflective band just posterior to the ELM (i.e., the mitochondrial configuration within photoreceptors per aspect ratio [MCP/AR]), and retinal laminar thickness were measured. Both wild-type substrains showed similar visual performance indices and dark-evoked ELM-RPE contraction. The lack of a light-dark change in B6NTac MCP/AR, unlike in B6J mice, is consistent with relatively greater mitochondrial efficiency. 5xFAD B6J mice, but not 5xFAD B6NTac mice, showed lower-than-WT CS. Light-adapted 5xFAD substrains both showed abnormal ELM-RPE contraction and greater-than-WT MCP/AR contraction. The inner retina and superior outer retina were thinner. Treating 5xFAD B6J mice with R-carvedilol + DMSO or DMSO alone corrected CS and ELM-RPE contraction but not supernormal MCP/AR contraction or laminar thinning. These results provide biomarker evidence for prodromal photoreceptor mitochondrial dysfunction/oxidative stress/oxidative damage, which is unrelated to visual performance, as well as the presence of the Nnt gene. This pathophysiology is druggable in 5xFAD mice.
Subject(s)
Dimethyl Sulfoxide , Mice, Inbred C57BL , Animals , Mice , Dimethyl Sulfoxide/pharmacology , Biomarkers/metabolism , Mice, Transgenic , Tomography, Optical Coherence , Retinal Rod Photoreceptor Cells/drug effects , Contrast Sensitivity/drug effects , Contrast Sensitivity/physiology , Disease Models, Animal , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/pathology , Retinal Pigment Epithelium/metabolism , Vision, Ocular/drug effects , Vision, Ocular/physiologyABSTRACT
Diabetic retinopathy (DR) is a secondary complication of diabetes that can lead to visual impairment and blindness. The retinal pigment epithelium (RPE) is a monolayer of pigment cells that forms the blood-retinal barrier (BRB) via tight junction (TJ) proteins and plays a crucial role in the physiological function of the retina. Hyperglycemia induces RPE death and BRB breakdown, which accelerates the process of DR. Curcumin, an active extract of Curcuma longa , has anti-inflammatory, antioxidant, antiapoptotic, and neuroprotective properties. However, the effect of Curcumin on the BRB under high glucose conditions remains unknown. This study aimed to investigate the protective effects of Curcumin on RPE physiology in vitro and in vivo . Curcumin significantly alleviated cell viability inhibition under high glucose conditions. Moreover, high glucose reduced extracellular signal-regulated kinase and Akt pathways activation to diminish RPE cell growth but reversed by Curcumin treatment. Curcumin protected not only TJ integrity but also retinoid regeneration through TJ proteins and isomerase modulation in diabetic retina. Furthermore, Curcumin decreased the expression of angiogenic factor to inhibit retinal neovascularization. Finally, Curcumin treatment markedly reduced apoptosis during hyperglycemia. In conclusion, Curcumin can alleviate the progression of DR by promoting RPE survival, TJ integrity, retinoid isomerase activity, RPE senescence inhibition, and neovascularization. Therefore, Curcumin exhibits high potential for use as a therapeutic agent for early DR.