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1.
Int J Mol Sci ; 22(6)2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33810153

RESUMO

Currently, retinal pigment epithelium (RPE) transplantation includes sheet and single-cell transplantation, the latter of which includes cell death and may be highly immunogenic, and there are some issues to be improved in single-cell transplantation. Y-27632 is an inhibitor of Rho-associated protein kinase (ROCK), the downstream kinase of Rho. We herein investigated the effect of Y-27632 in vitro on retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE cells), and also its effects in vivo on the transplantation of iPS-RPE cell suspensions. As a result, the addition of Y-27632 in vitro showed suppression of apoptosis, promotion of cell adhesion, and higher proliferation and pigmentation of iPS-RPE cells. Y-27632 also increased the viability of the transplant without showing obvious retinal toxicity in human iPS-RPE transplantation into monkey subretinal space in vivo. Therefore, it is possible that ROCK inhibitors can improve the engraftment of iPS-RPE cell suspensions after transplantation.


Assuntos
Sobrevivência de Enxerto/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Inibidores de Proteínas Quinases/farmacologia , Transplante de Células-Tronco , Quinases Associadas a rho/antagonistas & inibidores , Amidas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biomarcadores , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Humanos , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Macaca fascicularis , Piridinas/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo
2.
Adv Exp Med Biol ; 1256: 237-264, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33848005

RESUMO

Strong experimental evidence from studies in human donor retinas and animal models supports the idea that the retinal pathology associated with age-related macular degeneration (AMD) involves mitochondrial dysfunction and consequent altered retinal metabolism. This chapter provides a brief overview of mitochondrial structure and function, summarizes evidence for mitochondrial defects in AMD, and highlights the potential ramifications of these defects on retinal health and function. Discussion of mitochondrial haplogroups and their association with AMD brings to light how mitochondrial genetics can influence disease outcome. As one of the most metabolically active tissues in the human body, there is strong evidence that disruption in key metabolic pathways contributes to AMD pathology. The section on retinal metabolism reviews cell-specific metabolic differences and how the metabolic interdependence of each retinal cell type creates a unique ecosystem that is disrupted in the diseased retina. The final discussion includes strategies for therapeutic interventions that target key mitochondrial pathways as a treatment for AMD.


Assuntos
DNA Mitocondrial , Degeneração Macular , Animais , DNA Mitocondrial/metabolismo , Ecossistema , Humanos , Degeneração Macular/genética , Degeneração Macular/metabolismo , Mitocôndrias/genética , Retina , Epitélio Pigmentado da Retina/metabolismo
3.
Mol Med Rep ; 23(5)2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33760200

RESUMO

Proliferative vitreoretinopathy (PVR) is a disease leading to the formation of contractile preretinal membranes (PRMs) and is one of the leading causes of blindness. Connective tissue growth factor (CTGF) has been identified as a possible key determinant of progressive tissue fibrosis and excessive scarring. Therefore, the present study investigated the role and mechanism of action of CTGF in PVR. Immunohistochemical staining was performed to detect the expression of CTGF, fibronectin and collagen type III in PRMs from patients with PVR. The effects and mechanisms of recombinant human CTGF and its upstream regulator, TGF­ß1, on epithelial­mesenchymal transition (EMT) and the synthesis of extracellular matrix (ECM) by retinal pigment epithelium (RPE) cells were investigated using reverse transcription­quantitative PCR, western blotting and a [3H]proline incorporation assay. The data indicated that CTGF, fibronectin and collagen type III were highly expressed in PRMs. In vitro, CTGF significantly decreased the expression of the epithelial markers ZO­1 and E­cadherin and increased that of the mesenchymal markers fibronectin, N­cadherin and α­smooth muscle actin in a concentration­dependent manner. Furthermore, the expression of the ECM protein collagen type III was upregulated by CTGF. However, the trends in expression for the above­mentioned markers were reversed after knocking down CTGF. The incorporation of [3H]proline into RPE cells was also increased by CTGF. In addition, 8­Bromoadenosine cAMP inhibited CTGF­stimulated collagen synthesis and transient transfection of RPE cells with a CTGF antisense oligonucleotide inhibited TGF­ß1­induced collagen synthesis. The phosphorylation of PI3K and AKT in RPE cells was promoted by CTGF and TGF­ß1 and the latter promoted the expression of CTGF. The results of the present study indicated that CTGF may promote EMT and ECM synthesis in PVR via the PI3K/AKT signaling pathway and suggested that targeting CTGF signaling may have a therapeutic or preventative effect on PVR.


Assuntos
Fator de Crescimento do Tecido Conjuntivo/genética , Transição Epitelial-Mesenquimal/genética , Pigmentos da Retina/genética , Fator de Crescimento Transformador beta1/genética , Vitreorretinopatia Proliferativa/genética , Western Blotting , Movimento Celular/genética , Matriz Extracelular/genética , Fibronectinas/genética , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais/genética , Vitreorretinopatia Proliferativa/patologia
4.
Nat Metab ; 3(3): 366-377, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33758422

RESUMO

Macular telangiectasia type 2 (MacTel) is a progressive, late-onset retinal degenerative disease linked to decreased serum levels of serine that elevate circulating levels of a toxic ceramide species, deoxysphingolipids (deoxySLs); however, causal genetic variants that reduce serine levels in patients have not been identified. Here we identify rare, functional variants in the gene encoding the rate-limiting serine biosynthetic enzyme, phosphoglycerate dehydrogenase (PHGDH), as the single locus accounting for a significant fraction of MacTel. Under a dominant collapsing analysis model of a genome-wide enrichment analysis of rare variants predicted to impact protein function in 793 MacTel cases and 17,610 matched controls, the PHGDH gene achieves genome-wide significance (P = 1.2 × 10-13) with variants explaining ~3.2% of affected individuals. We further show that the resulting functional defects in PHGDH cause decreased serine biosynthesis and accumulation of deoxySLs in retinal pigmented epithelial cells. PHGDH is a significant locus for MacTel that explains the typical disease phenotype and suggests a number of potential treatment options.


Assuntos
Haploinsuficiência , Fosfoglicerato Desidrogenase/genética , Telangiectasia Retiniana/genética , Serina/biossíntese , Estudos de Coortes , Humanos , Fenótipo , Epitélio Pigmentado da Retina/metabolismo
5.
Biochem Biophys Res Commun ; 545: 8-13, 2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33545636

RESUMO

Dysregulation of Wnt signaling is implicated in multiple ocular disorders. The roles of Wnt co-receptors LRP5 and LRP6 in Wnt signaling regulation remain elusive, as most retinal cells express both of the co-receptors. To address this question, LRP5 and LRP6 were individually knocked-out in a human retinal pigment epithelium cell line using the CRISPR-Cas9 technology. Wnt signaling activity induced by various Wnt ligands was measured using wild-type and the KO cell lines. The results identified three groups of Wnt ligands based on their co-receptor specificity: 1) activation of Wnt signaling only through LRP6, 2) through both LRP5 and LRP6 and 3) predominantly through LRP5. These results indicate that LRP5 and LRP6 have differential roles in Wnt signaling regulation.


Assuntos
Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Via de Sinalização Wnt , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Meios de Cultivo Condicionados , Receptores Frizzled/metabolismo , Técnicas de Inativação de Genes , Humanos , Ligantes , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/deficiência , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/deficiência , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Epitélio Pigmentado da Retina/citologia , Retinoides/metabolismo , Retinoides/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3A/metabolismo
6.
Int J Mol Sci ; 22(4)2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567500

RESUMO

Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness worldwide in the elderly population. In our previous studies, we found that deficiencies in the nuclear factor, erythroid 2 like 2 (NFE2L2) and peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) genes caused AMD-like pathological phenotypes in mice. In the present work, we show hijacked epithelial-mesenchymal transition (EMT) due to the common loss of PGC-1α and NFE2L2 (double knock-out, dKO) genes in aged animals. The implanted area was assessed by histology, immunohistochemistry and transmission electron microscopy. Confocal microscopy revealed altered regions in the filamentous actin ring. This contrasted with hexagonal RPE morphology in wild-type mice. The ultrastructural RPE features here illustrated loss of apical microvilli, alteration of cell-cell contact, loss of basal in-folding with deposits on Bruch's membrane, and excessive lipofuscin deposition in dKO samples. We also found the expression of epithelial-mesenchymal transition transcription factors, such as Snail, Slug, collagen 1, vimentin and OB-cadherin, to be significantly different in dKO RPEs. An increased immunoreactivity of senescence markers p16, DEC1 and HMGB1 was also noted. These findings suggest that EMT and senescence pathways may intersect in the retinas of dKO mice. Both processes can be activated by damage to the RPE, which may be caused by increased oxidative stress resulting from the absence of NFE2L2 and PGC-1α genes, important for antioxidant defense. This dKO model may provide useful tools for studying AMD pathogenesis and evaluating novel therapies for this disease.


Assuntos
Senescência Celular , Transição Epitelial-Mesenquimal , Mitocôndrias/patologia , Fator 2 Relacionado a NF-E2/fisiologia , Estresse Oxidativo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/fisiologia , Epitélio Pigmentado da Retina/patologia , Animais , Degeneração Macular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fenótipo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais
7.
Int J Mol Sci ; 22(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33572787

RESUMO

Age-related macular degeneration (AMD) is the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris among elderly individuals and is the leading cause of blindness worldwide. Thus, a better understanding of the underlying mechanisms in retinal tissue activated by blue light exposure is important for developing novel treatment and intervention strategies. In this study, blue-light-emitting diodes with a wavelength of 440 nm were applied to RPE cells at a dose of 3.7 ± 0.75 mW/cm2 for 24 h. ARPE-19 cells were used to investigate the underlying mechanism induced by blue light exposure. A trypan blue exclusion assay was used for the cell viability determination. Flow cytometry was used for apoptosis rate detection and autophagy analysis. An immunofluorescence microscopy analysis was used to investigate cellular oxidative stress and DNA damage using DCFDA fluorescence staining and an anti-γH2AX antibody. Blue light exposure of zebrafish larvae was established to investigate the effect on retinal tissue development in vivo. To further demonstrate the comprehensive effect of blue light on ARPE-19 cells, next-generation sequencing (NGS) was performed for an ingenuity pathway analysis (IPA) to reveal additional related mechanisms. The results showed that blue light exposure caused a decrease in cell proliferation and an increase in apoptosis in ARPE-19 cells in a time-dependent manner. Oxidative stress increased during the early stage of 2 h of exposure and activated DNA damage in ARPE-19 cells after 8 h. Furthermore, autophagy was activated in response to blue light exposure at 24-48 h. The zebrafish larvae model showed the unfavorable effect of blue light in prohibiting retinal tissue development. The RNA-Seq results confirmed that blue light induced cell death and participated in tissue growth inhibition and maturation. The current study reveals the mechanisms by which blue light induces cell death in a time-dependent manner. Moreover, both the in vivo and NGS data uncovered blue light's effect on retinal tissue development, suggesting that exposing children to blue light could be relatively dangerous. These results could benefit the development of preventive strategies utilizing herbal medicine-based treatments for eye diseases or degeneration in the future.


Assuntos
Autofagia/efeitos da radiação , Dano ao DNA/efeitos da radiação , Luz/efeitos adversos , Degeneração Macular/etiologia , Estresse Oxidativo/efeitos da radiação , Epitélio Pigmentado da Retina/efeitos da radiação , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Peixe-Zebra
8.
J Vis Exp ; (168)2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33616098

RESUMO

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 µL of resuspension buffer and combined with 2 µL of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.


Assuntos
Elementos de DNA Transponíveis , Eletroporação/métodos , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Transgenes , Transposases/metabolismo , Proteínas do Olho/metabolismo , Humanos , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Transfecção , Transposases/genética
9.
Am J Pathol ; 191(4): 652-668, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33385343

RESUMO

Age-related macular degeneration (AMD) is a leading cause of visual impairment. Anti-vascular endothelial growth factor drugs used to treat AMD carry the risk of inducing subretinal fibrosis. We investigated the use of adrenomedullin (AM), a vasoactive peptide, and its receptor activity-modifying protein 2, RAMP2, which regulate vascular homeostasis and suppress fibrosis. The therapeutic potential of the AM-RAMP2 system was evaluated after laser-induced choroidal neovascularization (LI-CNV), a mouse model of AMD. Neovascular formation, subretinal fibrosis, and macrophage invasion were all enhanced in both AM and RAMP2 knockout mice compared with those in wild-type mice. These pathologic changes were suppressed by intravitreal injection of AM. Comprehensive gene expression analysis of the choroid after LI-CNV with or without AM administration revealed that fibrosis-related molecules, including Tgfb, Cxcr4, Ccn2, and Thbs1, were all down-regulated by AM. In retinal pigment epithelial cells, co-administration of transforming growth factor-ß and tumor necrosis factor-α induced epithelial-mesenchymal transition, which was also prevented by AM. Finally, transforming growth factor-ß and C-X-C chemokine receptor type 4 (CXCR4) inhibitors eliminated the difference in subretinal fibrosis between RAMP2 knockout and wild-type mice. These findings suggest the AM-RAMP2 system suppresses subretinal fibrosis in LI-CNV by suppressing epithelial-mesenchymal transition.


Assuntos
Adrenomedulina/metabolismo , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Proteína 2 Modificadora da Atividade de Receptores/metabolismo , Animais , Neovascularização de Coroide/metabolismo , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal/fisiologia , Fibrose/metabolismo , Humanos , Injeções Intravítreas/métodos , Camundongos Knockout , Proteína 2 Modificadora da Atividade de Receptores/genética , Epitélio Pigmentado da Retina/metabolismo
10.
Nat Commun ; 12(1): 662, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510165

RESUMO

Dynamic assembly and disassembly of primary cilia controls embryonic development and tissue homeostasis. Dysregulation of ciliogenesis causes human developmental diseases termed ciliopathies. Cell-intrinsic regulatory mechanisms of cilia disassembly have been well-studied. The extracellular cues controlling cilia disassembly remain elusive, however. Here, we show that lysophosphatidic acid (LPA), a multifunctional bioactive phospholipid, acts as a physiological extracellular factor to initiate cilia disassembly and promote neurogenesis. Through systematic analysis of serum components, we identify a small molecular-LPA as the major driver of cilia disassembly. Genetic inactivation and pharmacological inhibition of LPA receptor 1 (LPAR1) abrogate cilia disassembly triggered by serum. The LPA-LPAR-G-protein pathway promotes the transcription and phosphorylation of cilia disassembly factors-Aurora A, through activating the transcription coactivators YAP/TAZ and calcium/CaM pathway, respectively. Deletion of Lpar1 in mice causes abnormally elongated cilia and decreased proliferation in neural progenitor cells, thereby resulting in defective neurogenesis. Collectively, our findings establish LPA as a physiological initiator of cilia disassembly and suggest targeting the metabolism of LPA and the LPA pathway as potential therapies for diseases with dysfunctional ciliogenesis.


Assuntos
Cílios/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Neurogênese/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Transdução de Sinais , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cílios/genética , Cílios/metabolismo , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Lisofosfolipídeos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Ligação Proteica , Interferência de RNA , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo
11.
Nutrients ; 13(2)2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33503991

RESUMO

N-retinylidene-N-retinylethanolamine (A2E) accumulation in the retina is a prominent marker of retinal degenerative diseases. Blue light exposure is considered as an important factor contributing to dry age-related macular degeneration (AMD). Eggplant and its constituents have been shown to confer health benefits, but their therapeutic effects on dry AMD remain incompletely understood. In this study, we showed that an extract of Solanum melongena L. (EPX) protected A2E-laden ARPE-19 cells against blue light-induced cell death via attenuating reactive oxygen species. Transcriptomic analysis demonstrated that blue light modulated the expression of genes associated with stress response, inflammation, and cell death, and EPX suppressed the inflammatory pathway induced by blue light in A2E-laden ARPE-19 cells by inhibiting the nuclear translocation of nuclear factor kappa B and transcription of pro-inflammatory genes (CXCL8 and IL1B). The degradation of intracellular A2E was considered the major mechanism underlying the protective effect of EPX. Moreover, chlorogenic acid isolated from EPX exerted protective effects against blue light-induced cell damage in A2E-laden ARPE-19 cells. In vivo, EPX administration in BALB/c mice reduced the fundus damage and degeneration of the retinal layer in a blue light-induced retinal damage model. Collectively, our findings suggest the potential role of Solanum melongena L. extract for AMD treatment.


Assuntos
Dermatite Fototóxica/prevenção & controle , Extratos Vegetais/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Pigmentos da Retina/metabolismo , Solanum melongena , Animais , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Luz , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Extratos Vegetais/metabolismo , Epitélio Pigmentado da Retina/metabolismo
12.
Int J Biol Macromol ; 169: 342-351, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33347930

RESUMO

γD-crystallin is among the most abundant γ-crystallins in the human eye lens which are essential for preserving its transparency. Aging, and environmental changes, cause crystallins to lose their native soluble structure and aggregate, resulting in the formation of cataract. Current treatment of cataract is surgical removal which is costly. Pharmaceutical therapeutics of cataract is an unmet need. We report a screen for small molecules capable of inhibiting aggregation of human γD-crystallin. Using a highly amyloidogenic hexapeptide model 41GCWMLY46 derived from the full-length protein, we screened a library of 68 anthraquinone molecules using ThT fluorescence assay. A leading hit, the cochineal Carmine, effectively reduced aggregation of the model GDC6 peptide in dose dependent manner. Similar effect was observed toward aggregation of the full-length γD-crystallin. Transmission electron microscopy, intrinsic Tryptophan fluorescence and ANS fluorescence assays corroborated these results. Insights obtained from molecular docking suggested that Carmine interaction with monomeric GDC6 involved hydrogen bonding with Ace group, Cys, Met residues and hydrophobic contact with Trp residue. Carmine was non-toxic toward retinal cells in culture. It also reduced ex vivo the turbidity of human extracted cataract material. Collectively, our results indicate that Carmine could be used for developing new therapeutics to treat cataract.


Assuntos
Amiloide/metabolismo , Carmim/farmacologia , gama-Cristalinas/metabolismo , Proteínas Amiloidogênicas/metabolismo , Carmim/metabolismo , Catarata/metabolismo , Linhagem Celular , Humanos , Cristalino/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Peptídeos/metabolismo , Agregados Proteicos/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , gama-Cristalinas/química
13.
Carbohydr Polym ; 254: 117409, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33357895

RESUMO

Aiming to enhance therapeutic efficiency of lutein, lutein loaded chitosan-sodium alginate (CS-SA) based nanocarrier system (LNCs) were prepared and evaluated for lutein bioavailability and pharmacokinetics in diabetic rats in comparison to micellar lutein (control). Further, cytotoxicity, cellular uptake and protective activity against H2O2 induced oxidative stress in ARPE-19 cells were studied. Results revealed that LNCs displayed maximal lutein AUC in plasma, liver and eye respectively in normal (3.1, 2.7 and 5.2 folds) and diabetic (7.3, 3.4 and 2.8 folds) rats. Lutein from LNCs exhibited a higher half-life time, mean residence time and slow clearance from the plasma, indicating prolonged circulation compared to control. In ARPE-19 cells, pre-treatment with LNCs (10 µM) have significantly attenuated H2O2 induced cell death, intracellular ROS and mitochondrial membrane potential compared to control. In conclusion, LNCs improve the lutein bioavailability in conditions like diabetes, diabetic retinopathy and cataract to curtail oxidative stress in retinal cells.


Assuntos
Alginatos/química , Quitosana/química , Diabetes Mellitus Experimental/tratamento farmacológico , Portadores de Fármacos/química , Ácidos Graxos/química , Absorção Gastrointestinal/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Luteína/administração & dosagem , Luteína/farmacocinética , Nanopartículas/química , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/administração & dosagem , Substâncias Protetoras/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Animais , Disponibilidade Biológica , Linhagem Celular , Meia-Vida , Humanos , Luteína/sangue , Masculino , Micelas , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos , Ratos Wistar , Epitélio Pigmentado da Retina/metabolismo
14.
J Vis Exp ; (166)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33369607

RESUMO

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.


Assuntos
Elementos de DNA Transponíveis/genética , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Transfecção , Antioxidantes/farmacologia , Biomarcadores/metabolismo , Contagem de Células , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/química , Células Epiteliais/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/isolamento & purificação , Proteínas do Olho/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/isolamento & purificação , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/isolamento & purificação , Fatores de Crescimento Neural/metabolismo , Neuroproteção/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serpinas/genética , Serpinas/isolamento & purificação , Serpinas/metabolismo , Doadores de Tecidos , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismo
15.
PLoS One ; 15(12): e0244307, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33362238

RESUMO

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


Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Complemento C3/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Ribonucleosídeos/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Adenosina/metabolismo , Aminoimidazol Carboxamida/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Ativação do Complemento/efeitos dos fármacos , Complemento C3/efeitos dos fármacos , Humanos , Degeneração Macular/metabolismo , Fosforilação , Epitélio Pigmentado da Retina/efeitos dos fármacos , Pigmentos da Retina/metabolismo , Ribonucleosídeos/metabolismo , Ribonucleotídeos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo
16.
Yonsei Med J ; 61(9): 816-825, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32882766

RESUMO

PURPOSE: To understand the pathophysiology of Best disease (BD) and autosomal recessive bestrophinopathy (ARB) by establishing an in vitro model using human induced pluripotent stem cell (iPSC). MATERIALS AND METHODS: Human iPSC lines were generated from mononuclear cells in peripheral blood of one ARB patient, one autosomal dominant BD patient, and two normal controls. Immunocytochemistry and reverse transcriptase polymerase chain reaction in iPSC lines were conducted to demonstrate the pluripotent markers. After the differentiation of iPSC into functional retinal pigment epithelium (RPE), morphological characteristics of the RPE were evaluated using confocal microscopy and immunocytochemistry. The rates of fluid flow across iPSC-RPE monolayer were measured to compare apical to basal fluid transports by RPE. RNA sequencing was performed on iPSC-RPE to identify the differences in gene expression profiles, and specific gene sets were tested using Gene Set Enrichment Analysis. RESULTS: Morphological characteristics, gene expression, and epithelial integrity of ARB iPSC were comparable to those of BD patient or normal control. Fluid transport from apical to basal was significantly decreased in ARB iPSC-RPE compared with BD iPSC-RPE or control iPSC-RPE. Gene Set Enrichment Analysis confirmed that ARB iPSC-RPE exhibited significant enrichments of epithelial-mesenchymal transition gene set and TNF-α signaling via NF-κB gene set compared to control iPSC-RPE or BD iPSC-RPE. CONCLUSION: A human iPSC model of ARB showed a functional deficiency rather than anatomical defects. ARB may be caused by RPE dysfunction following BEST1 mutation.


Assuntos
Bestrofinas/genética , Oftalmopatias Hereditárias/genética , Células-Tronco Pluripotentes Induzidas , Doenças Retinianas/genética , Epitélio Pigmentado da Retina , Distrofia Macular Viteliforme/genética , Distrofia Macular Viteliforme/fisiopatologia , Diferenciação Celular , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Oftalmopatias Hereditárias/diagnóstico , Oftalmopatias Hereditárias/metabolismo , Oftalmopatias Hereditárias/patologia , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Doenças Retinianas/diagnóstico , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/fisiopatologia , Transtornos da Visão , Acuidade Visual , Distrofia Macular Viteliforme/metabolismo
17.
Life Sci ; 259: 118391, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32891610

RESUMO

AIMS: Dyslipidemia-associated diabetic retinopathy is featured by macular edema and retinal angiogenesis. This study investigated the in vitro lipotoxicity of free fatty acids and their modulatory roles in regulation of autophagy and angiogenic factor production in cultured human retinal pigment epithelium (RPE) ARPE-19 cells. MAIN METHODS: ARPE-19 cells were exposed to monounsaturated oleic acid (OA), saturated palmitic acid (PA), or both. Cell viability, cell cycle distribution, migration, and autophagy of the treated cells were monitored. Angiogenic factor production was determined by RT-qPCR and ELISA. KEY FINDINGS: OA, but not PA, at doses higher than 500 µM significantly induced cytostasis and lipotoxicity in ARPE-19 cells. OA exposure not only markedly enhanced autophagy flux, but also enhanced cell migration, while PA suppressed motility of RPE cells. Meanwhile, OA stimulated de novo synthesis of angiogenic factors including VEGF and bFGF in ARPE-19 cells. Mechanistically, OA treatment stimulated not only AMPK/mTOR/p70S6K signaling, but also induced hyperphosphorylation of MAPK pathway mediators, including ERK, JNK and p38 MAPK, as well as NF-κB activation. Kinase inhibition assays showed that blockade of PI3K/Akt, MAPK and NF-κB pathways prevented the OA-upregulated VEGF transcription and its peptide release. Comparatively, only NF-κB inhibition significantly suppressed bFGF peptide release from ARPE-19 cells. SIGNIFICANCE: Out findings support the OA-exhibited cytostasis, autophagy modulation and angiogenic factor production in RPE cells. This study sheds light on the interrelationship between metabolic disorder and retinopathy and provides molecular strategies for preventing and treating choroidal neovascularization in diabetic retinopathy.


Assuntos
Indutores da Angiogênese/metabolismo , Autofagia , Ácido Oleico/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Western Blotting , Movimento Celular , Proliferação de Células , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Sistema de Sinalização das MAP Quinases , Ácido Palmítico/metabolismo , Epitélio Pigmentado da Retina/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
18.
J Oleo Sci ; 69(9): 1095-1105, 2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32788523

RESUMO

Ayu sweetfish (Plecoglossus altivelis) is a diurnal freshwater fish that are surface swimmers and active under broad and short wavelength-dominated light. Biochemical analyses have shown that the ayu fish have abundant carotenoids including zeaxanthin in their integuments. Although zeaxanthin plays an important role in the physiological function of the retina, the amount and location of zeaxanthin in the ayu eye have not been accurately determined. In this study, circular dichroism spectral data and chiral high-performance liquid chromatography analysis showed that zeaxanthin was the primary carotenoid in the ayu eye, and the eye had the highest carotenoid content compared to those in the integuments, subcutaneous fat, and digestive tract. Interestingly, zeaxanthin in the ayu eyeball was expressed in the photoreceptor layer and near the retinal pigmented epithelium. In vitro assays showed that zeaxanthin could protect photoreceptors and retinal pigmented epithelial cell lines against the oxidative stress induced by exposure to L-buthionine-(S,R)-sulfoximine/glutamate. These findings indicate that zeaxanthin plays protective roles against oxidative stress in the vision of wild ayu.


Assuntos
Antioxidantes , Olho/metabolismo , Osmeriformes/metabolismo , Células Fotorreceptoras/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Zeaxantinas/metabolismo , Zeaxantinas/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Ácido Glutâmico/efeitos adversos , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Zeaxantinas/fisiologia
19.
Proc Natl Acad Sci U S A ; 117(33): 19629-19638, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759209

RESUMO

The visual phototransduction cascade begins with a cis-trans photoisomerization of a retinylidene chromophore associated with the visual pigments of rod and cone photoreceptors. Visual opsins release their all-trans-retinal chromophore following photoactivation, which necessitates the existence of pathways that produce 11-cis-retinal for continued formation of visual pigments and sustained vision. Proteins in the retinal pigment epithelium (RPE), a cell layer adjacent to the photoreceptor outer segments, form the well-established "dark" regeneration pathway known as the classical visual cycle. This pathway is sufficient to maintain continuous rod function and support cone photoreceptors as well although its throughput has to be augmented by additional mechanism(s) to maintain pigment levels in the face of high rates of photon capture. Recent studies indicate that the classical visual cycle works together with light-dependent processes in both the RPE and neural retina to ensure adequate 11-cis-retinal production under natural illuminances that can span ten orders of magnitude. Further elucidation of the interplay between these complementary systems is fundamental to understanding how cone-mediated vision is sustained in vivo. Here, we describe recent advances in understanding how 11-cis-retinal is synthesized via light-dependent mechanisms.


Assuntos
Retinaldeído/biossíntese , Visão Ocular , Animais , Humanos , Luz , Transdução de Sinal Luminoso , Opsinas/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Retinaldeído/química
20.
Chem Biol Interact ; 328: 109212, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32721430

RESUMO

Hydroxychloroquine (HCQ) is frequently used medications for many auto-immunity diseases. However, HCQ induced retinal toxicity, which might result in irreversible retinopathy, is one of the most important complications of HCQ. However, the molecular mechanism underlying the HCQ retinal toxicity is still not well known. Retinal pigment epithelium, in which HCQ is highly enriched due to the tissue-specific affinity of HCQ, is considered to play important role in HCQ retinopathy. Herein, we used a metabolomics approach based on liquid chromatography-mass spectrometry to investigate the metabolic changes in retinal pigment epithelial cells (ARPE-19) with HCQ exposure at 6 h and 24 h. ARPE-19 cells were treated with HCQ at sub-lethal concentration 20 (IC 20), which was determined with MTT assay. Untargeted metabolic profiling revealed 9 and 15 metabolites that were significantly different between control group and HCQ exposure group at 6 h and 24 h, respectively. Enrichment and pathway analysis highlighted ascorbate and aldarate metabolism, d-Glutamine and d-glutamate metabolism and C5-Branched dibasic acid metabolism were disturbed after HCQ exposure. These findings increased our knowledge about the metabolic perturbation induced by HCQ exposure and indicated that metabolic profiling in the ARPE-19 cells might be helpful in understanding the mechanism of HCQ retinal toxicity and exploring potential biomarker.


Assuntos
Células Epiteliais/metabolismo , Hidroxicloroquina/toxicidade , Redes e Vias Metabólicas , Metabolômica , Epitélio Pigmentado da Retina/metabolismo , Espectrometria de Massas em Tandem , Adulto , Biomarcadores/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida , Análise Discriminante , Células Epiteliais/efeitos dos fármacos , Humanos , Análise dos Mínimos Quadrados , Metaboloma/efeitos dos fármacos , Análise Multivariada , Análise de Componente Principal
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