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1.
Biomed Pharmacother ; 133: 111041, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33378949

RESUMO

Poly (ADP-ribose) polymerase 1 (PARP1)-dependent cell death in the retinal pigment epithelium (RPE) is implicated in dry age-related macular degeneration (AMD). Although PARP1 inhibitors are available for treating dry AMD, their delivery route is not ideal for patients. The aim of this study was to test the efficacy of a novel PARP1-inhibitory compound (PIC) in vitro and in vivo. This study presents PIC, a novel small molecule, with superior efficacy to PARP1 inhibitors in the market. PIC demonstrated a distinctive inhibitory profile against PARP isotypes than the FDA-approved PARP1 inhibitors. PIC inhibited PARP1 activation at an IC50 of 0.41 ± 0.15 nM in an enzyme-based assay in vitro and at IC50 and EC50 in ARPE-19 cells of 0.11 ± 0.02 nM and 0.22 ± 0.02 nM, respectively, upon H2O2 insult. PIC also moderated mitochondrial fission and depolarization and maintained cellular energy levels under oxidative stress in ARPE-19 cells. Furthermore, PIC demonstrated good corneal penetration in a rat model, presenting PIC as a promising candidate for eye drop therapeutics for dry AMD. When PIC was administered as an eye drop formulation, RPE morphology was preserved, maintaining the thickness of the outer nuclear layers under sodium iodate (SI) treatment in rats. In SI-treated rabbits, eye drop administration of PIC also retained the structural and functional integrity when analyzed using funduscopy and electroretinogram. Collectively, our data portray PIC as an attractive treatment measure for dry AMD.


Assuntos
Degeneração Macular/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Administração Oftálmica , Animais , Antioxidantes/farmacologia , Linhagem Celular , Modelos Animais de Doenças , Humanos , Iodatos , Degeneração Macular/induzido quimicamente , Degeneração Macular/enzimologia , Degeneração Macular/patologia , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Absorção Ocular , Soluções Oftálmicas , Estresse Oxidativo/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Coelhos , Ratos Sprague-Dawley , Epitélio Pigmentado da Retina/enzimologia , Epitélio Pigmentado da Retina/patologia
2.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32211891

RESUMO

Distal appendages (DAs) of the mother centriole are essential for the initial steps of ciliogenesis in G1/G0 phase of the cell cycle. DAs are released from centrosomes in mitosis by an undefined mechanism. Here, we show that specific DAs lose their centrosomal localization at the G2/M transition in a manner that relies upon Nek2 kinase activity to ensure low DA levels at mitotic centrosomes. Overexpression of active Nek2A, but not kinase-dead Nek2A, prematurely displaced DAs from the interphase centrosomes of immortalized retina pigment epithelial (RPE1) cells. This dramatic impact was also observed in mammary epithelial cells with constitutively high levels of Nek2. Conversely, Nek2 knockout led to incomplete dissociation of DAs and cilia in mitosis. As a consequence, we observed the presence of a cilia remnant that promoted the asymmetric inheritance of ciliary signaling components and supported cilium reassembly after cell division. Together, our data establish Nek2 as an important kinase that regulates DAs before mitosis.


Assuntos
Centríolos/enzimologia , Cílios/enzimologia , Células Epiteliais/enzimologia , Mitose , Quinases Relacionadas a NIMA/metabolismo , Epitélio Pigmentado da Retina/enzimologia , Animais , Sítios de Ligação , Linhagem Celular , Centríolos/genética , Cílios/genética , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular , Células-Tronco Hematopoéticas/enzimologia , Humanos , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/enzimologia , Camundongos , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Quinases Relacionadas a NIMA/genética , Ligação Proteica , Epitélio Pigmentado da Retina/citologia , Transdução de Sinais , Fatores de Tempo
3.
Exp Eye Res ; 190: 107884, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31786159

RESUMO

Proliferative vitreoretinopathy (PVR) is a blinding fibrotic eye disease that develops in 8-10% of patients who undergo primary retinal detachment-reparative surgery and in 40-60% of patients with open-globe injury. At present, there is no pharmacological treatment for this devastating disease. Vitreal growth factors activate their respective receptors of cells in the vitreous, trigger their downstream signaling transduction (e.g. phosphoinositide 3 kinases (PI3Ks)/Akt), and drive cellular responses intrinsic to the pathogenesis of PVR. PI3Ks play a central role in experimental PVR. However, which isoform(s) are involved in PVR pathogenesis remain unknown. Herein, we show that p110δ, a catalytic subunit of receptor-regulated PI3K isoform δ, is highly expressed in epiretinal membranes from patients with PVR, and that idelalisib, a specific inhibitor of PI3Kδ, effectively inhibits vitreous-induced Akt activation, proliferation, migration and contraction of retinal pigment epithelial cells derived from an epiretinal membrane of a PVR patient. Small molecules of kinase inhibitors have shown great promise as a class of therapeutics for a variety of human diseases. The data herein suggest that idelalisib is a promising PVR prophylactic.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Membrana Epirretiniana/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Purinas/farmacologia , Quinazolinonas/farmacologia , Epitélio Pigmentado da Retina/patologia , Corpo Vítreo/metabolismo , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Membrana Epirretiniana/enzimologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Epitélio Pigmentado da Retina/enzimologia , Transdução de Sinais , Vitreorretinopatia Proliferativa/tratamento farmacológico , Vitreorretinopatia Proliferativa/enzimologia
4.
Adv Exp Med Biol ; 1185: 341-346, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884635

RESUMO

Bisretinoid fluorophores are the major constituents of the lipofuscin of retinal pigment epithelium (RPE) that accumulates with age and contributes to retina disease. Knowledge of the burden placed on the RPE cell by the accumulation of these phototoxic retinaldehyde-adducts depends on the identification and quantitation of the various bisretinoid species that constitute this family of fluorophores. Here we report a previously unidentified fluorescent bisretinoid by UPLC coupled to photodiode array detection, fluorescence, and electrospray ionization mass spectrometry (UPLC-PDA-FLR-ESI-MS) (Kim HJ, Sparrow JR, J Lipid Res 59:1620-1629, 2018). This novel bisretinoid is 1-octadecyl-2-lyso-sn-glycero A2PE (alkyl ether lysoA2PE). The structural assignment was based on molecular weight (m/z 998), UV-visible absorbance maxima (340, 440 nm), and retention time (73 minutes) and was corroborated by biomimetic synthesis using all-trans-retinal and glycerophosphoethanolamine analogues as starting materials. In mechanistic studies, A2PE was hydrolyzed by PLA2, and plasmalogen lysoA2PE was cleaved under acidic conditions. Unprecedented UPLC detection of the bisretinoid alkyl ether lysoA2PE in human RPE but not in neural retina indicates that the phospholipase A2 activity that generates the latter bisretinoid resides in RPE.


Assuntos
Epitélio Pigmentado da Retina/enzimologia , Retinoides/química , Humanos , Lipofuscina/metabolismo , Fosfolipases A2/metabolismo , Retinaldeído/química
5.
Adv Exp Med Biol ; 1185: 377-382, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884641

RESUMO

Oxidative stress-mediated injury of the retinal pigment epithelium (RPE) can precede progressive retinal degeneration and ultimately lead to blindness (e.g., age-related macular degeneration (AMD)). The RPE expresses the PNPLA2 gene and produces its protein product PEDF-R that exhibits lipase activity. We have shown that transient PNPLA2 overexpression decreases dead-cell proteolytic activity and that synthetic peptides derived from a central region of PEDF-R efficiently protect ARPE-19 and pig primary RPE cells from oxidative stress. This study aims to evaluate the effect of loss of PNPLA2 in RPE cells undergoing oxidative stress. Loss of PNPLA2 conferred increased resistance to cells when subjected to oxidative stress.


Assuntos
Lipase/genética , Estresse Oxidativo , Epitélio Pigmentado da Retina/patologia , Animais , Epitélio Pigmentado da Retina/enzimologia , Suínos
6.
Adv Exp Med Biol ; 1185: 537-541, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31884667

RESUMO

RPE65, the retinal pigment epithelium (RPE) smooth endoplasmic reticulum (sER) membrane-associated retinoid isomerase, plays an indispensable role in sustaining visual function in vertebrates. An important aspect which has attracted considerable attention is the posttranslational modification by S-palmitoylation of RPE65. Some studies show that RPE65 is a palmitoylated protein, but others deny that conclusion. While it is considered to be mainly responsible for RPE65's membrane association, we still lack conclusive evidence about RPE65 palmitoylation. In this review, we provide an overview of the history and current understanding of RPE65 palmitoylation.


Assuntos
Proteínas do Olho/química , Lipídeos/química , Lipoilação , Processamento de Proteína Pós-Traducional , Epitélio Pigmentado da Retina/enzimologia , cis-trans-Isomerases/química , Animais , Retículo Endoplasmático , Humanos
7.
Nutrients ; 11(9)2019 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-31500218

RESUMO

Omega-3 polyunsaturated fatty acids (ω3-PUFAs) have potential protective activity in a variety of infectious diseases, but their actions and underlying mechanisms in Toxoplasma gondii infection remain poorly understood. Here, we report that docosahexaenoic acid (DHA) robustly induced autophagy in murine bone marrow-derived macrophages (BMDMs). Treatment of T. gondii-infected macrophages with DHA resulted in colocalization of Toxoplasma parasitophorous vacuoles with autophagosomes and reduced intracellular survival of T. gondii. The autophagic and anti-Toxoplasma effects induced by DHA were mediated by AMP-activated protein kinase (AMPK) signaling. Importantly, BMDMs isolated from Fat-1 transgenic mice, a well-known animal model capable of synthesizing ω3-PUFAs from ω6-PUFAs, showed increased activation of autophagy and AMPK, leading to reduced intracellular survival of T. gondii when compared with wild-type BMDMs. Moreover, Fat-1 transgenic mice exhibited lower cyst burden in the brain following infection with the avirulent strain ME49 than wild-type mice. Collectively, our results revealed mechanisms by which endogenous ω3-PUFAs and DHA control T. gondii infection and suggest that ω3-PUFAs might serve as therapeutic candidate to prevent toxoplasmosis and infection with other intracellular protozoan parasites.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Antiparasitários/farmacologia , Autofagia/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Macrófagos/efeitos dos fármacos , Toxoplasma/efeitos dos fármacos , Toxoplasmose Animal/prevenção & controle , Toxoplasmose Cerebral/prevenção & controle , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Encéfalo/parasitologia , Encéfalo/patologia , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Ativação Enzimática , Humanos , Macrófagos/enzimologia , Macrófagos/parasitologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/enzimologia , Epitélio Pigmentado da Retina/parasitologia , Transdução de Sinais , Toxoplasma/patogenicidade , Toxoplasmose Animal/enzimologia , Toxoplasmose Animal/parasitologia , Toxoplasmose Animal/patologia , Toxoplasmose Cerebral/enzimologia , Toxoplasmose Cerebral/parasitologia , Toxoplasmose Cerebral/patologia
8.
J Biol Chem ; 294(42): 15408-15417, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31467081

RESUMO

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that play a critical role in transmitting signals from cell-surface molecules to intracellular protein effectors. Key PI3Ks include PI3Kα, PI3Kß, and PI3Kδ, which are regulated by receptors. The signaling pathway comprising the PI3Ks, along with a Ser/Thr kinase (AKT), a proto-oncogene product (mouse double minute (MDM)2), and a tumor suppressor protein (p53), plays an essential role in experimental proliferative vitreoretinopathy (PVR), which is a fibrotic blinding eye disorder. However, which PI3K isoforms are involved in PVR is unknown. A major characteristic of PVR is the formation of epi (or sub)-retinal membranes that consist of extracellular matrix and cells, including retinal pigment epithelium (RPE) cells, glial cells, and macrophages. RPE cells are considered key players in PVR pathogenesis. Using immunoblotting and immunofluorescence analyses, we herein provide the evidence that PI3Kδ is highly expressed in human RPEs when it is primarily expressed in leukocytes. We also found that PI3Kδ inactivation through two approaches, CRISPR/Cas9-mediated depletion and a PI3Kδ-specific inhibitor (idelalisib), not only blocks vitreous-induced activation of AKT and MDM2 but also abrogates a vitreous-stimulated decrease in p53. Furthermore, we demonstrate that PI3Kδ inactivation prevents vitreous-induced proliferation, migration, and contraction of human RPEs. These results suggest that PI3Kδ may represent a potential therapeutic target for RPE-related eye diseases, including PVR.


Assuntos
Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Corpo Vítreo/metabolismo , Movimento Celular , Proliferação de Células , Classe I de Fosfatidilinositol 3-Quinases/genética , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/enzimologia , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Corpo Vítreo/enzimologia
9.
Mol Vis ; 25: 329-344, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31341381

RESUMO

Purpose: Systemic hypertension is a risk factor of age-related macular degeneration, a disease associated with chronic retinal inflammation. The main cause of acute hypertension in the elderly is consumption of dietary salt (NaCl) resulting in increased extracellular osmolarity. The aim of the present study was to determine whether extracellular osmolarity regulates the expression of cyclooxygenase (COX) genes in cultured human retinal pigment epithelial (RPE) cells, and whether COX activity is involved in mediating the osmotic expression of key inflammatory (NLRP3 and IL1B) and angiogenic factor (VEGFA) genes. Methods: Extracellular hyperosmolarity was induced by addition of NaCl or sucrose. Gene expression was determined with real-time reverse transcription (RT)-PCR. Cytosolic interleukin-1ß (IL-1ß) and extracellular vascular endothelial growth factor (VEGF) levels were evaluated with enzyme-linked immunosorbent assay (ELISA). Results: Extracellular hyperosmolarity induced a dose-dependent increase in COX2 gene expression when >10 mM NaCl was added to the culture medium, while COX1 gene expression was increased at higher doses (>50 mM of added NaCl). Extracellular hypo-osmolarity decreased COX2 gene expression. High extracellular osmolarity also induced increases in the COX2 protein level. NaCl-induced expression of COX2 was mediated by various intracellular signal transduction molecules (p38 mitogen-activated protein kinase [p38 MAPK], extracellular signal-regulated kinases 1 and 2 [ERK1/2], and phosphatidylinositol-3 kinase [PI3K]), intracellular calcium signaling involving activation of phospholipase Cγ (PLCγ) and protein kinase Cα/ß (PKCα/ß), and the activity of nuclear factor of activated T cell 5 (NFAT5). Inhibition of fibroblast growth factor (FGF), transforming growth factor-ß (TGF-ß), and interleukin-1 (IL-1) receptor activities decreased NaCl-induced COX2 gene expression. Selective inhibition of COX2 activity decreased osmotic expression of the VEGFA, IL1B, and NLRP3 genes, and blocked the NaCl-induced increase in the cytosolic IL-1ß level. Conclusions: The expression of COX2 in RPE cells is osmoresponsive, and depends on NFAT5. COX2 activity stimulates hyperosmotic expression of angiogenic (VEGFA) and inflammatory factor (IL1B and NLRP3) genes, and activation of the NLRP3 inflammasome in RPE cells.


Assuntos
Ciclo-Oxigenase 2/biossíntese , Inflamassomos/metabolismo , Osmose , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/genética , Indução Enzimática , Feminino , Humanos , Inflamação/genética , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/genética , Nitrobenzenos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Cloreto de Sódio/farmacologia , Sulfonamidas/farmacologia , Fatores de Transcrição/metabolismo , Adulto Jovem
10.
Med Microbiol Immunol ; 208(6): 811-824, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31267172

RESUMO

Human retinal pigment epithelial (hRPE) cells form a selectively permeable monolayer between the neural retina and the highly permeable choroidal vessels. Thus, hRPE cells bear important regulatory functions and are potential targets of pathogens in vivo. Endogenous bacterial endophthalmitis (EBE) is frequently caused by infections with the Gram-positive bacterium Staphylococcus aureus (S. aureus). Upon microbial infection, interferon gamma (IFN-γ), a major cytokine of the adaptive immune response, induces a broad spectrum of effector molecules, such as the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase-1 (IDO1). We stimulated human RPE (hRPE) cells in vitro with proinflammatory cytokines and analyzed the expression levels and enzymatic activities of IDO1 and inducible nitric oxide synthase (iNOS), another antimicrobial effector molecule. The antimicrobial capacity was analyzed in infection experiments using S. aureus and Toxoplasma gondii (T. gondii). Our aim was to characterize the particular importance of IDO1 and iNOS during EBE. We found that an IFN-γ stimulation of hPRE cells induced the expression of IDO1, which inhibited the growth of T. gondii and S. aureus. A co-stimulation with IFN-γ, interleukin-1 beta, and tumor necrosis factor alpha induced a strong expression of iNOS. The iNOS-derived nitric oxide production was dependent on cell-culture conditions; however, it could not cause antimicrobial effects. iNOS did not act synergistically with IDO1. Instead, iNOS activity inhibited IDO1-mediated tryptophan degradation and bacteriostasis. This effect was reversible by the addition of the iNOS inhibitor NG-monomethyl-L-arginine. In conclusion, iNOS mediates anti-inflammatory effects in hRPE cells stimulated with high amounts of IFN-γ together with tumor necrosis factor alpha and Interleukin-1 beta and prevents potential IDO1-dependent tissue damage.


Assuntos
Células Epiteliais/enzimologia , Células Epiteliais/imunologia , Fatores Imunológicos/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Linhagem Celular , Endoftalmite/imunologia , Humanos , Modelos Teóricos , Epitélio Pigmentado da Retina/enzimologia , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/imunologia , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/imunologia
11.
Nutrients ; 11(7)2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31277394

RESUMO

This study investigated the protective effect and the molecular mechanism of piceatannol on hydrogen peroxide (H2O2)-induced retinal pigment epithelium cell (ARPE-19) damage. Piceatannol treatment significantly inhibited H2O2-induced RPE cell death and reactive oxygen species (ROS) generation by 64.4% and 75.0%, respectively. Results of flow cytometry showed that H2O2-induced ARPE-19 cells apoptosis was ameliorated by piceatannol supplementation, along with decreased relative protein expressions of Bax/Bcl-2, Cleave-Caspase-3, and Cleave-PARP. Moreover, piceatannol treatment induced NF-E2-related factor 2 (Nrf2) signaling activation, which was evidenced by increased transcription of anti-oxidant genes, glutamate-cysteine ligase catalytic subunit (GCLc), SOD, and HO-1. Knockdown of Nrf2 through targeted siRNA alleviated piceatannol-mediated HO-1 transcription, and significantly abolished piceatannol-mediated cytoprotection. LY294002 (PI3K inhibitor) dramatically blocked piceatannol-mediated increasing of Nrf2 nuclear translocation, HO-1 expression, and cytoprotective activity, indicating the involvement of PI3K/Akt pathway in the cytoprotective effect of piceatannol. The results from this suggest the potential of piceatannol in reducing the risk of age-related macular degeneration.


Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Estilbenos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular , Citoproteção , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Heme Oxigenase-1/metabolismo , Humanos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Epitélio Pigmentado da Retina/enzimologia , Epitélio Pigmentado da Retina/patologia , Transdução de Sinais
12.
J Biochem Mol Toxicol ; 33(8): e22352, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31157476

RESUMO

Cynaroside is a flavonoid compound proved to possess antioxidant activity, but its protective effect on age-related macular degeneration still remains unclear. In this study, the protective effects of cynaroside on oxidative stress and apoptosis in retinal pigment epithelial (RPE) cells induced by hydrogen peroxide (H2 O2 ) were investigated. Results showed that cynaroside effectively attenuated the decrease of cell activity induced by H2 O2 . The total reactive oxygen species can be remitted by decreasing malondialdehyde level, as well as increasing glutathione level, and superoxide dismutase and catalase activities. In addition, Western blot analysis indicated that cynaroside protected ARPE-19 cells from apoptosis through downregulation of caspase-3 protein activation which was controlled by the upstream proteins Bcl-2 and Bax. It was finally proved that cynaroside could enhance the antioxidant and antiapoptotic ability in ARPE-19 cells by promoting the expression of p-Akt.


Assuntos
Glucosídeos/farmacologia , Luteolina/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Apoptose , Catalase/metabolismo , Linhagem Celular , Ativação Enzimática , Glutationa/metabolismo , Humanos , Malondialdeído/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/enzimologia , Epitélio Pigmentado da Retina/metabolismo , Superóxido Dismutase/metabolismo
13.
Peptides ; 119: 170108, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31247223

RESUMO

Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes. In the last years, several in vivo studies have demonstrated the protective role of pituitary adenylate cyclase-activating peptide (PACAP-38) to counteract several alterations occurring during DR. Recently, different studies have demonstrated that some PACAP-38 effects are mediated by EGFR trans-activation, although no data exist regarding the link between this peptide and EGFR in DR. The aim of the present study has been to investigate whether retinal effect of PACAP-38 against high glucose damage is mediated by EGFR phosphorylation. Diabetes was induced by a single injection of streptozotocin (STZ) in rats. After 1 week, a group of animals was treated with a single intravitreal injection of 100 µM PACAP-38 or saline solution. Immunohistochemistry and western blot analysis have demonstrated that intravitreal injection of PACAP-38 induced p-EGFR over-expression in retina of diabetic rats. Several pathogenic mechanisms may contribute to diabetic retinopathy including BRB alteration. To better clarify the relationship between PACAP-38 and EGFR, we have also carried out a study on ARPE-19 cells, representing a model in vitro of outer BRB. Our results have shown that PACAP-38 treatment improved cell viability in ARPE-19 cells exposed to hyperglycemic/hypoxic insult mimicking tissue microenvironment occurring in DR. Binding to PAC1R, peptide induces EGFR phosphorylation via PKA-signaling cascade stimulation. EGFR trans-activation triggers MAPK/ERK signaling pathway involved in cell survival and proliferation. In conclusion, data have suggested that PACAP-38 acts through EGFR phosphorylation in DR and this effect particularly occurs on RPE layer.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Retinopatia Diabética/prevenção & controle , Receptores ErbB/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Epitélio Pigmentado da Retina/enzimologia , Animais , Linhagem Celular , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/enzimologia , Retinopatia Diabética/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Epitélio Pigmentado da Retina/patologia
14.
Cell Death Dis ; 10(4): 293, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926772

RESUMO

Hydrogen sulfide (H2S) serves as a gasotransmitter in the regulation of organ development and maintenance of homeostasis in tissues. Its abnormal levels are associated with multiple human diseases, such as neurodegenerative disease, myocardial injury, and ophthalmic diseases. Excessive exposure to H2S could lead to cellular toxicity, orchestrate pathological process, and increase the risk of various diseases. Interestingly, under physiological status, H2S plays a critical role in maintaining cellular physiology and limiting damages to tissues. In mammalian species, the generation of H2S is catalyzed by cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE), 3-mercapto-methylthio pyruvate aminotransferase (3MST) and cysteine aminotransferase (CAT). These enzymes are found inside the mammalian eyeballs at different locations. Their aberrant expression and the accumulation of substrates and intermediates can change the level of H2S by orders of magnitude, causing abnormal structures or functions in the eyes. Detailed investigations have demonstrated that H2S donors' administration could regulate intraocular pressure, protect retinal cells, inhibit oxidative stress and alleviate inflammation by modulating the function of intra or extracellular proteins in ocular tissues. Thus, several slow-releasing H2S donors have been shown to be promising drugs for treating multiple diseases. In this review, we discuss the biological function of H2S metabolism and its application in ophthalmic diseases.


Assuntos
Retinopatia Diabética/metabolismo , Glaucoma/metabolismo , Sulfeto de Hidrogênio/farmacologia , Pressão Intraocular/efeitos dos fármacos , Degeneração Retiniana/metabolismo , Neurônios Retinianos/efeitos dos fármacos , Epitélio Pigmentado da Retina/enzimologia , Animais , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/metabolismo , AMP Cíclico/metabolismo , Retinopatia Diabética/enzimologia , Glaucoma/enzimologia , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/genética , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Sulfeto de Hidrogênio/metabolismo , Pressão Intraocular/genética , Estresse Oxidativo/efeitos dos fármacos , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/genética , Neurônios Retinianos/química , Neurônios Retinianos/enzimologia , Epitélio Pigmentado da Retina/irrigação sanguínea , Transplante de Células-Tronco
15.
J Cell Biol ; 218(4): 1164-1181, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30765437

RESUMO

Faithful mitotic chromosome segregation is required for the maintenance of genomic stability. We discovered the phosphorylation of histone H2B at serine 6 (H2B S6ph) as a new chromatin modification site and found that this modification occurs during the early mitotic phases at inner centromeres and pericentromeric heterochromatin. This modification is directly mediated by cyclin B1-associated CDK1, and indirectly by Aurora B, and is antagonized by PP1-mediated dephosphorylation. H2B S6ph impairs chromatin binding of the histone chaperone SET (I2PP2A), which is important for mitotic fidelity. Injection of phosphorylation-specific H2B S6 antibodies in mitotic cells caused anaphase defects with impaired chromosome segregation and incomplete cytokinesis. As H2B S6ph is important for faithful chromosome separation, this modification may contribute to the prevention chromosomal instability and aneuploidy which frequently occur in cancer cells.


Assuntos
Proteína Quinase CDC2/metabolismo , Núcleo Celular/enzimologia , Segregação de Cromossomos , Cromossomos Humanos , Histonas/metabolismo , Mitose , Proteína Quinase CDC2/genética , Núcleo Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Instabilidade Genômica , Células HCT116 , Células HEK293 , Células HeLa , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Humanos , Neoplasias/enzimologia , Neoplasias/genética , Fosforilação , Ligação Proteica , Epitélio Pigmentado da Retina/enzimologia , Serina , Transdução de Sinais
16.
J Cell Biol ; 218(4): 1218-1234, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30709852

RESUMO

Mitotic kinesins must be regulated to ensure a precise balance of spindle forces and accurate segregation of chromosomes into daughter cells. Here, we demonstrate that kinesin-binding protein (KBP) reduces the activity of KIF18A and KIF15 during metaphase. Overexpression of KBP disrupts the movement and alignment of mitotic chromosomes and decreases spindle length, a combination of phenotypes observed in cells deficient for KIF18A and KIF15, respectively. We show through gliding filament and microtubule co-pelleting assays that KBP directly inhibits KIF18A and KIF15 motor activity by preventing microtubule binding. Consistent with these effects, the mitotic localizations of KIF18A and KIF15 are altered by overexpression of KBP. Cells depleted of KBP exhibit lagging chromosomes in anaphase, an effect that is recapitulated by KIF15 and KIF18A overexpression. Based on these data, we propose a model in which KBP acts as a protein buffer in mitosis, protecting cells from excessive KIF18A and KIF15 activity to promote accurate chromosome segregation.


Assuntos
Anáfase , Núcleo Celular/enzimologia , Segregação de Cromossomos , Cromossomos Humanos , Cinesina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Núcleo Celular/genética , Células HeLa , Humanos , Cinesina/genética , Proteínas do Tecido Nervoso/genética , Epitélio Pigmentado da Retina/enzimologia , Transdução de Sinais
17.
J Cell Biol ; 218(4): 1182-1199, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30674582

RESUMO

Spindle checkpoint signaling is initiated by recruitment of the kinase MPS1 to unattached kinetochores during mitosis. We show that CDK1-CCNB1 and a counteracting phosphatase PP2A-B55 regulate the engagement of human MPS1 with unattached kinetochores by controlling the phosphorylation status of S281 in the kinetochore-binding domain. This regulation is essential for checkpoint signaling, since MPS1S281A is not recruited to unattached kinetochores and fails to support the recruitment of other checkpoint proteins. Directly tethering MPS1S281A to the kinetochore protein Mis12 bypasses this regulation and hence the requirement for S281 phosphorylation in checkpoint signaling. At the metaphase-anaphase transition, MPS1 S281 dephosphorylation is delayed because PP2A-B55 is negatively regulated by CDK1-CCNB1 and only becomes fully active once CCNB1 concentration falls below a characteristic threshold. This mechanism prolongs the checkpoint-responsive period when MPS1 can localize to kinetochores and enables a response to late-stage spindle defects. By acting together, CDK1-CCNB1 and PP2A-B55 thus create a spindle checkpoint-permissive state and ensure the fidelity of mitosis.


Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/enzimologia , Ciclina B1/metabolismo , Cinetocoros/enzimologia , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Aurora Quinase B/genética , Aurora Quinase B/metabolismo , Proteína Quinase CDC2/genética , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Ciclina B1/genética , Células HEK293 , Células HeLa , Humanos , Fosforilação , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Epitélio Pigmentado da Retina/enzimologia , Transdução de Sinais , Fatores de Tempo
18.
Pharmacol Rep ; 71(1): 175-182, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30554037

RESUMO

BACKGROUND: Diabetic retinopathy (DR) is one of the most common complications of diabetes and the leading cause of acquired blindness in adults. In diabetic patients hyperglycemia induces complex metabolic abnormalities affecting retinal homeostasis, and promotes retinal inflammation and angiogenesis. Incretin mimetic drugs such exenatide, are a relatively new group of drugs used in the treatment of diabetes. We investigated the potential direct effects of exenatide on human retinal pigment epithelium (HRPE). METHODS: cAMP production was measured after stimulation of HRPE cells with GLP-1 and exenatide. Intracellular signaling pathways were also examined. HRPE cells were stimulated with TNF-α and subsequently incubated with exenatide. The concentration of metalloproteinases, MMP-1, MMP-2 and MMP-9, and tissue inhibitors of metalloproteinases, TIMP-1, TIMP-2, and TIMP-3 were evaluated. Viability, cytotoxicity and caspase 3/7 activation were determined. Activity of dipeptidyl peptidase-4 (DPP-4), an enzyme involved in GLP-1 inactivation, was also determined. RESULTS: Both GLP-1 and exenatide stimulation in HRPE cells caused no effect in cAMP levels suggesting alternative signaling pathways. Signaling pathway analysis showed that exenatide reduced phosphorylation of Akt-Ser473, PRAS40, SAPK/JNK, Bad, and S6 proteins but not Akt-Thr308. Exenatide also decreased MMP-1, MMP-9, and TIMP-2 protein levels whereas MMP-2 level in HRPE cells was increased. Finally, we show that exenatide decreased the activity of DPP-4 in TNF-α stimulated HRPE cells. CONCLUSIONS: These findings indicate that exenatide modulates regulation of extracellular matrix components involved in retinal remodeling.


Assuntos
Colagenases/metabolismo , Células Epiteliais/efeitos dos fármacos , Exenatida/farmacologia , Hipoglicemiantes/farmacologia , Incretinas/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Inibidores Teciduais de Metaloproteinases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Linhagem Celular , Células Epiteliais/enzimologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/enzimologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Epitélio Pigmentado da Retina/enzimologia , Transdução de Sinais/efeitos dos fármacos , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo
19.
J Biol Chem ; 293(45): 17546-17558, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30228185

RESUMO

RAB28, a member of the RAS oncogene family, is a ubiquitous, farnesylated, small GTPase of unknown function present in photoreceptors and the retinal pigmented epithelium (RPE). Nonsense mutations of the human RAB28 gene cause recessive cone-rod dystrophy 18 (CRD18), characterized by macular hyperpigmentation, progressive loss of visual acuity, RPE atrophy, and severely attenuated cone and rod electroretinography (ERG) responses. In an attempt to elucidate the disease-causing mechanism, we generated Rab28 -/- mice by deleting exon 3 and truncating RAB28 after exon 2. We found that Rab28 -/- mice recapitulate features of the human dystrophy (i.e. they exhibited reduced cone and rod ERG responses and progressive retina degeneration). Cones of Rab28 -/- mice extended their outer segments (OSs) to the RPE apical processes and formed enlarged, balloon-like distal tips before undergoing degeneration. The visual pigment content of WT and Rab28 -/- cones was comparable before the onset of degeneration. Cone phagosomes were almost absent in Rab28 -/- mice, whereas rod phagosomes displayed normal levels. A protein-protein interaction screen identified several RAB28-interacting proteins, including the prenyl-binding protein phosphodiesterase 6 δ-subunit (PDE6D) and voltage-gated potassium channel subfamily J member 13 (KCNJ13) present in the RPE apical processes. Of note, the loss of PDE6D prevented delivery of RAB28 to OSs. Taken together, these findings reveal that RAB28 is required for shedding and phagocytosis of cone OS discs.


Assuntos
Fagocitose , Células Fotorreceptoras Retinianas Cones/enzimologia , Epitélio Pigmentado da Retina/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Distrofias de Cones e Bastonetes/enzimologia , Distrofias de Cones e Bastonetes/genética , Distrofias de Cones e Bastonetes/patologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/metabolismo , Camundongos , Camundongos Knockout , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Epitélio Pigmentado da Retina/patologia , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Células Fotorreceptoras Retinianas Bastonetes/patologia , Proteínas rab de Ligação ao GTP/genética
20.
Adv Exp Med Biol ; 1074: 351-357, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29721963

RESUMO

c-Jun N-terminal kinase (JNK), a member of stress-induced mitogen-activated protein (MAP) kinase family, has been shown to modulate a variety of biological processes associated with neurodegenerative pathology of the retina. In particular, various retinal cell culture and animal models related to glaucoma, age-related macular degeneration (AMD), and retinitis pigmentosa indicate that JNK signaling may contribute to disease pathogenesis. This mini-review discusses the impact of JNK signaling in retinal disease, with a focus on retinal ganglion cells (RGCs), photoreceptor cells, retinal pigment epithelial (RPE) cells, and animal studies, with particular attention to modulation of JNK signaling as a potential therapeutic target for the treatment of retinal disease.


Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Sistema de Sinalização das MAP Quinases , Degeneração Retiniana/enzimologia , Transtornos da Visão/enzimologia , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica/fisiologia , Glaucoma/enzimologia , Glaucoma/genética , Glaucoma/fisiopatologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/deficiência , Degeneração Macular/enzimologia , Degeneração Macular/genética , Degeneração Macular/fisiopatologia , Camundongos , Terapia de Alvo Molecular , Células Fotorreceptoras de Vertebrados/enzimologia , Células Fotorreceptoras de Vertebrados/fisiologia , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Epitélio Pigmentado da Retina/enzimologia , Epitélio Pigmentado da Retina/fisiologia , Transtornos da Visão/genética , Transtornos da Visão/terapia
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