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1.
BMC Ophthalmol ; 19(1): 168, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375076

RESUMO

BACKGROUND: Age-related macular degeneration (AMD) is the primary cause of blindness and severe vision loss in developed countries and is responsible for 8.7% of blindness globally. Ultraviolet radiation can induce DNA breakdown, produce reactive oxygen species, and has been implicated as a risk factor for AMD. This study investigated the effects of UVA radiation on Human retinal pigment epithelial cell (ARPE-19) growth and protein expression. METHODS: ARPE-19 cells were irradiated with a UVA lamp at different doses (5, 10, 20, 30 and 40 J/cm2) from 10 cm. Cell viability was determined by MTT assay. Visual inspection was first achieved with inverted light microscopy and then the DeadEnd™ Fluorometric TUNEL System was used to observe nuclear DNA fragmentation. Flow cytometry based-Annexin V-FITC/PI double-staining was used to further quantify cellular viability. Mitochondrial membrane potential was assessed with JC-1 staining. 2D electrophoresis maps of exposed cells were compared to nonexposed cells and gel images analyzed with PDQuest 2-D Analysis Software. Spots with greater than a 1.5-fold difference were selected for LC-MS/MS analysis and some confirmed by western blot. We further investigated whether caspase activation, apoptotic-related mitochondrial proteins, and regulators of ER stress sensors were involved in UVA-induced apoptosis. RESULTS: We detected 29 differentially expressed proteins (9 up-regulated and 20 down-regulated) in the exposed cells. Some of these proteins such as CALR, GRP78, NPM, Hsp27, PDI, ATP synthase subunit alpha, PRDX1, and GAPDH are associated with anti-proliferation, induction of apoptosis, and oxidative-stress protection. We also detected altered protein expression levels among caspases (caspase 3 and 9) and in the mitochondrial (cytosolic cytochrome C, AIF, Mcl-1, Bcl-2, Bcl-xl, Bax, Bad, and p-Bad) and ER stress-related (p-PERK, p-eIF2α, ATF4 and CHOP) apoptotic pathways. CONCLUSIONS: UVA irradiation suppressed the proliferation of ARPE-19 cells in a dose-dependent manner, caused quantitative loses in transmembrane potential (ΔΨm), and induced both early and late apoptosis.


Assuntos
Degeneração Macular/patologia , Estresse Oxidativo , Proteômica/métodos , Epitélio Pigmentado da Retina/metabolismo , Raios Ultravioleta , Apoptose , Sobrevivência Celular , Células Cultivadas , Cromatografia Líquida , Citocromos c/metabolismo , Humanos , Degeneração Macular/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/efeitos da radiação , Transdução de Sinais , Espectrometria de Massas em Tandem
2.
Cell Physiol Biochem ; 53(2): 400-412, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31403270

RESUMO

BACKGROUND/AIMS: Mutations in ABCA4 cause Stargardt macular degeneration, which invariably ends in legal blindness. We studied two common mutants, A1038V (in NBD1) and G1961E (in NBD2), with the purpose of exploring how they interact with the cell's quality control mechanism. The study was designed to determine how these mutants can be rescued. METHODS: We expressed wt and mutant ABCA4 in HEK293 cells and studied the effect of the mutations on trafficking and processing and the ability of correctors to rescue them. We used a combination of western blotting, confocal microscopy and surface biotinylation coupled with pulldown of plasma membrane proteins. RESULTS: G1961E is sensitive to inhibitors of the aggresome, tubacin and the lysosome, bafilomycin A. Both mutants cause a reduction in heat shock protein, Hsp27. Incubation of HEK293 cells expressing the mutants with VX-809, an FDA approved drug for the treatment of cystic fibrosis, increased the levels of A1038V and G1961E by 2- to 3-fold. Importantly, VX-809 increased the levels of both mutants at the plasma membrane suggesting that trafficking had been restored. Transfecting additional Hsp27 to the cells also increased the steady state levels of both mutants. However, in combination with VX-809 the addition of Hsp27 caused a dramatic increase in the protein expression particularly in the G1961 mutant which increased approximately 5-fold. CONCLUSION: Our results provide a new mechanism for the rescue of ABCA4 trafficking mutants based on the restoration of Hsp27. Our results provide a pathway for the treatment of Stargardt disease.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Transportadores de Cassetes de Ligação de ATP/genética , Aminopiridinas/uso terapêutico , Anilidas/farmacologia , Benzodioxóis/uso terapêutico , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Leupeptinas/farmacologia , Lisossomos/metabolismo , Degeneração Macular/congênito , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Mutação , Transporte Proteico/efeitos dos fármacos
3.
Invest Ophthalmol Vis Sci ; 60(6): 2049-2063, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31074760

RESUMO

Purpose: To assess the phenotypic variability and natural course of inherited retinal diseases (IRDs) caused by EYS mutations. Methods: Multiethnic cohort study (N = 30) with biallelic EYS variants from a clinical IRD database (retinitis pigmentosa [RP], N = 27; cone-rod dystrophy [CRD], N = 1; and macular dystrophy, N = 2). In vitro minigene splice assay was performed to determine the effect on EYS pre-mRNA splicing of the c.1299+5_1299+8del variant in macular dystrophy patients. Results: We found 27 different EYS variants in RP patients and 7 were novel. The rate of visual field loss of the V4e isopter area was -0.84 ± 0.44 ln(deg2) per year, and the rate of visual acuity loss was 0.75 Early Treatment Diabetic Retinopathy Study letters per year. Ellipsoid zone width was correlated with area of the hyperautofluorescent ring, with rs = 0.78 and P < 0.001. Rate of decline in ellipsoid zone width was -57 ± 17 µm per year (P < 0.01) (n = 14) or -3.69% ± 0.51% from baseline per year (P < 0.001). An isolated CRD patient carried a homozygous EYS variant (c.9405T>A), previously identified in RP patients. Two siblings with macular dystrophy carried compound heterozygous EYS variants: c.1299+5_1299+8del and c.6050G>T. The former was novel and shown to result in skipping of exon 8, and the latter was a known RP variant. Conclusions: We report on EYS-associated macular dystrophy, extending the spectrum of EYS-associated IRDs. We observed heterogeneity between RP patients in age of onset and disease progression. Identical EYS variants were found in cases with RP, CRD, and macular dystrophy. Screening for EYS variants in CRD and macular dystrophy patients might increase the diagnostic yield in previously unsolved cases.


Assuntos
Proteínas do Olho/genética , Degeneração Macular/genética , Mutação , RNA Mensageiro/genética , Retina/patologia , Retinite Pigmentosa/genética , Acuidade Visual , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , Eletrorretinografia , Proteínas do Olho/metabolismo , Feminino , Homozigoto , Humanos , Degeneração Macular/diagnóstico , Degeneração Macular/metabolismo , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Retina/metabolismo , Retina/fisiopatologia , Retinite Pigmentosa/diagnóstico , Retinite Pigmentosa/metabolismo , Tomografia de Coerência Óptica , Adulto Jovem
4.
J Opt Soc Am A Opt Image Sci Vis ; 36(4): B123-B131, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31044990

RESUMO

Macular pigments (MPs), by absorbing potentially toxic short-wavelength (400-500 nm) visible light, provide protection against photo-chemical damage thought to be relevant in the pathogenesis of age-related macular degeneration (AMD). A method of screening for low levels of MPs could be part of a prevention strategy for helping people to delay the onset of AMD. We introduce a new method for assessing MP density that takes advantage of the polarization-dependent absorption of blue light by MPs, which results in the entoptic phenomenon called Haidinger's brushes (HB). Subjects were asked to identify the direction of rotation of HB when presented with a circular stimulus illuminated with an even intensity of polarized white light in which the electric field vector was rotating either clockwise or anti-clockwise. By reducing the degree of polarization of the stimulus light, a threshold for perceiving HB (degree of polarization threshold) was determined and correlated (r2=0.66) to macular pigment optical density assessed using dual-wavelength fundus autofluoresence. The speed and ease of measurement of degree of polarization threshold makes it well suited for large-scale screening of macular pigmentation.


Assuntos
Pigmento Macular/metabolismo , Dispositivos Ópticos , Humanos , Degeneração Macular/metabolismo , Rotação , Fatores de Tempo
5.
Mol Vis ; 25: 174-182, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30996586

RESUMO

Purpose: To evaluate the efficacy of using a CRISPR/Cas-mediated strategy to correct a common high-risk allele that is associated with age-related macular degeneration (AMD; rs1061170; NM_000186.3:c.1204T>C; NP_000177.2:p.His402Tyr) in the complement factor H (CFH) gene. Methods: A human embryonic kidney cell line (HEK293A) was engineered to contain the pathogenic risk variant for AMD (HEK293A-CFH). Several different base editor constructs (BE3, SaBE3, SaKKH-BE3, VQR-BE3, and Target-AID) and their respective single-guide RNA (sgRNA) expression cassettes targeting either the pathogenic risk variant allele in the CFH locus or the LacZ gene, as a negative control, were evaluated head-to-head for the incidence of a cytosine-to-thymine nucleotide correction. The base editor construct that showed appreciable editing activity was selected for further assessment in which the base-edited region was subjected to next-generation deep sequencing to quantify on-target and off-target editing efficacy. Results: The tandem use of the Target-AID base editor and its respective sgRNA demonstrated a base editing efficiency of facilitating a cytosine-to-thymine nucleotide correction in 21.5% of the total sequencing reads. Additionally, the incidence of insertions and deletions (indels) was detected in only 0.15% of the sequencing reads with virtually no off-target effects evident across the top 11 predicted off-target sites containing at least one cytosine in the activity window (n = 3, pooled amplicons). Conclusions: CRISPR-mediated base editing can be used to facilitate a permanent and stably inherited cytosine-to-thymine nucleotide correction of the rs1061170 SNP in the CFH gene with minimal off-target effects.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , RNA Guia/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Citosina/metabolismo , Expressão Gênica , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Óperon Lac , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , RNA Guia/metabolismo , Timina/metabolismo
6.
Int J Mol Sci ; 20(7)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987401

RESUMO

The present review focuses on recent clinical trials that analyze the efficacy of intravitreal therapeutic agents for the treatment of dry age-related macular degeneration (AMD), such as neuroprotective drugs, and complement inhibitors, also called immunomodulatory or anti-inflammatory agents. A systematic literature search was performed to identify randomized controlled trials published prior to January 2019. Patients affected by dry AMD treated with intravitreal therapeutic agents were included. Changes in the correct visual acuity and reduction in geographic atrophy progression were evaluated. Several new drugs have shown promising results, including those targeting the complement cascade and neuroprotective agents. The potential action of the two groups of drugs is to block complement cascade upregulation of immunomodulating agents, and to prevent the degeneration and apoptosis of ganglion cells for the neuroprotectors, respectively. Our analysis indicates that finding treatments for dry AMD will require continued collaboration among researchers to identify additional molecular targets and to fully interrogate the utility of pluripotent stem cells for personalized therapy.


Assuntos
Atrofia Geográfica/tratamento farmacológico , Injeções Intravítreas/métodos , Degeneração Macular/tratamento farmacológico , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/uso terapêutico , Atrofia Geográfica/metabolismo , Humanos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/uso terapêutico , Degeneração Macular/metabolismo , Ensaios Clínicos Controlados Aleatórios como Assunto
7.
PLoS Genet ; 15(3): e1007873, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30889179

RESUMO

Autosomal recessive retinal degenerative diseases cause visual impairment and blindness in both humans and dogs. Currently, no standard treatment is available, but pioneering gene therapy-based canine models have been instrumental for clinical trials in humans. To study a novel form of retinal degeneration in Labrador retriever dogs with clinical signs indicating cone and rod degeneration, we used whole-genome sequencing of an affected sib-pair and their unaffected parents. A frameshift insertion in the ATP binding cassette subfamily A member 4 (ABCA4) gene (c.4176insC), leading to a premature stop codon in exon 28 (p.F1393Lfs*1395), was identified. In contrast to unaffected dogs, no full-length ABCA4 protein was detected in the retina of an affected dog. The ABCA4 gene encodes a membrane transporter protein localized in the outer segments of rod and cone photoreceptors. In humans, the ABCA4 gene is associated with Stargardt disease (STGD), an autosomal recessive retinal degeneration leading to central visual impairment. A hallmark of STGD is the accumulation of lipofuscin deposits in the retinal pigment epithelium (RPE). The discovery of a canine homozygous ABCA4 loss-of-function mutation may advance the development of dog as a large animal model for human STGD.


Assuntos
Membro 4 da Subfamília A de Transportadores de Cassetes de Ligação de ATP/genética , Doenças do Cão/genética , Degeneração Macular/congênito , Mutação , Membro 4 da Subfamília A de Transportadores de Cassetes de Ligação de ATP/química , Membro 4 da Subfamília A de Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Códon sem Sentido , Modelos Animais de Doenças , Doenças do Cão/metabolismo , Doenças do Cão/patologia , Cães , Feminino , Genes Recessivos , Homozigoto , Humanos , Lipofuscina/metabolismo , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/veterinária , Masculino , Microscopia de Fluorescência , Modelos Moleculares , Mutagênese Insercional , Linhagem , Conformação Proteica , Retina/metabolismo , Retina/patologia , Sequenciamento Completo do Genoma
8.
Artigo em Inglês | MEDLINE | ID: mdl-30857240

RESUMO

This study provides diverse lines of evidence demonstrating that fluoride (F) exposure contributes to degenerative eye diseases by stimulating or inhibiting biological pathways associated with the pathogenesis of cataract, age-related macular degeneration and glaucoma. As elucidated in this study, F exerts this effect by inhibiting enolase, τ-crystallin, Hsp40, Na⁺, K⁺-ATPase, Nrf2, γ -GCS, HO-1 Bcl-2, FoxO1, SOD, PON-1 and glutathione activity, and upregulating NF-κB, IL-6, AGEs, HsP27 and Hsp70 expression. Moreover, F exposure leads to enhanced oxidative stress and impaired antioxidant activity. Based on the evidence presented in this study, it can be concluded that F exposure may be added to the list of identifiable risk factors associated with pathogenesis of degenerative eye diseases. The broader impact of these findings suggests that reducing F intake may lead to an overall reduction in the modifiable risk factors associated with degenerative eye diseases. Further studies are required to examine this association and determine differences in prevalence rates amongst fluoridated and non-fluoridated communities, taking into consideration other dietary sources of F such as tea. Finally, the findings of this study elucidate molecular pathways associated with F exposure that may suggest a possible association between F exposure and other inflammatory diseases. Further studies are also warranted to examine these associations.


Assuntos
Catarata/metabolismo , Fluoretos/administração & dosagem , Glaucoma/metabolismo , Degeneração Macular/metabolismo , Saúde Pública , Antioxidantes/metabolismo , Biomarcadores/metabolismo , Catarata/induzido quimicamente , Catarata/fisiopatologia , Fluoretação , Fluoretos/efeitos adversos , Glaucoma/induzido quimicamente , Glaucoma/fisiopatologia , Humanos , Degeneração Macular/induzido quimicamente , Degeneração Macular/fisiopatologia , Saúde Bucal , Estresse Oxidativo , Abastecimento de Água
9.
Mol Vis ; 25: 106-117, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820146

RESUMO

Purpose: Inherited retinal diseases (IRDs) are clinically and genetically heterogeneous showing progressive retinal cell death which results in vision loss. IRDs include a wide spectrum of disorders, such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), cone-rod dystrophy (CRD), and Stargardt disease (STGD1). Methods: In this study, we performed targeted next-generation sequencing based on molecular inversion probes (MIPs) that allowed the sequence analysis of 108 IRD-associated genes in 50 Iranian IRD probands. Results: The sequencing and variant filtering led to the identification of putative pathogenic variants in 36 out of 50 (72%) probands. Among 36 unique variants, we identified 20 novel variants in 15 genes. Four out of 36 probands carry compound heterozygous variants, and 32 probands carry homozygous variants. Conclusions: Employing a cost-effective targeted next-generation sequencing procedure, we identified the genetic causes of different retinal disorders in the majority of Iranian families in this study.


Assuntos
Distrofias de Cones e Bastonetes/genética , Proteínas do Olho/genética , Amaurose Congênita de Leber/genética , Degeneração Macular/congênito , Mutação , Retinite Pigmentosa/genética , Adolescente , Adulto , Criança , Distrofias de Cones e Bastonetes/metabolismo , Distrofias de Cones e Bastonetes/patologia , Proteínas do Olho/metabolismo , Feminino , Expressão Gênica , Estudos de Associação Genética , Genótipo , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Homozigoto , Humanos , Irã (Geográfico) , Amaurose Congênita de Leber/metabolismo , Amaurose Congênita de Leber/patologia , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Masculino , Linhagem , Fenótipo , Retina/metabolismo , Retina/patologia , Retinite Pigmentosa/congênito , Retinite Pigmentosa/metabolismo , Retinite Pigmentosa/patologia
10.
Graefes Arch Clin Exp Ophthalmol ; 257(6): 1147-1157, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30903310

RESUMO

PURPOSE: To raise awareness of Stargardt disease (STGD1) patients without fundus abnormalities. METHODS: Medical records were evaluated for age at onset, initial symptoms and diagnosis, reason for delay of diagnosis, age at STGD1 diagnosis, best-corrected visual acuity (BCVA), ophthalmoscopy, fundus photography, fundus autofluorescence (FAF), fluorescein angiography (FA), spectral-domain optical coherence tomography (SD-OCT), full-field electroretinography (ffERG), color vision test, and the presence of ABCA4 variants. RESULTS: In 11.1% of our STGD1 cohort of 280 patients, no fundus abnormalities were observed at first ophthalmic consultation. The median age at onset was 8 years (range, 1-18). There was a median delay in diagnosis of 3 years (range, 0-19) in 27 out of 31 patients, which resulted in a median age at diagnosis of 12 years (range, 7-26). Patients were misdiagnosed with amblyopia, myopia, optic disk pathology, mental health problems, tension headache, neuritis bulbaris, and uveitis. Subtle abnormalities, such as lipofuscin accumulation, were seen on FAF at an earlier disease stage than in ophthalmoscopy. On SD-OCT, this included a thickened external limiting membrane. Color vision tests showed red-green insufficiency in 79% of patients. Reduced ERG amplitudes were only present in 26% (N = 8) and a dark choroid sign in 65% of the patients. Visual acuity considerably fluctuated in the first 5 years after onset. The majority of the patients (65%) carried a least one variant with a severe effect on ABCA4 function. CONCLUSIONS: Childhood-onset STGD1 patients were diagnosed with a delay of median 3 years. The presence of accurate competence, equipment, and the possibility for genetic screening is required; therefore, we recommend to refer children with visual complaints without initial fundus abnormalities to a specialized ophthalmologic center. In particular, to diagnose patients at an early stage of disease is of increased importance with the advent of new therapeutic possibilities.


Assuntos
Eletrorretinografia/métodos , Angiofluoresceinografia/métodos , Degeneração Macular/congênito , Epitélio Pigmentado da Retina/patologia , Tomografia de Coerência Óptica/métodos , Acuidade Visual , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Erros de Diagnóstico , Feminino , Fundo de Olho , Testes Genéticos , Humanos , Lactente , Degeneração Macular/diagnóstico , Degeneração Macular/genética , Degeneração Macular/metabolismo , Masculino , Oftalmoscopia , Adulto Jovem
11.
BMC Ophthalmol ; 19(1): 79, 2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30885167

RESUMO

BACKGROUND: Vitreomacular adhesion (VMA) has been reported to associated with age-related macular degeneration (AMD). Understanding the mechanisms underlying cyclic stretch induced in retinal pigment epithelial cells (RPE) may be important for the treatment of VMA-related AMD. METHOD: Cyclic stretch (1HZ, 20% elongation) was applied to cultured ARPE-19 cells for 15 min, 2 h, 6 h, 12 h, 24 h by flexcell FX-5000 Tension system. Total reactive oxygen species (ROS) were detected using DCFH-DA. Mitochondrial superoxide were detected using MitoSOX Red mitochondrial superoxide indicator. NADPH oxidases (NOX) and signaling pathways, such as p38 and PKC, were detected using western blot. Apocycin (Apo) were used as NOX inhibitors. RESULT: High levels of total ROS were detected from 15 min to 24 h, whereas mitochondrial superoxide were higher only in early time. NOX2 were significantly increased at 24 h. NOX4 were significantly increased at 2 h and reach its peak at 24 h. P-p38 was significantly increased at 12 h and 24 h. P-PKC was significantly increased at 15 min and kept a persistent high level. The upregulated expression of NOX4 by cyclic stretch can be significantly decreased under p-PKC inhibitor other than p-p38 inhibitor. CONCLUSION: Cyclic stretch induce oxidative stress from both mitochodrial and NADPH oxidase in RPE cells, which may prompt oxidative damage in VMA-related AMD.


Assuntos
Degeneração Macular , Mitocôndrias/metabolismo , NADPH Oxidases/fisiologia , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Doenças Retinianas/fisiopatologia , Epitélio Pigmentado da Retina/fisiopatologia , Estresse Mecânico , Fenômenos Biomecânicos , Células Cultivadas , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/fisiopatologia , NADPH Oxidase 4/metabolismo , NADPH Oxidases/metabolismo , Transdução de Sinais/fisiologia , Superóxidos/metabolismo , Vias Visuais/fisiologia
12.
Int J Mol Sci ; 20(3)2019 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-30754662

RESUMO

The retinal pigment epithelium (RPE) forms the outer blood⁻retina barrier and facilitates the transepithelial transport of glucose into the outer retina via GLUT1. Glucose is metabolized in photoreceptors via the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS) but also by aerobic glycolysis to generate glycerol for the synthesis of phospholipids for the renewal of their outer segments. Aerobic glycolysis in the photoreceptors also leads to a high rate of production of lactate which is transported out of the subretinal space to the choroidal circulation by the RPE. Lactate taken up by the RPE is converted to pyruvate and metabolized via OXPHOS. Excess lactate in the RPE is transported across the basolateral membrane to the choroid. The uptake of glucose by cone photoreceptor cells is enhanced by rod-derived cone viability factor (RdCVF) secreted by rods and by insulin signaling. Together, the three cells act as symbiotes: the RPE supplies the glucose from the choroidal circulation to the photoreceptors, the rods help the cones, and both produce lactate to feed the RPE. In age-related macular degeneration this delicate ménage à trois is disturbed by the chronic infiltration of inflammatory macrophages. These immune cells also rely on aerobic glycolysis and compete for glucose and produce lactate. We here review the glucose metabolism in the homeostasis of the outer retina and in macrophages and hypothesize what happens when the metabolism of photoreceptors and the RPE is disturbed by chronic inflammation.


Assuntos
Degeneração Macular/etiologia , Degeneração Macular/metabolismo , Retina/metabolismo , Animais , Sobrevivência Celular , Suscetibilidade a Doenças , Metabolismo Energético , Predisposição Genética para Doença , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Degeneração Macular/patologia , Oxirredução , Retina/patologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinite/complicações , Retinite/patologia
13.
Int J Mol Sci ; 20(1)2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30626110

RESUMO

Age-related macular degeneration (AMD) is a complex eye disease with many pathogenesis factors, including defective cellular waste management in retinal pigment epithelium (RPE). Main cellular waste in AMD are: all-trans retinal, drusen and lipofuscin, containing unfolded, damaged and unneeded proteins, which are degraded and recycled in RPE cells by two main machineries-the ubiquitin-proteasome system (UPS) and autophagy. Recent findings show that these systems can act together with a significant role of the EI24 (etoposide-induced protein 2.4 homolog) ubiquitin ligase in their action. On the other hand, E3 ligases are essential in both systems, but E3 is degraded by autophagy. The interplay between UPS and autophagy was targeted in several diseases, including Alzheimer disease. Therefore, cellular waste clearing in AMD should be considered in the context of such interplay rather than either of these systems singly. Aging and oxidative stress, two major AMD risk factors, reduce both UPS and autophagy. In conclusion, molecular mechanisms of UPS and autophagy can be considered as a target in AMD prevention and therapeutic perspective. Further work is needed to identify molecules and effects important for the coordination of action of these two cellular waste management systems.


Assuntos
Autofagia , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Humanos , Modelos Biológicos , Retina/patologia , Retina/fisiopatologia
14.
Nat Commun ; 10(1): 369, 2019 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-30664640

RESUMO

Choroidal neovascularization (CNV) is a major cause of visual impairment in patients suffering from wet age-related macular degeneration (AMD), particularly when refractory to intraocular anti-VEGF injections. Here we report that treatment with the oral mineralocorticoid receptor (MR) antagonist spironolactone reduces signs of CNV in patients refractory to anti-VEGF treatment. In animal models of wet AMD, pharmacological inhibition of the MR pathway or endothelial-specific deletion of MR inhibits CNV through VEGF-independent mechanisms, in part through upregulation of the extracellular matrix protein decorin. Intravitreal injections of spironolactone-loaded microspheres and systemic delivery lead to similar reductions in CNV. Together, our work suggests MR inhibition as a novel therapeutic option for wet AMD patients unresponsive to anti-VEGF drugs.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Neovascularização de Coroide/tratamento farmacológico , Degeneração Macular/tratamento farmacológico , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Receptores de Mineralocorticoides/genética , Espironolactona/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Animais , Corioide/efeitos dos fármacos , Corioide/metabolismo , Corioide/patologia , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Composição de Medicamentos/métodos , Feminino , Expressão Gênica , Humanos , Injeções Intravítreas , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Transgênicos , Microesferas , Projetos Piloto , Estudos Prospectivos , Ranibizumab/uso terapêutico , Ratos Long-Evans , Receptores de Mineralocorticoides/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
15.
Graefes Arch Clin Exp Ophthalmol ; 257(5): 931-952, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30693383

RESUMO

PURPOSE: To examine the reaction of microglial cells (MG) when incubated with lipofuscin (LP) in vitro with emphasis on the immunological reaction of the MG toward LP and the suppression of this reaction by immunomodulatory agents. MG are involved in the pathogenesis of degenerative eye disorders such as age-related macular degeneration (AMD). LP is a heterogeneous waste material that accumulates in the retinal pigment epithelium (RPE) cells with advancing age. LP is known to have toxic effects on RPE cells and therefore an elevated LP-derived fundus autofluorescence is a risk factor for AMD development. MG in the subretinal space have been reported in eyes affected by AMD. Moreover, in senescent mice, subretinal MG were found, which display an autofluorescence that may be derived from LP uptake. METHODS: In this study, we incubated MG (BV-2 cell line and primary cells from murine brain) in vitro with LP isolated from the human RPE. We observed phagocytosis, studied cell morphologies, and analyzed the cell culture supernatants. We also investigated the effect of the immunomodulatory agents hydrocortisone (HC), minocycline, and the tripeptide TKP. RESULTS: The MG phagocytosed the LP quickly and completely. We detected highly elevated levels of pro-inflammatory cytokines (especially of IL-6, IL-23p19, TNF-α, KC, RANTES, and IL-1α) in the cell culture supernatants. Furthermore, levels of vascular endothelial growth factor (VEGF) were raised in BV-2 cells. Anti-inflammatory agents added to the cell cultures inhibited the inflammatory reaction, in particular hydrocortisone (HC). Minocycline and TKP had less impact on the cytokine release. CONCLUSION: The interaction of MG and LP could play a role in the development of retinal degeneration by triggering an inflammatory reaction and angiogenesis.


Assuntos
Lipofuscina/farmacologia , Degeneração Macular/diagnóstico , Microglia/ultraestrutura , Epitélio Pigmentado da Retina/ultraestrutura , Idoso , Animais , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Degeneração Macular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microscopia Eletrônica , Epitélio Pigmentado da Retina/efeitos dos fármacos
16.
Biosci Rep ; 39(1)2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30563929

RESUMO

Stargardt disease (STGD1, OMIM 248200) is a common hereditary juvenile or early adult onset macular degeneration. It ultimately leads to progressive central vision loss. Here, we sought to identify gene mutations associated with STGD1 in a three-generation Han Chinese pedigree by whole exome sequencing and Sanger sequencing. Two novel potentially pathogenic variants in a compound heterozygous state, c.3607G>T (p.(Gly1203Trp)) and c.6722T>C (p.(Leu2241Pro)), in the ATP binding cassette subfamily A member 4 gene (ABCA4) were identified as contributing to the family's STGD1 phenotype. These variants may impact the ABCA4 protein structure and reduce the retinal-activated ATPase activity, leading to abnormal all-trans retinal accumulation in photoreceptor outer segments and in retinal pigment epithelium cells. The present study broadens the mutational spectrum of the ABCA4 responsible for STGD1. A combination of whole exome sequencing and Sanger sequencing is likely to be a time-saving and cost-efficient approach to screen pathogenic variants in genetic disorders caused by sizable genes, as well as avoiding misdiagnosis. These results perhaps refine genetic counseling and ABCA4-targetted treatments for families affected by STGD1.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Degeneração Macular/congênito , Polimorfismo Genético , Epitélio Pigmentado da Retina/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adulto , Sequência de Aminoácidos , Animais , Grupo com Ancestrais do Continente Asiático , Sequência de Bases , Estudos de Casos e Controles , Feminino , Expressão Gênica , Heterozigoto , Humanos , Degeneração Macular/etnologia , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Masculino , Modelos Moleculares , Linhagem , Fenótipo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Epitélio Pigmentado da Retina/patologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Sequenciamento Completo do Exoma
17.
Microvasc Res ; 123: 50-57, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30571950

RESUMO

Age-related macular degeneration (AMD) is a common cause of blindness worldwide. While recent studies have revealed that the loss of choroidal endothelial cells (ChECs) is critical to the disease pathogenesis of dry AMD, in vitro studies are needed to fully elucidate the disease mechanism. However, these studies remain hindered due to the lack of publically available human ChEC lines. To address this need, ChECs were harvested form donor tissue and enriched for by using magnetic cell separation using anti-CD31 conjugated microbeads. Next, lenti-viral vectors with endothelial-specific promoters driving genes necessary for immortalization, CDH5p-hTERT and CDH5p TAg, were generated. Stable integration of both gene cassettes allowed cells to maintain their proliferative state and yielded an immortalized cell line (iChEC-1). Immunocytochemical analysis of iChEC-1 confirmed the expression of important ChEC markers such as CA4, a marker of choriocapillaris endothelial cells, CDH5, and CD34, pan-endothelial cell markers. qRT-PCR analysis of expanded clones from iChEC-1 further showed that the line maintained expression of other important endothelial markers, vWF, PECAM1, and PLVAP, similar to primary cells. Functional responses were characterized by tube-forming assays and repopulation of decellularized choroid with the immortalized cell line. In conclusion, the iChEC-1 line presents a suitable immortalized human ChEC line for future in vitro studies of AMD.


Assuntos
Antígenos CD/genética , Caderinas/genética , Corioide/irrigação sanguínea , Células Endoteliais/fisiologia , Regiões Promotoras Genéticas , Antígenos Virais de Tumores/genética , Antígenos Virais de Tumores/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Genótipo , Humanos , Separação Imunomagnética , Recém-Nascido , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Degeneração Macular/fisiopatologia , Neovascularização Fisiológica , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Telomerase/genética , Telomerase/metabolismo , Transfecção
18.
Adv Gerontol ; 31(3): 339-344, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30584871

RESUMO

Age-related macular degeneration (AMD) is the leading cause of vision loss among the elderly. By clinical signs, there are two forms of AMD: the atrophic or dry (~ 90% of all cases) and wet or neovascular AMD (~10% of cases). Anti-vascular endothelial growth factor (VEGF) intravitreal agents are the only successful treatment for wet AMD. However, there are emerging signals that anti-VEGF treatment can potentially increase development of atrophic AMD. There is neither a treatment of the dry AMD due poor understanding of the pathogenesis and retina aging process in general. We have shown previously that senescence-accelerated OXYS rats are a suitable model of dry AMD. Signs of retinopathy in OXYS rats manifest themselves by age 3 months against the background of a decline in the number of retinal pigment epithelium (RPE) cells and an alteration of choroidal microcirculation. Herein, we compared retinal expression of proteins VEGF and PEDF (pigment epithelium-derived factor) between OXYS and Wistar rats (control). The amount of the VEGF protein increased with age in the retina of both rat strains from 3 months of age. From age 3 to 24 months, this parameter was significantly lower in OXYS rats than in Wistar rats. PEDF protein concentration was significant lower in the OXYS retina only at the age of 3 months. We can conclude that development of retinopathy in OXYS rats takes place at reduced concentrations of VEGF and PEDF. Because RPE cells control the VEGF-PEDF balance, an RPE-targeted approach is a logical choice for AMD treatment and for decreasing adverse effects of anti-VEGF treatment.


Assuntos
Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/metabolismo , Retina/metabolismo , Serpinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Envelhecimento/fisiologia , Animais , Modelos Animais de Doenças , Degeneração Macular/metabolismo , Ratos , Ratos Wistar
19.
Oxid Med Cell Longev ; 2018: 1610751, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30584457

RESUMO

The protection of retinal pigment epithelium (RPE) injury plays an important role in the prevention of or in delaying the pathological progress of retinal degeneration diseases, like age-related macular degeneration (AMD), diabetic retinopathy, and retinitis pigmentosa. Oxidative stress has been identified as a major inducer of RPE injury, which eventually could lead to a loss of vision. Kaempferol is a natural flavonoid widely distributed in many edible plants, fruits, and traditional medicines and has been reported to have antioxidant, anti-inflammatory, anticancer, and antimicrobial activities. The present study demonstrates that the total antioxidant capacity of kaempferol is approximately two times stronger than that of lutein which is also a natural antioxidant that is widely used in the prevention or treatment of AMD. Our data indicates that kaempferol protects human RPE cells (ARPE-19) from hydrogen peroxide- (H2O2-) induced oxidative cell damage and apoptosis through the signaling pathways involving Bax/Bcl-2 and caspase-3 molecules proofed by real-time PCR and Western blot results. Kaempferol also inhibits the upregulated vascular endothelial growth factor (VEGF) mRNA expression levels induced by H2O2 in ARPE-19 cells and affects the oxidation and antioxidant imbalanced system in ARPE-19 cells treated by H2O2 through the regulations of both the activities of reactive oxygen species (ROS) and superoxide dismutase (SOD). Furthermore, our in vivo experimental results show that in sodium iodate-induced retinal degeneration rat model, kaempferol could protect sodium iodate-induced pathological changes of retina tissue and retinal cells apoptosis as well as the upregulated VEGF protein expression in RPE cells. In summary, these novel findings demonstrate that kaempferol could protect oxidative stressed-human RPE cell damage through its antioxidant activity and antiapoptosis function, suggesting that kaempferol has a potential role in the prevention and therapeutic treatment of AMD or other retinal diseases mediated by oxidative stress.


Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Quempferóis/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/metabolismo , Epitélio Pigmentado da Retina/citologia , Animais , Humanos , Peróxido de Hidrogênio/farmacologia , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , RNA Mensageiro/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética
20.
Invest Ophthalmol Vis Sci ; 59(4): AMD182-AMD194, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30357337

RESUMO

AMD pathobiology was irreversibly changed by the recent discovery of extracellular cholesterol-containing deposits in the subretinal space, between the photoreceptors and retinal pigment epithelium (RPE), called subretinal drusenoid deposits (SDDs). SDDs strikingly mirror the topography of rod photoreceptors in human macula, raising the question of whether an equivalent process results in a deposition related to foveal cones. Herein we propose that AMD's pathognomonic lesion-soft drusen and basal linear deposit (BLinD, same material, diffusely distributed)-is the leading candidate. Epidemiologic, clinical, and histologic data suggest that these deposits are most abundant in the central macula, under the fovea. Strong evidence presented in a companion article supports the idea that the dominant ultrastructural component is large apolipoprotein B,E-containing lipoproteins, constitutively secreted by RPE. Lipoprotein fatty acids are dominated by linoleate (implicating diet) rather than docosahexaenoate (implicating photoreceptors); we seek within the retina cellular relationships and dietary drivers to explain soft druse topography. The delivery of xanthophyll pigments to highly evolved and numerous Müller cells in the human fovea, through RPE, is one strong candidate, because Müller cells are the main reservoir of these pigments, which replenish from diet. We propose that the evolution of neuroglial relations and xanthophyll delivery that underlie exquisite human foveal vision came with a price, that is, soft drusen and sequela, long after our reproductive years.


Assuntos
Apolipoproteínas B/metabolismo , Apolipoproteínas E/metabolismo , Macula Lutea/metabolismo , Degeneração Macular/metabolismo , Drusas Retinianas/metabolismo , Humanos , Degeneração Macular/fisiopatologia , Drusas Retinianas/fisiopatologia , Epitélio Pigmentado da Retina/metabolismo , Xantofilas/metabolismo
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