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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): 2208-2217, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31108551

RESUMO

Purpose: To identify the factors associated with retinal vessel diameters in the population of the Thessaloniki Eye Study. Methods: Cross-sectional population-based study (age ≥ 60 years). Subjects with glaucoma, late age-related macular degeneration, and diabetic retinopathy were excluded from the analyses. Retinal vessel diameters were measured using the IVAN software, and measurements were summarized to central retinal artery equivalent (CRAE), central retinal vein equivalent (CRVE), and arteriole to venule ratio (AVR). Results: The analysis included 1614 subjects. The hypertensive group showed lower values of CRAE (P = 0.033) and AVR (P = 0.0351) compared to the normal blood pressure (BP) group. On the contrary, the group having normal BP under antihypertensive treatment did not have different values compared to the normal BP group. Diastolic BP (per mm Hg) was negatively associated with CRAE (P < 0.0001) and AVR (P < 0.0001), while systolic BP (per mm Hg) was positively associated with CRAE (P = 0.001) and AVR (P = 0.0096). Other factors significantly associated included age, sex, alcohol, smoking, cardiovascular disease history, ophthalmic medication, weight, and IOP; differences were observed in a stratified analysis based on BP medication use. Conclusions: Our study confirms previous reports about the association of age and BP with vessel diameters. The negative correlation between BP and CRAE seems to be guided by the effect of diastolic BP as higher systolic BP is independently associated with higher values of CRAE. The association of BP status with retinal vessel diameters is determined by diastolic BP status in our population. Multiple other factors are also independently associated with retinal vessel diameters.


Assuntos
Envelhecimento/patologia , Retinopatia Diabética/patologia , Glaucoma/patologia , Hipertensão/patologia , Degeneração Macular/patologia , Vasos Retinianos/patologia , Idoso , Arteríolas/patologia , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Vênulas/patologia
4.
Invest Ophthalmol Vis Sci ; 60(5): 1581-1594, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30995315

RESUMO

Purpose: To systematically analyze thicknesses of retinal layers in an older population and their link to early age-related macular degeneration (AMD). Methods: In the AugUR baseline survey from a population aged ≥70 years, we conducted multimodal retinal imaging, including spectral-domain optical coherence tomography. Autosegmentation of eight distinct retinal layers was followed by manual correction of segmentation errors. AMD status was graded on color fundus images according to the Three Continent AMD Consortium Severity Scale. We tested the association of early AMD on retinal layer thicknesses by using linear mixed models and replicated significant results in independent data also from the AugUR platform. Results: When comparing layer thicknesses between early AMD and no AMD (822 eyes, 449 participants), the retinal pigment epithelium/Bruch's membrane complex demonstrated a statistically significant thickening (e.g., P = 6.41 × 10-92 for severe early versus no AMD) and photoreceptor layers showed a significant thinning. Autosegmented retinal layer thicknesses revealed similar associations as manually corrected values but underestimated some effects. Independent replication analysis in 1026 eyes (546 participants) confirmed associations (e.g., P = 9.38 × 10-36 for retinal pigment epithelium/Bruch's membrane complex, severe early versus no AMD). Conclusions: This first population-based study on spectral-domain optical coherence tomography-derived retinal layer thicknesses in a total of ∼1000 individuals provides insights into the reliability of autosegmentation and layer-specific reference values for an older population. Our findings show a difference in thicknesses between early AMD and no AMD for some retinal layers, suggesting these as potential imaging biomarkers. The thinning of photoreceptor layers substantiates a photoreceptor cell loss/damage already occurring in early AMD.


Assuntos
Degeneração Macular/patologia , Retina/patologia , Idoso , Idoso de 80 Anos ou mais , Lâmina Basilar da Corioide/patologia , Bases de Dados Factuais , Feminino , Alemanha , Humanos , Degeneração Macular/classificação , Degeneração Macular/diagnóstico por imagem , Masculino , Imagem Multimodal , Tamanho do Órgão , Células Fotorreceptoras de Vertebrados/patologia , Estudos Prospectivos , Retina/diagnóstico por imagem , Células Ganglionares da Retina/patologia , Epitélio Pigmentado da Retina/patologia , Tomografia de Coerência Óptica/métodos
5.
Invest Ophthalmol Vis Sci ; 60(5): 1394-1402, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30938774

RESUMO

Purpose: To analyze the frequency and phenotypic variation of AMD in subjects with high myopia (HM), and to describe the clinical course and response to treatment of neovascularization (NV). Methods: Patients with HM were identified at five retina tertiary referral centers. Inclusion criteria were myopic patients aged 55 years or more with axial lengths equal or greater than 25.5 mm. Results: A total of 874 eyes from 442 HM subjects older than 55 years were identified and 104 eyes of 54 patients (72 ± 11 years) were included in the study and followed up for 23.5 ± 19.5 months. The estimated AMD frequency in HM subjects over 55 years was 11.9% (95% confidence interval; 9.8%-14.0%). A total of 34 of 104 eyes were diagnosed with drusen, 22 with reticular pseudodrusen (RPD), 28 with both drusen and RPD, and 20 with geographic atrophy. Neovascularization was detected in 52 eyes (50%), and type 1 was the most frequent form (39 eyes, 75%). Overall, NV was treated with 4.6 ± 2.6 anti-VEGF injections. Eyes with treatment-naïve NV at baseline (n = 34) required 3.8 ± 1.5 anti-VEGF injections during the first year of treatment. This exceeded the injection number in the purely myopic population (1.8 to 3.6 injections for the first year). Conclusions: This study provides evidence to suggest that older patients with HM are at a significant risk of the dry and neovascular forms of AMD. NV in eyes with HM and AMD required more injections in the first year compared to NV in HM eyes without AMD.


Assuntos
Degeneração Macular , Idoso , Idoso de 80 Anos ou mais , Inibidores da Angiogênese/uso terapêutico , Neovascularização de Coroide/epidemiologia , Feminino , Atrofia Geográfica/epidemiologia , Humanos , Itália/epidemiologia , Degeneração Macular/tratamento farmacológico , Degeneração Macular/epidemiologia , Degeneração Macular/patologia , Masculino , Pessoa de Meia-Idade , Miopia Degenerativa/tratamento farmacológico , Miopia Degenerativa/epidemiologia , Miopia Degenerativa/patologia , Fenótipo , Prevalência , Drusas Retinianas/epidemiologia , Estudos Retrospectivos
6.
Oxid Med Cell Longev ; 2019: 9682318, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31019656

RESUMO

Age-related macular degeneration (AMD) is a multifactorial disease of the retina featured by degeneration and loss of photoreceptors and retinal pigment epithelium (RPE) cells with oxidative stress playing a role in its pathology. Although systematic reviews do not support the protective role of diet rich in antioxidants against AMD, dietary polyphenols (DPs) have been reported to have beneficial effects on vision. Some of them, such as quercetin and cyanidin-3-glucoside, can directly scavenge reactive oxygen species (ROS) due to the presence of two hydroxyl groups in their B ring structure. Apart from direct ROS scavenging, DPs can lower oxidative stress in several other pathways. Many DPs induce NRF2 (nuclear factor, erythroid 2-like 2) activation and expression of phase II enzymes that are under transcriptional control of this factor. DPs can inhibit A2E photooxidation in RPE cells, which is a source of oxidative stress. Anti-inflammatory action of DPs in RPE cells is associated with regulation of various interleukins and signaling pathways, including IL-6/JAK2 (Janus kinase 2)/STAT3. Some DPs can improve impaired cellular waste clearance, including AMD-specific deficient phagocytosis of the Aß42 peptide and autophagy.


Assuntos
Dieta , Degeneração Macular/tratamento farmacológico , Degeneração Macular/patologia , Estresse Oxidativo , Polifenóis/uso terapêutico , Substâncias Protetoras/uso terapêutico , Humanos , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Polifenóis/química , Polifenóis/farmacologia , Substâncias Protetoras/farmacologia
7.
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
8.
Mol Vis ; 25: 70-78, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820143

RESUMO

Purpose: To visualize and analyze ex vivo flatmounted human RPE morphology from patients with age-related macular degeneration (AMD), and to compare the morphology with histologic findings. To establish whether the sub-RPE structures identified en face in RPE flatmount preparations are drusen with histopathological registration in serial sections. To detect characteristic patterns found en face in RPE with the same structures in histological cross sections from eyes from cadavers of patients with AMD. Methods: Twenty-eight postmortem eyes from 14 patients (16 eyes with AMD and 12 age-matched control eyes) were oriented and microdissected yielding a RPE-choroid preparation. The tissues were flatmounted, stained with Alexa Fluor 635 Phalloidin (AF635-phalloidin) for f-actin and propidium iodide for DNA, and imaged using confocal microscopy. Portions of tissue from macular regions were processed for electron microscopic examination. After confocal imaging, the samples were remounted for histologic processing, embedded in paraffin, and serially sectioned perpendicular to the plane of the RPE-choroid sheet. Scaled two-dimensional (2D) maps of drusen locations found with the histological cross sections were constructed and correlated with the en face confocal microscopic images. Results: Twenty-eight postmortem eyes with a mean time of death to tissue preservation of 23.7 h (range 8.0-51 h) from 14 donors (seven women and seven men) with an average age of 78 years (range 60-93 years) were evaluated. Eight donors had AMD, and six served as controls. Scattered small, hard drusen were present in the periphery of the eyes with AMD and the healthy eyes. The macular region of the eyes with AMD contained small (<63 µm), medium (63.0-124 µm), and large ( 125 µm) drusen. The RPE was arranged in rosette-like structures overlying small drusen, attenuated overlying medium-sized drusen, and consisted of large multinucleated cells overlying large drusen. The RPE in the area of geographic atrophy was attenuated and depigmented. Conclusions: Confocal images of flatmounts from eyes with AMD showed RPE patterns overlying various types of drusen and geographic atrophy that correlated with histologic characteristics. We propose RPE repair mechanisms that may result in the patterns that we observed.


Assuntos
Atrofia Geográfica/patologia , Degeneração Macular/patologia , Drusas Retinianas/patologia , Epitélio Pigmentado da Retina/patologia , Idoso , Idoso de 80 Anos ou mais , Autopsia , Feminino , Atrofia Geográfica/diagnóstico por imagem , Humanos , Degeneração Macular/diagnóstico por imagem , Masculino , Microscopia Confocal , Microtomia , Pessoa de Meia-Idade , Drusas Retinianas/diagnóstico por imagem , Epitélio Pigmentado da Retina/diagnóstico por imagem , Técnicas de Cultura de Tecidos
10.
Mol Vis ; 25: 79-92, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820144

RESUMO

Purpose: Smoking and the incidence of age-related macular degeneration (AMD) have been linked to an overactive complement system. Here, we examined in a retrospective cohort study whether AMD-associated single nucleotide polymorphisms (SNPs), smoking, ethnicity, and disease status are correlated with blood complement levels. Methods: Population: The study involved 91 AMD patients and 133 controls, which included 73% Americans of European descent (EUR) and 27% Americans of African descent (AFR) in South Carolina. Readouts: Participants were genotyped for 10 SNPs and systemic levels of complement factor H (CFH) activity, and the complement activation products C3a, C5a, and Bb were assessed. Main Outcome Measures: Univariate and multivariable logistic regression models were used to examine associations between AMD status and distinct readouts. Results: AMD affects EUR individuals more than AFRs. EUR but not AFR AMD subjects revealed higher levels of Factors C3a and Bb. In all subjects, a 10-unit increase in C3a levels was associated with an approximately 10% increase in the odds of being AMD-positive, and C3a and Bb were associated with smoking. While CFH activity levels were not correlated with AMD, a significant interaction was evident between patient age and CFH activity. Finally, EURs had lower odds of AMD with enhanced copies of rs1536304 (VEGFA) and higher odds with more copy numbers of rs3766404 (CFH). Conclusions: Our results support previous studies of systemic complement components being potential biomarkers for AMD, but they suggest that smoking and disease do not synergistically affect complement levels. We also suggest a novel susceptibility and protective haplotypes in the South Carolinian AMD population. Our studies indicate that augmented complement activation associated with advanced AMD could be attributed to a decrease in CFH activity in younger patients.


Assuntos
Ativação do Complemento/genética , Fator H do Complemento/genética , Degeneração Macular/genética , Polimorfismo de Nucleotídeo Único , Fumar/genética , Grupo com Ancestrais do Continente Africano , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Complemento C3a/genética , Complemento C3a/imunologia , Complemento C5a/genética , Complemento C5a/imunologia , Fator B do Complemento/genética , Fator B do Complemento/imunologia , Fator H do Complemento/imunologia , Grupo com Ancestrais do Continente Europeu , Feminino , Expressão Gênica , Humanos , Modelos Logísticos , Degeneração Macular/etnologia , Degeneração Macular/imunologia , Degeneração Macular/patologia , Masculino , Estudos Retrospectivos , Fumar/etnologia , Fumar/imunologia , Fumar/fisiopatologia , South Carolina , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/imunologia
11.
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
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.
Proc Natl Acad Sci U S A ; 116(9): 3703-3711, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808757

RESUMO

One of the strongest susceptibility genes for age-related macular degeneration (AMD) is complement factor H (CFH); however, its impact on AMD pathobiology remains unresolved. Here, the effect of the principal AMD-risk-associated CFH variant (Y402H) on the development and progression of age-dependent AMD-like pathologies was determined in vivo. Transgenic mice expressing equal amounts of the full-length normal human CFH Y402 (CFH-Y/0) or the AMD-risk associated CFH H402 (CFH-H/H) variant on a Cfh -/- background were aged to 90 weeks and switched from normal diet (ND) to a high fat, cholesterol-enriched (HFC) diet for 8 weeks. The resulting phenotype was compared with age-matched controls maintained on ND. Remarkably, an AMD-like phenotype consisting of vision loss, increased retinal pigmented epithelium (RPE) stress, and increased basal laminar deposits was detected only in aged CFH-H/H mice following the HFC diet. These changes were not observed in aged CFH-Y/0 mice or in younger (36- to 40-week-old) CFH mice of both genotypes fed either diet. Biochemical analyses of aged CFH mice after HFC diet revealed genotype-dependent changes in plasma and eyecup lipoproteins, but not complement activation, which correlated with the AMD-like phenotype in old CFH-H/H mice. Specifically, apolipoproteins B48 and A1 are elevated in the RPE/choroid of the aged CFH-H/H mice compared with age-matched control CFH-Y/0 fed a HFC diet. Hence, we demonstrate a functional consequence of the Y402H polymorphism in vivo, which promotes AMD-like pathology development and affects lipoprotein levels in aged mice. These findings support targeting lipoproteins as a viable therapeutic strategy for treating AMD.


Assuntos
Ativação do Complemento/genética , Fator H do Complemento/genética , Lipoproteínas/genética , Degeneração Macular/genética , Animais , Dieta Hiperlipídica/efeitos adversos , Feminino , Genótipo , Humanos , Lipoproteínas/metabolismo , Degeneração Macular/patologia , Masculino , Camundongos , Camundongos Transgênicos/genética , Polimorfismo de Nucleotídeo Único/genética , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia
14.
Drug Dev Ind Pharm ; 45(5): 715-723, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30704311

RESUMO

OBJECTIVE: Polymeric nanoparticles (NPs) containing doxorubicin (DOX) were prepared for the inhibition of hypoxia-induced factor 1α (HIF-1α). SIGNIFICANCE: HIF-1α is responsible for the upregulation of several angiogenic factors, including vascular endothelial growth factor (VEGF). DOX inhibits HIF-1α but is highly toxic. By encapsulating DOX in NPs, drug delivery will be sustained and toxicity will be reduced without limiting efficacy. METHODS: DOX NPs were prepared using both polylactic coglycolic acid (PLGA) and chitosan. PLGA NPs were prepared via nanoprecipitation (NPC) and single and double emulsion diffusion (SE; DE). Chitosan NPs were formulated using ionic gelation (IG), and complex coacervation (CC). Size, polydispersity index (PDI), and zeta potential (ZP) were determined via dynamic light scattering (DLS) (n = 3). The encapsulation efficiency (EE), drug loading capacity (DLC) (n = 3) and in vitro drug release profiles (IVR) at 37 °C (n = 4) were analyzed via spectroscopy at 480 nm (λmax). The cytotoxicity of each formulation as well as free DOX solution in ARPE-19 cells was determined via MTT assay after 24 h (n = 3). HIF-1α and VEGF inhibition in ARPE-19 cells were measured via ELISA (n = 3). RESULTS: The results were consistent with the hypothesis; the NP formulations decreased HIF-1α and VEGF-A expression in ARPE-19 cells with reduced cytotoxicity. SE, DE, and CC demonstrated low ZP as well as the most rapid drug release of the tested formulations. FTIR confirmed the presence of DOX on the SE NP surface, indicating instability. CONCLUSIONS: SE, DE, and CC destabilized. NPC was the most efficient formulation for the nanodelivery of DOX for AMD.


Assuntos
Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Degeneração Macular/tratamento farmacológico , Linhagem Celular , Precipitação Química , Doxorrubicina/farmacocinética , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Emulsões , Células Epiteliais , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Degeneração Macular/patologia , Nanopartículas/química , Tamanho da Partícula , Polímeros/química , Epitélio Pigmentado da Retina/citologia , Fator A de Crescimento do Endotélio Vascular/metabolismo
15.
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
16.
PLoS Genet ; 15(1): e1007939, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30695061

RESUMO

The retinal pigment epithelium (RPE) is a specialized monolayer of pigmented cells within the eye that is critical for maintaining visual system function. Diseases affecting the RPE have dire consequences for vision, and the most prevalent of these is atrophic (dry) age-related macular degeneration (AMD), which is thought to result from RPE dysfunction and degeneration. An intriguing possibility for treating RPE degenerative diseases like atrophic AMD is the stimulation of endogenous RPE regeneration; however, very little is known about the mechanisms driving successful RPE regeneration in vivo. Here, we developed a zebrafish transgenic model (rpe65a:nfsB-eGFP) that enabled ablation of large swathes of mature RPE. RPE ablation resulted in rapid RPE degeneration, as well as degeneration of Bruch's membrane and underlying photoreceptors. Using this model, we demonstrate for the first time that zebrafish are capable of regenerating a functional RPE monolayer after RPE ablation. Regenerated RPE cells first appear at the periphery of the RPE, and regeneration proceeds in a peripheral-to-central fashion. RPE ablation elicits a robust proliferative response in the remaining RPE. Subsequently, proliferative cells move into the injury site and differentiate into RPE. BrdU incorporation assays demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological disruption using IWR-1, a Wnt signaling antagonist, significantly reduces cell proliferation in the RPE and impairs overall RPE recovery. These data demonstrate that the zebrafish RPE possesses a robust capacity for regeneration and highlight a potential mechanism through which endogenous RPE regenerate in vivo.


Assuntos
Degeneração Macular/genética , Regeneração/genética , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , cis-trans-Isomerases/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Apoptose/genética , Lâmina Basilar da Corioide/crescimento & desenvolvimento , Lâmina Basilar da Corioide/metabolismo , Diferenciação Celular/genética , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Humanos , Imidas/administração & dosagem , Larva/genética , Larva/crescimento & desenvolvimento , Degeneração Macular/patologia , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patologia , Quinolinas/administração & dosagem , Retina/crescimento & desenvolvimento , Retina/patologia , Epitélio Pigmentado da Retina/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
17.
Lipids Health Dis ; 18(1): 7, 2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30621701

RESUMO

BACKGROUND: Lipids are implicated in the pathogenesis of age-related macular degeneration (AMD). The relationship between systemic lipids and AMD has not been well characterized. The objective was to investigate the relationship between serum lipids and AMD in older adults using a lipidomic approach. METHODS: In a case-control study, 240 adults, aged ≥66 years, a third each having geographic atrophy, neovascular AMD, or no signs of AMD, were selected from a population-based sample of participants in the Age Gene/Environment Susceptibility-Reykjavik Study. The exposure was serum lipids and risk factors for AMD. The outcome was late AMD, assessed through fundus images taken through dilated pupils using a 45-degree digital camera and grading for neovascular AMD and geographic atrophy using the modified Wisconsin Age-Related Maculopathy Grading System. RESULTS: Of 177 serum lipid species measured, there were no significant differences in serum lipids between controls and those with geographic atrophy or neovascular AMD, respectively. Adults with neovascular AMD had higher total serum lysophosphatidylcholine (LPC) (P = 0.004) and serum LPC 18:0 (P = 0.0002) compared to those with geographic atrophy. CONCLUSION: Late AMD was not characterized by alterations in systemic lipids compared with normal controls. These findings suggest that there may be differences in the LPC pathway between adults with neovascular AMD and geographic atrophy.


Assuntos
Atrofia Geográfica/sangue , Degeneração Macular/sangue , Neovascularização Retiniana/sangue , Idoso , Idoso de 80 Anos ou mais , Proteína C-Reativa/metabolismo , Estudos de Casos e Controles , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Feminino , Atrofia Geográfica/diagnóstico , Atrofia Geográfica/patologia , Humanos , Lisofosfatidilcolinas/sangue , Degeneração Macular/diagnóstico , Degeneração Macular/patologia , Masculino , Neovascularização Retiniana/diagnóstico , Neovascularização Retiniana/patologia , Fatores de Risco , Índice de Gravidade de Doença
18.
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
19.
Mol Immunol ; 106: 119-126, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30594674

RESUMO

PURPOSE: To investigate the role of tissue inhibitor of metalloproteinases-3 (TIMP-3) as a key moderator of macrophage polarization in choroidal neovascularization (CNV) lesions of model mice and in bone marrow-derived macrophage (BMDM). METHOD: We used siR-TIMP-3 to transfect BMDM and gave an intravitreal injection of siR-TIMP-3 to laser-induced CNV mice model, real time-PCR and western blot were applied for detecting the expressions of TIMP-3 and macrophages' biomarker. Besides, CNV lesions in different treatment groups of animal model were examined by the optical coherence tomography angiography (OCTA). RESULTS: Our experimental data showed that lack of TIMP-3 stimulated M2 polarization proved by real time-PCR and western blot in BMDMs and CNV mice model. Moreover, intravitreal injection of siR-TIMP-3 accelerated CNV formation using OCTA, which indicated that TIMP-3 suppression is related to pro-angiogenesis of M2 macrophage. CONCLUSION: We showed that the absence of TIMP-3 leads to a more pro-angiogenic microenvironment, playing a key role in CNV formation by positively modulating M2 polarization. The role of TIMP-3 in the regulating inflammation and novel therapeutic target of nAMD needs to be further studied.


Assuntos
Microambiente Celular/imunologia , Neovascularização de Coroide/imunologia , Macrófagos/imunologia , Degeneração Macular/imunologia , Neovascularização Patológica/imunologia , Inibidor Tecidual de Metaloproteinase-3/antagonistas & inibidores , Animais , Microambiente Celular/genética , Neovascularização de Coroide/genética , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Macrófagos/patologia , Degeneração Macular/genética , Degeneração Macular/patologia , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/imunologia
20.
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
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