RESUMO
Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD, and IL18 and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.
Assuntos
Elementos Alu , RNA Helicases DEAD-box/metabolismo , Atrofia Geográfica/imunologia , Atrofia Geográfica/patologia , Inflamassomos/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Ribonuclease III/metabolismo , Animais , Proteínas de Transporte/metabolismo , Atrofia Geográfica/metabolismo , Humanos , Inflamassomos/metabolismo , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Epitélio Pigmentado da Retina/patologia , Receptores Toll-Like/metabolismoRESUMO
Because the selective estrogen receptor modulator tamoxifen was shown to be retina-protective in the light damage and rd10 models of retinal degeneration, the purpose of this study was to test whether tamoxifen is retina-protective in a model where retinal pigment epithelium (RPE) toxicity appears to be the primary insult: the sodium iodate (NaIO3) model. C57Bl/6J mice were given oral tamoxifen (in the diet) or the same diet lacking tamoxifen, then given an intraperitoneal injection of NaIO3 at 25 mg/kg. The mice were imaged a week later using optical coherence tomography (OCT). ImageJ with a custom macro was utilized to measure retinal thicknesses in OCT images. Electroretinography (ERG) was used to measure retinal function one week post-injection. After euthanasia, quantitative real-time PCR (qRT-PCR) was performed. Tamoxifen administration partially protected photoreceptors. There was less photoreceptor layer thinning in OCT images of tamoxifen-treated mice. qRT-PCR revealed, in the tamoxifen-treated group, less upregulation of antioxidant and complement factor 3 mRNAs, and less reduction in the rhodopsin and short-wave cone opsin mRNAs. Furthermore, ERG results demonstrated preservation of photoreceptor function for the tamoxifen-treated group. Cone function was better protected than rods. These results indicate that tamoxifen provided structural and functional protection to photoreceptors against NaIO3. RPE cells were not protected. These neuroprotective effects suggest that estrogen-receptor modulation may be retina-protective. The fact that cones are particularly protected is intriguing given their importance for human visual function and their survival until the late stages of retinitis pigmentosa. Further investigation of this protective pathway could lead to new photoreceptor-protective therapeutics.
Assuntos
Modelos Animais de Doenças , Eletrorretinografia , Iodatos , Camundongos Endogâmicos C57BL , Degeneração Retiniana , Tamoxifeno , Tomografia de Coerência Óptica , Animais , Iodatos/toxicidade , Camundongos , Tomografia de Coerência Óptica/métodos , Tamoxifeno/farmacologia , Degeneração Retiniana/prevenção & controle , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Reação em Cadeia da Polimerase em Tempo Real , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Rodopsina/metabolismo , Rodopsina/genética , Moduladores Seletivos de Receptor Estrogênico/farmacologia , RNA Mensageiro/genética , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/metabolismo , Opsinas de Bastonetes/metabolismoRESUMO
Sodium iodate (NaIO3) is a commonly used model for age-related macular degeneration (AMD), but its rapid and severe induction of retinal pigment epithelial (RPE) and photoreceptor degeneration can lead to the premature dismissal of potentially effective therapeutics. Additionally, little is known about how sex and age affect the retinal response to NaIO3. This study aims to establish a less severe yet reproducible regimen by testing low doses of NaIO3 while considering age- and sex-related effects, enabling a broader range of therapeutic evaluations. In this study, young (3-5 months) and old (18-24 months) male and female C57Bl/6J mice were given an intraperitoneal (IP) injection of 15, 20, or 25 mg/kg NaIO3. Damage assessment one week post-injection included in vivo imaging, histological examination, and qRT-PCR analysis. The results revealed that young mice showed no damage at 15 mg/kg IP NaIO3, with varying degrees of damage observed at 20 mg/kg. At 25 mg/kg, most young mice displayed widespread retinal damage, with females exhibiting less retinal thinning than males. In contrast, older mice at 20 and 25 mg/kg displayed a more patchy degeneration pattern, outer retinal undulations, and greater variability in degeneration than the young mice. The most effective model for minimizing damage while maintaining consistency utilizes young female mice injected with 25 mg/kg NaIO3. The observed sex- and age-related differences underscore the importance of considering these variables in research, aligning with the National Institutes of Health's guidance. While the model does not fully replicate the complexity of AMD, these findings enhance its utility as a valuable tool for testing RPE/photoreceptor protective or replacement therapies.
Assuntos
Degeneração Macular , Degeneração Retiniana , Feminino , Masculino , Camundongos , Animais , Retina/patologia , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/patologia , Degeneração Macular/tratamento farmacológico , Degeneração Macular/patologia , Iodatos/toxicidade , Camundongos Endogâmicos C57BL , Epitélio Pigmentado da Retina/patologia , Modelos Animais de DoençasRESUMO
Age-related macular degeneration (AMD) is one of the leading causes of vision loss in the elderly. This disease involves oxidative stress burden in the retina leading to death of retinal pigment epithelial (RPE) cells and photoreceptors. The retina is susceptible to oxidative stress, in part due to high metabolic activity and high concentration of polyunsaturated fatty acids that undergo lipid peroxidation chain reactions. Antioxidant enzymes exist in the retina to combat this stress, including glutathione peroxidase 4 (GPX4). GPX4 specifically reduces oxidized lipids, protecting against lipid peroxidation-induced oxidative stress, which is noted in dry AMD. We hypothesize that Gpx4 knockout within the RPE will result in an environment of chronic oxidative stress yielding degeneration akin to AMD. C57BL/6J mice with a floxed Gpx4 gene were mated with Rpe65Cre/ER mice. Offspring containing Rpe65Cre ± alleles and either Gpx4 WT or Gpx4 fl/fl alleles were administered tamoxifen to induce Gpx4 knockout in Gpx4 fl/fl mice. At sequential timepoints, retinal phenotypes were assessed via in vivo imaging utilizing confocal scanning laser ophthalmoscopy and optical coherence tomography (OCT), and visual function was probed by electroretinography. Retinas were studied post-mortem by immunohistochemical analyses, electron microscopy, plastic sectioning, and quantitative polymerase chain reaction and Western analyses. The RPE-specific Gpx4 knockout model was validated via Western analysis indicating diminished GPX4 protein only within the RPE and not the neural retina. Following Gpx4 knockout, RPE cells became dysfunctional and died, with significant cell loss occurring 2 weeks post-knockout. Progressive thinning of the photoreceptor layer followed RPE degeneration and was accompanied by loss of visual function. OCT and light microscopy showed hyperreflective foci and enlarged, pigmented cells in and above the RPE layer. Electron microscopy revealed decreased mitochondrial cristae and loss of basal and apical RPE ultrastructure. Finally, there was increased carboxyethylpyrrole staining, indicating oxidation of docosahexaenoic acid, and increased levels of mRNAs encoding oxidative stress-associated genes in the RPE and photoreceptors. Overall, we show that RPE-localized GPX4 is necessary for the health of the RPE and outer retina, and that knockout recapitulates phenotypes of dry AMD.
RESUMO
The ability of iron to transfer electrons enables the contribution of this metal to a variety of cellular activities even as the redox properties of iron are also responsible for the generation of hydroxyl radicals (â¢OH), the most destructive of the reactive oxygen species. We previously showed that iron can promote the oxidation of bisretinoid by generating highly reactive hydroxyl radical (â¢OH). Now we report that preservation of iron regulation in the retina is not sufficient to prevent iron-induced bisretinoid oxidative degradation when blood iron levels are elevated in liver-specific hepcidin knockout mice. We obtained evidence for the perpetuation of Fenton reactions in the presence of the bisretinoid A2E and visible light. On the other hand, iron chelation by deferiprone was not associated with changes in postbleaching recovery of 11-cis-retinal or dark-adapted ERG b-wave amplitudes indicating that the activity of Rpe65, a rate-determining visual cycle protein that carries an iron-binding domain, is not affected. Notably, iron levels were elevated in the neural retina and retinal pigment epithelial (RPE) cells of Abca4-/- mice. Consistent with higher iron content, ferritin-L immunostaining was elevated in RPE of a patient diagnosed with ABCA4-associated disease and in RPE and photoreceptor cells of Abca4-/- mice. In neural retina of the mutant mice, reduced Tfrc mRNA was also an indicator of retinal iron overload. Thus iron chelation may defend retina when bisretinoid toxicity is implicated in disease processes.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Genes Recessivos , Retinaldeído/metabolismo , Retinoides/metabolismo , Doença de Stargardt/metabolismo , cis-trans-Isomerases/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Camundongos , Camundongos Knockout , Oxirredução , Retinaldeído/genética , Retinoides/genética , Doença de Stargardt/genética , Doença de Stargardt/patologia , cis-trans-Isomerases/genéticaRESUMO
The ability of iron to transfer electrons enables the contribution of this metal to a variety of cellular activities even as the redox properties of iron are also responsible for the generation of hydroxyl radicals (â¢OH), the most destructive of the reactive oxygen species. We previously showed that iron can promote the oxidation of bisretinoid by generating highly reactive hydroxyl radical (â¢OH). Now we report that preservation of iron regulation in the retina is not sufficient to prevent iron-induced bisretinoid oxidative degradation when blood iron levels are elevated in liver-specific hepcidin knock-out mice. We obtained evidence for the perpetuation of Fenton reactions in the presence of the bisretinoid A2E and visible light. On the other hand, iron chelation by deferiprone was not associated with changes in post-bleaching recovery of 11-cis-retinal or dark-adapted ERG b-wave amplitudes indicating that the activity of Rpe65, a rate-determining visual cycle protein that carries an iron-binding domain is not affected. Notably, iron levels were elevated in the neural retina and RPE of Abca4-/- mice. Consistent with higher iron content, ferritin-L immunostaining was elevated in RPE of a patient diagnosed with ABCA4-associated disease and in RPE and photoreceptor cells of Abca4-/- mice. In neural retina of the mutant mice, reduced Tfrc mRNA was also an indicator of retinal iron overload. Thus iron chelation may defend retina when bisretinoid toxicity is implicated in disease processes.
RESUMO
Iron accumulation has been implicated in degenerative retinal diseases. It can catalyze the production of damaging reactive oxygen species. Previous work has demonstrated iron accumulation in multiple retinal diseases, including age-related macular degeneration and diabetic retinopathy. In mice, systemic knockout of the ferroxidases ceruloplasmin (Cp) and hephaestin (Heph), which oxidize iron, results in retinal iron accumulation and iron-induced degeneration. To determine the role of Heph in the retina, we generated a neural retina-specific Heph knockout on a background of systemic Cp knockout. This resulted in elevated neural retina iron. Conversely, retinal ganglion cells had elevated transferrin receptor and decreased ferritin, suggesting diminished iron levels. The retinal degeneration observed in systemic Cp-/-, Heph-/- mice did not occur. These findings indicate that Heph has a local role in regulating neural retina iron homeostasis, but also suggest that preserved Heph function in either the RPE or systemically mitigates the degeneration phenotype observed in the systemic Cp-/-, Heph-/- mice.
Assuntos
Degeneração Macular , Proteínas de Membrana , Animais , Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Homeostase , Ferro/metabolismo , Degeneração Macular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Retina/metabolismoRESUMO
Iron-induced oxidative stress can cause or exacerbate retinal degenerative diseases. Retinal iron overload has been reported in several mouse disease models with systemic or neural retina-specific knockout (KO) of homologous ferroxidases ceruloplasmin (Cp) and hephaestin (Heph). Cp and Heph can potentiate ferroportin (Fpn) mediated cellular iron export. Here, we used retina-specific Fpn KO mice to test the hypothesis that retinal iron overload in Cp/Heph DKO mice is caused by impaired iron export from neurons and glia. Surprisingly, there was no indication of retinal iron overload in retina-specific Fpn KO mice: the mRNA levels of transferrin receptor in the retina were not altered at 7-10-months age. Consistent with this, levels and localization of ferritin light chain were unchanged. To "stress the system", we injected iron intraperitoneally into Fpn KO mice with or without Cp KO. Only mice with both retina-specific Fpn KO and Cp KO had modestly elevated retinal iron levels. These results suggest that impaired iron export through Fpn is not sufficient to explain the retinal iron overload in Cp/Heph DKO mice. An increase in the levels of retinal ferrous iron caused by the absence of these ferroxidases, followed by uptake into cells by ferrous iron importers, is most likely necessary.
Assuntos
Proteínas de Transporte de Cátions , Sobrecarga de Ferro , Animais , Proteínas de Transporte de Cátions/genética , Ceruloplasmina/genética , Ceruloplasmina/metabolismo , Ferro/metabolismo , Camundongos , Camundongos Knockout , Retina/metabolismoRESUMO
PURPOSE: To determine associations of systemic medications with the incidence and growth of geographic atrophy (GA) in participants of the comparison of age-related macular degeneration treatments trials. METHODS: Participants of comparison of age-related macular degeneration treatments trials with new untreated choroidal neovascularization in the study eye (one study eye per participant) were randomized to receive treatment with bevacizumab or ranibizumab. Participants were released from clinical trial treatment at 2 years and examined at approximately 5 years. Color fundus photographs and fluorescein angiograms taken at baseline, Years 1, 2, and 5 were assessed for the presence and size of GA by two masked graders. Participants were interviewed about systemic medication use at baseline. Systemic medications previously reported to be associated with age-related macular degeneration were evaluated for associations with GA incidence in study eye using univariable and multivariable Cox models and for association with the GA growth using linear mixed effects models. RESULTS: In multivariable analysis of 1,011 study eyes without baseline GA, systemic medications, including cholinesterase inhibitors, angiotensin-converting enzyme inhibitors, calcium channel blockers, beta-blockers, diuretics, aspirin, steroids, statins, hormone replacement therapy, antacids, and drugs targeting G protein-coupled receptors, were not associated with GA incidence in the study eye (all adjusted hazard ratios ≤1.86, P ≥ 0.18). In multivariable analysis of 214 study eyes with longitudinal GA size measurements, calcium channel blockers were associated with a higher GA growth rate (0.40 vs. 0.30 mm/year, P = 0.02). CONCLUSION: None of the systemic medications analyzed were associated with GA incidence. However, calcium channel blockers were associated with a higher growth rate of GA in the study eye.
Assuntos
Bevacizumab/administração & dosagem , Atrofia Geográfica/tratamento farmacológico , Degeneração Macular/tratamento farmacológico , Ranibizumab/administração & dosagem , Acuidade Visual , Idoso , Idoso de 80 Anos ou mais , Inibidores da Angiogênese/administração & dosagem , Feminino , Angiofluoresceinografia/métodos , Seguimentos , Fundo de Olho , Atrofia Geográfica/diagnóstico , Atrofia Geográfica/epidemiologia , Humanos , Incidência , Injeções Intravítreas , Degeneração Macular/diagnóstico , Masculino , Fatores de Tempo , Tomografia de Coerência Óptica/métodos , Resultado do Tratamento , Estados Unidos/epidemiologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Intracellular Fe plays a key role in redox active energy and electron transfer. We sought to understand how Fe levels impact the retina, given that retinal pigment epithelial (RPE) cells are also challenged by accumulations of vitamin A aldehyde adducts (bisretinoid lipofuscin) that photogenerate reactive oxygen species and photodecompose into damaging aldehyde- and dicarbonyl-bearing species. In mice treated with the Fe chelator deferiprone (DFP), intracellular Fe levels, as reflected in transferrin receptor mRNA expression, were reduced. DFP-treated albino Abca4-/- and agouti wild-type mice exhibited elevated bisretinoid levels as measured by high-performance liquid chromatography or noninvasively by quantitative fundus autofluorescence. Thinning of the outer nuclear layer, a parameter indicative of the loss of photoreceptor cell viability, was also reduced in DFP-treated albino Abca4-/- In contrast to the effects of the Fe chelator, mice burdened with increased intracellular Fe in RPE due to deficiency in the Fe export proteins hephaestin and ceruloplasmin, presented with reduced bisretinoid levels. These findings indicate that intracellular Fe promotes bisretinoid oxidation and degradation. This interpretation was supported by experiments showing that DFP decreased the oxidative/degradation of the bisretinoid A2E in the presence of light and reduced cell death in cell-based experiments. Moreover, light-independent oxidation and degradation of A2E by Fenton chemistry products were evidenced by the consumption of A2E, release of dicarbonyls, and generation of oxidized A2E species in cell-free assays.
Assuntos
Epitélio Pigmentado da Retina/metabolismo , Retinoides/metabolismo , Transportadores de Cassetes de Ligação de ATP/deficiência , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Deferiprona , Ferro , Quelantes de Ferro/farmacologia , Lipofuscina/genética , Lipofuscina/metabolismo , Camundongos , Camundongos Knockout , Piridonas/farmacologia , Epitélio Pigmentado da Retina/patologia , Retinaldeído/genética , Retinaldeído/metabolismoRESUMO
Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss among the elderly population. Genetic studies in susceptible individuals have linked this ocular disease to deregulated complement activity that culminates in increased C3 turnover, retinal inflammation and photoreceptor loss. Therapeutic targeting of C3 has therefore emerged as a promising strategy for broadly intercepting the detrimental proinflammatory consequences of complement activation in the retinal tissue. In this regard, a PEGylated second-generation derivative of the compstatin family of C3-targeted inhibitors is currently in late-stage clinical development as a treatment option for geographic atrophy, an advanced form of AMD which lacks approved therapy. While efficacy has been strongly suggested in phase 2 clinical trials, crucial aspects still remain to be defined with regard to the ocular bioavailability, tissue distribution and residence, and dosing frequency of such inhibitors in AMD patients. Here we report the intraocular distribution and pharmacokinetic profile of the fourth-generation compstatin analog, Cp40-KKK in cynomolgus monkeys following a single intravitreal injection. Using a sensitive surface plasmon resonance (SPR)-based competition assay and ELISA, we have quantified both the amount of inhibitor and the concentration of C3 retained in the vitreous of Cp40-KKK-injected animals. Cp40-KKK displays prolonged intraocular residence, being detected at C3-saturating levels for over 3 months after a single intravitreal injection. Moreover, we have probed the distribution of Cp40-KKK within the ocular tissue by means of immunohistochemistry and highly specific anti-Cp40-KKK antibodies. Both C3 and Cp40-KKK were detected in the retinal tissue of inhibitor-injected animals, with prominent co-localization in the choroid one-month post intravitreal injection. These results attest to the high retinal tissue penetrance and target-driven distribution of Cp40-KKK. Given its subnanomolar binding affinity and prolonged ocular residence, Cp40-KKK constitutes a promising drug candidate for ocular pathologies underpinned by deregulated C3 activation.
Assuntos
Complemento C3/antagonistas & inibidores , Olho/química , Idoso , Animais , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Injeções Intravítreas , Macaca fascicularis , Retina/química , Fatores de Tempo , Distribuição TecidualRESUMO
The liver secretes hepcidin (Hepc) into the bloodstream to reduce blood iron levels. Hepc accomplishes this by triggering degradation of the only known cellular iron exporter ferroportin in the gut, macrophages, and liver. We previously demonstrated that systemic Hepc knockout (HepcKO) mice, which have high serum iron, develop retinal iron overload and degeneration. However, it was unclear whether this is caused by high blood iron levels or, alternatively, retinal iron influx that would normally be regulated by retina-produced Hepc. To address this question, retinas of liver-specific and retina-specific HepcKO mice were studied. Liver-specific HepcKO mice had elevated blood and retinal pigment epithelium (RPE) iron levels and increased free (labile) iron levels in the retina, despite an intact blood-retinal barrier. This led to RPE hypertrophy associated with lipofuscin-laden lysosome accumulation. Photoreceptors also degenerated focally. In contrast, there was no change in retinal or RPE iron levels or degeneration in the retina-specific HepcKO mice. These data indicate that high blood iron levels can lead to retinal iron accumulation and degeneration. High blood iron levels can occur in patients with hereditary hemochromatosis or result from use of iron supplements or multiple blood transfusions. Our results suggest that high blood iron levels may cause or exacerbate retinal disease.
Assuntos
Hepcidinas/fisiologia , Sobrecarga de Ferro/etiologia , Ferro/metabolismo , Fígado/metabolismo , Retina/metabolismo , Degeneração Retiniana/etiologia , Animais , Barreira Hematorretiniana , Feminino , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Retina/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologiaRESUMO
Single-nucleotide polymorphisms and rare mutations in factor H (FH; official name, CFH) are associated with age-related macular degeneration and atypical hemolytic uremic syndrome, a form of thrombotic microangiopathy. Mice with the FH W1206R mutation (FHR/R) share features with human atypical hemolytic uremic syndrome. Herein, we report that FHR/R mice exhibited retinal vascular occlusion and ischemia. Retinal fluorescein angiography demonstrated delayed perfusion and vascular leakage in FHR/R mice. Optical coherence tomography imaging of FHR/R mice showed retinal degeneration, edema, and detachment. Histologic analysis of FHR/R mice revealed retinal thinning, vessel occlusion, as well as degeneration of photoreceptors and retinal pigment epithelium. Immunofluorescence showed albumin leakage from blood vessels into the neural retina, and electron microscopy demonstrated vascular endothelial cell irregularity with narrowing of retinal and choroidal vessels. Knockout of C6, a component of the membrane attack complex, prevented the aforementioned retinal phenotype in FHR/R mice, consistent with membrane attack complex-mediated pathogenesis. Pharmacologic blockade of C5 also rescued retinas of FHR/R mice. This FHR/R mouse strain represents a model for retinal vascular occlusive disorders and ischemic retinopathy. The results suggest complement dysregulation can contribute to retinal vascular occlusion and that an anti-C5 antibody might be helpful for C5-mediated thrombotic retinal diseases.
Assuntos
Fator H do Complemento/fisiologia , Isquemia/etiologia , Mutação , Neovascularização Patológica/etiologia , Doenças Retinianas/etiologia , Epitélio Pigmentado da Retina/patologia , Trombose/etiologia , Animais , Fator H do Complemento/genética , Isquemia/metabolismo , Isquemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Epitélio Pigmentado da Retina/metabolismo , Trombose/metabolismo , Trombose/patologiaRESUMO
Glaucoma is a progressive neurodegenerative process affecting the retinal ganglion cells (RGCs) and the optic nerve. Oxidative stress has been implicated in glaucoma pathogenesis, and iron is a potent generator of oxidative stress. The oral iron chelator deferiprone (DFP) is protective against retinal degenerations associated with oxidative stress. To test whether DFP could be protective in glaucoma, we used microbead injections to induce elevated intraocular pressure (IOP) in a cohort of 3-month old C57BL/6J mice. One eye of each animal was injected with magnetic microbeads resulting in ocular hypertension for >7 weeks while the fellow eye was injected with saline and served as a normotensive internal control. While half of the cohort received oral DFP (1 mg/ml in the drinking water), the other half did not and served as controls. After 8 weeks, Brn3a immunolabeling of flat-mounted retinas was used for manual RGC quantification. Axon counts were obtained from thin sections of optic nerves using the AxonJ plugin for ImageJ. DFP administration was protective against RGC and optic nerve loss in the setting of elevated IOP. These results suggest that iron chelation by DFP may provide glaucoma neuroprotection.
Assuntos
Deferiprona/administração & dosagem , Glaucoma/complicações , Nervo Óptico/patologia , Degeneração Retiniana/prevenção & controle , Células Ganglionares da Retina/patologia , Administração Oral , Animais , Modelos Animais de Doenças , Feminino , Glaucoma/tratamento farmacológico , Glaucoma/patologia , Quelantes de Ferro/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nervo Óptico/efeitos dos fármacos , Estresse Oxidativo , Degeneração Retiniana/etiologia , Degeneração Retiniana/patologia , Células Ganglionares da Retina/efeitos dos fármacosRESUMO
Atypical hemolytic uremic syndrome (aHUS) is a form of thrombotic microangiopathy (TMA) caused by dysregulated complement activation. Clinically, aHUS is effectively treated by an anti-C5 monoclonal antibody (mAb) but whether the disease is mediated by the C5a receptor (C5aR) or C5b-9 pathway, or both, is unknown. Here we address this in a factor H mutant mouse (FHR/R) which developed complement-mediated TMA as well as macrovascular thrombosis caused by an aHUS-related factor H point mutation (mouse W1206R, corresponding to human W1183R). C5 deficiency and anti-C5 mAb treatment blocked all disease manifestations in FHR/R mice. C5aR1 gene deficiency prevented macrovascular thrombosis in various organs but did not improve survival or reduce renal TMA. Conversely, C6 or C9 deficiency significantly improved survival and markedly diminished renal TMA but did not prevent macrovascular thrombosis. Interestingly, as they aged both FHR/R C6-/- and FHR/R C9-/- mice developed glomerular disease reminiscent of C3 glomerulonephritis. Thus, C5aR and C5b-9 pathways drove different aspects of disease in FHR/R mice with the C5aR pathway being responsible for macrovascular thrombosis and chronic inflammatory injury while the C5b-9 pathway caused renal TMA. Our data provide new understanding of the pathogenesis of complement-mediated TMA and macrovascular thrombosis in FHR/R mice and suggest that C5 blockade is more effective for the treatment of aHUS than selectively targeting the C5aR or C5b-9 pathway alone.
Assuntos
Síndrome Hemolítico-Urêmica Atípica/imunologia , Fator H do Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/imunologia , Glomérulos Renais/patologia , Receptor da Anafilatoxina C5a/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Síndrome Hemolítico-Urêmica Atípica/tratamento farmacológico , Síndrome Hemolítico-Urêmica Atípica/genética , Síndrome Hemolítico-Urêmica Atípica/patologia , Ativação do Complemento/efeitos dos fármacos , Ativação do Complemento/genética , Ativação do Complemento/imunologia , Complemento C6/genética , Complemento C6/imunologia , Complemento C6/metabolismo , Fator H do Complemento/imunologia , Complexo de Ataque à Membrana do Sistema Complemento/genética , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/ultraestrutura , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Mutação Puntual , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismoRESUMO
PURPOSE: To describe changes in visual acuity (VA) and macular morphologic features at 5 years in eyes with nonfibrotic scar (NFS) identified at 1 year in the Comparison of Age-Related Macular Degeneration Treatments Trials (CATT). DESIGN: Prospective cohort study within a randomized clinical trial. PARTICIPANTS: Participants in CATT. METHODS: Participants assigned to ranibizumab or bevacizumab and to 1 of 3 dosing regimens were released from the clinical trial protocol after 2 years and recalled at 5 years. Nonfibrotic scar was identified on color images at year 1 as flat, small, well-circumscribed areas of pigmentation with varying degrees of central hypopigmentation without exposure of underlying choroidal vessels at the site of baseline choroidal neovascularization. Follow-up images were assessed for changes in and around NFS. MAIN OUTCOME MEASURES: Pigmentation changes, VA, development of fibrotic scar (FS), nongeographic atrophy (NGA), geographic atrophy (GA), retinal fluid on OCT, and fluorescein leakage. RESULTS: Among 474 eyes with images obtained at 1, 2, and 5 years, 39 (8.2%) showed NFS at 1 year with a mean VA of 80 letters (Snellen equivalent, 20/25). Among these eyes, FS developed in 5% at 2 years and 28% at 5 years. Nongeographic atrophy was observed in 34%, 47%, and 65% of eyes at 1, 2, and 5 years, respectively. Geographic atrophy developed in 5% of eyes at 2 years and 21% at 5 years. Among eyes with NFS, FS, or no scar at 1 year, mean VA at 5 years was 73 letters (20/32), 48 letters (20/100), and 62 letters (20/63), respectively. At 5 years, NFS eyes demonstrated less GA, less intraretinal fluid, more subretinal fluid, and less subretinal pigment epithelium fluid (all P < 0.01). Among NFS eyes, mean thickness of the retina, subretinal tissue complex, and total retina did not change across years 1 to 5 (P > 0.50). The proportion of eyes with fluid on OCT also did not change (P = 0.36). Subretinal hyperreflective material disappeared by 5 years in 40% of eyes with NFS. CONCLUSIONS: These results indicate that, on average, eyes with NFS after anti-VEGF treatment have good VA not only at 1 and 2 years, but also through 5 years.
Assuntos
Bevacizumab/administração & dosagem , Cicatriz/diagnóstico , Macula Lutea/patologia , Degeneração Macular/tratamento farmacológico , Ranibizumab/administração & dosagem , Acuidade Visual , Inibidores da Angiogênese/administração & dosagem , Cicatriz/etiologia , Progressão da Doença , Angiofluoresceinografia , Seguimentos , Fundo de Olho , Humanos , Injeções Intravítreas , Degeneração Macular/complicações , Degeneração Macular/diagnóstico , Prognóstico , Estudos Prospectivos , Fatores de Tempo , Tomografia de Coerência Óptica , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Chronic exposure of the retina to light and high concentrations of polyunsaturated fatty acid in photoreceptor cells make this tissue susceptible to oxidative damage. As retinal degenerative diseases are associated with photoreceptor degeneration, the antioxidant activity of both hydrogen sulfide (H2S) and glutathione (GSH) may play an important role in ameliorating disease progression. H2S production is driven by cystathionine-γ-lyase (CSE) and cystathionine-ß-synthase (CBS), the key enzymes that also drive transsulfuration pathway (TSP) necessary for GSH production. As it is currently unclear whether localized production of either H2S or GSH contributes to retinal homeostasis, we undertook a comparative analysis of CBS and CSE expression in canine, non-human primate (NHP) and human retinas to determine if these antioxidants could play a regulatory role in age-related or disease-associated retinal degeneration. Retinas from normal dogs, NHPs and humans were used for the study. Laser capture microdissection (LCM) was performed to isolate individual layers of the canine retina and analyze CBS and CSE gene expression by qRT-PCR. Immunohistochemistry and western blotting were performed for CBS and CSE labeling and protein expression in dog, NHP, and human retina, respectively. Using qRT-PCR, western blot, and immunohistochemistry (IHC), we showed that CBS and CSE are expressed in the canine, NHP, and human retina. IHC results from canine retina demonstrated increased expression levels of CBS but not CSE with post-developmental aging. IHC results also showed non-overlapping localization of both proteins with CBS presenting in rods, amacrine, horizontal, and nerve fiber cell layers while CSE was expressed by RPE, cones and MÏller cells. Finally, we demonstrated that these enzymes localized to all three layers of canine, NHP and human retina: photoreceptors, outer plexiform layer (OPL) and notably in the ganglion cells layer/nerve fiber layer (GCL/NFL). QRT-PCR performed using RNA extracted from tissues isolated from these cell layers using laser capture microdissection (LCM) confirmed that each of CBS and CSE are expressed equally in these three layers. Together, these findings reveal that CSE and CBS are expressed in the retina, thereby supporting further studies to determine the role of H2S and these proteins in oxidative stress and apoptosis in retinal degenerative diseases.
Assuntos
Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/metabolismo , Retina/metabolismo , Animais , Western Blotting , Cães , Imuno-Histoquímica , PrimatasRESUMO
Retinal iron accumulation has been implicated in the pathogenesis of age-related macular degeneration (AMD) and other neurodegenerative diseases. The retina and the brain are protected from the systemic circulation by the blood retinal barrier (BRB) and blood brain barrier (BBB), respectively. Iron levels within the retina and brain need to be tightly regulated to prevent oxidative injury. The method of iron entry through the retina and brain vascular endothelial cells (r&bVECs), an essential component of the BRB and BBB, is not fully understood. However, localization of the cellular iron exporter, ferroportin (Fpn), to the abluminal membrane of these cells, leads to the hypothesis that Fpn may play an important role in the import of iron across the BRB and BBB. To test this hypothesis, a mouse model with deletion of Fpn within the VECs in both the retina and the brain was developed through tail vein injection of AAV9-Ple261(CLDN5)-icre to both experimental Fpnf/f, and control Fpn+/+ mice at P21. Mice were aged to 9â¯mo and changes in retinal and brain iron distribution were observed. In vivo fundus imaging and quantitative serum iron detection were used for model validation. Eyes and brains were collected for immunofluorescence. Deletion of Fpn from the retinal and brain VECs leads to ferritin-L accumulation, an indicator of elevated iron levels, in the retinal and brain VECs. This occurred despite lower serum iron levels in the experimental mice. This result suggests that Fpn normally transfers iron from retinal and brain VECs into the retina and brain. These results help to better define the method of retina and brain iron import and will increase understanding of neurodegenerative diseases involving iron accumulation.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Circulação Cerebrovascular/fisiologia , Células Endoteliais/metabolismo , Ferritinas/metabolismo , Vasos Retinianos/metabolismo , Animais , Transporte Biológico , Barreira Hematoencefálica , Barreira Hematorretiniana , Claudina-5/genética , Dependovirus/genética , Técnica Indireta de Fluorescência para Anticorpo , Ferro/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The blood retinal barrier (BRB) closely regulates the retinal microenvironment. Its compromise leads to the accumulation of retinal fluid containing potentially harmful plasma components. While eyes with non-exudative age-related macular degeneration (AMD) were previously felt to have an intact BRB, we propose that the BRB in non-exudative AMD eyes may be subclinically compromised, allowing entry of retina-toxic plasma proteins. We test this hypothesis by measuring retinal levels of abundant plasma proteins that should not cross the intact BRB. Two cohorts of frozen, post mortem neurosensory retinas were studied by Western analysis. One cohort from Alabama had 4 normal controls and 4 eyes with various forms of AMD. Another cohort from Minnesota had 5 intermediate AMD eyes and 5 normals. Both cohorts were age/post mortem interval (PMI) matched. The non-exudative AMD retinas in the Alabama cohort had significantly higher levels of albumin and complement component 9 (C9) than normal controls. The positive control exudative AMD donor retina had higher levels of all but one serum protein. In both macular and peripheral neurosensory retina samples, intermediate AMD retinas in the Minnesota cohort had significantly higher levels of albumin, fibrinogen, IgG, and C9 than controls. Our results suggest that there may be moderate subclinical BRB leakage in non-exudative AMD. Potentially harmful plasma components including complement or iron could enter the neurosensory retina in AMD patients prior to advanced disease. Thus, therapies aiming to stabilize the BRB might have a role in the management of non-exudative AMD.
Assuntos
Proteínas Sanguíneas/metabolismo , Atrofia Geográfica/sangue , Retina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Barreira Hematorretiniana/fisiologia , Western Blotting , Complemento C9/metabolismo , Exsudatos e Transudatos , Feminino , Fibrinogênio/metabolismo , Humanos , Imunoglobulina G/metabolismo , Masculino , Albumina Sérica/metabolismoRESUMO
PURPOSE: To describe patterns of hypoautofluorescence in eyes with neovascular age-related macular degeneration occurring after subretinal hemorrhage. METHODS: This was a retrospective descriptive analysis of neovascular age-related macular degeneration eyes presenting with subretinal hemorrhage over the last 5 years that underwent serial multimodal imaging. A review of color fundus photographs, fundus autofluorescence, near-infrared reflectance, and optical coherence tomography was performed at baseline and all available follow-up visits to document the course and evolution of subretinal hemorrhage in these eyes. RESULTS: Eleven eyes of 10 patients (9 female, 1 male; mean age: 84.1 years, range: 72-99 years) with a mean follow-up of 19.8 months (range: 3-68 months) were included. Color fundus photographs showed subretinal hemorrhage that resolved over a mean of 5.5 months. During and after hemorrhage resolution, all eyes showed hypoautofluorescence, which appeared distinct from that due to retinal pigment epithelium loss. Discrete multifocal punctate hyperpigmented lesions were observed in 90% of eyes and were markedly hypoautofluorescent, producing a speckled pattern on fundus autofluorescence. CONCLUSION: Areas of hypoautofluorescence in the absence of retinal pigment epithelium atrophy, often with a speckled pattern, delineate areas of prior subretinal hemorrhage long after its resolution in patients with neovascular age-related macular degeneration. Potential mechanisms for the development of this pattern are proposed.