RESUMO
It has previously been reported that antioxidant vitamins can help reduce the risk of vision loss associated with progression to advanced age-related macular degeneration (AMD), a leading cause of visual impairment among the elderly. Nonetheless, how oxidative stress contributes to the development of choroidal neovascularization (CNV) in some AMD patients and geographic atrophy (GA) in others is poorly understood. Here, we provide evidence demonstrating that oxidative stress cooperates with hypoxia to synergistically stimulate the accumulation of hypoxia-inducible factor (HIF)-1α in the retinal pigment epithelium (RPE), resulting in increased expression of the HIF-1-dependent angiogenic mediators that promote CNV. HIF-1 inhibition blocked the expression of these angiogenic mediators and prevented CNV development in an animal model of ocular oxidative stress, demonstrating the pathological role of HIF-1 in response to oxidative stress stimulation in neovascular AMD. While human-induced pluripotent stem cell (hiPSC)-derived RPE monolayers exposed to chemical oxidants resulted in disorganization and disruption of their normal architecture, RPE cells proved remarkably resistant to oxidative stress. Conversely, equivalent doses of chemical oxidants resulted in apoptosis of hiPSC-derived retinal photoreceptors. Pharmacologic inhibition of HIF-1 in the mouse retina enhanced-while HIF-1 augmentation reduced-photoreceptor apoptosis in two mouse models for oxidative stress, consistent with a protective role for HIF-1 in photoreceptors in patients with advanced dry AMD. Collectively, these results suggest that in patients with AMD, increased expression of HIF-1α in RPE exposed to oxidative stress promotes the development of CNV, but inadequate HIF-1α expression in photoreceptors contributes to the development of GA.
Assuntos
Neovascularização de Coroide , Atrofia Geográfica , Degeneração Macular Exsudativa , Camundongos , Animais , Humanos , Idoso , Epitélio Pigmentado da Retina/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Inibidores da Angiogênese , Degeneração Macular Exsudativa/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Acuidade Visual , Neovascularização de Coroide/genética , Neovascularização de Coroide/prevenção & controle , Neovascularização de Coroide/metabolismo , Oxidantes/metabolismo , Hipóxia/metabolismoRESUMO
The advent of stem cell-derived retinal organoids has brought forth unprecedented opportunities for developmental and physiological studies, while presenting new therapeutic promise for retinal degenerative diseases. From a translational perspective, organoid systems provide exciting new prospects for drug discovery, offering the possibility to perform compound screening in a three-dimensional (3D) human tissue context that resembles the native histoarchitecture and to some extent recapitulates cellular interactions. However, inherent variability issues and a general lack of robust quantitative technologies for analyzing organoids on a large scale pose severe limitations for their use in translational applications. To address this need, we have developed a screening platform that enables accurate quantification of fluorescent reporters in complex human iPSC-derived retinal organoids. This platform incorporates a fluorescence microplate reader that allows xyz-dimensional detection and fine-tuned wavelength selection. We have established optimal parameters for fluorescent reporter signal detection, devised methods to compensate for organoid size variability, evaluated performance and sensitivity parameters, and validated this technology for functional applications.
Assuntos
Técnicas Genéticas , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/fisiologia , Retina/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Corantes Fluorescentes , Genes Reporter , Humanos , Microscopia de Fluorescência , Estresse Oxidativo , Transplante de Células-Tronco , Transgenes , Pesquisa Translacional BiomédicaRESUMO
The retinal pigment epithelium (RPE), a monolayer located between the photoreceptors and the choroid, is constantly damaged by oxidative stress, particularly because of reactive oxygen species (ROS). As the RPE, because of its physiological functions, is essential for the survival of the retina, any sustained damage may consequently lead to loss of vision. Exosomes are small membranous vesicles released into the extracellular medium by numerous cell types, including RPE cells. Their cargo includes genetic material and proteins, making these vesicles essential for cell-to-cell communication. Exosomes may fuse with neighbouring cells influencing their fate. It has been observed that RPE cells release higher amounts of exosomes when they are under oxidative stress. Exosomes derived from cultured RPE cells were isolated by ultracentrifugation and quantified by flow cytometry. VEGF receptors (VEGFR) were analysed by both flow cytometry and Western blot. RT-PCR and qPCR were conducted to assess mRNA content of VEGFRs in exosomes. Neovascularization assays were performed after applying RPE exosomes into endothelial cell cultures. Our results showed that stressed RPE cells released a higher amount of exosomes than controls, with a higher expression of VEGFR in the membrane, and enclosed an extra cargo of VEGFR mRNA. Angiogenesis assays confirmed that endothelial cells increased their tube formation capacity when exposed to stressed RPE exosomes.
Assuntos
Exossomos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica , Estresse Oxidativo , Epitélio Pigmentado da Retina/patologia , Linhagem Celular , Etanol/farmacologia , Exossomos/efeitos dos fármacos , Exossomos/ultraestrutura , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
We have explored the mechanisms underlying ethanol-induced mitochondrial dynamics disruption and mitophagy. Ethanol increases mitochondrial fission in a concentration-dependent manner through Drp1 mitochondrial translocation and OPA1 proteolytic cleavage. ARPE-19 (a human retinal pigment epithelial cell line) cells challenged with ethanol showed mitochondrial potential disruptions mediated by alterations in mitochondrial complex IV protein level and increases in mitochondrial reactive oxygen species production. In addition, ethanol activated the canonical autophagic pathway, as denoted by autophagosome formation and autophagy regulator elements including Beclin1, ATG5-ATG12 and P-S6 kinase. Likewise, autophagy inhibition dramatically increased mitochondrial fission and cell death, whereas autophagy stimulation rendered the opposite results, placing autophagy as a cytoprotective response aimed to remove damaged mitochondria. Interestingly, although ethanol induced mitochondrial Bax translocation, this episode was associated to cell death rather than mitochondrial fission or autophagy responses. Thus, Bax required 600 mM ethanol to migrate to mitochondria, a concentration that resulted in cell death. Furthermore, mouse embryonic fibroblasts lacking this protein respond to ethanol by undergoing mitochondrial fission and autophagy but not cytotoxicity. Finally, by using the specific mitochondrial-targeted scavenger MitoQ, we revealed mitochondria as the main source of reactive oxygen species that trigger autophagy activation. These findings suggest that cells respond to ethanol activating mitochondrial fission machinery by Drp1 and OPA1 rather than bax, in a manner that stimulates cytoprotective autophagy through mitochondrial ROS.
Assuntos
Etanol/farmacologia , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial , Mitofagia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 12 Relacionada à Autofagia , Proteína 5 Relacionada à Autofagia , Proteína Beclina-1 , Linhagem Celular , Dinaminas/metabolismo , Fibroblastos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/citologia , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Despite the suggestion that impulsivity plays a central role in the transfer from a recreational drug use to a substance use disorder, very few studies focused on neurobiological markers for addiction. This study aimed to identify volumetric alterations in a sample of patients with mild alcohol use disorder with a short history of alcohol use, compared with a control group, and also focused on its association with impulsivity levels. Most magnetic resonance imaging studies have focused on severe alcohol use disorder, formerly called alcohol-dependent patients, showing alcohol-related structural alterations and their association with alcohol use history variables but not with personality parameters like impulsivity. Our hypothesis is that our group of alcohol users may already display structural alterations especially in brain regions related to inhibitory control like medial-prefrontal regions, and that those structural alterations could be more associated to personality traits like impulsivity than to drug use variables. Our results clearly demonstrate that our population showed lower regional grey and white matter volumes in the medial-prefrontal and orbitofrontal cortices, as well as higher regional white matter volume in the ventral striatum and the internal capsule. Volumetric alterations were associated to the Barratt's impulsivity score: the more impulsive the subjects, the lower the medial-prefrontal cortex grey matter volume.
Assuntos
Alcoolismo/patologia , Mapeamento Encefálico/métodos , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Comportamento Impulsivo , Imageamento por Ressonância Magnética/métodos , Adolescente , Adulto , Humanos , Processamento de Imagem Assistida por Computador/métodos , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos/estatística & dados numéricos , Tamanho do Órgão , Adulto JovemRESUMO
Diabetes and alcohol misuse are two of the major challenges in health systems worldwide. These two diseases finally affect several organs and systems including the central nervous system. Hippocampus is one of the most relevant structures due to neurogenesis and memory-related processing among other functions. The present review focuses on the common profile of diabetes and ethanol exposure in terms of oxidative stress and proinflammatory and prosurvival recruiting transcription factors affecting hippocampal neurogenesis. Some aspects around antioxidant strategies are also included. As a global conclusion, the present review points out some common hits on both diseases giving support to the relations between alcohol intake and diabetes.
Assuntos
Alcoolismo/metabolismo , Alcoolismo/fisiopatologia , Inflamação/fisiopatologia , Neurogênese/fisiologia , Estresse Oxidativo/fisiologia , Animais , Humanos , Inflamação/metabolismoRESUMO
Non-neovascular or dry age-related macular degeneration (AMD) is a multi-factorial disease with degeneration of the aging retinal-pigmented epithelium (RPE). Lysosomes play a crucial role in RPE health via phagocytosis and autophagy, which are regulated by transcription factor EB/E3 (TFEB/E3). Here, we find that increased AKT2 inhibits PGC-1α to downregulate SIRT5, which we identify as an AKT2 binding partner. Crosstalk between SIRT5 and AKT2 facilitates TFEB-dependent lysosomal function in the RPE. AKT2/SIRT5/TFEB pathway inhibition in the RPE induced lysosome/autophagy signaling abnormalities, disrupted mitochondrial function and induced release of debris contributing to drusen. Accordingly, AKT2 overexpression in the RPE caused a dry AMD-like phenotype in aging Akt2 KI mice, as evident from decline in retinal function. Importantly, we show that induced pluripotent stem cell-derived RPE encoding the major risk variant associated with AMD (complement factor H; CFH Y402H) express increased AKT2, impairing TFEB/TFE3-dependent lysosomal function. Collectively, these findings suggest that targeting the AKT2/SIRT5/TFEB pathway may be an effective therapy to delay the progression of dry AMD.
Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Lisossomos , Degeneração Macular , Proteínas Proto-Oncogênicas c-akt , Epitélio Pigmentado da Retina , Transdução de Sinais , Sirtuínas , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Animais , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sirtuínas/metabolismo , Sirtuínas/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Degeneração Macular/genética , Humanos , Camundongos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Lisossomos/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Modelos Animais de Doenças , Células-Tronco Pluripotentes Induzidas/metabolismo , MasculinoRESUMO
Tight glycemic control (TGC), the cornerstone of diabetic management, reduces the incidence and progression of diabetic microvascular disease. However, TGC can also lead to transient episodes of hypoglycemia, which have been associated with adverse outcomes in patients with diabetes. Here, we demonstrate that low glucose levels result in hypoxia-inducible factor (HIF)-1-dependent expression of the glucose transporter, Glut1, in retinal cells. Enhanced nuclear accumulation of HIF-1α was independent of its canonical post-translational stabilization but instead dependent on stimulation of its translation and nuclear localization. In the presence of hypoxia, this physiologic response to low glucose resulted in a marked increase in the secretion of the HIF-dependent vasoactive mediators that promote diabetic retinopathy. Our results provide a molecular explanation for how early glucose control, as well as glycemic variability (i.e., oscillating serum glucose levels), contributes to diabetic eye disease. These observations have important implications for optimizing glucose management in patients with diabetes.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Hipoglicemia , Humanos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Retinopatia Diabética/metabolismo , Glucose/metabolismo , Hipoglicemia/complicações , Hipóxia , Subunidade alfa do Fator 1 Induzível por HipóxiaRESUMO
Usher syndrome type 1B (USH1B) is a deaf-blindness disorder, caused by mutations in the MYO7A gene, which encodes the heavy chain of an unconventional actin-based motor protein. Here, we examined the two retinal isoforms of MYO7A, IF1 and IF2. We compared 3D models of the two isoforms and noted that the 38-amino acid region that is present in IF1 but absent from IF2 affects the C lobe of the FERM1 domain and the opening of a cleft in this potentially important protein binding domain. Expression of each of the two isoforms of human MYO7A and pig and mouse Myo7a was detected in the RPE and neural retina. Quantification by qPCR showed that the expression of IF2 was typically â¼ 7-fold greater than that of IF1. We discuss the implications of these findings for any USH1B gene therapy strategy. Given the current incomplete knowledge of the functions of each isoform, both isoforms should be considered for targeting both the RPE and the neural retina in gene augmentation therapies.
Assuntos
Síndromes de Usher , Humanos , Camundongos , Animais , Suínos , Síndromes de Usher/genética , Síndromes de Usher/terapia , Síndromes de Usher/metabolismo , Miosina VIIa/genética , Miosina VIIa/metabolismo , Retina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Mutação , Terapia GenéticaRESUMO
Many patients with diabetic eye disease respond inadequately to anti-VEGF therapies, implicating additional vasoactive mediators in its pathogenesis. We demonstrate that levels of angiogenic proteins regulated by HIF-1 and -2 remain elevated in the eyes of people with diabetes despite treatment with anti-VEGF therapy. Conversely, by inhibiting HIFs, we normalized the expression of multiple vasoactive mediators in mouse models of diabetic eye disease. Accumulation of HIFs and HIF-regulated vasoactive mediators in hyperglycemic animals was observed in the absence of tissue hypoxia, suggesting that targeting HIFs may be an effective early treatment for diabetic retinopathy. However, while the HIF inhibitor acriflavine prevented retinal vascular hyperpermeability in diabetic mice for several months following a single intraocular injection, accumulation of acriflavine in the retina resulted in retinal toxicity over time, raising concerns for its use in patients. Conversely, 32-134D, a recently developed HIF inhibitor structurally unrelated to acriflavine, was not toxic to the retina, yet effectively inhibited HIF accumulation and normalized HIF-regulated gene expression in mice and in human retinal organoids. Intraocular administration of 32-134D prevented retinal neovascularization and vascular hyperpermeability in mice. These results provide the foundation for clinical studies assessing 32-134D for the treatment of patients with diabetic eye disease.
Assuntos
Diabetes Mellitus Experimental , Retinopatia Diabética , Neovascularização Retiniana , Humanos , Camundongos , Animais , Acriflavina/metabolismo , Acriflavina/farmacologia , Acriflavina/uso terapêutico , Diabetes Mellitus Experimental/metabolismo , Retina/metabolismo , Neovascularização Retiniana/metabolismo , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismoRESUMO
Human retinal organoid transplantation could potentially be a treatment for degenerative retinal diseases. How the recipient retina regulates the survival, maturation, and proliferation of transplanted organoid cells is unknown. We transplanted human retinal organoid-derived cells into photoreceptor-deficient mice and conducted histology and single-cell RNA sequencing alongside time-matched cultured retinal organoids. Unexpectedly, we observed human cells that migrated into all recipient retinal layers and traveled long distances. Using an unbiased approach, we identified these cells as astrocytes and brain/spinal cord-like neural precursors that were absent or rare in stage-matched cultured organoids. In contrast, retinal progenitor-derived rods and cones remained in the subretinal space, maturing more rapidly than those in the cultured controls. These data suggest that recipient microenvironment promotes the maturation of transplanted photoreceptors while inducing or facilitating the survival of migratory cell populations that are not normally derived from retinal progenitors. These findings have important implications for potential cell-based treatments of retinal diseases.
Assuntos
Degeneração Retiniana , Análise da Expressão Gênica de Célula Única , Humanos , Camundongos , Animais , Diferenciação Celular/fisiologia , Retina , Células Fotorreceptoras Retinianas Cones , Degeneração Retiniana/terapia , Organoides/transplanteRESUMO
Age-related macular degeneration (AMD), the leading cause of geriatric blindness, is a multi-factorial disease with retinal-pigmented epithelial (RPE) cell dysfunction as a central pathogenic driver. With RPE degeneration, lysosomal function is a core process that is disrupted. Transcription factors EB/E3 (TFEB/E3) tightly control lysosomal function; their disruption can cause aging disorders, such as AMD. Here, we show that induced pluripotent stem cells (iPSC)-derived RPE cells with the complement factor H variant [ CFH (Y402H)] have increased AKT2, which impairs TFEB/TFE3 nuclear translocation and lysosomal function. Increased AKT2 can inhibit PGC1α, which downregulates SIRT5, an AKT2 binding partner. SIRT5 and AKT2 co-regulate each other, thereby modulating TFEB-dependent lysosomal function in the RPE. Failure of the AKT2/SIRT5/TFEB pathway in the RPE induced abnormalities in the autophagy-lysosome cellular axis by upregulating secretory autophagy, thereby releasing a plethora of factors that likely contribute to drusen formation, a hallmark of AMD. Finally, overexpressing AKT2 in RPE cells in mice led to an AMD-like phenotype. Thus, targeting the AKT2/SIRT5/TFEB pathway could be a potential therapy for atrophic AMD.
RESUMO
Transplantation of stem cell-derived retinal pigment epithelium (RPE) cells is a promising potential therapy for currently incurable retinal degenerative diseases like advanced dry age-related macular degeneration. In this study, we designed a set of clinically applicable devices for subretinal implantation of RPE grafts, towards the overarching goal of establishing enabling technologies for cell-based therapeutic approaches to regenerate RPE cells. This RPE transplant kit includes a custom-designed trephine for the production of RPE transplants, a carrier for storage and transportation, and a surgical device for subretinal delivery of RPE transplants. Cell viability assay confirmed biocompatibility of the transplant carrier and high preservation of RPE transplants upon storage and transportation. The transplant surgical device combines foldable technology that minimizes incision size, controlled delivery speed, no fluid reflux, curved translucent tip, usability of loading and in vivo reloading, and ergonomic handle. Furthermore, the complementary design of the transplant carrier and the delivery device resulted in proper grasping, loading, and orientation of the RPE transplants into the delivery device. Proof-of-concept transplantation studies in a porcine model demonstrated no damage or structural change in RPE transplants during surgical manipulation and subretinal deployment. Post-operative assessment confirmed that RPE transplants were delivered precisely, with no damage to the host retina or choroid, and no significant structural change to the RPE transplants. Our novel surgical kit provides a comprehensive set of tools encompassing RPE graft manufacturing to surgical implantation rendering key enabling technologies for pre-clinical and clinical phases of stem cell-derived RPE regenerative therapies.
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Introduction: Diabetic Retinopathy (DR) is a potentially blinding retinal disorder that develops through the pathogenesis of diabetes. The lack of disease predictors implies a poor prognosis with frequent irreversible retinal damage and vision loss. Extracellular Vesicles (EVs) present a novel opportunity for pre-symptomatic disease diagnosis and prognosis, both severely limited in DR. All biological fluids contain EVs, which are currently being studied as disease biomarkers. EV proteins derived from urine have emerged as potential noninvasive biomarkers. Methods: In this study, we isolated EVs from DR retinal tissue explants and from DR patients' urine, and characterized the vesicles, finding differences in particle number and size. Next, we performed proteomic analysis on human explanted DR retinal tissue conditioned media, DR retinal EVs and DR urinary EVs and compared to normal human retinal tissue, retinal EVs, and urinary EVs, respectively. Results: Our system biology analysis of DR tissue and EV expression profiles revealed biological pathways related to cell-to-cell junctions, vesicle biology, and degranulation processes. Junction Plakoglobin (JUP), detected in DR tissue-derived EVs and DR urinary EVs, but not in controls, was revealed to be a central node in many identified pathogenic pathways. Proteomic results were validated by western blot. Urinary EVs obtained from healthy donors and diabetic patient without DR did not contain JUP. Conclusion: The absence of JUP in healthy urinary EVs provide the basis for development of a novel Diabetic Retinopathy biomarker, potentially facilitating diagnosis.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Vesículas Extracelulares , Doenças Retinianas , Humanos , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/metabolismo , Proteômica , Retina/metabolismo , Biomarcadores/metabolismo , Vesículas Extracelulares/metabolismo , Diabetes Mellitus/metabolismoRESUMO
For patients with proliferative diabetic retinopathy (PDR) who do not respond adequately to pan-retinal laser photocoagulation (PRP) or anti-vascular endothelial growth factor (VEGF) therapies, we hypothesized that vascular cells within neovascular tissue secrete autocrine/paracrine angiogenic factors that promote disease progression. To identify these factors, we performed multiplex ELISA angiogenesis arrays on aqueous fluid from PDR patients who responded inadequately to anti-VEGF therapy and/or PRP and identified plasminogen activator inhibitor-1 (PAI-1). PAI-1 expression was increased in vitreous biopsies and neovascular tissue from PDR eyes, limited to retinal vascular cells, regulated by the transcription factor hypoxia-inducible factor (HIF)-2α, and necessary and sufficient to stimulate angiogenesis. Using a pharmacologic inhibitor of HIF-2α (PT-2385) or nanoparticle-mediated RNA interference targeting Pai1, we demonstrate that the HIF-2α/PAI-1 axis is necessary for the development of retinal neovascularization in mice. These results suggest that targeting HIF-2α/PAI-1 will be an effective adjunct therapy for the treatment of PDR patients.
Assuntos
Diabetes Mellitus , Retinopatia Diabética , Neovascularização Retiniana , Indutores da Angiogênese/uso terapêutico , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/etiologia , Retinopatia Diabética/metabolismo , Humanos , Camundongos , Neovascularização Patológica , Inibidor 1 de Ativador de Plasminogênio/genética , Neovascularização Retiniana/etiologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The nuclear factor-erythroid 2-related factor-2 (Nrf2), a major antioxidant transcription factor, is decreased in several age-related diseases including age-related macular degeneration (AMD), the most common cause of blindness among the elderly in western society. Since Nrf2's mito-protective response is understudied, we investigated its antioxidant response on mitochondria. Control and Nrf2-deficient retinal pigmented epithelial (RPE) cells were compared after treating with cigarette smoke extract (CSE). Mitochondrial antioxidant abundance and reactive oxygen species (ROS) were quantified. Mitochondrial function was assessed by TMRM assay, NADPH, electron transport chain activity, and Seahorse. Results were corroborated in Nrf2-/- mice and relevance to AMD was provided by immunohistochemistry of human globes. CSE induced mitochondrial ROS to impair mitochondrial function. H2 O2 increase in particular, was magnified by Nrf2 deficiency, and corresponded with exaggerated mitochondrial dysfunction. While Nrf2 did not affect mitochondrial antioxidant abundance, oxidized PRX3 was magnified by Nrf2 deficiency due to decreased NADPH from decreased expression of IDH2 and pentose phosphate pathway (PPP) genes. With severe CSE stress, intrinsic apoptosis was activated to increase cell death. PPP component TALDO1 immunolabeling was decreased in dysmorphic RPE of human AMD globes. Despite limited regulation of mitochondrial antioxidant expression, Nrf2 influences PPP and IDH shuttle activity that indirectly supplies NADPH for the TRX2 system. These results provide insight into how Nrf2 deficiency impacts the mitochondrial antioxidant response, and its role in AMD pathobiology.
Assuntos
Isocitrato Desidrogenase/metabolismo , Mitocôndrias/metabolismo , NADP/metabolismo , Fator 2 Relacionado a NF-E2/deficiência , Estresse Oxidativo/fisiologia , Epitélio Pigmentado da Retina/metabolismo , Animais , Humanos , Células-Tronco Pluripotentes Induzidas , Camundongos , Via de Pentose Fosfato , Espécies Reativas de Oxigênio/metabolismoRESUMO
Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen-associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen-associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely-tuned mechanism regulating directional apical:basal sorting and secretion of drusen-associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE-derived EVs as a key source of drusen proteins and important contributors to drusen development and growth.
Assuntos
Polaridade Celular/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Degeneração Macular/complicações , Degeneração Macular/metabolismo , Proteínas/metabolismo , Drusas Retinianas/complicações , Drusas Retinianas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Nicotina/farmacologia , Organoides/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Secretoma/metabolismoRESUMO
Therapies targeting VEGF have proven only modestly effective for the treatment of proliferative sickle cell retinopathy (PSR), the leading cause of blindness in patients with sickle cell disease. Here, we shift our attention upstream from the genes that promote retinal neovascularization (NV) to the transcription factors that regulate their expression. We demonstrated increased expression of HIF-1α and HIF-2α in the ischemic inner retina of PSR eyes. Although both HIFs participated in promoting VEGF expression by hypoxic retinal Müller cells, HIF-1 alone was sufficient to promote retinal NV in mice, suggesting that therapies targeting only HIF-2 would not be adequate to prevent PSR. Nonetheless, administration of a HIF-2-specific inhibitor currently in clinical trials (PT2385) inhibited NV in the oxygen-induced retinopathy (OIR) mouse model. To unravel these discordant observations, we examined the expression of HIFs in OIR mice and demonstrated rapid but transient accumulation of HIF-1α but delayed and sustained accumulation of HIF-2α; simultaneous expression of HIF-1α and HIF-2α was not observed. Staggered HIF expression was corroborated in hypoxic adult mouse retinal explants but not in human retinal organoids, suggesting that this phenomenon may be unique to mice. Using pharmacological inhibition or an in vivo nanoparticle-mediated RNAi approach, we demonstrated that inhibiting either HIF was effective for preventing NV in OIR mice. Collectively, these results explain why inhibition of either HIF-1α or HIF-2α is equally effective for preventing retinal NV in mice but suggest that therapies targeting both HIFs will be necessary to prevent NV in patients with PSR.
Assuntos
Anemia Falciforme/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Regulação da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Neovascularização Retiniana/metabolismo , Vasos Retinianos/metabolismo , Anemia Falciforme/complicações , Anemia Falciforme/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Modelos Animais de Doenças , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Indanos/farmacologia , Camundongos , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/genética , Sulfonas/farmacologiaRESUMO
The mechanisms underlying retinal development have not been completely elucidated. Extracellular vesicles (EVs) are novel essential mediators of cell-to-cell communication with emerging roles in developmental processes. Nevertheless, the identification of EVs in human retinal tissue, characterization of their cargo, and analysis of their potential role in retina development has not been accomplished. Three-dimensional retinal tissue derived from human induced pluripotent stem cells (hiPSC) provide an ideal developmental system to achieve this goal. Here we report that hiPSC-derived retinal organoids release exosomes and microvesicles with small noncoding RNA cargo. EV miRNA cargo-predicted targetome correlates with Gene Ontology (GO) pathways involved in mechanisms of retinogenesis relevant to specific developmental stages corresponding to hallmarks of native human retina development. Furthermore, uptake of EVs by human retinal progenitor cells leads to changes in gene expression correlated with EV miRNA cargo predicted gene targets, and mechanisms involved in retinal development, ganglion cell and photoreceptor differentiation and function.
Assuntos
Comunicação Celular , Vesículas Extracelulares/metabolismo , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Organoides/metabolismo , Retina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/citologia , Retina/citologiaRESUMO
The retinal pigment epithelium (RPE) plays a key role in retinal health, being essential for the protection against reactive oxygen species (ROS). Nevertheless, excessive oxidative stress can induce RPE dysfunction, promoting visual loss. Our aim is to clarify the possible implication of CYP2E1 in ethanol (EtOH)-induced oxidative stress in RPE alterations. Despite the increase in the levels of ROS, measured by fluorescence probes, the RPE cells exposed to the lowest EtOH concentrations were able to maintain cell survival, measured by the Cell Proliferation Kit II (XTT). However, EtOH-induced oxidative stress modified inflammation and angiogenesis biomarkers, analyzed by proteome array, ELISA, qPCR and Western blot. The highest EtOH concentration used stimulated a large increase in ROS levels, upregulating the cytochrome P450-2E1 (CYP2E1) and promoting cell death. The use of antioxidants such as N-acetylcysteine (NAC) and diallyl sulfide (DAS), which is also a CYP2E1 inhibitor, reverted cell death and oxidative stress, modulating also the upstream angiogenesis and inflammation regulators. Because oxidative stress plays a central role in most frequent ocular diseases, the results herein support the proposal that CYP2E1 upregulation could aggravate retinal degeneration, especially in those patients with high baseline oxidative stress levels due to their ocular pathology and should be considered as a risk factor.