RESUMO
Subretinal fibrosis is a major untreatable cause of poor outcomes in neovascular age-related macular degeneration. Mouse models of subretinal fibrosis all possess a degree of invasiveness and tissue damage not typical of fibrosis progression. This project characterises JR5558 mice as a model to study subretinal fibrosis. Fundus and optical coherence tomography (OCT) imaging was used to non-invasively track lesions. Lesion number and area were quantified with ImageJ. Retinal sections, wholemounts and Western blots were used to characterise alterations. Subretinal lesions expand between 4 and 8 weeks and become established in size and location around 12 weeks. Subretinal lesions were confirmed to be fibrotic, including various cell populations involved in fibrosis development. Müller cell processes extended from superficial retina into subretinal lesions at 8 weeks. Western blotting revealed increases in fibronectin (4 wk and 8 wk, p < 0.001), CTGF (20 wks, p < 0.001), MMP2 (12 wks and 20 wks p < 0.05), αSMA (12 wks and 20 wks p < 0.05) and GFAP (8 wk and 12 wk, p ≤ 0.01), consistent with our immunofluorescence results. Intravitreal injection of Aflibercept reduced subretinal lesion growth. Our study provides evidence JR5558 mice have subretinal fibrotic lesions that grow between 4 and 8 weeks and confirms this line to be a good model to study subretinal fibrosis development and assess treatment options.
Assuntos
Modelos Animais de Doenças , Fibrose , Retina , Tomografia de Coerência Óptica , Animais , Camundongos , Tomografia de Coerência Óptica/métodos , Retina/patologia , Retina/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fibronectinas/metabolismo , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fator de Crescimento do Tecido Conjuntivo/genética , Degeneração Macular/patologia , Degeneração Macular/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Injeções Intravítreas , Proteína Glial Fibrilar Ácida/metabolismo , Actinas/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de FusãoRESUMO
The common final pathway to blindness in many forms of retinal degeneration is the death of the light-sensitive primary retinal neurons. However, the normally light-insensitive second- and third-order neurons persist optogenetic gene therapy aims to restore sight by rendering such neurons light-sensitive. Here, we investigate whether bReaChES, a newly described high sensitivity Type I opsin with peak sensitivity to long-wavelength visible light, can restore vision in a murine model of severe early-onset retinal degeneration. Intravitreal injection of an adeno-associated viral vector carrying the sequence for bReaChES downstream of the calcium calmodulin kinase IIα promoter resulted in sustained retinal expression of bReaChES. Retinal ganglion cells (RGCs) expressing bReaChES generated action potentials at light levels consistent with bright indoor lighting (from 13.6 log photons cm-2 s-1). They could also detect flicker at up to 50 Hz, which approaches the upper temporal limit of human photopic vision. Topological response maps of bReaChES-expressing RGCs suggest that optogenetically activated RGCs may demonstrate similar topographical responses to RGCs stimulated by photoreceptor activation. Furthermore, treated dystrophic mice displayed restored cortical neuronal activity in response to light and rescued behavioral responses to a looming stimulus that simulated an aerial predator. Finally, human surgical retinal explants exposed to the bReaChES treatment vector demonstrated transduction. Together, these findings suggest that intravitreal gene therapy to deliver bReaChES to the retina may restore vision in human retinal degeneration in vivo at ecologically relevant light levels with spectral and temporal response characteristics approaching those of normal human photopic vision.
Assuntos
Degeneração Retiniana , Camundongos , Humanos , Animais , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Degeneração Retiniana/metabolismo , Optogenética/métodos , Opsinas de Bastonetes/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
The importance of Müller glia for retinal homeostasis suggests that they may have vulnerabilities that lead to retinal disease. Here, we studied the effect of selectively knocking down key metabolic genes in Müller glia on photoreceptor health. Immunostaining indicated that murine Müller glia expressed insulin receptor (IR), hexokinase 2 (HK2) and phosphoglycerate dehydrogenase (PHGDH) but very little pyruvate dehydrogenase E1 alpha 1 (PDH-E1α) and lactate dehydrogenase A (LDH-A). We crossed Müller glial cell-CreER (MC-CreER) mice with transgenic mice carrying a floxed IR, HK2, PDH-E1α, LDH-A, or PHGDH gene to study the effect of selectively knocking down key metabolic genes in Müller glia cells on retinal health. Selectively knocking down IR, HK2, or PHGDH led to photoreceptor degeneration and reduced electroretinographic responses. Supplementing exogenous l-serine prevented photoreceptor degeneration and improved retinal function in MC-PHGDH knockdown mice. We unexpectedly found that the levels of retinal serine and glycine were not reduced but, on the contrary, highly increased in MC-PHGDH knockdown mice. Moreover, dietary serine supplementation, while rescuing the retinal phenotypes caused by genetic deletion of PHGDH in Müller glial cells, restored retinal serine and glycine homeostasis probably through regulation of serine transport. No retinal abnormalities were observed in MC-CreER mice crossed with PDH-E1α- or LDH-A-floxed mice despite Cre expression. Our findings suggest that Müller glia do not complete glycolysis but use glucose to produce serine to support photoreceptors. Supplementation with exogenous serine is effective in preventing photoreceptor degeneration caused by PHGDH deficiency in Müller glia.
Assuntos
Células Fotorreceptoras , Degeneração Retiniana , Animais , Células Ependimogliais/metabolismo , Camundongos , Neuroglia/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Degeneração Retiniana/metabolismoRESUMO
PURPOSE: To describe the novel observation of spontaneously migrating retinal cells from living donor surgical retinal explants that express progenitor cell markers in the absence of exogenous growth factors. METHODS: Surgical retinal explants were harvested from 5 consecutive patients undergoing 23 G pars plana vitrectomy for the management of rhegmatogenous detachment. During surgery, equatorial flap tears were trimmed with the vitreous cutter and aspirated. Excised tissue was then regurgitated into a syringe containing balanced salt solution and immediately transferred to tissue culture. Migrating cells subsequently underwent immunohistochemical staining and their characteristics were compared with those of a spontaneously immortalized Müller stem cell line. RESULTS: Spontaneously migrating cells were observed from samples taken from all 5 patients from Day 2 to 10 after transfer to culture. These cells were found to express embryonic cell markers, including paired box 6 (Pax6), sex-determining region Y-box 2 (Sox-2), nestin, cone-rod homeobox, and cyclin-dependent kinase inhibitor 1B (p27Kip1) as well as proteins consistent with early or retained differentiation down the Müller cell lineage, including glial fibrillary acidic protein and glutamine synthetase. CONCLUSION: After injury, the human equatorial retina is capable of spontaneously producing cells that demonstrate migration and that express progenitor cell markers. In addition, these cells express proteins consistent with Müller cell lineage. These initial observations support the assertion that the human retina may possess the potential for regeneration and that surgical retinal explants could also act as a ready source of retinal progenitor cells.
Assuntos
Células Ependimogliais/patologia , Retina/patologia , Descolamento Retiniano/diagnóstico , Células-Tronco/citologia , Idoso , Diferenciação Celular , Linhagem Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Retina/cirurgia , Descolamento Retiniano/cirurgia , VitrectomiaRESUMO
Mitochondrial respiration in mammalian cells not only generates ATP to meet their own energy needs but also couples with biosynthetic pathways to produce metabolites that can be exported to support neighboring cells. However, how defects in mitochondrial respiration influence these biosynthetic and exporting pathways remains poorly understood. Mitochondrial dysfunction in retinal pigment epithelium (RPE) cells is an emerging contributor to the death of their neighboring photoreceptors in degenerative retinal diseases including age-related macular degeneration. In this study, we used targeted-metabolomics and 13C tracing to investigate how inhibition of mitochondrial respiration influences the intracellular and extracellular metabolome. We found inhibition of mitochondrial respiration strikingly influenced both the intracellular and extracellular metabolome in primary RPE cells. Intriguingly, the extracellular metabolic changes sensitively reflected the intracellular changes. These changes included substantially enhanced glucose consumption and lactate production; reduced release of pyruvate, citrate, and ketone bodies; and massive accumulation of multiple amino acids and nucleosides. In conclusion, these findings reveal a metabolic signature of nutrient consumption and release in mitochondrial dysfunction in RPE cells. Testing medium metabolites provides a sensitive and noninvasive method to assess mitochondrial function in nutrient utilization and transport.
Assuntos
Mitocôndrias , Epitélio Pigmentado da Retina , Animais , Humanos , Nutrientes , Respiração , Retina/metabolismoRESUMO
Photoreceptors, the primary site of phototransduction in the retina, require energy and metabolites to constantly renew their outer segments. They preferentially consume most glucose through aerobic glycolysis despite possessing abundant mitochondria and enzymes for oxidative phosphorylation (OXPHOS). Exactly how photoreceptors balance aerobic glycolysis and mitochondrial OXPHOS to regulate their survival is still unclear. We crossed rhodopsin-Cre mice with hexokinase 2 (HK2)-floxed mice to study the effect of knocking down HK2, the first rate-limiting enzyme in glycolysis, on retinal health and metabolic remodeling. Immunohistochemistry and Western blots were performed to study changes in photoreceptor-specific proteins and key enzymes in glycolysis and the tricarboxylic acid (TCA) cycle. Changes in retinal structure and function were studied by optical coherence tomography and electroretinography. Mass spectrometry was performed to profile changes in 13C-glucose-derived metabolites in glycolysis and the TCA cycle. We found that knocking down HK2 in rods led to age-related photoreceptor degeneration, evidenced by reduced expression of photoreceptor-specific proteins, age-related reductions of the outer nuclear layer, photoreceptor inner and outer segments and impaired electroretinographic responses. Loss of HK2 in rods led to upregulation of HK1, phosphorylation of pyruvate kinase muscle isozyme 2, mitochondrial stress proteins and enzymes in the TCA cycle. Mass spectrometry found that the deletion of HK2 in rods resulted in accumulation of 13C-glucose along with decreased pyruvate and increased metabolites in the TCA cycle. Our data suggest that HK2-mediated aerobic glycolysis is indispensable for the maintenance of photoreceptor structure and function and that long-term inhibition of glycolysis leads to photoreceptor degeneration.
Assuntos
Hexoquinase/metabolismo , Mitocôndrias/metabolismo , Degeneração Retiniana/metabolismo , Fatores Etários , Animais , Ciclo do Ácido Cítrico/fisiologia , Hexoquinase/genética , Camundongos Transgênicos , Proteínas Mitocondriais/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Tomografia de Coerência Óptica/métodosRESUMO
AIMS/HYPOTHESIS: Diabetic macular oedema (DME) is the leading cause of visual impairment in people with diabetes. Intravitreal injections of vascular endothelial growth factor inhibitors or corticosteroids prevent loss of vision by reducing DME, but the injections must be given frequently and usually for years. Here we report laboratory and clinical studies on the safety and efficacy of 670 nm photobiomodulation (PBM) for treatment of centre-involving DME. METHODS: The therapeutic effect of PBM delivered via a light-emitting diode (LED) device was tested in transgenic mice in which induced Müller cell disruption led to photoreceptor degeneration and retinal vascular leakage. We also developed a purpose-built 670 nm retinal laser for PBM to treat DME in humans. The effect of laser-delivered PBM on improving mitochondrial function and protecting against oxidative stress was studied in cultured rat Müller cells and its safety was studied in pigmented and non-pigmented rat eyes. We then used the retinal laser to perform PBM in an open-label, dose-escalation Phase IIa clinical trial involving 21 patients with centre-involving DME. Patients received 12 sessions of PBM over 5 weeks for 90 s per treatment at a setting of 25, 100 or 200 mW/cm2 for the three sequential cohorts of 6-8 patients each. Patients were recruited from the Sydney Eye Hospital, over the age of 18 and had centre-involving DME with central macular thickness (CMT) of >300 µm with visual acuity of 75-35 Log minimum angle of resolution (logMAR) letters (Snellen visual acuity equivalent of 20/30-20/200). The objective of this trial was to assess the safety and efficacy of laser-delivered PBM at 2 and 6 months. The primary efficacy outcome was change in CMT at 2 and 6 months. RESULTS: LED-delivered PBM enhanced photoreceptor mitochondrial membrane potential, protected Müller cells and photoreceptors from damage and reduced retinal vascular leakage resulting from induced Müller cell disruption in transgenic mice. PBM delivered via the retinal laser enhanced mitochondrial function and protected against oxidative stress in cultured Müller cells. Laser-delivered PBM did not damage the retina in pigmented rat eyes at 100 mW/cm2. The completed clinical trial found a significant reduction in CMT at 2 months by 59 ± 46 µm (p = 0.03 at 200 mW/cm2) and significant reduction at all three settings at 6 months (25 mW/cm2: 53 ± 24 µm, p = 0.04; 100 mW/cm2: 129 ± 51 µm, p < 0.01; 200 mW/cm2: 114 ± 60 µm, p < 0.01). Laser-delivered PBM was well tolerated in humans at settings up to 200 mW/cm2 with no significant side effects. CONCLUSIONS/INTERPRETATION: PBM results in anatomical improvement of DME over 6 months and may represent a safe and non-invasive treatment. Further testing is warranted in randomised clinical trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT02181400 Graphical abstract.
Assuntos
Retinopatia Diabética/radioterapia , Células Ependimogliais/efeitos da radiação , Terapia com Luz de Baixa Intensidade/métodos , Edema Macular/radioterapia , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Mitocôndrias/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Ratos , Tomografia de Coerência ÓpticaRESUMO
Rationale: The Notch and transforming growth factor-ß (TGFß) signaling pathways are two intracellular mechanisms that control fibrosis in general but whether they play a major role in retinal fibrosis is less clear. Here we study how these two signaling pathways regulate Müller cell-dominated retinal fibrosis in vitro and in vivo. Methods: Human MIO-M1 Müller cells were treated with Notch ligands and TGFß1, either alone or in combination. Western blots were performed to study changes in γ-secretase proteases, Notch downstream effectors, endogenous TGFß1, phosphorylated Smad3 (p-Smad3) and extracellular matrix (ECM) proteins. We also studied the effects of RO4929097, a selective γ-secretase inhibitor, on expression of ECM proteins after ligand stimulation. Müller cell viability was studied by AlamarBlue and cytotoxicity by lactate cytotoxicity assays. Finally, we studied changes in Notch and TGFß signaling and tested the effect of intravitreal injections of the Notch pathway inhibitor RO4929097 on retinal fibrosis resulted from Sodium iodate (NaIO3)-induced retinal injury in mice. We also studied the safety of intravitreal injections of RO4929097 in normal mice. Results: Treatment of Müller cells with Notch ligands upregulated γ-secretase proteases and Notch downstream effectors, with increased expression of endogenous TGFß1, TGFß receptors and p-Smad3. TGFß1 upregulated the expression of proteins associated with both signaling pathways in a similar manner. Notch ligands and TGFß1 had additive effects on overexpression of ECM proteins in Müller cells which were inhibited by RO4929097. Notch and TGFß ligands stimulated Müller cell proliferation which was inhibited by RO4929097 without damaging the cells. NaIO3-induced retinal injury activated both Notch and TGFß signaling pathways in vivo. Intravitreal injection of RO4929097 prevented Müller cell gliosis and inhibited overexpression of ECM proteins in this murine model. We found no safety concerns for up to 17 days after an intravitreal injection of RO4929097. Conclusions: Inhibiting Notch signaling might be an effective way to prevent retinal fibrosis. This study is of clinical significance in developing a treatment for preventing fibrosis in proliferative vitreoretinopathy, proliferative diabetic retinopathy and wet age-related macular degeneration.
Assuntos
Benzazepinas/farmacologia , Células Ependimogliais/patologia , Gliose/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Animais , Benzazepinas/uso terapêutico , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Células Ependimogliais/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibrose , Gliose/induzido quimicamente , Gliose/patologia , Humanos , Injeções Intravítreas , Iodatos/administração & dosagem , Iodatos/toxicidade , Masculino , Camundongos , Receptores Notch/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Vitreorretinopatia Proliferativa/tratamento farmacológico , Vitreorretinopatia Proliferativa/patologia , Degeneração Macular Exsudativa/tratamento farmacológico , Degeneração Macular Exsudativa/patologiaRESUMO
Dysfunction of retinal glial cells, particularly Müller cells, has been implicated in several retinal diseases. Despite their important contribution to retinal homeostasis, a specific way to differentiate retinal glial cells from human pluripotent stem cells has not yet been described. Here, we report a method to differentiate retinal glial cells from human embryonic stem cells (hESCs) through promoting the Notch signaling pathway. We first generated retinal progenitor cells (RPCs) from hESCs then promoted the Notch signaling pathway using Notch ligands, including Delta-like ligand 4 and Jagged-1. We validated glial cell differentiation with qRT-PCR, immunocytochemistry, western blots and fluorescence-activated cell sorting as we promoted Notch signaling in RPCs. We found that promoting Notch signaling in RPCs for 2 weeks led to upregulation of glial cell markers, including glial fibrillary acidic protein (GFAP), glutamine synthetase, vimentin and cellular retinaldehyde-binding protein (CRALBP). Of these markers, we found the greatest increase in expression of the pan glial cell marker, GFAP. Conversely, we also found that inhibition of Notch signaling in RPCs led to upregulation of retinal neuronal markers including cone-rod homeobox (CRX) and orthodenticle homeobox 2 (OTX2) but with little expression of GFAP. This retinal glial differentiation method will help advance the generation of stem cell disease models to study the pathogenesis of retinal diseases associated with glial dysfunction such as macular telangiectasia type 2. This method may also be useful for the development of future therapeutics such as drug screening and gene editing using patient-derived retinal glial cells.
RESUMO
BACKGROUND: Retinal pigment epithelium (RPE) is known to secrete factors important for retinal homeostasis. How this secretome changes in diabetic eyes treated with anti-vascular endothelial growth factor (VEGF) inhibitors is unclear. METHODS: Diabetic conditions were simulated in vitro using ARPE-19 cell-line culture, with high glucose (25 mM) culture media, and hypoxia was chemically induced using cobalt chloride. Stress was assessed using cell viability assays as well as Western blots and enzyme-linked immunosorbent assay (ELISA) for production of HIF-1a and VEGF-A. Production of neurotrophic factors was quantified once conditions were established using ELISA under stress with and without the addition of VEGF inhibitors. Changes were analysed with one-way ANOVA. RESULTS: Hypoxia downregulated pigment epithelium-derived factor (PEDF) expression. The addition of bevacizumab, ranibizumab and aflibercept in normoxic conditions all led to a significant downregulation of PEDF. Glucose concentration had no effect on secretion of PEDF. Brain-derived neurotrophic factor (BDNF) secretion was downregulated in high glucose and was upregulated in hypoxia. Placental growth factor (PlGF) secretion by ARPE-19 was undetectable by ELISA. CONCLUSIONS: We found that hypoxia, high glucose or VEGF inhibitors affected secretion of neurotrophic factors. This variation under different conditions may influence neuron and photoreceptor survival in the diabetic state and VEGF inhibitor treated eyes.
Assuntos
Inibidores da Angiogênese/farmacologia , Glucose/farmacologia , Hiperglicemia/patologia , Hipóxia/patologia , Fatores de Crescimento Neural/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Bevacizumab/farmacologia , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Linhagem Celular , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Proteínas do Olho/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Membrana/metabolismo , Ranibizumab/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão/farmacologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Serpinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The human macula is more susceptible than the peripheral retina to developing blinding conditions such as age-related macular degeneration, diabetic retinopathy. A key difference between them may be the nature of their Müller cells. We found primary cultured Müller cells from macula and peripheral retina display significant morphological and transcriptomic differences. Macular Müller cells expressed more phosphoglycerate dehydrogenase (PHGDH, a rate-limiting enzyme in serine synthesis) than peripheral Müller cells. The serine synthesis, glycolytic and mitochondrial function were more activated in macular than peripheral Müller cells. Serine biosynthesis is critical in defending against oxidative stress. Intracellular reactive oxygen species and glutathione levels were increased in primary cultured macular Müller cells which were more susceptible to oxidative stress after inhibition of PHGDH. Our findings indicate serine biosynthesis is a critical part of the macular defence against oxidative stress and suggest dysregulation of this pathway as a potential cause of macular pathology.
Assuntos
Células Ependimogliais/metabolismo , Estresse Oxidativo/fisiologia , Retina/metabolismo , Serina/biossíntese , Células Ependimogliais/citologia , Regulação da Expressão Gênica , Glutationa/metabolismo , Humanos , Mitocôndrias/metabolismo , Fosfoglicerato Desidrogenase , Espécies Reativas de Oxigênio/metabolismo , Serina/genética , TranscriptomaRESUMO
BACKGROUND AND PURPOSE: Simvastatin is a 3-hydroxy-3-methylglutaryl CoA reductase inhibitor with multiple targets and effects. It protects neurons in the brain, but its protective effects on photoreceptors are unclear. In this study, we evaluated the neuroprotective effect of simvastatin on photoreceptors exposed to stress induced by all-trans-retinal (atRAL). EXPERIMENTAL APPROACH: AlamarBlue and LDH assays were used to evaluate the viability and metabolic activity of Y79 cells (a retinoblastoma cell line) exposed to atRAL-induced stress with or without simvastatin pretreatment. Changes in cellular ROS were evaluated using flow cytometry and mitochondrial stress markers JC-1 and HSP60. Changes in levels of two photoreceptor-specific markers, cone-rod homeobox protein (CRX) and interphotoreceptor retinoid binding protein (IRBP), were evaluated with western blot. The results were validated in ex vivo human retinal explants and a mouse model of photoreceptor degeneration. KEY RESULTS: Simvastatin improved mitochondrial function, alleviated oxidative stress and up-regulated the photoreceptor-specific markers IRBP and its upstream regulator CRX in Y79 cells and ex vivo human retinal explants under atRAL-induced stress. Simvastatin attenuated photoreceptor degeneration in association with up-regulation of IRBP and CRX expression after knockdown of IRBP in a murine model. CONCLUSION AND IMPLICATIONS: Our findings suggest that simvastatin has a novel role in protecting photoreceptors from atRAL-induced stress. Simvastatin treatment resulted in up-regulation of IRBP and its upstream transcription factor CRX in Y79 cells, ex vivo human retinal explants, and murine retinas in vivo. Further studies of simvastatin to treat photoreceptor degeneration are warranted.
Assuntos
Substâncias Protetoras/farmacologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Proteínas de Ligação ao Retinol/metabolismo , Sinvastatina/farmacologia , Animais , Antineoplásicos Hormonais/administração & dosagem , Antineoplásicos Hormonais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Injeções Intraperitoneais , Camundongos , Camundongos Transgênicos , Estrutura Molecular , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/administração & dosagem , Células Fotorreceptoras Retinianas Cones/metabolismo , Sinvastatina/administração & dosagem , Relação Estrutura-Atividade , Tamoxifeno/administração & dosagem , Tamoxifeno/farmacologia , Tretinoína/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
AIMS/HYPOTHESIS: A major feature of diabetic retinopathy is breakdown of the blood-retinal barrier, resulting in macular oedema. We have developed a novel oligonucleotide-based drug, CD5-2, that specifically increases expression of the key junctional protein involved in barrier integrity in endothelial cells, vascular-endothelial-specific cadherin (VE-cadherin). CD5-2 prevents the mRNA silencing by the pro-angiogenic microRNA, miR-27a. CD5-2 was evaluated in animal models of ocular neovascularisation and vascular leak to determine its potential efficacy for diabetic retinopathy. METHODS: CD5-2 was tested in three mouse models of retinal dysfunction: conditional Müller cell depletion, streptozotocin-induced diabetes and oxygen-induced retinopathy. Vascular permeability in the Müller cell-knockout model was assessed by fluorescein angiography. The Evans Blue leakage method was used to determine vascular permeability in streptozotocin- and oxygen-induced retinopathy models. The effects of CD5-2 on retinal neovascularisation, inter-endothelial junctions and pericyte coverage in streptozotocin- and oxygen-induced retinopathy models were determined by staining for isolectin-B4, VE-cadherin and neural/glial antigen 2 (NG2). Blockmir CD5-2 localisation in diseased retina was determined using fluorescent in situ hybridisation. The effects of CD5-2 on VE-cadherin expression and in diabetic retinopathy-associated pathways, such as the transforming growth factor beta (TGF-ß) and wingless/integrated (WNT) pathway, were confirmed using western blot of lysates from HUVECs, a mouse brain endothelial cell line and a VE-cadherin null mouse endothelial cell line. RESULTS: CD5-2 penetrated the vasculature of the eye in the oxygen-induced retinopathy model. Treatment of diseased mice with CD5-2 resulted in reduced vascular leak in all three animal models, enhanced expression of VE-cadherin in the microvessels of the eye and improved pericyte coverage of the retinal vasculature in streptozotocin-induced diabetic models and oxygen-induced retinopathy models. Further, CD5-2 reduced the activation of retinal microglial cells in the streptozotocin-induced diabetic model. The positive effects of CD5-2 seen in vivo were further confirmed in vitro by increased protein expression of VE-cadherin, SMAD2/3 activity, and platelet-derived growth factor B (PDGF-B). CONCLUSIONS/INTERPRETATION: CD5-2 has therapeutic potential for individuals with vascular-leak-associated retinal diseases based on its ease of delivery and its ability to reverse vascular dysfunction and inflammatory aspects in three animal models of retinopathy.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Retinopatia Diabética/tratamento farmacológico , Oligonucleotídeos/uso terapêutico , Animais , Barreira Hematorretiniana/metabolismo , Permeabilidade Capilar , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Camundongos , Retina/metabolismo , Vasos Retinianos/metabolismoRESUMO
Purpose: Subretinal fibroneovascularization is one of the most common causes of vision loss in neovascular AMD (nAMD). Anti-VEGF therapy effectively inhibits vascular leak and neovascularization but has little effect on fibrosis. This study aimed to identify a combination therapy to concurrently inhibit subretinal neovascularization and prevent fibrosis. Methods: We generated transgenic mice in which induced disruption of Müller cells leads to subretinal neovascularization, which is reliably accompanied by subretinal fibrosis. We conducted Western blots and immunohistochemistry to study changes in transforming growth factor-ß (TGFß) signaling including endoglin, a coreceptor essential for TGFß signaling, and then tested the effects of monthly intravitreal injection of anti-VEGF-A and anti-endoglin, either alone or in combination, on the development of subretinal fibroneovascularization in our transgenic mice. Results: Müller cell disruption increased expression of TGFß1, TGFß type 1 receptor, and phosphorylated-Smad3. Endoglin was strongly expressed in subretinal fibroneovascular tissue. Fluorescein angiography and measurements of retinal vascular permeability indicated that intravitreal anti-VEGF-A in combination with anti-endoglin treatment more efficiently inhibited vascular leak compared with either monotherapy. Immunostaining of retinal wholemounts with antibodies against glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 indicated that the combination therapy also effectively prevented subretinal fibrosis and inhibited microglial activation. Luminex cytokine assays indicated that intravitreal anti-VEGF-A and anti-endoglin treatment, either alone or in combination, reduced the production of IL33 and macrophage inflammatory protein-3α. Conclusions: Our findings offer a potentially novel combination approach to concurrently managing subretinal neovascularization and fibrosis in nAMD.
Assuntos
Anticorpos Monoclonais/farmacologia , Endoglina/imunologia , Células Ependimogliais/patologia , Retina/patologia , Neovascularização Retiniana/prevenção & controle , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Western Blotting , Proteínas de Ligação ao Cálcio/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Combinação de Medicamentos , Células Ependimogliais/metabolismo , Fibrose/metabolismo , Fibrose/prevenção & controle , Angiofluoresceinografia , Proteína Glial Fibrilar Ácida/metabolismo , Imuno-Histoquímica , Injeções Intravítreas , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Fosforilação , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Retina/metabolismo , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/metabolismo , Vasos Retinianos , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Endothelial progenitor cells (EPCs) are a group of rare cells that play an important role in the repair of injured vascular endothelial cells and assist in reperfusion of ischemic tissue. Decreased production and/or loss of function of EPCs are associated with diabetic vasculopathy. The molecular mechanisms by which diabetes impairs EPCs remain unclear. We conducted microarray experiments followed by integrative regulatory analysis on cells isolated from Akita diabetic mice (18-weeks after onset of diabetes) and age-matched non-diabetic controls. Two types of cells were isolated from mice bone marrow; Lin+ cells and Lin-/VEGF-R2+ EPCs. RNA was hybridized to mouse WG-6 V2 beadchips followed by comprehensive gene network analysis and computational validation of the obtained results. In total, 80 genes were exclusively DE between non-diabetic Lin-/VEGF-R2+ EPCs and diabetic Lin-/VEGF-R2+ EPCs, of which the 3 genes Clcnka, Pik3c2a, and Ptf1a are known to be associated with diabetic complications. Further analysis led to the establishment of a TF-miRNA mediated regulatory network specific to diabetic Lin-/VEGF-R2+ EPCs and to identify 11 central-hub TFs (Tbp, Ahr, Trp53, Gata1, Foxo1, Foxo4, Yy1, Max, Pparg, Myc, Cebpa), and 2 miRNAs (mir-139-5p, mir-709) that might act as putative genomic drivers of diabetic pathogenesis in Lin-/VEGF-R2+ EPCs. Moreover, we identified multiple TF-miRNA co-regulatory network motifs for which we validated their contribution to diabetic Lin-/VEGF-R2+ EPCs in terms of statistical significance and relevance to biological evidence. Our findings suggest that diabetic Lin-/VEGF-R2+ EPCs have specifically altered signature genes and miRNAs that render their capacity to proliferate and differentiate.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Células Progenitoras Endoteliais/metabolismo , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Células da Medula Óssea/metabolismo , Células Progenitoras Endoteliais/citologia , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Masculino , Camundongos TransgênicosRESUMO
Müller cells are the primary glia in the retina, playing a critical role in retinal homeostasis and retinal pathology. This study evaluated the canonical Wnt signalling pathway and its downstream effects on retinal degeneration in a transgenic mouse model of inducible Müller cell disruption. Increased expression of the LacZ reporter gene in the retina suggested Wnt signalling had been activated after induced Müller cell disruption. Activation was validated by observing nuclear translocation of ß-Catenin. The mRNA expression of 80 Wnt related genes were assessed using real-time PCR. The Wnt signalling inhibitors Dkk1, Dkk3 and sFRP3 were significantly downregulated. Furthermore, the ubiquitin-mediated ß-Catenin proteolysis genes ß-TrCP and SHFM3, were also significantly downregulated. The downstream target genes of the Wnt signalling, including Fra1, CyclinD2 and C-Myc were upregulated. The changes of these genes at the protein level were validated by Western blot. Their distributions in the retina were evaluated by immunofluorescent staining. Our findings indicate that Müller cells are involved in retinal Wnt signalling. Activation of Wnt signalling and its downstream target genes may play important roles in photoreceptor degeneration and neovascularization occurring in the retina after induced disruption of Müller cells.
Assuntos
Células Ependimogliais/patologia , Regulação da Expressão Gênica/fisiologia , Degeneração Retiniana/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Western Blotting , Quimiocinas , Proteínas F-Box/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/patologia , Proteínas Contendo Repetições de beta-Transducina/genéticaRESUMO
De novo serine synthesis plays important roles in normal mitochondrial function and cellular anti-oxidative capacity. It is reported to be mainly activated in glial cells of the central nervous system, but its role in retinal Müller glia remains unclear. In this study, we inhibited de novo serine synthesis using CBR-5884, a specific inhibitor of phosphoglycerate dehydrogenase (PHGDH, a rate limiting enzyme in de novo serine metabolism) in MIO-M1 cells (immortalized human Müller cells) and huPMCs (human primary Müller cells) under mild oxidative stress. Alamar blue and LDH (lactate dehydrogenase) assays showed significantly reduced metabolic activities and increased cellular damage of Müller cells, when exposed to CBR-5884 accompanied by mild oxidative stress; however, CBR-5884 alone had little effect. The increased cellular damage was partially reversed by supplementation with exogenous serine/glycine. HSP72 (an oxidative stress marker) and reactive oxygen species (ROS) levels were significantly increased; glutathione and NADPH/NADP+ levels were pronouncedly reduced under PHGDH inhibition accompanied by oxidative stress. JC-1 staining and Seahorse respiration experiments showed that inhibition of de novo serine synthesis in Müller cells can also increase mitochondrial stress and decrease mitochondrial ATP production. qPCR and Western blot demonstrated an increased expression of HSP60 (a key mitochondrial stress-related gene), and this was further validated in human retinal explants. Our study suggests that de novo serine synthesis is important for Müller cell survival, particularly when they are exposed to mild oxidative stress, possibly by maintaining mitochondrial function and generating glutathione and NADPH to counteract ROS.
Assuntos
Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Mitocôndrias/patologia , Estresse Oxidativo , Serina/biossíntese , Trifosfato de Adenosina/metabolismo , Idoso , Chaperonina 60/metabolismo , Células Ependimogliais/enzimologia , Glutationa/metabolismo , Proteínas de Choque Térmico HSP72/metabolismo , Humanos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , NADP/metabolismo , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Fosfoglicerato Desidrogenase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
Purpose: Retinal iron accumulation is observed in a wide range of retinal degenerative diseases, including AMD. Previous work suggests that Müller glial cells may be important mediators of retinal iron transport, distribution, and regulation. A transgenic model of Müller cell loss recently demonstrated that primary Müller cell ablation leads to blood-retinal barrier leakage and photoreceptor degeneration, and it recapitulates clinical features observed in macular telangiectasia type 2 (MacTel2), a rare human disease that features Müller cell loss. We used this mouse model to determine the effect of Müller cell loss on retinal iron homeostasis. Methods: Changes in total retinal iron levels after Müller cell ablation were measured using inductively coupled plasma mass spectrometry. Corresponding changes in the expression of iron flux and iron storage proteins were determined using quantitative PCR, Western analysis, and immunohistochemistry. Results: Müller cell loss led to blood-retinal barrier breakdown and increased iron levels throughout the neurosensory retina. There were corresponding changes in mRNA and/or protein levels of ferritin, transferrin receptor, ferroportin, Zip8, and Zip14. There were also increased iron levels within the RPE of retinal sections from a patient with MacTel2 and both RPE and neurosensory retina of a patient with diabetic retinopathy, which, like MacTel2, causes retinal vascular leakage. Conclusion: This study shows that Müller cells and the blood-retinal barrier play pivotal roles in the regulation of retinal iron homeostasis. The retinal iron accumulation resulting from blood-retinal barrier dysfunction may contribute to retinal degeneration in this model and in diseases such as MacTel2 and diabetic retinopathy.
Assuntos
Modelos Animais de Doenças , Células Ependimogliais/patologia , Ferro/metabolismo , Retina/metabolismo , Telangiectasia Retiniana/metabolismo , Idoso , Animais , Barreira Hematorretiniana/metabolismo , Barreira Hematorretiniana/patologia , Western Blotting , Permeabilidade Capilar , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Feminino , Ferritinas/genética , Ferritinas/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Pessoa de Meia-Idade , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Telangiectasia Retiniana/genéticaRESUMO
Anti-vascular endothelial growth factor (VEGF) therapy has revolutionized the treatment of retinal vascular diseases. However, constitutive VEGF also acts as a trophic factor on retinal nonvascular cells. We have studied the effects of aflibercept and ranibizumab on human Müller cells and photoreceptors exposed to starvation media containing various concentrations of glucose, with or without CoCl2-induced hypoxia. Cell survival was assessed by calcein-AM cell viability assays. Expression of heat shock proteins (Hsp) and redox proteins thioredoxin 1 and 2 (TRX1, TRX2) was studied by Western blots. The production of neurotrophic factors in Müller cells and interphotoreceptor retinoid-binding protein (IRBP) in photoreceptors was measured by enzymelinked immunosorbent assays. Aflibercept and ranibizumab did not affect the viability of both types of cells. Neither aflibercept nor ranibizumab affected the production of neurotrophic factors or expression of Hsp60 and Hsp90 in Müller cells. However, aflibercept but not ranibizumab affected the expression of Hsp60, Hsp9, TRX1 and TRX2 in photoreceptors. Aflibercept and ranibizumab both inhibited the production of IRBP in photoreceptors, aflibercept more so than ranibizumab. Our data indicates that the potential influence of aflibercept and ranibizumab on photoreceptors should be specifically monitored in clinical studies.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/metabolismo , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/metabolismo , Ranibizumab/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Estresse Fisiológico , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas do Olho/metabolismo , Expressão Gênica , Glucose/farmacologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Hipóxia/metabolismo , Fatores de Crescimento Neural/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas de Ligação ao Retinol/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
Vascular changes and photoreceptor degeneration are features of age-related macular degeneration, diabetic retinopathy and macular telangiectasis. We have profiled the differential expression of microRNAs and analysed their target genes in transgenic mice in which induced Müller cell disruption results in photoreceptor degeneration, vascular leak and deep retinal neovascularisation. We identified 9 miRNAs which were differentially expressed during the development of retinal neovascularization and chose miR-200b and its target genes for further study. Using qRT-PCR and western blot analysis, we found that downregulation of miR-200b was negatively correlated with its target genes, including zinc finger E-box binding homeobox (ZEB) 1 and 2 and vascular endothelial growth factor receptor 1. Double immunofluorescence labelling revealed that the newly formed vessels in the outer retina were positive for ZEB2. Furthermore, intravitreal injections of a miR-200b-mimic and anti-miR-200b confirmed the negative correlation of miR-200b and its target gene expression. We also found that the miR-200b-mimic inhibited vascular leak in the established mild vascular lesions, whereas anti-miR-200b promoted it. Taken together, these data suggest that miR-200b may play a role in the development of intraretinal neovascularisation.