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In recent decades, the role of tumor biomechanics on cancer cell behavior at the primary site has been increasingly appreciated. However, the effect of primary tumor biomechanics on the latter stages of the metastatic cascade, such as metastatic seeding of secondary sites and outgrowth remains underappreciated. This work sought to address this in the context of triple negative breast cancer (TNBC), a cancer type known to aggressively disseminate at all stages of disease progression. Using mechanically tuneable model systems, mimicking the range of stiffness's typically found within breast tumors, it is found that, contrary to expectations, cancer cells exposed to softer microenvironments are more able to colonize secondary tissues. It is shown that heightened cell survival is driven by enhanced metabolism of fatty acids within TNBC cells exposed to softer microenvironments. It is demonstrated that uncoupling cellular mechanosensing through integrin ß1 blocking antibody effectively causes stiff primed TNBC cells to behave like their soft counterparts, both in vitro and in vivo. This work is the first to show that softer tumor microenvironments may be contributing to changes in disease outcome by imprinting on TNBC cells a greater metabolic flexibility and conferring discrete cell survival advantages.
Assuntos
Ácidos Graxos , Neoplasias de Mama Triplo Negativas , Microambiente Tumoral , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Humanos , Feminino , Ácidos Graxos/metabolismo , Camundongos , Linhagem Celular Tumoral , Animais , Fenômenos Biomecânicos , Modelos Animais de Doenças , Metástase NeoplásicaRESUMO
The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma.
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Pancreatopatias , Neoplasias Pancreáticas , Humanos , Gencitabina , Proteína-Lisina 6-Oxidase , Neoplasias Pancreáticas/tratamento farmacológicoRESUMO
Rationale: Müller cells play an essential role in maintaining the health of retinal photoreceptors. Dysfunction of stressed Müller cells often results in photoreceptor degeneration. However, how these cells communicate under stress and the signalling pathways involved remain unclear. In this study, we inhibited the MAPK (ERK1/2) signalling, mainly activated in Müller cells, evaluated the protective effects on the photoreceptors and further explored the signalling communication between stressed Müller cells and degenerating photoreceptors. Methods: We evaluated the changes of MAPK (ERK1/2) signalling and its downstream targets in human retinal explants treated with PD98059, a specific phosphorylated ERK1/2 inhibitor, by western blot and immunostaining. We further assessed photoreceptor degeneration by TUNEL staining and outer nuclear layer thickness. We also injected PD98059 into the eyes of mice exposed to photo-oxidative stress. We evaluated the protective effects on photoreceptor degeneration by optical coherence tomography (OCT) and electroretinography (ERG). The crosstalk between Müller cells and photoreceptors was further dissected based on the changes of transcription factors by RNA sequencing and protein profiles of multiple signalling pathways. Results: We found that MAPK (ERK1/2) signalling was mainly activated in Müller cells under stress, both ex vivo and in vivo. PD98059 inhibited the phosphorylation of ERK1/2, reduced expression of the gliotic marker glial fibrillary acidic protein (GFAP) in Müller cells and increased levels of the neuroprotective factor, interphotoreceptor retinoid-binding protein (IRBP) in photoreceptors. Inhibition of pERK1/2 also reduced retinal photo-oxidative damage in mice retinas assessed by OCT and ERG. We also identified that the JAK/STAT3 signalling pathway might mediate signalling transduction from Müller cells to photoreceptors. Conclusion: MAPK (ERK1/2) deactivation through chemical inhibition, mainly in stressed Müller cells, can alleviate gliosis in Müller cells and restore the expression of IRBP in photoreceptors, which appears to prevent retinal degeneration. Our findings suggested a new way to prevent photoreceptor degeneration by manipulating the stress response in Müller cells.
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Degeneração Retiniana , Animais , Células Ependimogliais , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Degeneração Retiniana/genética , Fatores de Transcrição/metabolismoRESUMO
Isocitrate dehydrogenase 3 (IDH3) is a key enzyme in the mitochondrial tricarboxylic acid (TCA) cycle, which catalyzes the decarboxylation of isocitrate into α-ketoglutarate and concurrently converts NAD+ into NADH. Dysfunction of IDH3B, the ß subunit of IDH3, has been previously correlated with retinal degeneration and male infertility in humans, but tissue-specific effects of IDH3 dysfunction are unclear. Here, we generated Idh3b-KO mice and found that IDH3B is essential for IDH3 activity in multiple tissues. We determined that loss of Idh3b in mice causes substantial accumulation of isocitrate and its precursors in the TCA cycle, particularly in the testes, whereas the levels of the downstream metabolites remain unchanged or slightly increased. However, the Idh3b-KO mice did not fully recapitulate the defects observed in humans. Global deletion of Idh3b only causes male infertility but not retinal degeneration in mice. Our investigation showed that loss of Idh3b causes an energetic deficit and disrupts the biogenesis of acrosome and flagellum, resulting in spermiogenesis arrestment in sperm cells. Together, we demonstrate that IDH3B controls its substrate levels in the TCA cycle, and it is required for sperm mitochondrial metabolism and spermiogenesis, highlighting the importance of the tissue-specific function of the ubiquitous TCA cycle.
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Infertilidade Masculina , Isocitrato Desidrogenase , Degeneração Retiniana , Espermatogênese , Animais , Ciclo do Ácido Cítrico , Humanos , Infertilidade Masculina/genética , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Isocitratos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Masculino , Camundongos , NAD/metabolismo , Sêmen/metabolismoRESUMO
The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance. Cancer-associated fibroblasts (CAFs) coexist within breast cancer tissues and play both pro- and anti-tumourigenic roles through remodelling the ECM. Here, using temporal proteomic profiling of decellularized tumours, we interrogate the evolving matrisome during breast cancer progression. We identify 4 key matrisomal clusters, and pinpoint collagen type XII as a critical component that regulates collagen type I organisation. Through combining our proteomics with single-cell transcriptomics, and genetic manipulation models, we show how CAF-secreted collagen XII alters collagen I organisation to create a pro-invasive microenvironment supporting metastatic dissemination. Finally, we show in patient cohorts that collagen XII may represent an indicator of breast cancer patients at high risk of metastatic relapse.
Assuntos
Neoplasias da Mama , Colágeno Tipo XII/metabolismo , Metástase Neoplásica , Microambiente Tumoral , Neoplasias da Mama/patologia , Colágeno , Colágeno Tipo I , Matriz Extracelular/patologia , Feminino , Humanos , Metástase Neoplásica/patologia , Recidiva Local de Neoplasia/patologia , ProteômicaRESUMO
The Pentose Phosphate Pathway (PPP), a metabolic offshoot of the glycolytic pathway, provides protective metabolites and molecules essential for cell redox balance and survival. Transketolase (TKT) is the critical enzyme that controls the extent of "traffic flow" through the PPP. Here, we explored the role of TKT in maintaining the health of the human retina. We found that Müller cells were the primary retinal cell type expressing TKT in the human retina. We further explored the role of TKT in human Müller cells by knocking down its expression in primary cultured Müller cells (huPMCs), isolated from the human retina (11 human donors in total), under light-induced oxidative stress. TKT knockdown and light stress reduced TKT enzymatic activities and the overall metabolic activities of huPMCs with no detectable cell death. TKT knockdown restrained the PPP traffic flow, reduced the expression of NAD(P)H Quinone Dehydrogenase 1 (NQO1), impaired the antioxidative response of NRF2 to light stress and aggravated the endoplasmic reticulum (ER) stress. TKT knockdown also inhibited overall glucose intake, reduced expression of Dihydrolipoamide dehydrogenase (DLD) and impaired the energy supply of the huPMCs. In summary, Müller cell-mediated TKT activity plays a critical protective role in the stressed retina. Knockdown of TKT disrupted the PPP and impaired overall glucose utilisation by huPMCs and rendered huPMCs more vulnerable to light stress by impairing energy supply and antioxidative NRF2 responses.
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Fator 2 Relacionado a NF-E2 , Transcetolase , Células Ependimogliais/metabolismo , Glucose/metabolismo , Humanos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Via de Pentose Fosfato , Pentoses , Fosfatos , Transcetolase/genética , Transcetolase/metabolismoRESUMO
Muller cells, the major glial cells of the retina, play vital roles in maintaining redox homeostasis and retinal metabolism. An immortalized human Muller cell line (MIO-M1) is widely used as an in vitro model to study Muller cells' function, but they may not be exactly the same as primarily cultured human Muller cells. The use of human primary Muller cells (huPMCs) in culture has been limited by the requirement for complicated culture systems or particular age ranges of donors. We have successfully grown huPMCs using our established protocol. The cell type was pure, and cultured cells expressed Muller cell-specific markers strongly. The cultured huPMCs were used for morphologic, metabolic, transcriptomic, and functional studies. Graphic abstract: Timeline for human primary Muller cell (huPMC) culture.
RESUMO
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.
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Células Fotorreceptoras , Degeneração Retiniana , Animais , Células Ependimogliais/metabolismo , Camundongos , Neuroglia/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Degeneração Retiniana/metabolismoRESUMO
BACKGROUND: The use of in vitro cell cultures is a powerful tool for obtaining key insights into the behaviour and response of cells to interventions in normal and disease situations. Unlike in vivo settings, in vitro experiments allow a fine-tuned control of a range of microenvironmental elements independently within an isolated setting. The recent expansion in the use of three-dimensional (3D) in vitro assays has created a number of representative tools to study cell behaviour in a more physiologically 3D relevant microenvironment. Complex 3D in vitro models that can recapitulate human tissue biology are essential for understanding the pathophysiology of disease. AIM: The development of the 3D coculture collagen contraction and invasion assay, the "organotypic assay," has been widely adopted as a powerful approach to bridge the gap between standard two-dimensional tissue culture and in vivo mouse models. In the cancer setting, these assays can then be used to dissect how stromal cells, such as cancer-associated fibroblasts (CAFs), drive extracellular matrix (ECM) remodelling to alter cancer cell behaviour and response to intervention. However, to date, many of the published organotypic protocols are low-throughput, time-consuming (up to several weeks), and work-intensive with often limited scalability. Our aim was to develop a fast, high-throughput, scalable 3D organotypic assay for use in oncology screening and drug development. METHODS AND RESULTS: Here, we describe a modified 96-well organotypic assay, the "Mini-Organo," which can be easily completed within 5 days. We demonstrate its application in a wide range of mouse and human cancer biology approaches including evaluation of stromal cell 3D ECM remodelling, 3D cancer cell invasion, and the assessment of efficacy of potential anticancer therapeutic targets. Furthermore, the organotypic assay described is highly amenable to customisation using different cell types under diverse experimental conditions. CONCLUSIONS: The Mini-Organo high-throughput 3D organotypic assay allows the rapid screening of potential cancer therapeutics in human and mouse models in a time-efficient manner.
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Técnicas de Cocultura/métodos , Desenvolvimento de Medicamentos/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Animais , Fibroblastos Associados a Câncer/fisiologia , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Invasividade Neoplásica , RatosRESUMO
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.
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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
Congenital heart disease (CHD) is the most common birth defect and brings with it significant mortality and morbidity. The application of exome and genome sequencing has greatly improved the rate of genetic diagnosis for CHD but the cause in the majority of cases remains uncertain. It is clear that genetics, as well as environmental influences, play roles in the aetiology of CHD. Here we address both these aspects of causation with respect to the Notch signalling pathway. In our CHD cohort, variants in core Notch pathway genes account for 20% of those that cause disease, a rate that did not increase with the inclusion of genes of the broader Notch pathway and its regulators. This is reinforced by case-control burden analysis where variants in Notch pathway genes are enriched in CHD patients. This enrichment is due to variation in NOTCH1. Functional analysis of some novel missense NOTCH1 and DLL4 variants in cultured cells demonstrate reduced signalling activity, allowing variant reclassification. Although loss-of-function variants in DLL4 are known to cause Adams-Oliver syndrome, this is the first report of a hypomorphic DLL4 allele as a cause of isolated CHD. Finally, we demonstrate a gene-environment interaction in mouse embryos between Notch1 heterozygosity and low oxygen- or anti-arrhythmic drug-induced gestational hypoxia, resulting in an increased incidence of heart defects. This implies that exposure to environmental insults such as hypoxia could explain variable expressivity and penetrance of observed CHD in families carrying Notch pathway variants.
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Interação Gene-Ambiente , Predisposição Genética para Doença , Genômica/métodos , Cardiopatias Congênitas/patologia , Mutação , Receptor Notch1/genética , Animais , Estudos de Casos e Controles , Feminino , Cardiopatias Congênitas/etiologia , Cardiopatias Congênitas/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sequenciamento do ExomaRESUMO
The retinal pigment epithelium (RPE) is a monolayer of pigmented cells between the choroid and the retina. RPE dysfunction underlies many retinal degenerative diseases, including age-related macular degeneration, the leading cause of age-related blindness. To perform its various functions in nutrient transport, phagocytosis of the outer segment, and cytokine secretion, the RPE relies on an active energy metabolism. We previously reported that human RPE cells prefer proline as a nutrient and transport proline-derived metabolites to the apical, or retinal, side. In this study, we investigated how RPE utilizes proline in vivo and why proline is a preferred substrate. By using [13C]proline labeling both ex vivo and in vivo, we found that the retina rarely uses proline directly, whereas the RPE utilizes it at a high rate, exporting proline-derived mitochondrial intermediates for use by the retina. We observed that in primary human RPE cell culture, proline is the only amino acid whose uptake increases with cellular maturity. In human RPE, proline was sufficient to stimulate de novo serine synthesis, increase reductive carboxylation, and protect against oxidative damage. Blocking proline catabolism in RPE impaired glucose metabolism and GSH production. Notably, in an acute model of RPE-induced retinal degeneration, dietary proline improved visual function. In conclusion, proline is an important nutrient that supports RPE metabolism and the metabolic demand of the retina.
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Metabolismo Energético/efeitos dos fármacos , Prolina/administração & dosagem , Retina/metabolismo , Degeneração Retiniana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Radioisótopos de Carbono/análise , Diferenciação Celular , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Oxirredução , Prolina/farmacologia , Retina/efeitos dos fármacos , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/etiologia , Epitélio Pigmentado da Retina/efeitos dos fármacosRESUMO
Glucose metabolism in vertebrate retinas is dominated by aerobic glycolysis (the "Warburg Effect"), which allows only a small fraction of glucose-derived pyruvate to enter mitochondria. Here, we report evidence that the small fraction of pyruvate in photoreceptors that does get oxidized by their mitochondria is required for visual function, photoreceptor structure and viability, normal neuron-glial interaction, and homeostasis of retinal metabolism. The mitochondrial pyruvate carrier (MPC) links glycolysis and mitochondrial metabolism. Retina-specific deletion of MPC1 results in progressive retinal degeneration and decline of visual function in both rod and cone photoreceptors. Using targeted-metabolomics and 13C tracers, we found that MPC1 is required for cytosolic reducing power maintenance, glutamine/glutamate metabolism, and flexibility in fuel utilization.
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Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Retina/metabolismo , Visão Ocular/genética , Animais , Glucose/metabolismo , Glicólise/genética , Humanos , Camundongos , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Transportadores de Ácidos Monocarboxílicos , Ácido Pirúvico/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologiaRESUMO
Congenital heart disease (CHD) is the most common type of birth defect. In recent years, research has focussed on identifying the genetic causes of CHD. However, only a minority of CHD cases can be attributed to single gene mutations. In addition, studies have identified different environmental stressors that promote CHD, but the additive effect of genetic susceptibility and environmental factors is poorly understood. In this context, we have investigated the effects of short-term gestational hypoxia on mouse embryos genetically predisposed to heart defects. Exposure of mouse embryos heterozygous for Tbx1 or Fgfr1/Fgfr2 to hypoxia in utero increased the incidence and severity of heart defects while Nkx2-5+/- embryos died within 2 days of hypoxic exposure. We identified the molecular consequences of the interaction between Nkx2-5 and short-term gestational hypoxia, which suggest that reduced Nkx2-5 expression and a prolonged hypoxia-inducible factor 1α response together precipitate embryo death. Our study provides insight into the causes of embryo loss and variable penetrance of monogenic CHD, and raises the possibility that cases of foetal death and CHD in humans could be caused by similar gene-environment interactions.
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Interação Gene-Ambiente , Cardiopatias Congênitas/genética , Coração/embriologia , Proteína Homeobox Nkx-2.5/genética , Proteínas de Homeodomínio/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Animais , Apoptose , Proliferação de Células , Embrião de Mamíferos/metabolismo , Feminino , Predisposição Genética para Doença , Coração/diagnóstico por imagem , Heterozigoto , Proteína Homeobox Nkx-2.5/fisiologia , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxigênio/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Proteínas com Domínio T/genética , Fatores de TempoRESUMO
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.
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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
Aging is a major risk factor for age-related ocular diseases including age-related macular degeneration in the retina and retinal pigment epithelium (RPE), cataracts in the lens, glaucoma in the optic nerve, and dry eye syndrome in the cornea. We used targeted metabolomics to analyze metabolites from young (6 weeks) and old (73 weeks) eyes in C57 BL6/J mice. Old mice had diminished electroretinogram responses and decreased number of photoreceptors in their retinas. Among the 297 detected metabolites, 45-114 metabolites are significantly altered in aged eye tissues, mostly in the neuronal tissues (retina and optic nerve) and less in cornea, RPE/choroid, and lens. We noted that changes of metabolites in mitochondrial metabolism and glucose metabolism are common features in the aged retina, RPE/choroid, and optic nerve. The aging retina, cornea, and optic nerve also share similar changes in Nicotinamide adenine dinucleotide (NAD), 1-methylnicotinamides, 3-methylhistidine, and other methylated metabolites. Metabolites in taurine metabolism are strikingly influenced by aging in the cornea and lens. In conclusion, the aging eye has both common and tissue-specific metabolic signatures. These changes may be attributed to dysregulated mitochondrial metabolism, reprogrammed glucose metabolism and impaired methylation in the aging eye. Our findings provide biochemical insights into the mechanisms of age-related ocular changes.
Assuntos
Envelhecimento/metabolismo , Olho/metabolismo , Animais , Corioide/metabolismo , Córnea/metabolismo , Eletrorretinografia , Feminino , Masculino , Metabolômica , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Nervo Óptico/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismoRESUMO
Metabolomics studies in the retina and retinal pigment epithelium (RPE) in animal models or postmortem donors are essential to understanding the retinal metabolism and to revealing the underlying mechanisms of retinal degenerative diseases. We have studied how different methods of euthanasia (CO2 or cervical dislocation) different isolation procedures and postmortem delay affect metabolites in mouse retina and RPE/choroid using LC MS/MS and GC MS. Compared with cervical dislocation, CO2 exposure for 5â¯min dramatically degrades ATP and GTP into purine metabolites in the retina while raising intermediates in glucose metabolism and amino acids in the RPE/choroid. Isolation in cold buffer containing glucose has the least change in metabolites. Postmortem delay time-dependently and differentially impacts metabolites in the retina and RPE/choroid. In the postmortem retina, 18% of metabolites were changed at 0.5â¯h (h), 41% at 4â¯h and 51% at 8â¯h. However, only 6% of metabolites were changed in the postmortem RPE/choroid and it steadily increased to 20% at 8â¯h. Notably, both postmortem retina and RPE/choroid tissue showed increased purine metabolites. Storage of eyes in cold nutrient-rich medium substantially blocked the postmortem change in the retina and RPE/choroid. In conclusion, our study provides optimized methods to prepare fresh or postmortem retina and RPE/choroid tissue for metabolomics studies.
Assuntos
Dióxido de Carbono/farmacologia , Corioide , Dissecação , Eutanásia , Metaboloma/efeitos dos fármacos , Epitélio Pigmentado da Retina , Trifosfato de Adenosina/metabolismo , Animais , Corioide/efeitos dos fármacos , Corioide/metabolismo , Cromatografia Líquida , Modelos Animais de Doenças , Glucose/metabolismo , Guanosina Trifosfato/metabolismo , Camundongos , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Espectrometria de Massas em Tandem , Fatores de TempoRESUMO
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
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.