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In a 1-year survey of wild terrestrial predators in northern Germany, we found that 5 of 110 foxes were infected with contemporary avian influenza A(H5N1) viruses, forming a temporal cluster during JanuaryâMarch 2023. Encephalitis and strong cerebral virus replication but only sporadic mammalian-adaptive viral polymerase basic 2 protein E627K mutations were seen.
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Subtipo H5N1 del Virus de la Influenza A , Gripe Aviar , Animales , Humanos , Gripe Aviar/epidemiología , Subtipo H5N1 del Virus de la Influenza A/genética , Zorros , Proteínas Virales/genética , Alemania/epidemiologíaRESUMEN
Hematopoietic development is spatiotemporally tightly regulated by defined cell-intrinsic and extrinsic modifiers. The role of cytokines has been intensively studied in adult hematopoiesis; however, their role in embryonic hematopoietic specification remains largely unexplored. Here, we used induced pluripotent stem cell (iPSC) technology and established a 3-dimensional, organoid-like differentiation system (hemanoid) maintaining the structural cellular integrity to evaluate the effect of cytokines on embryonic hematopoietic development. We show, that defined stages of early human hematopoietic development were recapitulated within the generated hemanoids. We identified KDR+/CD34high/CD144+/CD43-/CD45- hemato-endothelial progenitor cells (HEPs) forming organized, vasculature-like structures and giving rise to CD34low/CD144-/CD43+/CD45+ hematopoietic progenitor cells. We demonstrate that the endothelial to hematopoietic transition of HEPs is dependent on the presence of interleukin 3 (IL-3). Inhibition of IL-3 signalling blocked hematopoietic differentiation and arrested the cells in the HEP stage. Thus, our data suggest an important role for IL-3 in early human hematopoiesis by supporting the endothelial to hematopoietic transition of hemato-endothelial progenitor cells and highlight the potential of a hemanoid-based model to study human hematopoietic development.
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Células Madre Pluripotentes Inducidas , Interleucina-3 , Células Madre Pluripotentes , Adulto , Diferenciación Celular , Hematopoyesis , HumanosRESUMEN
BACKGROUND: The transcription factor GATA2 orchestrates the expression of many endothelial-specific genes, illustrating its crucial importance for endothelial cell function. The capacity of this transcription factor in orchestrating endothelial-important microRNAs (miRNAs/miR) is unknown. METHODS: Endothelial GATA2 was functionally analyzed in human endothelial cells in vitro. Endogenous short interfering RNA-mediated knockdown and lentiviral-based overexpression were applied to decipher the capacity of GATA2 in regulating cell viability and capillary formation. Next, the GATA2-dependent miR transcriptome was identified by using a profiling approach on the basis of quantitative real-time polymerase chain reaction. Transcriptional control of miR promoters was assessed via chromatin immunoprecipitation, luciferase promoter assays, and bisulfite sequencing analysis of sites in proximity. Selected miRs were modulated in combination with GATA2 to identify signaling pathways at the angiogenic cytokine level via proteome profiler and enzyme-linked immunosorbent assays. Downstream miR targets were identified via bioinformatic target prediction and luciferase reporter gene assays. In vitro findings were translated to a mouse model of carotid injury in an endothelial GATA2 knockout background. Nanoparticle-mediated delivery of proangiogenic miR-126 was tested in the reendothelialization model. RESULTS: GATA2 gain- and loss-of-function experiments in human umbilical vein endothelial cells identified a key role of GATA2 as master regulator of multiple endothelial functions via miRNA-dependent mechanisms. Global miRNAnome-screening identified several GATA2-regulated miRNAs including miR-126 and miR-221. Specifically, proangiogenic miR-126 was regulated by GATA2 transcriptionally and targeted antiangiogenic SPRED1 and FOXO3a contributing to GATA2-mediated formation of normal vascular structures, whereas GATA2 deficiency led to vascular abnormalities. In contrast to GATA2 deficiency, supplementation with miR-126 normalized vascular function and expression profiles of cytokines contributing to proangiogenic paracrine effects. GATA2 silencing resulted in endothelial DNA hypomethylation leading to induced expression of antiangiogenic miR-221 by GATA2-dependent demethylation of a putative CpG island in the miR-221 promoter. Mechanistically, a reverted GATA2 phenotype by endogenous suppression of miR-221 was mediated through direct proangiogenic miR-221 target genes ICAM1 and ETS1. In a mouse model of carotid injury, GATA2 was reduced, and systemic supplementation of miR-126-coupled nanoparticles enhanced miR-126 availability in the carotid artery and improved reendothelialization of injured carotid arteries in vivo. CONCLUSIONS: GATA2-mediated regulation of miR-126 and miR-221 has an important impact on endothelial biology. Hence, modulation of GATA2 and its targets miR-126 and miR-221 is a promising therapeutic strategy for treatment of many vascular diseases.
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Enfermedades de las Arterias Carótidas/terapia , Factor de Transcripción GATA2/metabolismo , MicroARNs/uso terapéutico , Regiones no Traducidas 3' , Proteínas Adaptadoras Transductoras de Señales , Animales , Antagomirs/metabolismo , Secuencia de Bases , Enfermedades de las Arterias Carótidas/patología , Modelos Animales de Enfermedad , Proteína Forkhead Box O3/antagonistas & inhibidores , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Factor de Transcripción GATA2/antagonistas & inhibidores , Factor de Transcripción GATA2/genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Molécula 1 de Adhesión Intercelular/química , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lentivirus/genética , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/antagonistas & inhibidores , MicroARNs/metabolismo , Nanopartículas/química , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Alineación de SecuenciaRESUMEN
AIMS: Osteopontin (OPN) is a multifunctional cytokine critically involved in cardiac fibrosis. However, the underlying mechanisms are unresolved. Non-coding RNAs are powerful regulators of gene expression and thus might mediate this process. METHODS AND RESULTS: OPN and miR-21 were significantly increased in cardiac biopsies of patients with myocardial fibrosis. Ang II infusion via osmotic minipumps led to specific miRNA regulations with miR-21 being strongly induced in wild-type (WT) but not OPN knockout (KO) mice. This was associated with enhanced cardiac collagen content, myofibroblast activation, ERK-MAP kinase as well as AKT signalling pathway activation and a reduced expression of Phosphatase and Tensin Homologue (PTEN) as well as SMAD7 in WT but not OPN KO mice. In contrast, cardiotropic AAV9-mediated overexpression of OPN in vivo further enhanced cardiac fibrosis. In vitro, Ang II induced expression of miR-21 in WT cardiac fibroblasts, while miR-21 levels were unchanged in OPN KO fibroblasts. As pri-miR-21 was also increased by Ang II, we studied potential involved upstream regulators; Electrophoretic Mobility Shift and Chromatin Immunoprecipitation analyses confirmed activation of the miR-21 upstream-transcription factor AP-1 by Ang II. Recombinant OPN directly activated miR-21, enhanced fibrosis, and activated the phosphoinositide 3-kinase pathway. Locked nucleic acid-mediated miR-21 silencing ameliorated cardiac fibrosis development in vivo. CONCLUSION: In cardiac fibrosis related to Ang II, miR-21 is transcriptionally activated and targets PTEN/SMAD7 resulting in increased fibroblast survival. OPN KO animals are protected from miR-21 increase and fibrosis development due to impaired AP-1 activation and fibroblast activation.
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Angiotensina II/fisiología , MicroARNs/genética , Miocardio/patología , Osteopontina/fisiología , Adenoviridae , Anciano , Animales , Supervivencia Celular , Células Cultivadas , Colágeno/metabolismo , Femenino , Fibrosis/genética , Silenciador del Gen , Vectores Genéticos/administración & dosificación , Humanos , Técnicas In Vitro , Masculino , Ratones Noqueados , MicroARNs/metabolismo , Miofibroblastos/fisiología , Osteopontina/farmacología , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Recombinantes/farmacología , Factores de TranscripciónRESUMEN
BACKGROUND: Safety is an important consideration for the clinical application of dendritic cells (DC) loaded with autologous tumor lysate (TL). Thus, avitalization of TL from living autologous tumor tissue has to be guaranteed. METHODS: Composition of TL was investigated by static image analysis (SIA) with the Morphologi G3 device, which simultaneously measures size and shape of up to 100,000 particles within one sample run. This approach was compared with sample characterization by high-resolution automated cell counting, trypan blue staining, and ATP quantification. RESULTS: Using SIA, we only detected fragmented, non-cellular structures in completely avitalized TL, indicating complete destruction of living cells. Analysis of particle size distribution by SIA as well as CASY cell counter showed that 95% of particles had a diameter of <10 µm as a sign of cell fragmentation. Complete avitalization of TL was confirmed with trypan blue staining and ATP analysis. CONCLUSION: Regarding generation of DC vaccines, the proof of avitality of TL from living tumor tissue can clearly be achieved by SIA alone or in combination with standard assays. Our data show that SIA is a highly precise method for TL characterization. The SIA device complies with FDA regulation and, therefore, might be suitable for characterization of cellular therapy medicinal products.
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The human polycistronic miRNA cluster miR-17-92 is frequently overexpressed in hematopoietic malignancies and cancers. Its transcription is in part controlled by an E2F-regulated host gene promoter. An intronic A/T-rich region directly upstream of the miRNA coding region also contributes to cluster expression. Our deletion analysis of the A/T-rich region revealed a strong dependence on c-Myc binding to the functional E3 site. Yet, constructs lacking the 5'-proximal ~1.3 kb or 3'-distal ~0.1 kb of the 1.5 kb A/T-rich region still retained residual specific promoter activity, suggesting multiple transcription start sites (TSS) in this region. Furthermore, the protooncogenic kinase, Pim-1, its phosphorylation target HP1γ and c-Myc colocalize to the E3 region, as inferred from chromatin immunoprecipitation. Analysis of pri-miR-17-92 expression levels in K562 and HeLa cells revealed that silencing of E2F3, c-Myc or Pim-1 negatively affects cluster expression, with a synergistic effect caused by c-Myc/Pim-1 double knockdown in HeLa cells. Thus, we show, for the first time, that the protooncogene Pim-1 is part of the network that regulates transcription of the human miR-17-92 cluster.
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Regulación de la Expresión Génica , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Transcripción Genética , Secuencia Rica en At/genética , Sitios de Unión/genética , Proteínas Cromosómicas no Histona/metabolismo , Sitios Genéticos , Genoma Humano , Células HeLa , Humanos , Intrones/genética , Células K562 , MicroARNs/genética , Unión Proteica/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Largo no CodificanteRESUMEN
MicroRNAs (miRNAs) are small non-coding RNAs that control expression of complementary target mRNAs. A growing number of miRNAs has been implicated in the pathogenesis of cardiac diseases, mostly based not on functional data, but on the observation that they are dysregulated in diseased myocardium. Consequently, our knowledge regarding a potential cardiac role of the majority of miRNAs is limited. Here, we report the development of an assay format that allows the simultaneous analysis of several hundred molecules with regard to their phenotypic effect on primary rat cardiomyocytes. Using automated microscopy and an edge detection algorithm, this assay achieved high reproducibility and a robust assessment of cardiomyocyte size as a key parameter. Screening a library of synthetic miRNAs revealed several miRNAs previously not recognized as pro- or anti-hypertrophic. Out of these, we selected nine miRNAs and confirmed the pro-hypertrophic potential of miR-22, miR-30c, miR-30d, miR-212, miR-365 and the anti-hypertrophic potential of miR-27a, miR-27b and miR-133a. Quantitative analysis of the expression level of pro-hypertrophic miRNAs in primary cardiomyocytes indicated a rather low level of correlation of the phenotypic effects of individual miRNAs and their expression level. This assay allows the automated determination of cell size in primary cardiomyocytes and permitted the identification of a set of miRNAs capable of regulating cardiomyocyte hypertrophy. Elucidating their mechanism of action should provide insight into mechanisms underlying the cardiomyocyte hypertrophic response. This article is part of a Special Issue entitled 'Possible Editorial'.
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Ensayos Analíticos de Alto Rendimiento , MicroARNs/genética , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Fenotipo , Animales , Aumento de la Célula , Separación Celular , Células Cultivadas , Perfilación de la Expresión Génica , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , TransfecciónRESUMEN
BACKGROUND: Myocardial infarction leads to cardiac remodeling and development of heart failure. Insufficient myocardial capillary density after myocardial infarction has been identified as a critical event in this process, although the underlying mechanisms of cardiac angiogenesis are mechanistically not well understood. METHODS AND RESULTS: Here, we show that the small noncoding RNA microRNA-24 (miR-24) is enriched in cardiac endothelial cells and considerably upregulated after cardiac ischemia. MiR-24 induces endothelial cell apoptosis, abolishes endothelial capillary network formation on Matrigel, and inhibits cell sprouting from endothelial spheroids. These effects are mediated through targeting of the endothelium-enriched transcription factor GATA2 and the p21-activated kinase PAK4, which were identified by bioinformatic predictions and validated by luciferase gene reporter assays. Respective downstream signaling cascades involving phosphorylated BAD (Bcl-XL/Bcl-2-associated death promoter) and Sirtuin1 were identified by transcriptome, protein arrays, and chromatin immunoprecipitation analyses. Overexpression of miR-24 or silencing of its targets significantly impaired angiogenesis in zebrafish embryos. Blocking of endothelial miR-24 limited myocardial infarct size of mice via prevention of endothelial apoptosis and enhancement of vascularity, which led to preserved cardiac function and survival. CONCLUSIONS: Our findings indicate that miR-24 acts as a critical regulator of endothelial cell apoptosis and angiogenesis and is suitable for therapeutic intervention in the setting of ischemic heart disease.
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Células Endoteliales/metabolismo , MicroARNs/fisiología , Infarto del Miocardio/fisiopatología , Animales , Apoptosis/efectos de los fármacos , Arteriolas/patología , Capilares/patología , Hipoxia de la Célula , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Colágeno , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos , Células Endoteliales/patología , Factor de Transcripción GATA2/biosíntesis , Factor de Transcripción GATA2/genética , Perfilación de la Expresión Génica , Insuficiencia Cardíaca/etiología , Hemo-Oxigenasa 1/biosíntesis , Hemo-Oxigenasa 1/genética , Laminina , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Infarto del Miocardio/complicaciones , Infarto del Miocardio/genética , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , Oligorribonucleótidos/farmacología , Proteoglicanos , Interferencia de ARN , ARN Interferente Pequeño/farmacología , ARN Interferente Pequeño/uso terapéutico , Esferoides Celulares , Remodelación Ventricular , Pez Cebra/embriología , Proteínas de Pez Cebra/biosíntesis , Proteínas de Pez Cebra/genética , Quinasas p21 Activadas/biosíntesis , Quinasas p21 Activadas/genéticaRESUMEN
BACKGROUND: Long noncoding ribonucleic acids (lncRNAs) are a subclass of regulatory noncoding ribonucleic acids for which expression and function in human endothelial cells and angiogenic processes is not well studied. OBJECTIVES: The authors discovered hypoxia-sensitive human lncRNAs via next-generation ribonucleic acid sequencing and microarray approaches. To address their functional importance in angiogenic processes, several endothelial lncRNAs were characterized for their angiogenic characteristics in vitro and ex vivo. METHODS: Ribonucleic acid sequencing and microarray-derived data showed specific endothelial lncRNA expression changes after hypoxia. Validation experiments confirmed strong hypoxia-dependent activation of 2 intergenic lncRNAs: LINC00323 and MIR503HG. RESULTS: Silencing of these lncRNA transcripts led to angiogenic defects, including repression of growth factor signaling and/or the key endothelial transcription factor GATA2. Endothelial loss of these hypoxia-driven lncRNAs impaired cell-cycle control and inhibited capillary formation. The potential clinical importance of these endothelial lncRNAs to vascular structural integrity was demonstrated in an ex vivo model of human induced pluripotent stem cell-based engineered heart tissue. CONCLUSIONS: The authors report an expression atlas of human hypoxia-sensitive lncRNAs and identified 2 lncRNAs with important functions to sustain endothelial cell biology. LncRNAs hold great promise to serve as important future therapeutic targets of cardiovascular disease.
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Hipoxia de la Célula/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Neovascularización Patológica , Interferencia de ARN , ARN Largo no Codificante/genética , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Análisis de Secuencia de ARN , Transducción de Señal , Ingeniería de Tejidos/métodosRESUMEN
UNLABELLED: Smooth muscle cells (SMCs) are key components within the vasculature. Dependent on the stimulus, SMC can either be in a proliferative (synthetic) or differentiated state. Alterations of SMC phenotype also appear in several disease settings, further contributing to disease progression. AIMS: Here, we asked whether microRNAs (miRNAs, miRs), which are strong posttranscriptional regulators of gene expression, could alter SMC proliferation. Results and Innovation: Employing a robotic-assisted high-throughput screening method using miRNA libraries, we identified hypoxia-regulated miR-24 as a master regulator of SMC proliferation. Proteome profiling showed a strong miR-24-dependent impact on cellular stress-associated factors, overall resulting in reduced stress resistance. In vitro, synthetic miR-24 overexpression had detrimental effects on SMC functional capacity inducing apoptosis, migration defects, enhanced autophagy, and loss of contractile marker genes. Impaired SMC function was mediated in part by the herein identified direct target gene heme oxygenase 1. Ex vivo, miR-24 was shown to inhibit the development of vasculature in a model of engineered heart tissue. CONCLUSION: Collectively, we report the identification of the hypoxamir-24 as an inhibitor of SMC proliferation, contributing to loss of vascularization.
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MicroARNs/fisiología , Miocitos del Músculo Liso/fisiología , Animales , Aorta/citología , Secuencia de Bases , Sitios de Unión , Proliferación Celular , Células Cultivadas , Represión Enzimática , Biblioteca de Genes , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Humanos , Ratones Transgénicos , Neovascularización Fisiológica , Interferencia de ARN , Técnicas de Cultivo de TejidosRESUMEN
Asthma is a chronic condition with unknown pathogenesis, and recent evidence suggests that enhanced airway epithelial chloride (Cl-) secretion plays a role in the disease. However, the molecular mechanism underlying Cl- secretion and its relevance in asthma pathophysiology remain unknown. To determine the role of the solute carrier family 26, member 9 (SLC26A9) Cl- channel in asthma, we induced Th2-mediated inflammation via IL-13 treatment in wild-type and Slc26a9-deficient mice and compared the effects on airway ion transport, morphology, and mucus content. We found that IL-13 treatment increased Cl- secretion in the airways of wild-type but not Slc26a9-deficient mice. While IL-13-induced mucus overproduction was similar in both strains, treated Slc26a9-deficient mice exhibited airway mucus obstruction, which did not occur in wild-type controls. In a study involving healthy children and asthmatics, a polymorphism in the 3' UTR of SLC26A9 that reduced protein expression in vitro was associated with asthma. Our data demonstrate that the SLC26A9 Cl- channel is activated in airway inflammation and suggest that SLC26A9-mediated Cl- secretion is essential for preventing airway obstruction in allergic airway disease. These results indicate that SLC26A9 may serve as a therapeutic target for airway diseases associated with mucus plugging.
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Obstrucción de las Vías Aéreas/prevención & control , Antiportadores/fisiología , Asma/genética , Bronquitis/fisiopatología , Cloruros/metabolismo , Transporte Iónico/fisiología , Moco/metabolismo , Traqueítis/fisiopatología , Regiones no Traducidas 3' , Obstrucción de las Vías Aéreas/etiología , Obstrucción de las Vías Aéreas/fisiopatología , Animales , Antiportadores/deficiencia , Antiportadores/genética , Asma/fisiopatología , Bronquitis/inducido químicamente , Bronquitis/genética , Bronquitis/inmunología , Calcio/farmacología , Niño , AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/deficiencia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Predisposición Genética a la Enfermedad , Humanos , Interleucina-13/toxicidad , Pulmón/patología , Ratones , Ratones Noqueados , Transportadores de Sulfato , Células Th2/inmunología , Traqueítis/inducido químicamente , Traqueítis/genética , Traqueítis/inmunologíaRESUMEN
MicroRNAs (miRNAs) are small non-coding RNAs, that control diverse cellular functions by either promoting degradation or inhibition of target messenger RNA translation. An aberrant expression profile of miRNAs has been linked to human diseases, including cardiovascular dysfunction. This review summarizes the latest insights in the identification of vascular-specific miRNAs and their targets, as well as their roles and mechanisms in the vasculature. Furthermore, we discuss how manipulation of these miRNAs could represent a novel therapeutic approach in the treatment of vascular dysfunction.