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Since emergence of the highly pathogenic avian influenza virus (HPAIV) H5 A/Goose/Guangdong/1/1996 (gs/GD) lineage in 1996, successors continue to circulate in waves around the world, leading to massive losses in [...]

Introduction The clinical syndrome of asthma has evolved as one of the most common chronic diseases in children and adults, but its pathogenesis remains incompletely understood. Th2-mediated inflammation triggered by [...]

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 endothelialimportant 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 realtime 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 miRNAnomescreening 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. [ABSTRACT FROM AUTHOR]

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. © 2015 S. Karger GmbH, Freiburg [ABSTRACT FROM AUTHOR]

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 HP1? 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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’. [Copyright &y& Elsevier]

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.