Embryonic stem cell differentiation into smooth muscle cells is mediated by Nox4-produced H2O2

NADPH oxidase (Nox4) produces reactive oxygen species (ROS) that are important for vascular smooth muscle cell (SMC) behavior, but the potential impact of Nox4 in stem cell differentiation is unknown. When mouse embryonic stem (ES) cells were plated on collagen IV-coated dishes/flasks, a panel of SMC-specific genes was significantly and consistently upregulated. Nox4 expression was markedly correlated with such a gene induction as confirmed by real-time PCR, immunofluorescence, and Western blot analysis. Overexpression of Nox4 specifically resulted in increased SMC marker production, whereas knockdown of Nox4 induced a decrease. Furthermore, SMC-specific transcription factors, including serum response factor (SRF) and myocardin were activated by Nox4 gene expression. Moreover, Nox4 was demonstrated to drive SMC differentiation through generation of H(2)O(2). Confocal microscopy analysis indicates that SRF was translocated into the nucleus during SMC differentiation in which SRF was phosphorylated. Additionally, autosecreted transforming growth factor (TGF)-beta(1) activated Nox4 and promoted SMC differentiation. Interestingly, cell lines generated from stem cells by Nox4 transfection and G418 selection displayed a characteristic of mature SMCs, including expression of SMC markers and cells with contractile function. Thus we demonstrate for the first time that Nox4 is crucial for SMC differentiation from ES cells, and enforced Nox4 expression can maintain differentiation status and functional features of stem cell-derived SMCs, highlighting its impact on vessel formation in vivo and vascular tissue engineering in the future.

HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells

Reendothelialization involves endothelial progenitor cell (EPC) homing, proliferation, and differentiation, which may be influenced by fluid shear stress and local flow pattern. This study aims to elucidate the role of laminar flow on embryonic stem (ES) cell differentiation and the underlying mechanism. We demonstrated that laminar flow enhanced ES cell-derived progenitor cell proliferation and differentiation into endothelial cells (ECs). Laminar flow stabilized and activated histone deacetylase 3 (HDAC3) through the Flk-1-PI3K-Akt pathway, which in turn deacetylated p53, leading to p21 activation. A similar signal pathway was detected in vascular endothelial growth factor-induced EC differentiation. HDAC3 and p21 were detected in blood vessels during embryogenesis. Local transfer of ES cell-derived EPC incorporated into injured femoral artery and reduced neointima formation in a mouse model. These data suggest that shear stress is a key regulator for stem cell differentiation into EC, especially in EPC differentiation, which can be used for vascular repair, and that the Flk-1-PI3K-Akt-HDAC3-p53-p21 pathway is crucial in such a process.

The biosynthesis and biological role of lipopolysaccharide O-antigens of pathogenic Yersiniae

Lipopolysaccharide (LPS) is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria. The LPS molecule is composed of two biosynthetic entities: the lipid A--core and the O-polysaccharide (O-antigen). Most biological effects of LPS are due to the lipid A part, however, there is an increasing body of evidence indicating that O-antigen (O-ag) plays an important role in effective colonization of host tissues, resistance to complement-mediated killing and in the resistance to cationic antimicrobial peptides that are key elements of the innate immune system. In this review, we will discuss: (i) the work done on the genetics and biosynthesis of the O-ags in the genus Yersinia; (ii) the role of O-ag in virulence of these bacteria; (iii) the work done on regulation of the O-ag gene cluster expression and; (iv) the impact that the O-ag expression has on other bacterial surface and membrane components.

Molecular similarity between myelodysplastic form of chronic myelomonocytic leukemia and refractory anemia with ring sideroblasts

Chronic myelomonocytic leukemia is similar to but a separate entity from both myeloproliferative neoplasms and myelodysplastic syndromes, and shows either myeloproliferative or myelodysplastic features. We ask whether this distinction may have a molecular basis. We established the gene expression profiles of 39 samples of chronic myelomonocytic leukemia (including 12 CD34-positive) and 32 CD34-positive samples of myelodysplastic syndromes by using Affymetrix microarrays, and studied the status of 18 genes by Sanger sequencing and array-comparative genomic hybridization in 53 samples. Analysis of 12 mRNAS from chronic myelomonocytic leukemia established a gene expression signature of 122 probe sets differentially expressed between proliferative and dysplastic cases of chronic myelomonocytic leukemia. As compared to proliferative cases, dysplastic cases over-expressed genes involved in red blood cell biology. When applied to 32 myelodysplastic syndromes, this gene expression signature was able to discriminate refractory anemias with ring sideroblasts from refractory anemias with excess of blasts. By comparing mRNAS from these two forms of myelodysplastic syndromes we derived a second gene expression signature. This signature separated the myelodysplastic and myeloproliferative forms of chronic myelomonocytic leukemias. These results were validated using two independent gene expression data sets. We found that myelodysplastic chronic myelomonocytic leukemias are characterized by mutations in transcription/epigenetic regulators (ASXL1, RUNX1, TET2) and splicing genes (SRSF2) and the absence of mutations in signaling genes. Myelodysplastic chronic myelomonocytic leukemias and refractory anemias with ring sideroblasts share a common expression program suggesting they are part of a continuum, which is not totally explained by their similar but not, however, identical mutation spectrum. © 2013 Ferrata Storti Foundation.

Chronic IL9 and IL-13 Exposure Leads to an Altered Differentiation of Ciliated Cells in a Well-Differentiated Paediatric Bronchial Epithelial Cell Model

Asthma is a chronic inflammatory disease characterised by airways remodelling. In mouse models IL-9 and IL-13 have been implicated in airways remodelling including mucus hypersecretion and goblet cell hyperplasia. Their role, especially that of IL-9, has been much less studied in authentic human ex vivo models of the bronchial epithelium from normal and asthmatic children. We assessed the effects of IL-9, IL-13 and an IL-9/IL-13 combination, during differentiation of bronchial epithelial cells from normal (n?=?6) and asthmatic (n?=?8) children. Cultures were analysed for morphological markers and factors associated with altered differentiation (MUC5AC, SPDEF and MMP-7). IL-9, IL-9/IL-13 combination and IL-13 stimulated bronchial epithelial cells from normal children had fewer ciliated cells [14.8% (SD 8.9), p?=?0.048, 12.4 (SD 6.1), p?=?0.016 and 7.3% (SD 6.6), p?=?0.031] respectively compared with unstimulated [(21.4% (SD 9.6)]. IL-9 stimulation had no effect on goblet cell number in either group whereas IL-9/IL-13 combination and IL-13 significantly increased goblet cell number [24.8% (SD 8.8), p?=?0.02), 32.9% (SD 8.6), p?=?0.007] compared with unstimulated normal bronchial cells [(18.6% (SD 6.2)]. All stimulations increased MUC5AC mRNA in bronchial epithelial cells from normal children and increased MUC5AC mucin secretion. MMP-7 localisation was dysregulated in normal bronchial epithelium stimulated with Th2 cytokines which resembled the unstimulated bronchial epithelium of asthmatic children. All stimulations resulted in a significant reduction in transepithelial electrical resistance values over time suggesting a role in altered tight junction formation. We conclude that IL-9 does not increase goblet cell numbers in bronchial epithelial cell cultures from normal or asthmatic children. IL-9 and IL-13 alone and in combination, reduce ciliated cell numbers and transepithelial electrical resistance during differentiation of normal epithelium, which clinically could inhibit mucociliary clearance and drive an altered repair mechanism. This suggests an alternative role for IL-9 in airways remodelling and reaffirms IL-9 as a potential therapeutic target.© 2013 Parker et al.

BRCA1 is a key regulator of breast differentiation through activation of Notch signalling with implications for anti-endocrine treatment of breast cancers

Here, we show for the first time, that the familial breast/ovarian cancer susceptibility gene BRCA1 activates the Notch pathway in breast cells by transcriptional upregulation of Notch ligands and receptors in both normal and cancer cells. We demonstrate through chromatin immunoprecipitation assays that BRCA1 is localized to a conserved intronic enhancer region within the Notch ligand Jagged-1 (JAG1) gene, an event requiring ΔNp63. We propose that this BRCA1/ΔNp63-mediated induction of JAG1 may be important the regulation of breast stem/precursor cells, as knockdown of all three proteins resulted in increased tumoursphere growth and increased activity of stem cell markers such as Aldehyde Dehydrogenase 1 (ALDH1). Knockdown of Notch1 and JAG1 phenocopied BRCA1 knockdown resulting in the loss of Estrogen Receptor-α (ER-α) expression and other luminal markers. A Notch mimetic peptide could activate an ER-α promoter reporter in a BRCA1-dependent manner, whereas Notch inhibition using a γ-secretase inhibitor reversed this process. We demonstrate that inhibition of Notch signalling resulted in decreased sensitivity to the anti-estrogen drug Tamoxifen but increased expression of markers associated with basal-like breast cancer. Together, these findings suggest that BRCA1 transcriptional upregulation of Notch signalling is a key event in the normal differentiation process in breast tissue.

Sirolimus Stimulates Vascular Stem/Progenitor Cell Migration and Differentiation Into Smooth Muscle Cells via Epidermal Growth Factor Receptor/Extracellular Signal-Regulated Kinase/β-Catenin Signaling Pathway

Sirolimus-eluting stent therapy has achieved considerable success in overcoming coronary artery restenosis. However, there remain a large number of patients presenting with restenosis after the treatment, and the source of its persistence remains unclarified. Although recent evidence supports the contribution of vascular stem/progenitor cells in restenosis formation, their functional and molecular responses to sirolimus are largely unknown.

Constitutive expression of human keratin 14 gene in mouse lung induces premalignant lesions and squamous differentiation

Squamous cell carcinoma accounts for 20% of all human lung cancers and is strongly linked to cigarette smoking. It develops through premalignant changes that are characterized by high levels of keratin 14 (K14) expression in the airway epithelium and evolve through basal cell hyperplasia, squamous metaplasia and dysplasia to carcinoma in situ and invasive carcinoma. In order to explore the impact of K14 in the pulmonary epithelium that normally lacks both squamous differentiation and K14 expression, human keratin 14 gene hK14 was constitutively expressed in mouse airway progenitor cells using a mouse Clara cell specific 10 kDa protein (CC10) promoter. While the lungs of CC10-hK14 transgenic mice developed normally, we detected increased expression of K14 and the molecular markers of squamous differentiation program such as involucrin, loricrin, small proline-rich protein 1A, transglutaminase 1 and cholesterol sulfotransferase 2B1. In contrast, wild-type lungs were negative. Aging CC10-hK14 mice revealed multifocal airway cell hyperplasia, occasional squamous metaplasia and their lung tumors displayed evidence for multidirectional differentiation. We conclude that constitutive expression of hK14 initiates squamous differentiation program in the mouse lung, but fails to promote squamous maturation. Our study provides a novel model for assessing the mechanisms of premalignant lesions in vivo by modifying differentiation and proliferation of airway progenitor cells. © The Author 2008. Published by Oxford University Press. All rights reserved.

Use of immunosorbent-purified antigens of Fasciola hepatica in enzyme immunoassays

A monoclonal antibody specific for the T1 tegumental antigen of Fasciola hepatica was used as a solid-phase immunosorbent for the purification of T1 antigen from homogenised mature F hepatica. Material fractionated by this technique was successfully used in enzyme-linked immunoassays to detect antibodies to F hepatica in sera from sheep and cattle. Species differences in response to infection by F hepatica were demonstrated.

Fasciola hepatica:development of monoclonal antibodies against somatic antigens and their characterization by ultrastructural localization of antibody binding

A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized.

Endothelial lineage differentiation from induced pluripotent stem cells is regulated by microRNA-21 and transforming growth factor beta2 (TGF-β2) pathways

Finding a suitable cell source for endothelial cells (ECs) for cardiovascular regeneration is a challenging issue for regenerative medicine. In the paper we describe a novel mechanism regulating induced pluripotent stem cells (iPSC) differentiation into ECs, with a particular focus on miRNAs and their targets. We first established a protocol using collagen IV and VEGF to drive the functional differentiation of iPSCs into ECs and compared the miRNA signature of differentiated and undifferentiated cells. Among the miRNAs overrepresented in differentiated cells, we focused on microRNA-21 (miR-21) and studied its role in iPSC differentiation. Overexpression of miR-21 in pre-differentiated iPSCs induced EC marker upregulation and in vitro and in vivo capillary formation; accordingly, inhibition of miR-21 produced the opposite effects. Importantly, miR-21 overexpression increased TGF-β2 mRNA and secreted protein level, consistent with the strong upregulation of TGF-β2 during iPSC differentiation. Indeed, treatment of iPSCs with TGFβ-2 induced EC marker expression and in vitro tube formation. Inhibition of SMAD3, a downstream effector of TGFβ-2, strongly decreased VE-cadherin expression. Furthermore, TGFβ-2 neutralization and knockdown inhibited miR-21-induced EC marker expression. Finally, we confirmed the PTEN/Akt pathway as a direct target of miR-21 and we showed that PTEN knockdown is required for miR-21 mediated endothelial differentiation. In conclusion, we elucidated a novel signaling pathway that promotes the differentiation of iPSC into functional ECs suitable for regenerative medicine applications.