title: Involvement of phosphoinositide 3-kinase class IA (PI3K 110α) and NADPH oxidase 1 (NOX1) in regulation of vascular differentiation induced by vascular endothelial growth factor (VEGF) in mouse embryonic stem cells.
abstract: The impact of reactive oxygen species and phosphoinositide 3-kinase (PI3K) in differentiating embryonic stem (ES) cells is largely unknown. Here, we show that the silencing of the PI3K catalytic subunit p110α and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) by short hairpin RNA or pharmacological inhibition of NOX and ras-related C3 botulinum toxin substrate 1 (Rac1) abolishes superoxide production by vascular endothelial growth factor (VEGF) in mouse ES cells and in ES-cell-derived fetal liver kinase-1(+) (Flk-1(+)) vascular progenitor cells, whereas the mitochondrial complex I inhibitor rotenone does not have an effect. Silencing p110α or inhibiting Rac1 arrests vasculogenesis at initial stages in embryoid bodies, even under VEGF treatment, as indicated by platelet endothelial cell adhesion molecule-1 (PECAM-1)-positive areas and branching points. In the absence of p110α, tube-like structure formation on matrigel and cell migration of Flk-1(+) cells in scratch migration assays are totally impaired. Silencing NOX1 causes a reduction in PECAM-1-positive areas, branching points, cell migration and tube length upon VEGF treatment, despite the expression of vascular differentiation markers. Interestingly, silencing p110α but not NOX1 inhibits the activation of Rac1, Ras homologue gene family member A (RhoA) and Akt leading to the abrogation of VEGF-induced lamellipodia structure formation. Thus, our data demonstrate that the PI3K p110α-Akt/Rac1 and NOX1 signalling pathways play a pivotal role in VEGF-induced vascular differentiation and cell migration. Rac1, RhoA and Akt phosphorylation occur downstream of PI3K and upstream of NOX1 underscoring a role of PI3K p110α in the regulation of cell polarity and migration.
authors.affiliation: University Heart Center, Clinic of Internal Medicine I, Department of Cardiology, Friedrich Schiller University Jena, Erlanger Allee 101, 07743, Jena, Germany. Mohamed.el_Saied@med.uni-jena.de.|Department of Zoology, Faculty of Science, Tanta University, Tanta, 31527, Egypt. Mohamed.el_Saied@med.uni-jena.de.
authors.forename: Mohamed M
authors.initials: MM
authors.lastname: Bekhite
authors.affiliation: University Heart Center, Clinic of Internal Medicine I, Department of Cardiology, Friedrich Schiller University Jena, Erlanger Allee 101, 07743, Jena, Germany.
authors.forename: Veronika
authors.initials: V
authors.lastname: Müller
authors.affiliation: University Heart Center, Clinic of Internal Medicine I, Department of Cardiology, Friedrich Schiller University Jena, Erlanger Allee 101, 07743, Jena, Germany.
authors.forename: Sebastian H
authors.initials: SH
authors.lastname: Tröger
authors.affiliation: Institute of Molecular Cell Biology, Center for Molecular Biomedicine, Friedrich Schiller University Jena, Jena, Germany.
authors.forename: Jörg P
authors.initials: JP
authors.lastname: Müller
authors.affiliation: University Heart Center, Clinic of Internal Medicine I, Department of Cardiology, Friedrich Schiller University Jena, Erlanger Allee 101, 07743, Jena, Germany.
authors.forename: Hans-Reiner
authors.initials: HR
authors.lastname: Figulla
authors.affiliation: Department of Physiology, Faculty of Medicine, Justus Liebig University, Giessen, Germany.
authors.forename: Heinrich
authors.initials: H
authors.lastname: Sauer
authors.affiliation: University Heart Center, Clinic of Internal Medicine I, Department of Cardiology, Friedrich Schiller University Jena, Erlanger Allee 101, 07743, Jena, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Wartenberg
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D058534:Class I Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; C000614711:NOX1 protein, mouse; D058534:Class I Phosphatidylinositol 3-Kinases
keywords: Mouse embryonic stem cells; NADPH oxidase; Phosphoinositide 3-kinase; Rac1; Vascular differentiation