title: NOX2, NOX4, and mitochondrial-derived reactive oxygen species contribute to angiopoietin-1 signaling and angiogenic responses in endothelial cells.
abstract: Angiopoietin-1 (Ang-1) is a ligand of Tie-2 receptors that promotes survival, migration, and differentiation of endothelial cells. Several studies have linked reactive oxygen species (ROS) to Ang-1 signaling and distinct angiogenic responses, but the molecular sources of these ROS have never been clearly identified. In this study, we have identified source-specific contributions of ROS to Ang-1/Tie 2 signaling and angiogenic responses in human umbilical vein endothelial cells (HUVECs), specifically the differential contributions of mitochondrial ROS (mtROS) and ROS from two isoforms of NADPH oxidase (NOX2, NOX4). We demonstrate that: 1) Ang-1 induces significant increases in mtROS production under normal conditions but does not when cells are pre-incubated with mitochondrial antioxidants; 2) Ang-1 induces rapid Tie-2-dependent increases in cytosolic ROS production but does not when NOX2 and NOX4 are knocked down; 3) Ang-1 induces simultaneous increases in phosphorylation of AKT, ERK1/2, p38, and SAPK/JNK proteins within a few minutes of exposure, but this response is strongly and selectively attenuated when NOX2 and NOX4 are knocked down or cells are pre-treated with mitochondrial antioxidants; 4) Ang-1 exerts a strong effect on HUVEC survival in serum-deprived medium and enhances cell migration and capillary tube formation, but the survival response is inhibited by NOX2 knockdown and the migration and tube formation responses are entirely absent with NOX4 knockdown or pre-treatment with mitochondrial antioxidants. We conclude that Ang-1 triggers NOX2, NOX4, and the mitochondria to release ROS and that ROS derived from these sources play distinct roles in the regulation of the Ang-1/Tie 2 signaling pathway and pro-angiogenic responses.
authors.affiliation: Meakins-Christie Laboratories and Translational Research in Respiratory Diseases Program, Research Institute of the McGill University Health Centre, Department of Critical Care Medicine, McGill University Health Centre, Montréal, Québec, Canada.
authors.forename: Sharon
authors.initials: S
authors.lastname: Harel
authors.affiliation: Meakins-Christie Laboratories and Translational Research in Respiratory Diseases Program, Research Institute of the McGill University Health Centre, Department of Critical Care Medicine, McGill University Health Centre, Montréal, Québec, Canada.
authors.forename: Dominique
authors.initials: D
authors.lastname: Mayaki
authors.affiliation: Meakins-Christie Laboratories and Translational Research in Respiratory Diseases Program, Research Institute of the McGill University Health Centre, Department of Critical Care Medicine, McGill University Health Centre, Montréal, Québec, Canada.
authors.forename: Veronica
authors.initials: V
authors.lastname: Sanchez
authors.affiliation: Meakins-Christie Laboratories and Translational Research in Respiratory Diseases Program, Research Institute of the McGill University Health Centre, Department of Critical Care Medicine, McGill University Health Centre, Montréal, Québec, Canada. Electronic address: sabah.hussain@muhc.mcgill.ca.
authors.forename: Sabah N A
authors.initials: SNA
authors.lastname: Hussain
mesh_terms: D043925:Angiogenesis Inducing Agents / Q000494:pharmacology*; D042683:Angiopoietin-1 / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism*; D042787:Receptor, TIE-2 / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D014162:Transfection
chemical_list: C467630:ANGPT1 protein, human; D043925:Angiogenesis Inducing Agents; D042683:Angiopoietin-1; D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D042787:Receptor, TIE-2; C505407:CASP3 protein, human; D053148:Caspase 3
keywords: Angiogenesis; Angiopoietin-1; Apoptosis; Endothelial cells; Mitochondrial reactive oxygen species; Mitogen-activated protein kinases; NADPH oxidase; Reactive oxygen species