Harel Sharon, Mayaki Dominique, Sanchez Veronica, Hussain Sabah N A
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.
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.
Vascul Pharmacol. 2017 May;92:22-32. doi: 10.1016/j.vph.2017.03.002. Epub 2017 Mar 27.
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.
血管生成素-1（Ang-1）是Tie-2受体的配体，可促进内皮细胞的存活、迁移和分化。多项研究已将活性氧（ROS）与Ang-1信号传导及不同的血管生成反应联系起来，但这些ROS的分子来源从未被明确鉴定。在本研究中，我们确定了ROS在人脐静脉内皮细胞（HUVECs）中对Ang-1/Tie 2信号传导和血管生成反应的来源特异性贡献，特别是线粒体ROS（mtROS）以及NADPH氧化酶两种同工型（NOX2、NOX4）产生的ROS的不同贡献。我们证明：1）在正常条件下，Ang-1可诱导mtROS产生显著增加，但当细胞预先用线粒体抗氧化剂孵育时则不会；2）Ang-1可诱导依赖Tie-2的胞质ROS产生迅速增加，但当NOX2和NOX4被敲低时则不会；3）Ang-1在暴露后几分钟内可诱导AKT、ERK1/2、p38和SAPK/JNK蛋白的磷酸化同时增加，但当NOX2和NOX4被敲低或细胞预先用线粒体抗氧化剂处理时，这种反应会强烈且选择性地减弱；4）Ang-1对血清饥饿培养基中的HUVEC存活有强烈影响，并增强细胞迁移和毛细血管管形成，但NOX2敲低会抑制存活反应，而NOX4敲低或用线粒体抗氧化剂预处理则完全消除迁移和管形成反应。我们得出结论，Ang-1触发NOX2、NOX4和线粒体释放ROS，并且源自这些来源的ROS在Ang-1/Tie 2信号通路的调节和促血管生成反应中发挥不同作用。
