title: Nox4- and Nox2-dependent oxidant production is required for VEGF-induced SERCA cysteine-674 S-glutathiolation and endothelial cell migration.
abstract: Endothelial cell (EC) migration in response to vascular endothelial growth factor (VEGF) is a critical step in both physiological and pathological angiogenesis. Although VEGF signaling has been extensively studied, the mechanisms by which VEGF-dependent reactive oxygen species (ROS) production affects EC signaling are not well understood. The aim of this study was to elucidate the involvement of Nox2- and Nox4-dependent ROS in VEGF-mediated EC Ca(2+) regulation and migration. VEGF induced migration of human aortic ECs into a scratch wound over 6 h, which was inhibited by overexpression of either catalase or superoxide dismutase (SOD). EC stimulation by micromolar concentrations of H2O2 was inhibited by catalase, but also unexpectedly by SOD. Both VEGF and H2O2 increased S-glutathiolation of SERCA2b and increased Ca(2+) influx into EC, and these events could be blocked by overexpression of catalase or overexpression of SERCA2b in which the reactive cysteine-674 was mutated to a serine. In determining the source of VEGF-mediated ROS production, our studies show that specific knockdown of either Nox2 or Nox4 inhibited VEGF-induced S-glutathiolation of SERCA, Ca(2+) influx, and EC migration. Treatment with H2O2 induced S-glutathiolation of SERCA and EC Ca(2+) influx, overcoming the knockdown of Nox4, but not Nox2, and Amplex red measurements indicated that Nox4 is the source of H2O2. These results demonstrate that VEGF stimulates EC migration through increased S-glutathiolation of SERCA and Ca(2+) influx in a Nox4- and H2O2-dependent manner, requiring Nox2 downstream.
authors.affiliation: Vascular Biology Section Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Alicia M
authors.initials: AM
authors.lastname: Evangelista
authors.affiliation: Vascular Biology Section Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Melissa D
authors.initials: MD
authors.lastname: Thompson
authors.affiliation: Ion Channel and Calcium Signaling Unit, Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Victoria M
authors.initials: VM
authors.lastname: Bolotina
authors.affiliation: Vascular Biology Section Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Xiaoyong
authors.initials: X
authors.lastname: Tong
authors.affiliation: Vascular Biology Section Boston University School of Medicine, Boston, MA 02118, USA. Electronic address: racohen@bu.edu.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Cohen
mesh_terms: D020013:Calcium Signaling; D002374:Catalase / Q000378:metabolism; D002465:Cell Movement; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D042783:Endothelial Cells / Q000502:physiology*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000378:metabolism*; D010084:Oxidation-Reduction; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology*
chemical_list: D008562:Membrane Glycoproteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D013481:Superoxides; D006861:Hydrogen Peroxide; D002374:Catalase; D013482:Superoxide Dismutase; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; D005978:Glutathione; D003545:Cysteine
keywords: Calcium; Endothelial cells; Free radicals; Hydrogen peroxide; Nox2; Nox4; SERCA; VEGF