title: Nox4 is a major source of superoxide production in human brain pericytes.
abstract: BACKGROUND
  Pericytes are multifunctional cells surrounding capillaries and postcapillary venules. In brain microvasculature, pericytes play a pivotal role under physiological and pathological conditions by producing reactive oxygen species (ROS). The aims of this study were to elucidate the source of ROS and its regulation in human brain pericytes.
  
  
  METHODS
  The expression of Nox enzymes in the cells was evaluated using RT-PCR and western blot. Superoxide production was determined by superoxide dismutase-inhibitable chemiluminescence. Silencing of Nox4 was performed using RNAi, and cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.
  
  
  RESULTS
  Nox4 was predominant among the Nox family in human brain pericytes. Membrane fractions of cells produced superoxide in the presence of NAD(P)H. Superoxide production was almost abolished with diphenileneiodonium, a Nox inhibitor; however, inhibitors of other possible superoxide-producing enzymes had no effect on NAD(P)H-dependent superoxide production. Pericytes expressed angiotensin II (Ang II) receptors, and Ang II upregulated Nox4 expression. Hypoxic conditions also increased the Nox4 expression. Silencing of Nox4 significantly reduced ROS production and attenuated cell proliferation.
  
  
  CONCLUSIONS
  Our study showed that Nox4 is a major superoxide-producing enzyme and that its expression is regulated by Ang II and hypoxic stress in human brain pericytes. In addition, Nox4 may promote cell growth.
authors.affiliation: Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Junya
authors.initials: J
authors.lastname: Kuroda
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Ago
authors.forename: Ataru
authors.initials: A
authors.lastname: Nishimura
authors.forename: Kuniyuki
authors.initials: K
authors.lastname: Nakamura
authors.forename: Ryu
authors.initials: R
authors.lastname: Matsuo
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Wakisaka
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kamouchi
authors.forename: Takanari
authors.initials: T
authors.lastname: Kitazono
mesh_terms: D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000098:blood supply*; D015687:Cell Hypoxia; D002462:Cell Membrane / Q000201:enzymology; D049109:Cell Proliferation; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020244:Infarction, Middle Cerebral Artery / Q000201:enzymology; D008297:Male; D051379:Mice; D055806:Microvessels / Q000201:enzymology; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020286:Pericytes / Q000187:drug effects / Q000201:enzymology*; D034622:RNA Interference; D011945:Receptors, Angiotensin / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
chemical_list: D004791:Enzyme Inhibitors; D011945:Receptors, Angiotensin; D013481:Superoxides; D000804:Angiotensin II; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C422943:Nox4 protein, mouse