Essential role of the NADPH oxidase subunit p47phox in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor-α

被引:268
作者
Li, JM
Mullen, AM
Yun, S
Wientjes, F
Brouns, GY
Thrasher, AJ
Shah, AM
机构
[1] Kings Coll London, GKT Sch Med, Dept Cardiol, London SE5 9PJ, England
[2] UCL, Dept Med, London WC1E 6BT, England
[3] Inst Child Hlth, Mol Immunol Unit, London, England
关键词
knockout mice; p47(phox); endothelial cells; NADPH oxidase; reactive oxygen species;
D O I
10.1161/hh0202.103615
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
A phagocyte-type NADPH oxidase complex is a major source of endothelial reactive oxygen species (ROS) production, but its biochemical function and regulation remain unclear. In neutrophils, the p47(phox) subunit is centrally involved in oxidase activation in response to agonists such as phorbol-12-myristate-13-acetate (PMA). We investigated the role of p47(phox) in endothelial cell ROS production in response to PMA or tumor necrosis factor-alpha (TNFalpha) stimulation. To specifically address the role of p47(phox), we studied coronary microvascular endothelial cells (CMECs) isolated from p47(phox-/-) mice and wild-type controls. p47(phox) was absent in hearts of knockout mice whereas the essential oxidase subunit, p22(phox), was expressed in both groups. In the absence of agonist stimulation, the lack of p47(phox) did not result in a reduction in NADPH-dependent ROS production in p47(phox-/-) CMECs compared with wild-type CMECs. Prestimulation with PMA (100 ng/mL) or TNFalpha (100 U/mL) for 10 minutes significantly increased NADPH-dependent O-2(-) production in wild-type CMECs, assessed either by lucigenin (5 mumol/L) chemiluminescence or dichlorohydrofluorescein (DCF) fluorescence. This response was completely lost in p47(phox-/-) cells. Transfection of the full-length p47(phox) cDNA into p47(phox-/-) CMECs caused expression of p47(phox) protein and restoration of the O-2(-) response to PMA and TNFalpha. In wild-type CMECs, transfection of antisense p47(phox) cDNA substantially reduced p47(phox) expression and caused loss of the O-2(-) response to PMA and TNFalpha. These data show that endothelial cell p47(phox) is critical in the upregulation of NADPH oxidase activity by PMA and TNFalpha.
引用
收藏
页码:143 / 150
页数:8
相关论文
共 38 条
  • [1] ABO A, 1992, J BIOL CHEM, V267, P16767
  • [2] NADPH oxidase: An update
    Babior, BM
    [J]. BLOOD, 1999, 93 (05) : 1464 - 1476
  • [3] Expression of a functional neutrophil-type NADPH oxidase in cultured rat coronary microvascular endothelial cells
    Bayraktutan, U
    Draper, N
    Lang, D
    Shah, AM
    [J]. CARDIOVASCULAR RESEARCH, 1998, 38 (01) : 256 - 262
  • [4] Molecular characterization and localization of the NAD(P)H oxidase components gp91-phox and p22-phox in endothelial cells
    Bayraktutan, U
    Blayney, L
    Shah, AM
    [J]. ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY, 2000, 20 (08) : 1903 - 1911
  • [5] Endothelial-derived superoxide anions in pig coronary arteries: Evidence from lucigenin chemiluminescence and histochemical techniques
    Brandes, RP
    Barton, M
    Philippens, KMH
    Schweitzer, G
    Mugge, A
    [J]. JOURNAL OF PHYSIOLOGY-LONDON, 1997, 500 (02): : 331 - 342
  • [6] Virulence of catalase-deficient Aspergillus nidulans in p47phox-/- mice -: Implications for fungal pathogenicity and host defense in chronic granulomatous disease
    Chang, YC
    Segal, BH
    Holland, SM
    Miller, GF
    Kwon-Chung, KJ
    [J]. JOURNAL OF CLINICAL INVESTIGATION, 1998, 101 (09) : 1843 - 1850
  • [7] Oscillatory and steady laminar shear stress differentially affect human endothelial redox state - Role of a superoxide-producing NADH oxidase
    De Keulenaer, GW
    Chappell, DC
    Ishizaka, N
    Nerem, RM
    Alexander, RW
    Griendling, KK
    [J]. CIRCULATION RESEARCH, 1998, 82 (10) : 1094 - 1101
  • [8] NADPH oxidase activity is independent of p47(phox) in vitro
    Freeman, JL
    Lambeth, JD
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (37) : 22578 - 22582
  • [9] A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall
    Görlach, A
    Brandes, RP
    Nguyen, K
    Amidi, M
    Dehghani, F
    Busse, R
    [J]. CIRCULATION RESEARCH, 2000, 87 (01) : 26 - 32
  • [10] NAD(P)H oxidase - Role in cardiovascular biology and disease
    Griendling, KK
    Sorescu, D
    Ushio-Fukai, M
    [J]. CIRCULATION RESEARCH, 2000, 86 (05) : 494 - 501