Modulation of reactive oxygen species in endothelial cells by peroxynitrite-treated lipoproteins.
Peroxynitrite has been implicated in the oxidative modification of low-density lipoprotein (LDL) particles, and nitrotyrosine residues in the LDL have been detected in atherosclerotic plaques. Studies have suggested that lipoproteins modified by peroxynitrite lead to the onset of atherosclerotic vascular disease. We therefore prepared in vitro lipoproteins oxidatively modified by peroxynitrite (NO(2)-lipoprotein) and investigated the effect of NO(2)-lipoprotein on the viability of cultured endothelial cells. After exposure of a high-density lipoprotein (HDL) to peroxynitrite, some intermolecular complexes of apolipoproteins in HDL were detected on immunoblotting with monoclonal antibodies against apolipoprotein AI and AII, suggesting that nitration of HDL by peroxynitrite causes intermolecular cross-linking of the apolipoproteins in the particles. Treatment with 1 mM peroxynitrite increased the 3-nitrotyrosine level to 28.5 mmol/mol of tyrosine residues in the prepared NO(2)-HDL, as quantitated by HPLC, and the amount in NO(2)-lipoprotein depended on the peroxynitrite concentration. HDL exhibited a shorter lag phase and the reaction plateaued more rapidly than that with LDL. To clarify whether or not NO(2)-lipoproteins affect the function of endothelial cells, we first examined the viability of cultured human aortic endothelial cells (HAECs) exposed to NO(2)-lipoproteins. Incubation with either NO(2)-HDL or NO(2)-LDL significantly reduced the HAEC viability at 72 h. The results of RT-PCR and Western blotting showed that NO(2)-HDL markedly suppressed at 48 h not only the expressed levels of mRNA and protein but also the activity of catalase in HAECs. In contrast, NO(2)-LDL significantly reduced the expression and activity of Cu(2+),Zn(2+)-superoxide dismutase (CuZn-SOD) in the cells. Neither NO(2)-HDL nor NO(2)-LDL interfered with nitric oxide production or expression of cyclooxygenases and NADPH oxidase in HAECs. Increased radical production in NO(2)-lipoprotein-treated HAECs implied that reactive oxygen species such as superoxide anions and hydroxyl radicals may contribute to the mechanism of the toxic effect induced in endothelial cells by NO(2)-lipoprotein. Overall, NO(2)-lipoprotein may lead to deterioration of the vascular function through these endothelial cell responses.
Matsunaga
T
T
The First Department of Biochemistry and The Fourth Department of Internal Medicine, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan. t_matsu@saitama-med.ac.jp
Nakajima
T
T
Sonoda
M
M
Koyama
I
I
Kawai
S
S
Inoue
I
I
Katayama
S
S
Hirano
K
K
Hokari
S
S
Komoda
T
T
D001161:Arteriosclerosis / Q000378:metabolism; D002374:Catalase / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000378:metabolism; D006801:Humans; D008074:Lipoproteins / Q000378:metabolism* / Q000494:pharmacology; D008075:Lipoproteins, HDL / Q000378:metabolism / Q000494:pharmacology; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D016166:Free Radical Scavengers; D008074:Lipoproteins; D008075:Lipoproteins, HDL; D017382:Reactive Oxygen Species; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D002374:Catalase; D019001:Nitric Oxide Synthase; D011451:Prostaglandin-Endoperoxide Synthases; D013482:Superoxide Dismutase; D019255:NADPH Oxidases