Modulation of reactive oxygen species in endothelial cells by peroxynitrite-treated lipoproteins

Peroxynitrite has been implicated in the oxidative modification of low-dens ity lipoprotein (LDL) particles, and nitrotyrosine residues in the LDL have been detected in atherosclerotic plaques. Studies have suggested that lipo proteins modified by peroxynitrite lead to the onset of atherosclerotic vas cular disease. We therefore prepared in vitro lipoproteins oxidatively modi fied by peroxynitrite (NO2-lipoprotein) and investigated the effect of NO2- lipoprotein on the viability of cultured endothelial cells. After exposure of a high-density lipoprotein (HDL) to peroxynitrite, some intermolecular c omplexes of apolipoproteins in HDL were detected on immunoblotting with mon oclonal antibodies against apolipoprotein AI and AII, suggesting that nitra tion of HDL by peroxynitrite causes intermolecular cross-linking of the apo lipoproteins in the particles. Treatment with 1 mM peroxynitrite increased the 3-nitrotyrosine level to 28.5 mmol/mol of tyrosine residues in the prep ared NO2-HDL, as quantitated by HPLC, and the amount in NO2-lipoprotein dep ended on the peroxynitrite concentration. HDL exhibited a shorter lag phase and the reaction plateaued more rapidly than that with LDL. To clarify whe ther or not NO2-lipoproteins affect the function of endothelial cells, we f irst examined the viability of cultured human aortic endothelial cells (HAE Cs) exposed to NO2-lipoproteins. Incubation with either NO2-HDL or NO2-LDL significantly reduced the HAEC viability at 72 h. The results of RT-PCR and Western blotting showed that NO2-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, NO2-LDL significantly reduced the expression and act ivity Of Cu2+ Zn2+ -superoxide dismutase (CuZn-SOD) in the cells. Neither N O2-HDL nor NO2-LDL interfered with nitric oxide production or expression of cyclooxygenases and NADPH oxidase in HAECs. Increased radical production i n NO2-lipoprotein-treated HAECs implied that reactive oxygen species such a s superoxide anions and hydroxyl radicals may contribute to the mechanism o f the toxic effect induced in endothelial cells by NO2-lipoprotein. Overall , NO2-lipoprotein may lead to deterioration of the vascular function throug h these endothelial cell responses.