Angiotensin II-Induced Oxidative Stress in Human Endothelial Cells: Modification of Cellular Molecules through Lipid...

Angiotensin (Ang) II is a major bioactive peptide of the renin/angiotensin system and is involved in various cardiovascular functions and diseases. Ang II type 1 (AT₁) receptor mediates most of the physiological effects of Ang II. Previous studies have revealed that the lipid peroxidation products 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal (HNE) readily modify the N-terminus and Asp¹, Arg², and His⁶ residues of Ang II, and these modifications alter the biological activities of Ang II. Ang II is known to stimulate the formation of reactive oxygen species (ROS) that mediate cardiovascular remodeling. Another major consequence of ROS-derived damage is lipid peroxidation, which generates genotoxic aldehydes such as ONE and HNE. This study demonstrated that Ang II induced lipid peroxidation-derived modifications of cellular molecules in EA.hy926 cells, a human vascular endothelial cell line. Ang P (ONE- and ROS-derived N-terminal pyruvamide Ang II) and [His⁶(HNE)]-Ang II were detected in the medium of EA.hy926 cells incubated with Ang II, and their concentrations increased dose-dependently upon the addition of ascorbic acid (AscA) and CuSO₄. Cells were then subjected to metabolic labeling using SILFAC (stable isotope labeling by fatty acids in cell culture) with [¹³C₁₈]-linoleic acid. Analysis of cellular phospholipids indicated over 90% labeling. [¹³C₉]-Thiadiazabicyclo-ONE-glutathione adduct as well as Ang P and [His⁶([¹³C₉]-HNE)]-Ang II was detected in the labeled cells upon treatment with Ang II and their concentrations increased in an Ang II dose-dependent manner. Incubation of the labeled cells with losartan, an AT₁ receptor blocker, inhibited the formation of modified Ang IIs in a dose-dependent manner. These results indicate that Ang II induces lipid peroxidation and modification of various cellular molecules and these reactions are mediated by the activation of AT₁ receptor. Therefore, lipid peroxidation could be one mechanism by which Ang II contributes to cardiovascular dysfunction.