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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2007;27:2340.)
© 2007 American Heart Association, Inc.

Vascular Biology

4-Hydroxy-2-Nonenal Increases Superoxide Anion Radical in Endothelial Cells via Stimulated GTP Cyclohydrolase Proteasomal Degradation

Jennifer Whitsett; Matthew J. Picklo, Sr; Jeannette Vasquez-Vivar

From the Department of Biophysics (J.W.) and Free Radical Research Center (J.V.V.), Medical College of Wisconsin, Milwaukee; and the Department of Pharmacology, Physiology, and Therapeutics (M.J.P.), University of North Dakota, Grand Forks.

Correspondence to Jeannette Vasquez-Vivar, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226. E-mail jvvivar{at}mcw.edu

Objective— 4-Hydroxy-2-nonenal (4-HNE) is an abundant electrophilic lipid that mediates oxidative stress in endothelium by mechanisms that remain controversial. This study examines the effects of 4-HNE on nitric oxide (NO) and superoxide levels in bovine aorta endothelial cells (BAECs).

Methods and Results— Exposure of BAECs to 4-HNE caused a dose-dependent inhibition of NO that correlated with losses of hsp90 and phosphorylated eNOS-serine1179 but not eNOS protein levels. 4-HNE failed to inhibit NO production in sepiapterin and ascorbate supplemented cells suggesting that tetrahydrobiopterin (BH₄) is a limiting factor in non supplemented cells. This was verified by quantification of BH₄ by high-performance liquid chromatography analysis with electrochemical detection and by examining GTP cyclohydrolase I (GTPCH) protein levels and activity all of which were diminished by 4-HNE treatment. Analysis of 2-hydroxyethidium indicated that 4-HNE increased superoxide release in BAECs. The effects of 4-HNE on GTPCH and hsp90 were efficiently counteracted by proteasomal inhibition, indicating that depletion of BH₄ by 4-HNE is attributable to specific mechanisms involving protein degradation.

Conclusions— 4-HNE by altering BH₄ homeostasis mediates eNOS-uncoupling and superoxide generation in BAECs. By also decreasing phosphorylation of eNOS-serine 1179 4-HNE may specifically regulate NO/reactive oxygen species fluxes in the endothelium with important consequences to redox signaling.

4-Hydroxy-2-nonenal (4-HNE) mediates oxidative stress in the endothelium by controversial mechanisms. This study shows that 4-HNE uncouples eNOS by promoting GTPCH degradation by the proteasome. Sepiapterin and to a lesser extent ascorbate counteracted loss of NO and superoxide increase. Preventing eNOS uncoupling may be important in decreasing 4-HNE cytotoxicity.

Key Words: tetrahydrobiopterin • eNOS phosphorylation • 2-hydroxyethidium • glutathione • ascorbate