Role of Increased Production of Superoxide Anions by NAD(P)H Oxidase and Xanthine Oxidase in Prolonged Endotoxemia

Abstract

Abstract-Superoxide anions (O 2 Ϫ) are supposedly involved in the pathogenesis of endothelial dysfunction. We investigated whether the enhanced formation of O 2 Ϫ is involved in the attenuation of endothelium-dependent relaxation induced by lipopolysaccharide (LPS). Rats were injected with LPS (10 mg/kg IP), the aorta was removed after 12 or 30 hours, and generation of O 2 Ϫ , H 2 O 2 , and ONOO Ϫ was measured using chemiluminescence assays. Protein tyrosine nitration and expression of xanthine oxidase (XO), NAD(P)H oxidase, and manganese superoxide dismutase were determined by Western or Northern blotting, and endothelium-dependent relaxation in aortic rings was studied. LPS treatment increased vascular O 2 Ϫ (from 35Ϯ2 cpm/ring at baseline to 166Ϯ21 cpm/ring at 12 hours and 225Ϯ16 cpm/ring at 30 hours) and H 2 O 2 formation, which was partially sensitive to the NAD(P)H oxidase inhibitor diphenylene iodonium at both time points studied and to the XO inhibitor oxypurinol only 30 hours after LPS treatment. Expression of XO and NAD(P)H oxidase (p22phox, p67phox, and gp91phox) were increased by LPS in a time-dependent manner, as were protein tyrosine nitration and ONOO Ϫ formation. LPS also induced expression of the oxidative stress-sensitive protein manganese superoxide dismutase. Endotheliumdependent relaxation was impaired after LPS treatment and could not be restored by inhibition of inducible NO synthase. Inhibition of O 2Ϫ with superoxide dismutase, oxypurinol, tiron, or the superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride did not restore but further deteriorated the relaxation of LPS-treated rings. In summary, treatment of rats with LPS enhances vascular expression of XO and NAD(P)H oxidase and increases formation of O 2 Ϫ and ONOO Ϫ is at least partially due to activation of vascular NAD(P)H oxidase and xanthine oxidase (XO). 1 Severe hypotension and hyporesponsiveness to vasoconstrictors and endothelium-dependent dilator agents are hallmarks of sepsis, eg, after exposure to bacterial lipopolysaccharide (LPS). 2 Although LPS-induced vascular hyporesponsiveness to constrictor agents may be partially due to excessive generation of NO by inducible NO synthase 2 (iNOS), the mechanism underlying LPS-induced endothelial dysfunction is not clear. 3 We investigated whether enhanced formation of O 2 Ϫ could be involved in the development of endothelial dysfunction after exposure to LPS. Methods MaterialsDiphenylene iodonium (DPI), U44069, and aminohydromethylthiazine (AMT) were obtained from Alexis. Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) was obtained from Calbiochem. Recombinant human superoxide dismutase (rhSOD) was a gift from Grünenthal Inc. Rat vascular smooth muscle cell p22phox cDNA was a kind gift from K.K. Griendling, Emory University. XO cDNA was provided by M. Saksela, University of Helsinki. The mouse

Gene transfer of extracellular superoxide dismutase improves relaxation of aorta after treatment with endotoxin

“…Previous studies (4,15,20) suggest that levels of ROS, including superoxide, are elevated in blood vessels after LPS. Pharmacological reduction of superoxide in arteries improves endothelium-dependent relaxation after LPS (15), but at least one study suggests that administration of CuZnSOD and SODmimetics after LPS does not improve endothelium-dependent relaxation in rat aorta (4). Thus the importance of elevated levels of superoxide and the role of SOD in vascular dysfunction after LPS is not clear.…”

“…Elevated levels of ROS are thought to contribute to vasomotor dysfunction during inflammation (4,15,26). Previous studies (4,15,20) suggest that levels of ROS, including superoxide, are elevated in blood vessels after LPS.…”

“…Elevated levels of ROS are thought to contribute to vasomotor dysfunction during inflammation (4,15,26). Previous studies (4,15,20) suggest that levels of ROS, including superoxide, are elevated in blood vessels after LPS. Pharmacological reduction of superoxide in arteries improves endothelium-dependent relaxation after LPS (15), but at least one study suggests that administration of CuZnSOD and SODmimetics after LPS does not improve endothelium-dependent relaxation in rat aorta (4).…”

“…We conclude that after gene transfer in vivo, binding of ECSOD to arteries effectively decreases the numbers of adherent leukocytes and levels of superoxide and improves impaired endothelium-dependent relaxation produced by LPS. acetylcholine; adenovirus; rats; hydroethidine LPS PRODUCES IMPAIRMENT OF endothelium-dependent vascular relaxation (3,4,15). Several studies suggest that endothelial dysfunction in LPS-treated vessels may be produced, in part, by increased levels of reactive oxygen species (ROS), especially superoxide, in blood vessels (15,21).…”

Superoxide as a Messenger of Endothelial Function

“…The latter cause of conversion of endogenous xanthine dehydrogenase to its oxidase form could have been triggered by NAD(P)H-oxidase (Zhang et al, 1999;Brandes et al, 1999;Lassegue et al, 2001). …”