Augmented superoxide production by Nox2-containing NADPH oxidase causes cerebral artery dysfunction during hypercholesterolemia.
OBJECTIVE
We tested the hypothesis that elevated superoxide production by Nox2-NADPH oxidase occurs in cerebral arteries during hypercholesterolemia and causes decreased nitric oxide function.


METHODS
Wild-type (WT), apolipoprotein E-deficient (ApoE(-/-)) and Nox2(-/-)/ApoE(-/-) mice were fed a high-fat diet for 7 to 14 weeks. Basal superoxide production by cerebral arteries was measured using L-012 (100 micromol/L)-enhanced chemiluminescence. Nitric oxide function was assessed in isolated middle cerebral arteries through the constrictor response to N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 micromol/L). Western blotting was used to measure protein expression of Nox2, p47phox, endothelial nitric oxide synthase, and superoxide dismutases (1-3).


RESULTS
Morphology of cerebral arteries was similar in WT and ApoE(-/-) mice. In ApoE(-/-), but not Nox2(-/-)/ApoE(-/-) mice, superoxide production by cerebral arteries was approximately 50% greater than in WT mice (P<0.05). Moreover, the magnitude of L-NAME-induced contractions of isolated middle cerebral arteries from ApoE(-/-) mice was <50% of that in WT mice (P<0.05), whereas in Nox2(-/-)/ApoE(-/-) mice, the contractile response was comparable to WT responses. In the presence of the superoxide scavenger, tempol (1 mmol/L), L-NAME-induced contractions of middle cerebral arteries were similar between WT and ApoE(-/-) mice. Expression of p47phox was approximately 2-fold higher in ApoE(-/-) versus WT mice, whereas Nox2, endothelial nitric oxide synthase, and superoxide dismutase isoforms were unchanged.


CONCLUSIONS
Elevated superoxide production and reduced basal nitric oxide-mediated relaxation occur in cerebral arteries of hypercholesterolemic mice even in the absence of lesions. These changes appear to be exclusively due to increased activity of Nox2-NADPH oxidase, possibly through increased expression of its regulatory subunit p47phox.
Miller
Alyson A
AA
Department of Pharmacology, Monash University, Clayton, Victoria, Australia. alyson.miller@med.monash.edu.au
De Silva
T Michael
TM
Judkins
Courtney P
CP
Diep
Henry
H
Drummond
Grant R
GR
Sobey
Christopher G
CG
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001011:Aorta / Q000033:anatomy & histology / Q000473:pathology; D001057:Apolipoproteins E / Q000235:genetics / Q000378:metabolism; D002536:Cerebral Arteries / Q000033:anatomy & histology / Q000187:drug effects / Q000378:metabolism* / Q000503:physiopathology*; D002784:Cholesterol / Q000097:blood; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004041:Dietary Fats; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006937:Hypercholesterolemia / Q000378:metabolism / Q000503:physiopathology; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D013113:Spin Labels; D013481:Superoxides / Q000378:metabolism*; D014661:Vasoconstriction / Q000187:drug effects / Q000502:physiology
D000975:Antioxidants; D001057:Apolipoproteins E; D003497:Cyclic N-Oxides; D004041:Dietary Fats; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D013113:Spin Labels; D013481:Superoxides; D009569:Nitric Oxide; D002784:Cholesterol; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C001803:tempol; D019331:NG-Nitroarginine Methyl Ester