Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II

Abstract

Hypertension caused by angiotensin II is dependent on vascular superoxide (O2*-) production. The nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase is a major source of vascular O2*- and is activated by angiotensin II in vitro. However, its role in angiotensin II-induced hypertension in vivo is less clear. In the present studies, we used mice deficient in p47(phox), a cytosolic subunit of the NADPH oxidase, to study the role of this enzyme system in vivo. In vivo, angiotensin II infusion (0.7 mg/kg per day for 7 days) increased systolic blood pressure from 105+/-2 to 151+/-6 mm Hg and increased vascular O2*- formation 2- to 3-fold in wild-type (WT) mice. In contrast, in p47(phox-/-) mice the hypertensive response to angiotensin II infusion (122+/-4 mm Hg; P<0.05) was markedly blunted, and there was no increase of vascular O2*- production. In situ staining for O2*- using dihydroethidium revealed a marked increase of O2*-production in both endothelial and vascular smooth muscle cells of angiotensin II-treated WT mice, but not in those of p47(phox-/-) mice. To directly examine the role of the NAD(P)H oxidase in endothelial production of O2*-, endothelial cells from WT and p47(phox-/-) mice were cultured. Western blotting confirmed the absence of p47(phox) in p47(phox-/-) mice. Angiotensin II increased O2*- production in endothelial cells from WT mice, but not in those from p47(phox-/-) mice, as determined by electron spin resonance spectroscopy. These results suggest a pivotal role of the NAD(P)H oxidase and its subunit p47(phox) in the vascular oxidant stress and the blood pressure response to angiotensin II in vivo.

Figure 1

Effect of angiotensin II (Ang II) infusion on vascular superoxide production in mouse aortas from wild-type (C57/BL6) and p47^phox−/− mice. a, Superoxide production in sham and Ang II–treated mice as determined with lucigenin-enhanced chemiluminescence (5 μmol/L; n=5 to 9). b, In situ detection of superoxide production with dihydroethidium (HE) in sham and Ang II–treated mice. Data are representative of 3 separate experiments.

Figure 2

Effect of Ang II infusion on systolic blood pressure in wild-type (C57/BL6) and p47^phox−/− mice (n=5 to 9). *P<0.05 (ang II-infused wild-type vs ang II-infused p47^phox−/− mice).

Figure 3

Determination of superoxide formation in cultured aortic endothelial cells from wild-type and p47^phox−/− mice using ESR spectroscopy. a, Increase of CP-H oxidation by endothelial cells in response to Ang II; effect of superoxide dismutase (50 U PEG-SOD). b, Representative “time scan” of CP-H oxidation in wild-type and p47^phox-deficient endothelial cells stimulated with Ang II. c, Western blot analysis of p47^phox expression in endothelial cells from wild-type (WT) and p47^phox−/− mice. Protein extracted from mouse macrophage lysates was used as a positive control. d, Western blot analysis of AT₁-receptor expression in endothelial cells from WT and p47^phox−/−mice. Data are representative of 3 separate experiments.