Deletion of p47phox attenuates angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein E-deficient mice

Abstract

Background: Angiotensin II (Ang II) contributes to vascular pathology in part by stimulating NADPH oxidase activity, leading to increased formation of superoxide (O2-). We reported that O2- levels, NADPH oxidase activity, and expression of the p47phox subunit of NADPH oxidase are increased in human abdominal aortic aneurysms (AAAs). Here, we tested the hypothesis that deletion of p47phox will attenuate oxidative stress and AAA formation in Ang II-infused apoE-/- mice.

Methods and results: Male apoE-/- and apoE-/-p47phox-/- mice received saline or Ang II (1000 ng x kg(-1) x min(-1)) infusion for 28 days, after which abdominal aortic weight and maximal diameter were determined. Aortic tissues and blood were examined for parameters of aneurysmal disease and oxidative stress. Ang II infusion induced AAAs in 90% of apoE-/- versus 16% of apo-/-p47phox-/- mice (P < 0.05). Abdominal aortic weight (14.1 +/- 3.2 versus 35.6 +/- 9.0 mg), maximal aortic diameter (1.5 +/- 0.2 versus 2.4 +/- 0.4 mm), aortic NADPH oxidase activity, and parameters of oxidative stress were reduced in apoE-/-p47phox-/- mice compared with apoE-/- mice (P < 0.05). In addition, aortic macrophage infiltration and matrix metalloproteinase-2 activity were reduced in apoE-/-p47phox-/- mice compared with apoE-/- mice. Deletion of p47phox attenuated the pressor response to Ang II; however, coinfusion of phenylephrine with Ang II, which restored the Ang II pressor response, did not alter the protective effects of p47phox deletion on AAA formation.

Conclusions: Deletion of p47phox attenuates Ang II-induced AAA formation in apoE-/- mice, suggesting that NADPH oxidase plays a critical role in AAA formation in this model.

Figure 1

Ang II–induced hypertensive effect was blunted in mice lacking p47^phox. SBP was measured with a tail-cuff system (Visitech 2000). There was a significant and sustained increase in SBP in apoE^–/– mice (n=10) that was markedly inhibited in apoE^–/–p47^phox–/– mice (n=6). The magnitude of the difference in the pressor response to Ang II between the 2 experimental groups diminished over time but remained significant throughout the duration of the study. *P<0.05 vs apoE^–/–p47^phox–/– mice.

Figure 2

Gross morphology and incidence of AAAs. A, Representative photographs showing macroscopic features of aneurysms induced by Ang II. No animals infused with NS developed aneurysms (left specimen). Infusion of Ang II in apoE^–/– animals led to the development of AAA and less frequently thoracic aortic aneurysms (middle specimens). The incidence of both types of aneurysms was markedly decreased by inactivation of the p47^phox gene (right specimens). B, Incidence of aneurysms expressed as percent of animals in apoE^–/– (n=10) vs apoE^–/–p47^phox–/– (n=6) mice. *P<0.05 vs apoE^–/–p47^phox+/+.

Figure 3

Ang II–induced AAA size was markedly reduced in mice lacking p47^phox. Abdominal aortic diameter (A) and weight (B) in saline-infused apoE^–/– mice (n=7), Ang II–infused apoE^–/– mice (n=10), and Ang II–infused apoE^–/–p47^phox–/– mice (n=6). *P<0.05 vs saline infusion; #P<0.05 vs Ang II–infused apoE^–/–p47^phox–/–.

Figure 4

Aortic superoxide production and leukocyte respiratory burst are diminished in mice lacking p47^phox. Aortic membrane homogenates and peritoneal leukocyte suspensions were prepared as described in Methods. In apoE^–/–p47^phox–/– animals, both aortic superoxide production (A) and leukocyte respiratory burst (B) were markedly suppressed compared with apoE^–/– animals. *P<0.05 vs NS; #P<0.05 vs apoE^–/–; n=4 per group.

Figure 5

Ang II–induced nitrotyrosine formation and macrophage infiltration were prominently diminished in mice lacking p47^phox. Abdominal aortic tissues were harvested, and transversal sections were prepared and immunostained as described. Representative pictures from Ang II–treated, apoE^–/– (top row), and apoE^–/–p47^phox–/– (bottom row) animals are shown, demonstrating that in the absence of functional p47^phox gene, inflammatory infiltrate is reduced (left, hematoxylin and eosin stain), nitrotyrosine formation is diminished (middle), and macrophage infiltration is attenuated (right). Arrows point to prominent, mostly adventitial, inflammatory infiltrates noted in the apoE^–/– group.

Figure 6

Ang II–induced MMP-2 proteolytic activity was inhibited in mice lacking p47^phox. Shown is a representative zymogram of aortic tissue homogenates demonstrating that in apoE^–/–p47^phox–/– (right 4 lanes), MMP-2 activity (normalized per 1 mg protein) is significantly decreased compared with apoE^–/– animals (left 4 lanes) (A, B). B, Densitometric quantification of MMP-2 gelatinolytic activity in the 2 groups of animals (n=4 per group). *P<0.05 vs apoE^–/–.

Figure 7

Attenuation of Ang II–induced AAA formation in mice lacking p47^phox was not due to reduced SBP. ApoE^–/– mice were infused with Ang II (1000 ng · kg^–1 · min^–1), whereas apoE^–/–p47^phox–/– mice were infused with Ang II plus phenylephrine (1000 and 12 500 ng · kg^–1 · min^–1, respectively) for 2 weeks (n=4 per group). SBP (A) was measured by the tail-cuff method, as described in Figure 1. After 2 weeks, mice were euthanized, abdominal aortic weight and diameter were measured (B; n=4 per group), and aortic oxidative stress was quantified by protein carbonyl formation (C) and compared with control apoE^–/– mice that were not infused with Ang II (n=3 per group). #P<0.05 vs apoE^–/–; *P<0.05 vs control.