p47phox is required for atherosclerotic lesion progression in ApoE(-/-) mice

Abstract

NADPH oxidase is upregulated in smooth muscle cells (SMCs) in response to growth factor stimulation, concomitant with increased reactive oxygen species (ROS) production. We investigated the role of ROS production by NADPH oxidase in SMC responses to growth factors and in atherosclerotic lesion formation in ApoE(-/-) mice. SMCs from wild-type, p47phox(-/-), and gp91phox(-/-) mice differed markedly with respect to growth factor responsiveness and ROS generation. p47phox(-/-) SMCs had diminished superoxide production and a decreased proliferative response to growth factors compared with wild-type cells, whereas the response of gp91phox(-/-) SMCs was indistinguishable from that of wild-type SMCs. The relevance of these in vitro observations was tested by measuring atherosclerotic lesion formation in genetically modified (wild-type, p47phox(-/-), ApoE(-/-), and ApoE(-/-)/p47phox(-/-)) mice. ApoE(-/-)/p47phox(-/-) mice had less total lesion area than ApoE(-/-) mice, regardless of whether mice were fed standard chow or a high-fat diet. Together, these studies provide convincing support for the hypothesis that superoxide generation in general, and NADPH oxidase in particular, have a requisite role in atherosclerotic lesion formation, and they provide a rationale for further studies to dissect the contributions of ROS to vascular lesion formation.

Figure 1

(a) RT-PCR of p47phox and β-actin in wild-type SMCs after thrombin stimulation (2 U/ml). There is induction of p47phox mRNA by 15 minutes after treatment. The first lane is control RNA from mouse spleen lysate. Results were quantified by densitometry and expressed as the mean ± SD of three experiments. *P < 0.05 compared with untreated SMCs. (b) Western blot analysis of p47phox protein from membrane and cytosolic fractions of SMCs following thrombin stimulation. Results were quantified by densitometry and expressed as the mean ± SD from three experiments. *P < 0.05 compared with untreated cytoplasmic fractions. **P < 0.05 compared with untreated membrane fractions.

Figure 2

Proliferative capacity is decreased in p47phox^–/– SMCs compared with wild-type SMCs after growth factor stimulation. (a) Wild-type, p47phox-, or gp91phox-deficient SMCs were treated with the indicated concentrations of thrombin or serum, and uptake experiments were performed using thymidine as a marker for DNA synthesis. Results are shown as the mean of six wells ± SD, and are representative of three separate experiments. *P < 0.05 compared with wild-type SMCs. (b) Growth curve analysis of wild-type, p47phox-, or gp91phox-deficient SMCs plated at equivalent densities and allowed to proliferate for the indicated time periods. Results are shown as the mean of three wells ± SD and are representative of three separate experiments.

Figure 3

Intracellular ROS production is decreased in p47phox^–/– SMCs compared with wild-type SMCs. (a) p47phox-deficient SMCs have significantly less O₂^– production, as reflected by attenuation of the decrease in aconitase activity, following thrombin and serum stimulation (**P < 0.05) than do wild-type SMCs. There is no difference in O₂^– production between wild-type and gp91phox-deficient cells. The reduction in aconitase activity is reversed by pretreatment with polyethylene glycol–conjugated superoxide dismutase (PEG-SOD). (b) Fumarase activity was similar in wild-type, p47phox^–/–, and gp91phox^–/– SMCs, demonstrating the specificity of the aconitase assays described in a.

Figure 4

Expression of p47phox in wild-type and ApoE^–/– atherosclerotic arteries. Representative arterial sections from wild-type (a) and ApoE^–/– (b–d) mice. Sections were stained for antibodies against p47phox alone (a and b, brown chromogen), or simultaneously with antibodies against p47phox (blue) and α-actin (brown). Yellow arrows indicate cells in the plaque staining positive for p47phox alone, and red arrows indicate neointimal cells staining positive for both p47phox and α-actin.

Figure 5

DHE staining is decreased in p47phox-deficient aortas compared with wild- type. (a) Fresh-frozen wild-type (left panels) and p47phox-deficient (right panels) aortas were stained for 10 minutes with DHE. Results are typical of staining of sections from four different aortas. (b) Digital scans of intimal regions of DHE-stained aortas from wild-type and p47phox-deficient mice were quantified using NIH Image software. Results shown are mean ± SD. *P < 0.05 compared with wild-type aortas.

Figure 6

Comparison of oil red O–positive lesion size in mice on chow diet. Lesions were measured in whole aortas (a) and the aortic sinus (b). Aortas taken from 30-week-old ApoE^–/–/p47phox^–/– male mice maintained on a normal chow diet had decreased total aortic lesion size compared with ApoE^–/– mice of the same age. There is a decrease of approximately 75% in lesion size in ApoE^–/–/p47phox^–/– mice compared with ApoE^–/– mice at this age. There is no detectable difference in atherosclerotic lesion size between these two genotypes when the aortic sinus lesions are compared (b). Representative oil red O–stained aortic specimens are shown in (c). **P < 0.005.

Figure 7

Comparison of oil red O–positive lesion size in mice on a high-fat diet. Lesions were measured from whole aortas (a) and the aortic sinus (b). Aortas from 18-week-old ApoE^–/–/p47phox^–/– male mice maintained on a high-fat diet for 10 weeks had decreased total aortic lesion size compared with ApoE^–/– mice of the same age. There is an approximately 67% decrease in lesion size in ApoE^–/–/p47phox^–/– mice compared with ApoE^–/– mice on a high-fat diet. There is no detectable difference between these two genotypes when the aortic sinus lesions are compared (b). **P < 0.05.

Figure 8

Comparison of ability of wild-type and p47phox^–/– macrophages (MO) to home to atherosclerotic lesions in plaques, at the luminal surface, or in total, in ApoE^–/– mice. No differences were observed in homing of macrophages of either genotype to lesions in ApoE^–/– mice.