Mechanisms contributing to cerebral infarct size after stroke: gender, reperfusion, T lymphocytes, and Nox2-derived superoxide.

Cerebral infarct volume is typically smaller in premenopausal females than in age-matched males after ischemic stroke, but the underlying mechanisms are poorly understood. In this study we provide evidence in mice that this gender difference only occurs when the ischemic brain is reperfused. The limited tissue salvage achieved by reperfusion in male mice is associated with increased expression of proinflammatory proteins, including cyclooxygenase-2 (Cox-2), Nox2, and vascular cell adhesion molecule-1 (VCAM-1), and infiltration of Nox2-containing T lymphocytes into the infarcted brain, whereas such changes are minimal in female mice after ischemia-reperfusion (I-R). Infarct volume after I-R was no greater at 72 h than at 24 h in either gender. Infarct development was Nox2 dependent in male but not in female mice, and Nox2 within the infarct was predominantly localized in T lymphocytes. Stroke resulted in an approximately 15-fold increase in Nox2-dependent superoxide production by circulating, but not spleen-derived, T lymphocytes in male mice, and this was approximately sevenfold greater than in female mice. These circulating immune cells may thus represent a major and previously unrecognized source of superoxide in the acutely ischemic and reperfused brain of males (and potentially in postmenopausal females). Our findings provide novel insights into mechanisms that could be therapeutically targeted in acute ischemic stroke patients who receive thrombolysis therapy to induce cerebral reperfusion.

Direct evidence of a role for Nox2 in superoxide production, reduced nitric oxide bioavailability, and early atherosclerotic plaque formation in ApoE-/- mice.

The Nox family NADPH oxidases are reactive oxygen species (ROS)-generating enzymes that are strongly implicated in atherogenesis. However, no studies have examined which Nox isoform(s) are involved. Here we investigated the role of the Nox2-containing NADPH oxidase in atherogenesis in apolipoprotein E-null (ApoE(-/-)) mice. Wild-type (C57Bl6/J), ApoE(-/-), and Nox2(-/y)/ApoE(-/-) mice were maintained on a high-fat (21%) diet from 5 wk of age until they were 12 or 19 wk old. Mice were euthanized and their aortas removed for measurement of Nox2 expression (Western blot analysis and immunohistochemistry), ROS production (L012-enhanced chemiluminescence), nitric oxide (NO) bioavailability (contractions to N(omega)-nitro-L-arginine), and atherosclerotic plaque development along the aorta and in the aortic sinus. Nox2 expression was upregulated in the aortic endothelium of ApoE(-/-) mice before the appearance of lesions, and this was associated with elevated ROS levels. Within developing plaques, macrophages were also a prominent source of Nox2. The absence of Nox2 in Nox2(-/y)/ApoE(-/-) double-knockout mice had minimal effects on plasma lipids or lesion development in the aortic sinus in animals up to 19 wk of age. However, an en face examination of the aorta from the arch to the iliac bifurcation revealed a 50% reduction in lesion area in Nox2(-/y)/ApoE(-/-) versus ApoE(-/-) mice, and this was associated with a marked decrease in aortic ROS production and an increased NO bioavailability. In conclusion, this is the first demonstration of a role for Nox2-NADPH oxidase in vascular ROS production, reduced NO bioavailability, and early lesion development in ApoE(-/-) mice, highlighting this Nox isoform as a potential target for future therapies for atherosclerosis.

NADPH oxidase activity is higher in cerebral versus systemic arteries of four animal species: role of Nox2.

We previously reported that NADPH oxidase activity is greater in intracranial cerebral versus systemic arteries of the rat. Here, we first tested whether NADPH oxidase activity is also greater in intracranial cerebral than systemic arteries of three other animal species, i.e., mouse, rabbit, and pig. Second, using Nox2-deficient mice, we evaluated the involvement of Nox2-containing NADPH oxidases in any such regional differences. NADPH-stimulated superoxide (O(2)(-)) production by basilar, middle cerebral arteries (MCA), and common carotid arteries (CA) and thoracic aorta (AO) from rat, mouse, rabbit, and pig was measured using lucigenin-enhanced chemiluminescence. Basal production of O(2)(-) and hydrogen peroxide (H(2)O(2)) by cerebral arteries, AO, and CA from wild-type (WT) and Nox2(-/-) mice was measured using L-012-enhanced chemiluminescence and Amplex Red fluorescence, respectively. Western blotting was used to measure Nox2 and SOD1-3 protein expression, and immunofluorescence was used to localize Nox2, in mouse arteries. In rats, WT mice, rabbits, and pigs, NADPH-stimulated O(2)(-) production by cerebral arteries was up to 40-fold greater than that in AO and CA. In WT mice, basal O(2)(-) and H(2)O(2) production by cerebral arteries was ninefold and approximately 2.5-fold higher, respectively, than that in AO and CA and was associated with approximately 40% greater expression of Nox2 protein. Nox2 immunofluorescence was localized to the endothelium, and to a lesser extent the adventitia, in all mouse arteries and appeared to be more intense in endothelium of MCA than AO or CA. In Nox2(-/-) mice, NADPH-stimulated O(2)(-) production by cerebral arteries was approximately 35% lower than that in WT mice, whereas Nox2 deletion had no significant effect on O(2)(-) production by AO or CA. Thus NADPH oxidase activity is greater in intracranial cerebral versus systemic arteries of several animal species and is associated with higher cerebrovascular expression and activity of Nox2.

Effect of gender on NADPH-oxidase activity, expression, and function in the cerebral circulation: role of estrogen.

BACKGROUND AND PURPOSE: This study tested whether NADPH-oxidase activity, expression, and functional effects on vascular tone are influenced by gender in the rat cerebral circulation and whether such differences are estrogen-dependent. METHODS: NADPH-stimulated superoxide production by cerebral (basilar [BA]; middle cerebral) arteries from male and female Sprague-Dawley rats was measured using lucigenin-enhanced chemiluminescence and dihydroethidium. Protein expression of Nox1, Nox2, Nox4, superoxide dismutase 1 (SOD1), SOD2, and SOD3 was measured using Western blotting. Vascular responses of BA to NADPH were assessed in a myograph. Some female rats were ovariectomized and treated with either vehicle (dimethyl sulfoxide) or 17beta-estradiol. RESULTS: NADPH-stimulated superoxide production by BA and middle cerebral arteries from males was approximately 2-fold greater than vessels from females. Superoxide production was virtually abolished by the NADPH-oxidase inhibitor, diphenyleneiodonium. Protein expression of Nox1 and Nox4 in BA was also higher in males than in females (2.4- and 2.8-fold, respectively), whereas Nox2, SOD1, SOD2, and SOD3 expression did not differ between genders. NADPH induced greater vasorelaxant effects in BA from males versus females (P<0.05). The hydrogen peroxide scavenger, catalase, abolished these NADPH-induced relaxations. NADPH-stimulated superoxide production by BA from ovariectomized rats treated with vehicle was 3-fold greater than levels in intact females. Treatment of ovariectomized rats with 17beta-estradiol decreased superoxide production (P<0.05). NADPH-induced relaxations of BA were smaller in 17beta-estradiol-treated than in vehicle-treated ovariectomized rats (P<0.05). CONCLUSIONS: NADPH-oxidase activity and function are lower in cerebral arteries of female rats. These gender differences are estrogen-dependent and are associated with lower Nox1 and Nox4 expression.