Oxidative stress is involved in many hypertension-related vascular diseases in the brain, including stroke and dementia. Thus, we examined the role of genetic deficiency of NADPH oxidase subunit Nox2 in the function and structure of cerebral arterioles during hypertension. Arterial pressure was increased in right-sided cerebral arterioles with transverse aortic banding for 4 weeks in 8-week-old wild-type (WT) and Nox2-deficient (-/y) mice. Mice were given
Chronic hypertension is a major risk factor of vascular disorders. Profound vascular functional and structural changes occur in many disease states, and emerging evidence suggests that oxidative stress has a major role in mediating these changes [
Although there are many sources of reactive oxygen species (ROS), the primary source of superoxide production in the vascular wall is thought to be NADPH oxidase [
Cerebral resistance arteries and arterioles play critical roles in controlling local cerebral blood flow [
Nitric oxide (NO) is a major mediator of endothelium-dependent dilation and inhibits mitogenesis and proliferation of vascular smooth muscle cells [
Nox2-/y and wild-type (WT) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Animals were housed in pathogen-free facility at 24°C, exposed to 12 hours of light (lights on at 06:00, off at 18:00) and allowed free access of food and fluid. All animals were studied at 13 to 15 weeks of age. Procedures followed in this study were approved by the Institutional Animal Care and Use Committee of the University of Iowa.
Increased pressure in the proximal aorta in all animals was induced by means of thoracic aortic banding using the method described previously [
L-NAME (10 mg/kg/day, 4 weeks) was given in drinking water to WT (
Systemic arterial blood pressures were measured in 6 mice from each group using an automated tail-cuff device (Visitech Systems BP-2000, Apex, NC, USA). Mice were placed in specifically designed mouse holders that allow measurement of systolic blood pressure under resting conditions. Mice were trained for 5 days, and then blood pressure was measured at days 0 (baseline), 7, 14, 21, and 28 of treatment. Each day, 30 measurements were made and averaged for each mouse.
Four weeks after aortic banding, we measured diameter in first-order arterioles on the surfaces of the right and left cerebral hemispheres through an open skull preparation as described in detail previously (
To determine whether increases in arterial pressure that result from transverse aortic banding are limited to the right side of the brain, pressure was measured in right- and left-sided first-order cerebral arterioles in a separate group of anesthetized WT mice (
In another set of animals (
In another set of animals (
ACh, SNP, and L-NAME were purchased from Sigma (St. Louis, MO, USA). ACh and SNP were dissolved in artificial CSF. L-NAME was dissolved in distilled water.
Analysis of variance was used to compare blood pressure, cerebral arteriolar diameters, cross-sectional areas, and superoxide levels of the vessel wall. Probability values were calculated using Graph Pad Prism 5 (Graph Pad Software, Inc., San Diego, CA, USA). Values were presented in mean ± SEM and were considered different when
To determine if Nox2-containing NADPH oxidase is responsible for hypertension-induced superoxide production, levels of superoxide were determined in cerebral arterioles from WT and Nox2-/y mice by ethidium fluorescence. Representative micrographs show that fluorescence of ethidium was higher in right- than left-sided cerebral arterioles in WT mice (Figure
Representative micrographs of superoxide levels determined by hydroethidium fluorescence of WT left-sided, WT right-sided, Nox2-/y left-sided, and Nox2-/y right-sided cerebral arterioles (highlighted in yellow) (a). Graphs showing relative staining density of WT (b) and Nox2-/y mice (c) (
SDs measured under conscious conditions by a tail-cuff method prior to L-NAME treatment were similar in WT and Nox2-/y mice (Figure
Graph showing systolic blood pressures measured by tail-cuff method. Results were the averages of thirty measurements made on days 0, 7, 14, and 28 of 6 individual mice.
Pressures were measured in right and left carotid arteries in anesthetized mice to confirm that transverse aortic banding was successful. SP and PP, but not DP and MP, were significantly increased by similar levels in right- compared to left-sided carotids in untreated WT and untreated Nox2-/y mice (Table
Summary of blood pressures (measured by carotid catheters in anesthetized mice) and arterial blood gases in WT and Nox2-/y mice.
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14 | 17 | 17 | 15 |
To test whether endothelial dysfunction induced by hypertension is Nox2-dependent, dilator responses to ACh and SNP were studied. Dilator response to ACh was significantly decreased in right-sided cerebral arterioles relative to left-sided arterioles in untreated WT mice, suggesting an endothelial dysfunction on the hypertensive side (Figure
Graph showing endothelium-dependent and -independent vasodilation in cerebral arterioles. Acetylcholine (Ach,
To determine whether Nox2 contributes to hypertension-induced hypertrophy in cerebral arterioles, we measured CSA of the arteriolar wall. CSA of the arteriolar wall was greater in right-, than in left-sided, cerebral arterioles in untreated WT mice, but not in untreated Nox2-/y mice (Figure
Graph showing cross-sectional areas of maximally dilated cerebral arterioles (a) and carotid arteries (
Chronic hypertension has profound impacts on the vasculature and is a known risk factor for stroke and dementia. It is important to understand the mechanism of vascular changes during chronic hypertension, particularly in smaller resistance arterioles because they provide substantial vascular resistance and are important in controlling local blood flow [
We used ethidium fluorescence to examine the effects of Nox2 deficiency on hypertension-induced production of superoxide in cerebral arterioles. Being aware of potential problems with this method, matched pairs of hypertensive (right-sided) and normotensive (left-sided) cerebral arterioles from each mouse were examined in parallel using the same reagents and laser settings. In addition, we have shown previously that incubation with PEG-SOD, a scavenger of superoxide, abolishes ethidium fluorescence in aorta of mice that overexpresses human renin and human angiotensinogen [
Chronic hypertension is well known to increase vascular production of ROS. Using a model of abdominal aortic banding, it was shown previously that superoxide levels are elevated in noncerebral vessels [
It is still debatable as to whether Nox2-derived ROS play a role in the development of hypertension. For example, the pressor response to angiotensin II was found to be reduced in Nox2-/y mice in one study [
Endothelial dysfunction caused by hypertension in the cerebral circulation has been previously demonstrated in various animal models [
Hypertension-induced impairment of endothelium-dependent dilatation is thought to result from reduced availability of NO due either to its destruction by NADPH-derived superoxide or to an uncoupling of eNOS, which results in the production of superoxide instead of NO. Our finding that Nox2 deficiency protected against impairment in cerebral arteriolar dilatation during hypertension produced with transverse aortic banding, but not L-NAME, supports the concept that hypertension impairs endothelial-dependent dilatation of cerebral arterioles through the destruction of NO by NADPH-derived superoxide, and not by uncoupling of eNOS.
Cerebral vascular hypertrophy is a well-known consequence of hypertension [
One mechanism by which superoxide may promote vascular hypertrophy is through the destruction of NO [
While we cannot exclude the possibility that ROS downstream of superoxide, such as peroxynitrite or hydrogen peroxide, contribute to the hypertrophic process, we believe that superoxide plays a more important and direct role in causing cerebral vascular hypertrophy. We base this speculation on two observations. First, we showed in this study that L-NAME inhibition, which supposedly limits the interaction of superoxide and NO to form peroxynitrite, does not attenuate the degree of cerebral arteriolar hypertrophy induced by transverse aortic banding in WT mice. Second, we showed in a previous study that the deficiency of copper-zinc superoxide dismutase, which leads to reduced conversion of superoxide to hydrogen peroxide, nevertheless causes hypertrophy in cerebral arterioles [
The present study demonstrated that ROS derived from Nox2-containing NADPH oxidase are critical in hypertension-mediated cerebral arteriolar vascular dysfunction and hypertrophy. This may lead to reduction of dilator capacity and the ability to control local cerebral blood flow during hypertension.
The authors would like to thank Mr. Thomas Gerhold (aortic banding procedure) and Ms. Shams Ghoneim (ethidium fluorescence) for their excellent technical assistance. This work was supported by the American Heart Association Heartland Affiliate Postdoctoral Fellowship no. 0725668Z (S.-L. Chan) and National Institutes of Health Grants HL-22149, NS72628, NS24621, and NS-62461. (G. L. Baumbach and S.-L. Chan).