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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:826.)
© 2006 American Heart Association, Inc.

Vascular Biology

Angiotensin II Attenuates Endothelium-Dependent Responses in the Cerebral Microcirculation Through Nox-2–Derived Radicals

Helene Girouard; Laibaik Park; Josef Anrather; Ping Zhou; Costantino Iadecola

From the Division of Neurobiology, Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, NY.

Correspondence to Costantino Iadecola, MD, Division of Neurobiology, Weill Medical College of Cornell University, 411 East 69^th Street; KB410, New York, NY 10021. E-mail coi2001{at}med.cornell.edu

	Abstract

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Objective— Angiotensin II (Ang II) exerts deleterious effect on the cerebral circulation through production of reactive oxygen species (ROS). However, the enzymatic source of the ROS has not been defined. We tested the hypothesis that Ang II impairs endothelium-dependent responses in the cerebral microcirculation through ROS generated in cerebrovascular cells by the enzyme NADPH oxidase.

Methods and Results— Cerebral blood flow (CBF) was monitored by laser Doppler flowmetry in anesthetized mice equipped with a cranial window. Ang II (0.25±0.02 µg/kg per minute for 30 to 45 minutes) attenuated the CBF increase produced by the endothelium-dependent vasodilators acetylcholine (–42±5%; P<0.05), bradykinin (–53±5%; P<0.05), and A23187 (–43±4%; P<0.05), and induced cerebrovascular ROS production, assessed by hydroethidine fluoromicrography. These actions of Ang II were prevented by losartan, by the ROS scavenger Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (100 µmol/L), or by the NADPH oxidase peptide inhibitor gp91ds-tat (1 µmol/L), and were not observed in mice lacking the NADPH oxidase subunit gp91^phox (nox-2).

Conclusions— Ang II impairs the endothelial regulation of the cerebral microcirculation through AT1 receptor-mediated cerebrovascular oxidative stress. The source of the ROS is a nox-2-containing NADPH oxidase. These effects of Ang II could threaten the cerebral blood supply and contribute to the increased susceptibility to stroke and dementia associated with hypertension.

We investigated the mechanism of the effect of angiotensin II on the increase in cerebral blood flow (CBF) produced by endothelium-dependent vasodilators. We found that the attenuation of endothelium-dependent responses induced by Ang II is mediated by free radicals produced by a NADPH oxidase containing nox-2 as the catalytic subunit.

Key Words: cerebral blood flow • gp91^phox • laser Doppler flowmetry • NADPH oxidase • reactive oxygen species

	Introduction

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Hypertension has profound effects on the brain and its circulation.¹ Hypertension alters the structure of cerebral blood vessels and disrupts the mechanisms regulating cerebral blood flow (CBF).² For example, hypertension alters cerebrovascular autoregulation, the ability of cerebral blood vessels to maintain CBF in the face of changes in mean arterial pressure (MAP), and impairs the regulation of CBF by vasodilators released from the endothelium.^2,3 These alterations compromise the blood supply of the brain and could underlie the increased susceptibility to stroke and dementia associated with hypertension.^4,5 Angiotensin II (Ang II) has emerged as a critical factor in the deleterious cerebrovascular effects of hypertension.^6,7 Ang II produces cerebrovascular inflammation and remodeling, and impairs CBF regulation.^8–11 Importantly, Ang II attenuates the CBF increase produced by endothelium-dependent vasodilators, such as acetylcholine (ACh) and bradykinin (BK).^12,13 Thus, Ang II may interfere with cerebrovascular regulatory processes that are critical for maintaining an adequate cerebral perfusion.

The mechanisms of the Ang II-induced alteration in endothelium-dependent vasodilation have not been fully elucidated. Ang II attenuates endothelium-dependent responses in mouse carotid arteries or rabbit pial arterioles through production of reactive oxygen species (ROS),^13,14 but the sources of the ROS have not been determined. Ang II exerts some of its cellular effects via AT1 receptors through production of ROS by the enzyme NADPH oxidase.⁷ NADPH oxidase is a multiunit enzyme comprised of membrane-bound (nox and p22^phox) and cytoplasmic components (p47^phox, p67^phox, p40^phox, rac1, or rac2).¹⁵ The catalytic subunit of the enzyme exists in different isoforms termed nox-1 through nox-5.¹⁶ Activation of AT1 receptors initiates intracellular signaling that leads to phosphorylation of p47^phox, which, in turn, results in assembly of the subunits and production of superoxide.¹⁷ Although Ang II attenuates endothelium-dependent responses in brain through production of ROS,^13,14 it is not known whether the ROS are produced by NADPH oxidase. Furthermore, although the NADPH oxidase subunit gp91^phox (nox-2) has been found in cerebral endothelial cells of mouse pial arterioles,¹⁸ the role of nox-2 in the effect of Ang II on endothelium-dependent responses in cerebral microvessels remains to be established. Therefore, in this study we investigated whether the ROS responsible for the effects of Ang II on endothelium-dependent relaxation originate from a NADPH oxidase containing nox-2.

	Materials and Methods

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Mice
We used male mice aged 2 to 3 months. Mice lacking the gp91^phox subunit of NADPH oxidase¹⁹ were obtained from an in house colony.^18,20,21 Null mice were congenic with the C57Bl/6J strain and were genotyped as described.^18,20,21 C57Bl/6J mice (Jackson Laboratories, Bar Harbor, Me) served as wild-type controls.

General Surgical Procedures
All experimental procedures were approved by the Institutional Animal Use and Care Committee. As described in detail elsewhere,^18,20–22 mice were anesthetized with isoflurane (induction, 5%; maintenance, 1% to 2%). The femoral vessels were cannulated for recording of arterial pressure, collection of blood samples, and intravenous administration of Ang II or vehicle. Mice were intubated and artificially ventilated to control blood gases (Table I, available online at http://atvb.ahajournals.org). Rectal temperature was maintained at 37°C. After surgery, isoflurane was discontinued and anesthesia was maintained with urethane (750 mg/kg; intraperitoneal) and -chloralose (50 mg/kg; intraperitoneal). The level of anesthesia was monitored by testing corneal reflexes and motor responses to tail pinch.

Monitoring CBF
A 2x2-mm craniotomy was performed to expose the somatosensory cortex, the dura was removed, and the site was superfused with a modified Ringer solution (37°C; pH, 7.3 to 7.4).^18,20–22 CBF was continuously monitored at the site of superfusion with a laser Doppler probe (Vasamedic, St. Paul, Minn) connected to a computerized data acquisition system (Mac Laboratory). CBF values were expressed as percent increase relative to the resting level.

Detection of ROS by Hydroethidine
The hydroethidine (HE) method was used to assess ROS production because it permits to identify the cellular source of ROS and can be combined with immunocytochemistry.^23,24 HE (Dihydroethidium; Molecular Probes, Eugene, Ore; 2 µmol/L) in Ringer’s solution was superfused on the somatosensory cortex for 60 minutes, as previously described.^20,21 Next, the brain was removed and frozen. Cryostat brain sections (thickness 20 µm) were collected at 100-µm intervals throughout the region exposed by the cranial window. Fluorescence intensity was measured using methods previously published.^20,21 Briefly, brain sections were examined under a fluorescence microscope (Nikon, Melville, NY) equipped with an ethidium filter set (Chroma Technology, No. 31014). Images were acquired with a digital camera (Coolsnap; Roper Scientific, Trenton, NJ). Fluorescence intensity was assessed in the brain area superfused with HE. The analysis of ROS production in the different conditions studied (see Experimental Protocol) was performed in a blinded fashion using the IPLab software (Scanalytics, Fairfax, Va).^20,21 Fluorescence intensities of all sections (30 per animal) were averaged and expressed as relative fluorescence units (RFU).^20,21 The contralateral somatosensory cortex not superfused with HE served as control for background fluorescence. For simultaneous visualization of the endothelial marker CD31, sections of HE-treated mice were post-fixed and incubated with a rat anti-mouse CD31 monoclonal antibody (1:200; BD Biosciences, San Diego, Calif), followed by a fluorescein isothiocyanate (FITC) goat anti-rat secondary antibody (1:200; Molecular Probes). The specificity of the immunostain was verified by omitting the primary antibody. Sections were viewed with a fluorescence microscope equipped with ethidium and FITC filter sets and photographed with a digital camera.

Microvessel Isolation and Quantitative Real-Time Polymerase Chain Reaction
Microvessels (capillaries, arterioles, and venules; diameter 6 to 100 µm) were isolated from the neocortex as described.²⁵ Total RNA was prepared from the microvessels (n=6 from 2 isolations) and gene expression levels were assessed by real-time polymerase chain reaction (PCR) using a Chromo 4 detector (MJ Research, Waltam, Mass), as previously described.^26,27 The PCR primers are presented in Table II (available online at http://atvb.ahajournals.org).

Experimental Protocols
All pharmacological agents studied were dissolved in Ringer’s solution.

Effect of Ang II on CBF Responses to Endothelium-Dependent Vasodilators
ACh (10 µmol/L; Sigma), BK (50 µmol/L; Sigma), or the calcium ionophore A23187 (3 µmol/L; Sigma) were superfused topically for 5 minutes and CBF responses were recorded. In some mice, the CBF increase produced by the NO donor S-nitroso-N-acetylpenicillamine (SNAP; 50 µmol/L; Sigma) was also examined. After testing CBF responses, Ang II (Ang II acetate; Sigma) or vehicle (saline) were administrated intravenously. The Ang II infusion was adjusted to elevate MAP by 20 to 25 mm Hg gradually over 10 to 15 minutes until a stable increase was obtained. At this time, the infusion rate was 0.25±0.02 µg/kg per minute, which results in high physiological levels of circulating Ang II.^10,18 The CBF response to ACh, BK, A231987, or SNAP was tested again after 30 to 45 minutes of Ang II infusion. CBF responses were also tested in gp91^–/– mice before and after intravenous infusion of Ang II. In some experiments, Ang II or vehicle (Ringer) was topically applied to the neocortex at a concentration (50 nmol) that does not alter resting CBF¹⁰ and CBF responses tested 30 minutes later.

Effect of Losartan or MnTBAP
In separate mice, the effect of the AT1 receptor inhibitor losartan (5 µmol/L), or the free radical scavenger Mn(III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) (100 µmol/L) was tested. CBF responses were first examined during superfusion Ringer’s solution. Then, the superfusion was switched to Ringer containing losartan or MnTBAP and the infusion of Ang II or vehicle was started 5 minutes later. CBF responses were tested again 30 to 45 minutes later.

Effect of the NADPH Oxidase Peptide Inhibitor gp91ds-tat
The NADPH oxidase peptide inhibitor gp91ds-tat²⁸ (YGRKKRRQRRRCSTRIRRQL-NH₂; Bio·Synthesis, Lewisville, Tex), which includes the HIV tat sequence to facilitate intracellular penetration, was used as previously described.^20,21 A peptide in which the active sequence was scrambled (YGRKKRRQRRRCLRITRQSR-NH₂) served as control. The effect of Ang II on CBF responses was tested with or without superfusion with gp91ds-tat (1 µmol/L) or the scrambled peptide (1 µmol/L).

Assessment of ROS Production by HE
The protocol for these experiments was identical to those in which the effect of Ang II infusion on CBF was studied. Ringer’s solution containing HE alone or HE plus losartan (5 µmol/L), MnTBAP (100 µmol/L), gp91ds-tat (1 µmol/L), or the scrambled peptide (1 µmol/L) was superfused for 30 minutes, followed by intravenous infusion of Ang II or vehicle. In some experiments, the SOD inhibitor diethyldithiocarbamate (DDC) (10 mmol/L; Sigma)²⁹ or the glutamate receptor agonist N-methyl-D-aspartic acid (NMDA) (40 µmol/L; Sigma) were superfused with HE for 30 minutes without Ang II. At the end of superfusion brains were removed and processed for ROS assessment as described.

Data Analysis
Data are expressed as mean±SEM. Two-group comparisons were analyzed by the 2-tailed t test. Multiple comparisons were evaluated by ANOVA and Tukey or Dunnet tests, as indicated. Statistical significance was considered for P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ang II Attenuates Endothelium-Dependent Responses of the Cerebral Circulation Via AT1 Receptors
We examined whether systemic administration of Ang II attenuates the increase in CBF produced by endothelium-dependent vasodilators or the NO donor SNAP. With vehicle (saline) infusion, neocortical application of ACh, BK, and A23187 increased CBF (Figure 1). Infusion of Ang II increased MAP but did not reduce resting CBF (Figure 1A and 1B; P>0.05; n=5 to 6 per group). However, the CBF response to ACh, BK and A23187 was markedly attenuated (Figure 1C, 1D, and 1E; P<0.05). In contrast, Ang II did not attenuate the increase in CBF produced by SNAP (Figure 1F; P>0.05). Topical application of losartan did not affect resting CBF or the increase in MAP induced by systemic administration of Ang II (Figure 1A and 1B; P>0.05), but it prevented the attenuation in endothelium-dependent responses (Figure 1C, 1D, and 1E). Losartan did not affect the increase in CBF-produced SNAP (Figure 1F; P>0.05). Topical application of Ang II (50 nmol) produced cerebrovascular effects comparable to those of systemic administration (Figure I, available online at http://atvb.ahajournals.org).

View larger version (27K): [in this window] [in a new window]   Figure 1. Effect of intravenous infusion of Ang II with and without neocortical superfusion of losartan (5 µmol/L) on MAP (A), resting CBF (B), and on the increase in CBF produced by ACh (10 µmol/L) (C), BK (50 µmol/L) (D), A23187 (3 µmol/L) (E), and SNAP (50 µmol/L)(F). *P<0.05 from saline; analysis of variance and Tukey test; n=5 to 6 per group.

Effect of Ang II on Endothelium-Dependent Responses Is Attenuated by MnTBAP
We next used the ROS scavenger MnTBAP to determine whether the cerebrovascular effects of Ang II are mediated by ROS. Topical application of MnTBAP did not affect the increase in MAP produced by Ang II or resting CBF (Figure 2A and 2B; P>0.05; n=5 to 6 per group). However, MnTBAP blocked the attenuation by Ang II of CBF responses to ACh, BK, and A23187 (Figure 2C, 2D, and 2E). MnTBAP did not alter the CBF response to SNAP (Figure 2F; P>0.05).

View larger version (26K): [in this window] [in a new window]   Figure 2. Effect of intravenous infusion of Ang II with and without neocortical superfusion of MnTBAP (100 µmol/L) on MAP (A), resting CBF (B), and on the increase in CBF produced by ACh (C), BK (D), A23187 (E), and SNAP (F). *P<0.05 from saline; analysis of variance and Tukey test; n=5 to 6 per group.

Effect of Ang II on Endothelium-Dependent Responses Is Attenuated by gp91ds-tat
We then used a peptide inhibitor of NAPDH oxidase (gp91ds-tat)²⁸ to provide evidence that the effect of Ang II on endothelium-dependent responses was mediated by ROS derived from NADPH oxidase. Application of gp91ds-tat or the scrambled peptide did not affect the increase in MAP produced by Ang II or resting CBF (Figure 3A and 3B; P>0.05; n=5 per group). However, gp91ds-tat, but not the scrambled peptide, prevented the effect of Ang II on endothelium-dependent responses (Figure 3C, 3D, and 3E). The peptides had no effect on the increase in CBF produced by SNAP (Figure 3F; P>0.05).

View larger version (27K): [in this window] [in a new window]   Figure 3. Effect of intravenous infusion of Ang II with and without neocortical superfusion of gp91ds-tat (gp91ds; 1 µmol/L) or its scrambled control (scr gp91ds; 1 µmol/L) on MAP (A), resting CBF (B), and on the increase in CBF produced by ACh (C), BK (D), A23187 (E), and SNAP (F). *P<0.05 from saline; analysis of variance and Tukey test; n=5 per group.

Effect of Ang II on Endothelium-Dependent Responses Is Not Observed in gp91^phox Null Mice
To provide further evidence that the effects of Ang II are mediated by NADPH oxidase, we used mice lacking the gp91^phox (nox-2) subunit of the enzyme.¹⁹ MAP and cerebrovascular responses to ACh, BK, A23187, and SNAP did not differ between gp91^+/+ and gp91^–/– mice (Figure 4; P>0.05; n=5 to 6 per group). However, Ang II did not attenuate endothelium-dependent responses in gp91^–/– mice (Figure 4B, 4C, 4D; P>0.05 from saline). Responses to SNAP were also not affected (Figure 4F).

View larger version (23K): [in this window] [in a new window]   Figure 4. Effect of intravenous infusion of Ang II on MAP (A), and on the increase in CBF produced by ACh (B), BK (C), A23187 (D), and SNAP (E) in wild-type mice (gp91+/+) or mice lacking gp91phox (gp91–/–). *P<0.05 from saline; analysis of variance and Tukey test; n=5 per group.

Systemic Administration of Ang II Increases ROS in Cerebral Blood Vessels
We used in situ HE microfluorography to determine whether systemic administration of Ang II increases ROS production in cerebral blood vessels. To identify blood vessels, brain sections of mice treated with HE (n=3 per group) were immunostained with the endothelial marker CD31. Administration of saline did not alter the fluorescent signal in the neocortex (Figure 5A). Ang II increased the ROS signal in cells that also stained positive for CD31, attesting to their endothelial nature (Figure 5A). As a control, we superfused the cortex with the glutamate receptor agonist NMDA, which is well known to increase ROS production in neurons.³⁰ NMDA induced an increase in ROS signal in cells that did not express CD31, most likely neurons, as well as in cerebrovascular cells (Figure 5A).

View larger version (24K): [in this window] [in a new window]   Figure 5. A, Effect of intravenous infusion of Ang II or topical neocortical application of NMDA (40 µmol/L) on ROS production assessed by in situ HE microfluorography. We used the endothelial marker CD31 to identify the cell type producing ROS. The brain surface is at the top of each panel. Large arrows indicate CD31-positive pial vessels displaying ROS signal. Small arrows in upper and middle panels indicate CD31-positive capillaries also displaying ROS signal. The bottom panels show NMDA-induced ROS production in CD31-positive vascular cells (large arrow) and in non-CD31-positive cells (asterisk), most likely neurons. B, Effect of intravenous infusion of Ang II on ROS production assessed by HE microfluorography. 1, saline infusion control; 2, Ang II infusion; 3, Ang II+losartan (5 µmol/L); 4, Ang II+MnTBAP (100 µmol/L); 5, Ang II+scrambled gp91ds-tat (1 µmol/L); 6, Ang II+gp91ds-tat (1 µmol/L); 7, Ang II in gp91–/– mice. RFU indicates relative fluorescence units; *P<0.05 from saline; analysis of variance and Dunnet test. C, Effect of the SOD inhibitor DDC (10 mmol/L) on ROS production in gp91+/+ and gp91–/– mice.

The Increase in ROS-Induced by Ang II Is Blocked by Losartan, MnTBAP, gp91ds-tat, and Was Not Observed in gp91^–/– Mice
The increase in ROS signal produced by Ang II (Figure 5A and 5B) was blocked by topical superfusion of the AT1 receptor antagonist losartan or by the free radical scavenger MnTBAP (Figure 5B; P>0.05 from vehicle; n=5 per group). Furthermore, the increase in ROS was blocked by gp91ds-tat, but not by its scrambled version (figure 5B; P>0.05 from vehicle; n=5 per group). Ang II did not increase ROS production in gp91^–/– mice (Figure 5B; P>0.05 from vehicle; n=5 per group). In contrast to Ang II, the increase in ROS signal produced by topical superfusion of the SOD inhibitor DDC was not attenuated in gp91^–/– mice (Figure 5C; P>0.05 from gp91^+/+ mice; n=5 per group). Finally, to verify whether NADPH oxidase subunits are expressed in neocortical microvessels we used real-time PCR. In agreement with previous reports,^18,31,32 we found that nox-1, nox-2, nox-4, p22, p47, and p67 are present in neocortical microvessels (Table II and Figure II, available online at http://atvb.ahajournals.org).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have demonstrated that systemic administration of Ang II attenuates the increase in CBF produced by neocortical application of the endothelium-dependent vasodilators ACh, BK, and A23187. The attenuation is specific for endothelium-dependent responses because the increase in CBF elicited by the NO donor SNAP was not affected. The attenuation by Ang II of endothelium-dependent responses was blocked by the AT1 receptor inhibitor losartan and by the ROS scavenger MnTBAP, indicating that the effect of Ang II is mediated by AT1 receptors via production of ROS. This conclusion is reinforced by our finding that Ang II increases ROS production in cerebrovascular cells, an effect also blocked by losartan and MnTBAP. We then examined the source of cerebrovascular oxidative stress induced by Ang II. We found that the effect of Ang II on endothelium-dependent responses and ROS was blocked by the NADPH oxidase peptide inhibitor gp91ds-tat, indicating an involvement of NADPH oxidase. Furthermore, Ang II failed to impair endothelium-dependent responses and to increase ROS in gp91^–/– mice. These findings, collectively, provide the first demonstration that systemic administration of Ang II attenuates endothelium-dependent responses by inducing cerebrovascular oxidative stress through activation of nox-2.

The findings of the present study cannot result from differences in MAP and blood gases, because these parameters were carefully controlled and did not differ among groups. We used in situ HE microfluorography to assess ROS production. This method was selected because it can be applied in vivo, provides cellular resolution and can be effectively combined with immunohistochemistry to identify the cell type in which ROS are produced. The validity of HE to detect ROS in our preparation was previously demonstrated¹⁸ and is confirmed by our observation that the increase in signal was blocked by MnTBAP and by inhibition of NADPH oxidase. Furthermore, the ROS signal was not observed in gp91^–/– mice treated with Ang II, but it was present when oxidative stress was induced with the SOD inhibitor DDC. This observation not only attests to the validity of HE to assess in vivo ROS production, but also rules out that the lack of ROS production in gp91^–/– mice is because of a generalized failure of ROS generation.

We found that systemic administration of Ang II increases ROS production in cerebrovascular cells. The ROS increase is blocked by losartan or gp91ds-tat and is not observed in gp91^phox null mice. It is therefore likely that Ang II activates AT1 receptors on endothelial cells leading to NADPH oxidase-dependent ROS production in these cells. This conclusion is supported by our previous observation that mice pial arterioles are endowed with AT1 receptors and gp91^phox(nox-2).¹⁸ However, it is unclear whether the Ang II-induced increase in ROS occurs also in the adventitia, where AT1 receptors and gp91^phox are also present.¹⁸ Similarly, although we cannot rule out ROS production in smooth muscle cells, this possibility seem unlikely because we did not observe AT1 receptors and nox-2 in smooth muscle cells of cerebral arterioles.¹⁸ In contrast to Ang II, neocortical superfusion with the glutamate receptor agonist NMDA increases ROS production in brain parenchyma and cerebral blood vessels. Although it is well established that activation of NMDA receptors in neurons leads to ROS production,³⁰ it had not been previously reported that NMDA increases cerebrovascular ROS production as well. The mechanisms of this effect remain to be established. Because endothelial cells probably do not have NMDA receptors,³³ direct effects of NMDA on endothelial cells are unlikely. Superoxide, the main radical detected by the HE microfluorography, does not cross cell membranes easily.²⁴ Therefore, it does not seem plausible that superoxide diffuses from neurons to blood vessels to induce oxidative stress. However, H₂O₂, formed by the dismutation of superoxide, could diffuse out of the cell and generate superoxide in vessels via the Haber-Weiss reaction²⁴ or by activating NADPH oxidase.³⁴ The mechanisms of vascular ROS production by NMDA and their functional role in vessels remain to be defined.

The mechanisms of the attenuation of endothelium-dependent responses by Ang II have not been established. The CBF response to ACh is mediated by NO, whereas responses to BK and A23187 are mediated by COX-1–derived ROS.²² Therefore, Ang II-induced oxidative stress is likely to act via multiple factors. These may include reduction of NO bioavailability and/or peroxynitrite-mediated activation of the DNA repair enzyme poly(ADP)ribose polymerase in vascular cells, which leads to dysfunction through energy depletion.³⁵ However, the attenuation by oxidative stress of ROS-mediated responses, such as BK and A23187, is more difficult to explain. Attenuation of responses to BK and A23187 is also observed in the cerebrovascular dysfunction produced by ß-amyloid (Aß), the peptide that accumulates in the brain of patients with Alzheimer disease and produces oxidative stress.^21,36,37 Perhaps the ROS induced by Ang II or Aß are able to counteract the vasodilatatory effects of COX-1–derived ROS, but experimental evidence supporting this hypothesis is lacking.

Ang II also impairs the mechanisms coupling neural activity to CBF. Ang II infusion attenuates the increase in somatosensory cortex blood flow produced by stimulation of the facial whiskers.¹⁰ Like the impairment of endothelium-dependent responses, the attenuation of functional hyperemia by Ang II is mediated by NADPH oxidase-derived ROS.¹⁸ The findings of the present study, in concert with these previous observations, indicate that Ang II exerts widespread effects on the regulation of cerebral blood vessels. However, Ang II does not impair all aspects of cerebrovascular regulation, because the CBF increase induced by hypercapnia is not affected.¹⁰ The effects of Ang II on cerebrovascular regulation are similar to those of Aß. Aß attenuates endothelium-dependent responses and functional hyperemia without reducing responses to hypercapnia.^36–38 Both Ang II and Aß act on cerebral blood vessels via NADPH oxidase-derived radicals (present study).²¹ Furthermore, Ang II, like Aß, increases the susceptibility of the brain to cerebral ischemia.^6,39 The similarities between the cerebrovascular effects of Ang II and Aß support the hypothesis that vascular oxidative stress is a final common pathway in the brain dysfunction associated with hypertension and Alzheimer disease.³⁵

In conclusion, we have demonstrated that systemic administration of Ang II induces cerebrovascular oxidative stress and attenuates endothelium-dependent responses in the cerebral circulation. The effect is blocked by the AT1 receptor inhibitor losartan, the free radical scavenger MnTBAP, the NADPH oxidase peptide inhibitor gp91ds-tat, and is not observed in mice lacking the NADPH oxidase subunit nox-2. These data provide evidence that Ang II impairs cerebrovascular reactivity by disrupting the endothelial regulation of the cerebral circulation through AT1 receptor-mediated activation of nox-2 in cerebrovascular cells. The findings support the concept that cerebrovascular oxidative stress mediates the powerful effects of Ang II on the cerebral circulation, which may contribute to the susceptibility to ischemic injury and dementia associated with hypertension.

	Acknowledgments

 
This work was supported by NIH grant HL18974. Losartan was a generous gift of Merck and DuPont.

Received October 14, 2005; accepted January 13, 2006.

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