Increased NADPH-Oxidase Activity and Nox4 Expression During Chronic Hypertension Is Associated With Enhanced Cerebral Vasodilatation to NADPH In Vivo
Background and Purpose— We examined the importance of NADPH-oxidase in reactive oxygen species production in cerebral arteries and its effect on vascular tone in vivo. Furthermore, we investigated whether chronic hypertension affects function or expression of this enzyme in cerebral vessels.
Methods— Superoxide generation was detected in isolated rat basilar arteries with the use of lucigenin-enhanced chemiluminescence. mRNA expression of NADPH-oxidase subunits was assessed by real-time polymerase chain reaction. Basilar artery diameter was measured with the use of a cranial window preparation in anesthetized rats.
Results— NADPH-stimulated superoxide production was 2.3-fold higher in arteries from spontaneously hypertensive rats (SHR) versus normotensive Wistar-Kyoto rats (WKY) and could be blocked by the NADPH-oxidase inhibitor diphenyleneiodonium. Higher NADPH-oxidase activity was also reflected at the molecular level as mRNA expression of the NADPH-oxidase subunit Nox4 was 4.1-fold higher in basilar arteries from SHR versus WKY. In contrast, expression of Nox1, gp91phox, p22phox, and p47phox did not differ between strains. Application of NADPH to basilar arteries caused larger vasodilatation in SHR than WKY. Vasodilatation to NADPH could be attenuated by diphenyleneiodonium, as well as diethyldithiocarbamate (Cu2+/Zn2+–superoxide dismutase inhibitor), catalase (H2O2 scavenger), or tetraethylammonium (BKCa channel inhibitor).
Conclusions— Activation of NADPH-oxidase in cerebral arteries generates superoxide, which is dismutated by Cu2+/Zn2+–superoxide dismutase to H2O2. H2O2 then elicits vasodilatation via activation of BKCa channels. Upregulation of Nox4 during chronic hypertension is associated with elevated cerebral artery NADPH-oxidase activity.
Reactive oxygen species (ROS) such as superoxide (O2−·) and hydrogen peroxide (H2O2) play important roles in regulating normal vascular function.1,2 Although ROS generally cause an increase in vascular tone via inactivation of endothelium-derived nitric oxide (NO), in cerebral microvessels ROS can decrease artery tone under physiological conditions.3 Specifically, H2O2 has been shown to activate large-conductance calcium-activated K+ (BKCa) channels in cerebral arteries, leading to hyperpolarization and vasodilatation.4 Chronic hypertension is associated with a number of alterations to cerebral artery function and structure5 and increased vascular ROS generation in systemic blood vessels.6 However, there is presently no information regarding whether ROS levels are higher in cerebral vessels during hypertension and, if so, how this may affect regulation of cerebrovascular tone.
NADPH-oxidases are a major enzymatic source of ROS in the systemic vasculature and are present in all major cell types that make up the vessel wall (ie, endothelial cells, smooth muscle cells, and adventitial fibroblasts).7 These enzymes transfer electrons to molecular O2 via a flavin-containing subunit, the identity of which varies between isoforms. In different types of vascular cells, gp91phox and the homologues Nox1 and Nox4 have been identified as electron-transferring subunits.8 In cerebral arteries, NADPH- and NADH-induced production of ROS are reported to be mediated by a flavin-containing enzyme, most likely NADPH-oxidase.9 Moreover, activation of this enzyme was associated with dilatation of cerebral vessels in vitro and in vivo.9 There is no information available on the role of this enzyme in cerebral vessels during pathophysiological conditions. The present study tested the hypothesis that activation of NADPH-oxidase in cerebral blood vessels causes H2O2-mediated opening of BKCa channels, leading to cerebral vasodilatation in vivo. Furthermore, we examined whether chronic hypertension affects the activity, function, and molecular expression of NADPH-oxidase in cerebral arteries.
Materials and Methods
All procedures were approved by the institutional animal ethics committee. In total, 80 male Wistar-Kyoto rats (WKY) (weight, 332±12 g; mean arterial pressure=92±2 mm Hg) and 54 male spontaneously hypertensive rats (SHR) (weight, 460±22 g; mean arterial pressure=165±6 mm Hg) up to 10 months old were studied. In specific experimental subgroups in which comparisons were made between WKY and SHR, rats from the 2 strains were age matched.
Measurement of O2−· Production by Isolated Cerebral Arteries
WKY (n=9) and SHR (n=11) were anesthetized by inhalation of 80% CO2/20% O2 and killed by decapitation. Basilar arteries were excised and cut into two 5-mm ring segments, and O2−· production was then measured by 5 μmol/L lucigenin-enhanced chemiluminescence as described previously.10
Measurement of Cerebral Artery Diameter In Vivo
WKY (n=47) and SHR (n=19) were anesthetized with pentobarbital sodium (50 mg/kg IP). Supplemental pentobarbital was administered via a femoral vein cannula (10 to 20 mg/kg per hour). Blood gases were monitored and adjusted (pH 7.39±0.01, Pco2=37±1 mm Hg, Po2=162±12 mm Hg) throughout each experiment. As described previously,11 a craniotomy was performed over the ventral brain stem, the basilar artery was exposed, and the cranial window was superfused with artificial cerebrospinal fluid at 2 mL/min. Cerebrospinal fluid sampled from the cranial window had the following pH and gases: pH 7.44±0.01, Pco2=31±1 mm Hg, Po2=136±1 mm Hg. Basilar artery diameter was monitored with a microscope coupled to a video monitor and measured with a computer-based tracking program (Diamtrak, Montech).
In Vivo Protocol
After surgical preparation, a 20-minute stabilization period was allowed before topical application of drugs. Artery diameter was measured under baseline conditions and again when responses to drugs were stable. A 15- to 20-minute period was allowed between applications of vasodilators. Vascular responses were expressed as percent change in artery diameter compared with baseline.
Responses of the basilar artery to the following agonists were investigated: NADPH (10 and 100 μmol/L), H2O2 (100 and 300 μmol/L), sodium nitroprusside (SNP) (10 and 100 nmol/L), papaverine (10 and 100 μmol/L), and aprikalim (10 and 30 μmol/L). Responses were examined in the absence and presence of diphenyleneiodonium (DPI) (5 μmol/L), catalase (1000 U/mL), diethyldithiocarbamate (DETCA) (3 mmol/L), or tetraethylammonium (TEA) (1 mmol/L).
In all experiments, control responses to each agonist were obtained before topical application of inhibitor drugs. After 20-minute treatment with 1 of these inhibitors, responses to vasoactive agents were reexamined in the continued presence of the inhibitor. Only 1 inhibitor was investigated per animal.
Reverse Transcription Reaction
For each mRNA sample, basilar arteries were pooled from 3 WKY or 3 SHR, and total RNA was extracted with the use of a commercially available kit (SV Total RNA Isolation System, Promega) and quantified spectrophotometrically (absorbance at 260 nm). Total RNA was subsequently used for reverse transcription (RT) in a final reaction volume of 100 μL as described previously.10
Real-Time Polymerase Chain Reaction
Real-time polymerase chain reaction (PCR) and the ΔΔCt method were used as previously described to examine mRNA expression of the NADPH-oxidase subunits (gp91phox, Nox1, Nox4, p22phox, p47phox) and Cu2+/Zn2+–superoxide dismutase (SOD), relative to a “reference” sample, in basilar arteries from WKY and SHR.10,12 Primers and probes for target genes were designed with the use of Primer Express software (PE Biosystems) and the published mRNA sequences (Table, available online at http://stroke.ahajournals.org). 18S rRNA was used as an internal standard and was measured with the use of commercially available primers and probe (PE Biosystems; Table, available online at http://stroke.ahajournals.org). Probes were labeled at the 5′ end with the fluorescent dyes FAM (for target genes) and VIC (for 18S) and at the 3′ end with the quencher molecule TAMRA.12
H2O2 was obtained from Merck, and all other drugs were from Sigma. DPI was prepared at 10 mmol/L in dimethyl sulfoxide and diluted in saline (in vivo experiments) or Krebs-HEPES (in vitro experiments) such that the final concentration of dimethyl sulfoxide was ≤0.05%. All other drugs were dissolved and diluted in saline or Krebs-HEPES as appropriate.
All results are expressed as mean±SEM. Statistical comparisons were made with the use of Student’s paired or unpaired t tests or ANOVA as appropriate. A value of P<0.05 was considered significant.
Basilar Artery O2−· Production by NADPH-Oxidase
O2−· was not detected under basal conditions in basilar arteries from either WKY (aged 26±4 weeks; n=9) or SHR (aged 26±3 weeks; n=11) (Figure 1a). Treatment with NADPH increased O2−· production to a similar extent in each strain (Figure 1a). To determine whether any differences in cerebral artery O2−· production between the 2 strains were being masked by endogenous Cu2+/Zn2+-SOD activity, arteries were treated with the Cu2+-chelating agent DETCA (3 mmol/L). Although we were still unable to detect a chemiluminescent signal in arteries from WKY, DETCA treatment unmasked a small O2−· signal in rings from SHR (Figure 1b). Furthermore, in DETCA-treated SHR arteries, NADPH-stimulated O2−· production was 2.3-fold higher than in similarly treated WKY rings (Figure 1b; n=8 to 9; P<0.05). The NADPH-oxidase inhibitor DPI abolished NADPH/DETCA-stimulated O2−· generation by basilar arteries from both WKY and SHR (Figure 1b), suggesting that NADPH-oxidase was the primary source of cerebral vessel O2−· in both strains.
mRNA Expression of NADPH-Oxidase Subunits and Cu2+/Zn2+-SOD
To determine whether increased NADPH-oxidase activity was reflected at the molecular level, real-time PCR was used to compare mRNA expression of the NADPH-oxidase subunits gp91phox, Nox1, Nox4, p22phox, and p47phox in basilar arteries from WKY (aged 30±1 weeks) and SHR (aged 30±1 weeks). Relative expression of Nox4 was 4.1-fold higher in SHR (ΔCt=19.41±0.25) compared with WKY (ΔCt=21.45±0.26; n=4; Figure 2a). In contrast, there were no differences in relative expression of other NADPH-oxidase subunits (each n=4 from 12 rats; Figure 2b to 2e), nor was there any change in expression of Cu2+/Zn2+-SOD (Figure 2f).
Cerebral Vasodilatation In Vivo
NADPH (10 and 100 μmol/L) elicited concentration-dependent dilatation of the basilar artery in WKY (Figure 3a and 3b). In most animals (approximately 70%), NADPH elicited a transient peak dilatation at either or both concentrations, followed by a sustained steady state response (Figure 3a). Peak responses to 10 and 100 μmol/L NADPH were 16±4% (n=10) and 29±3% (n=23), respectively. However, since this peak response did not occur in all animals, we have focused on the steady state responses that were reproducible within the same animal (time control steady state responses to 10 and 100 μmol/L NADPH in WKY: first=8±2% and 17±6%; second=8±1% and 14±1%; n=4). Overall, steady state responses to 10 and 100 μmol/L NADPH in WKY were 7±1% and 13±1% (n=30), respectively.
NADPH-Induced Vasodilatation Is Enhanced in Hypertension
NADPH-induced vasodilatation was approximately 70% greater in age-matched SHR (24±2 weeks; n=9) than WKY (24±1 weeks; n=18; Figure 3b). This enhancement was selective for NADPH since responses to SNP were similar in WKY and SHR (Figure 3c).
NADPH-Induced Vasodilation Is Mediated by NADPH-Oxidase, H2O2, and BKCa Channels
We performed further studies to elucidate features of the mechanism of NADPH-induced vasodilatation. Treatment of the cranial window with DPI or catalase had no significant effect on baseline artery diameter in WKY (−0.7±2.5% [n=7] and 2.6±4.2% [n=8], respectively). However, both DPI and catalase caused a substantial constriction in SHR (−8.4±1.6% [n=6] and −8.7±2.3% [n=5], respectively; both P<0.05). DPI inhibited NADPH-induced vasodilator responses by approximately 50% in both WKY (Figure 4a) and SHR (not shown), without affecting responses to a nonspecific vasodilator, papaverine (Figure 4b), or an endothelial NO-dependent vasodilator, acetylcholine (Figure 4c). Combined treatment of the cranial window with N-nitro-l-arginine methyl ester (30 μmol/L) and indomethacin (10 μmol/L) had no effect on responses to NADPH but abolished vasodilatation to acetylcholine (n=6; data not shown), confirming no contribution of NO synthase or cyclooxygenase to the response to NADPH.
We next tested whether vasodilator responses to NADPH may be mediated by endogenous H2O2. First, we established that exogenous H2O2 elicits concentration-dependent dilatation of the basilar artery, which was reproducible within the same animal (time control responses to 100 and 300 μmol/L H2O2 in WKY: first=9±3% and 31±12%; second=9±6% and 31±7%; n=3). Second, we found that catalase (1000 U/mL) inhibited vasodilatation to both NADPH (Figure 5a) and H2O2 (Figure 5b) without altering responses to SNP (Figure 5c), suggesting that NADPH-induced vasodilatation is mediated at least in part by NADPH-oxidase–derived H2O2. Catalase also inhibited NADPH-induced responses by approximately 50% in SHR (not shown). Finally, inactivation of Cu2+/Zn2+-SOD with DETCA (3 mmol/L) also inhibited NADPH-induced vasodilatation (Figure 6a), whereas vasodilator responses to exogenous H2O2 were unaltered (Figure 6b).
Inhibition of BKCa channels with 1 mmol/L TEA attenuated responses to NADPH (eg, response to 100 μmol/L NADPH: control=12±2%; TEA-treated=5±2%; n=6; P<0.05) and also H2O2 (eg, response to 300 μmol/L H2O2: control=38±6%; TEA-treated=19±7%; n=6; P<0.05) without affecting responses to the ATP-sensitive K+ (KATP) channel opener aprikalim (not shown). Neither catalase, DETCA, nor TEA altered the baseline diameter of the WKY basilar artery.
ROS are established to elicit cerebral vasodilatation under both normal and disease conditions.3 This study demonstrates that activation of NADPH-oxidase in cerebral arteries in vivo causes generation of O2−·, which is converted to H2O2 by Cu2+/Zn2+-SOD. H2O2 then elicits vasodilatation via opening of BKCa channels in vascular smooth muscle cells. Importantly, Nox4 mRNA expression was 4.1-fold higher in basilar arteries from SHR versus WKY, and this was associated with greater NADPH-oxidase–dependent O2−· production and vasodilatation in SHR. To our knowledge this is the first study to demonstrate a functional consequence of increased vascular expression and activity of a Nox4-containing NADPH-oxidase in chronic hypertension.
NADPH-Oxidase Activity and Expression in Cerebral Vessels
It is well established that NADPH-oxidase is a major source of ROS in the systemic vasculature and that its activity is upregulated in pathophysiological states such as hypertension.7 Although we were unable to detect O2−· in isolated basilar arteries from WKY under basal conditions, ex vivo treatment with NADPH caused a marked increase in O2−· generation. Furthermore, NADPH-stimulated O2−· production was inhibited by DPI, indicating an involvement of NADPH-oxidase. Messenger RNA for Nox4, gp91phox, Nox1, p22phox, and p47phox was found to be present in the WKY basilar artery. Thus, together with recent findings by others,9 there is now strong evidence that NADPH-oxidase is expressed and functional in cerebral blood vessels. Moreover, our present findings strongly suggest that activity of NADPH-oxidase is augmented in cerebral arteries during chronic hypertension. Inactivation of endogenous Cu2+/Zn2+-SOD with DETCA not only unmasked a small basal O2−· signal in basilar arteries from some SHR but also revealed that NADPH-stimulated O2−· production was 2.3-fold higher in these tissues than in those from age-matched WKY.
Our finding that higher O2−· production in cerebral arteries from SHR versus WKY was only observed after DETCA treatment suggests that Cu2+/Zn2+-SOD adequately disposes of the excess O2−· produced during hypertension. However, this is not to say that levels of ROS downstream from O2−· remain unaltered. Dismutation of O2−· by SOD yields H2O2, a membrane-permeable ROS that elicits a range of cellular responses. Although H2O2 is normally rapidly metabolized by enzyme systems such as catalase and glutathione peroxidase, increased dismutation of O2−· in the absence of compensatory catalase and/or peroxidase activity will lead to higher levels of H2O2 in the cerebral vasculature during hypertension. Indeed, a recent clinical study provided direct evidence that activities of catalase and glutathione peroxidase in whole blood are reduced in hypertensive patients,13 which probably contributes to the elevated plasma H2O2 levels in hypertensive patients compared with normotensive controls.14 Because H2O2 is a powerful cerebral vasodilator, elevated NADPH-oxidase–dependent O2−· production in the presence of normal Cu2+/Zn2+-SOD expression and activity may therefore have important consequences for cerebral vascular tone.
Functional Consequences of NADPH-Oxidase Activity in Cerebral Arteries
In the systemic vasculature, ROS generally cause vasoconstriction, mainly via the O2−·-mediated inactivation of endothelial NO.15,16 In contrast, a number of ROS have been reported to dilate cerebral arteries.3 In particular, both exogenous and endogenous H2O2 dilate rat cerebral arterioles in vivo via activation of BKCa channels.4,17 Having first confirmed that exogenous H2O2 similarly dilates the rat basilar artery, we tested whether activation of NADPH-oxidase elicits vasodilatation and, if so, whether this effect is ultimately mediated via the generation of H2O2 and activation of BKCa channels. In support of this proposal, NADPH elicited dilator responses of the basilar artery in vivo that were sensitive to treatment with the NADPH-oxidase inhibitor DPI at a concentration that did not affect responses to the NO synthase–dependent vasodilator acetylcholine. Vasodilator responses to NADPH were also inhibited by the H2O2 scavenger catalase and the inhibitor of BKCa channels TEA. Furthermore, NADPH-induced vasodilatation could also be inhibited by the Cu2+/Zn2+-SOD inhibitor DETCA, suggesting that SOD activity is required to generate H2O2 at a sufficient rate from NADPH-oxidase–derived O2−· to enable accumulation of H2O2 and decreased vascular tone. Interestingly, none of these inhibitor treatments had any significant effect on baseline diameter of the normotensive WKY basilar artery, suggesting that NADPH-oxidase activation may not play a major role in regulating basal cerebral artery tone under physiological conditions.
Chronic hypertension is a major risk factor for stroke. We found that vasodilatation in response to NADPH was significantly greater in chronically hypertensive rats than in age-matched normotensive controls. It is unclear what consequences (beneficial or detrimental) higher activity of NADPH-oxidase may have for the regulation of cerebral vascular tone during hypertension. However, ROS-mediated vasodilatation, which appears to represent a physiological mechanism in cerebral vessels during normotension,4,17 could be beneficial in hypertension by maintaining cerebral blood flow, particularly during cerebral ischemic episodes common in stroke. Evidence from the present study consistent with such a protective effect is that DPI and catalase each constricted the basilar artery of SHR but not WKY. In further support of a protective role for NADPH-oxidase in the cerebral circulation is the recent finding that in a Japanese population, the C242T polymorphism of p22phox is a novel risk factor for ischemic cerebrovascular disease.18 In a separate study, Guzik et al19 found that the C242T polymorphism is independently associated with decreased NADPH-oxidase activity. Taken together, our findings and these recent epidemiological data are compatible with an important protective role for NADPH-oxidase in the cerebral circulation.
Increased Cerebral Artery Nox4 Expression in Hypertension
To shed some light on which isoform of NADPH-oxidase is involved in augmented cerebrovascular O2−· production in hypertension, we measured mRNA expression of gp91phox and the putative vascular homologues of this catalytic subunit, Nox1 and Nox4. Interestingly, while levels of gp91phox and Nox1 were not different, expression of Nox4 was markedly higher in basilar arteries from SHR. This finding is somewhat at odds with previous studies examining the effects of pathophysiological stimuli on Nox4 expression. For example, in vitro treatment of rat aortic smooth muscle cells with angiotensin II downregulates Nox4 expression,8 while in aortas of renin-overexpressing20 or angiotensin II–treated21 hypertensive rats, Nox4 mRNA levels were only modestly higher (ie, <70%) than in normotensive controls. Indeed, we too found that aortic expression of Nox4 was unaltered in SHR (not shown), which may suggest that this subunit is selectively upregulated in certain vascular beds under pathophysiological conditions to act as a protective mechanism against reduced blood flow.
In summary, this study demonstrates that activation of NADPH-oxidase in cerebral arteries in vivo causes vasodilatation via the generation of H2O2 and opening of BKCa channels. Importantly, during chronic hypertension NADPH-oxidase activity is elevated in cerebral vessels, and this is presumably the result of increased expression of the catalytic subunit Nox4. The functional consequence of this is enhanced vasodilator responses on activation of the enzyme in vivo. It is presently unclear whether the consequences of increased NADPH-oxidase activity in the cerebral circulation are likely to be mainly beneficial or detrimental. However, if it were beneficial, caution should be taken in future attempts to inhibit effects of ROS in systemic vascular diseases so as not to inadvertently compromise cerebral blood flow.
This work was supported by funds from a grant-in-aid (G00M0659) from the National Heart Foundation of Australia and a project grant (208969) from the National Health and Medical Research Council of Australia (NHMRC). Dr Sobey is supported by a NHMRC R.D. Wright career development award (209160). Dr Drummond is supported by a NHMRC Peter Doherty postdoctoral fellowship (007044). T. Paravicini and S. Chrissobolis are supported by Australian postgraduate awards.
- Received August 4, 2003.
- Revision received October 20, 2003.
- Accepted October 24, 2003.
Irani K. Oxidant signaling in vascular cell growth, death, and survival: a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptotic signaling. Circ Res. 2000; 87: 179–183.
Wolin MS. Interactions of oxidants with vascular signaling systems. Arterioscler Thromb Vasc Biol. 2000; 20: 1430–1442.
Sobey CG, Heistad DD, Faraci FM. Mechanisms of bradykinin-induced cerebral vasodilatation in rats: evidence that reactive oxygen species activate K+ channels. Stroke. 1997; 28: 2290–2294.
Faraci FM, Heistad DD. Regulation of the cerebral circulation: role of endothelium and potassium channels. Physiol Rev. 1998; 78: 53–97.
McIntyre M, Bohr DF, Dominiczak AF. Endothelial function in hypertension: the role of superoxide anion. Hypertension. 1999; 34: 539–545.
Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.
Lassegue B, Sorescu D, Szocs K, et al. Novel gp91(phox) homologues in vascular smooth muscle cells: Nox1 mediates angiotensin II–induced superoxide formation and redox-sensitive signaling pathways. Circ Res. 2001; 88: 888–894.
Didion SP, Faraci FM. Effects of NADH and NADPH on superoxide levels and cerebral vascular tone. Am J Physiol. 2002; 282: H688–H695.
Paravicini TM, Gulluyan LM, Dusting GJ, et al. Increased NADPH oxidase activity, gp91phox expression, and endothelium-dependent vasorelaxation during neointima formation in rabbits. Circ Res. 2002; 91: 54–61.
Redon J, Oliva MR, Tormos C, et al. Antioxidant activities and oxidative stress byproducts in human hypertension. Hypertension. 2003; 41: 1096–1101.
Di Wang H, Hope S, Du Y, et al. Paracrine role of adventitial superoxide anion in mediating spontaneous tone of the isolated rat aorta in angiotensin II–induced hypertension. Hypertension. 1999; 33: 1225–1232.
Souza HP, Laurindo FR, Ziegelstein RC, et al. Vascular NAD(P)H oxidase is distinct from the phagocytic enzyme and modulates vascular reactivity control. Am J Physiol. 2001; 280: H658–H667.
Ito D, Murata M, Watanabe K, et al. C242T polymorphism of NADPH oxidase p22 PHOX gene and ischemic cerebrovascular disease in the Japanese population. Stroke. 2000; 31: 936–939.
Guzik TJ, West NE, Black E, et al. Functional effect of the C242T polymorphism in the NAD(P)H oxidase p22phox gene on vascular superoxide production in atherosclerosis. Circulation. 2000; 102: 1744–1747.
Mollnau H, Wendt M, Szocs K, et al. Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling. Circ Res. 2002; 90: e58–e65.