Upregulation of Nox1 in vascular smooth muscle leads to impaired ...

Am J Physiol Heart Circ Physiol 299: H673–H679, 2010. First published July 16, 2010; doi:10.1152/ajpheart.00242.2010.

Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling Anna E. Dikalova,1 María Carolina Góngora,1 David G. Harrison,1 J. David Lambeth,2 Sergey Dikalov,1 and Kathy K. Griendling1 Departments of 1Medicine and 2Pathology, Emory University, Atlanta, Georgia Submitted 9 March 2010; accepted in final form 13 July 2010

Dikalova AE, Góngora MC, Harrison DG, Lambeth JD, Dikalov S, Griendling KK. Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling. Am J Physiol Heart Circ Physiol 299: H673–H679, 2010. First published July 16, 2010; doi:10.1152/ajpheart.00242.2010.— Recent work has made it clear that oxidant systems interact. To investigate potential cross talk between NADPH oxidase (Nox) 1 upregulation in vascular smooth muscle and endothelial function, transgenic mice overexpressing Nox1 in smooth muscle cells (TgSMCnox1) were subjected to angiotensin II (ANG II)-induced hypertension. As expected, NADPH-dependent superoxide generation was increased in aortas from Nox1-overexpressing mice. Infusion of ANG II (0.7 mg·kg⫺1·day⫺1) for 2 wk potentiated NADPH-dependent superoxide generation and hydrogen peroxide production compared with similarly treated negative littermate controls. Endothelium-dependent relaxation was impaired in transgenic mice, and bioavailable nitric oxide was markedly decreased. To test the hypothesis that eNOS uncoupling might contribute to endothelial dysfunction, the diet was supplemented with tetrahydrobiopterin (BH4). BH4 decreased aortic superoxide production, partially restored bioavailable nitric oxide in aortas of ANG II-treated TgSMCnox1 mice, and significantly improved endothelium-dependent relaxation in these mice. Western blot analysis revealed less dimeric eNOS in TgSMCnox1 mice compared with the wild-type mice; however, total eNOS was equivalent. Pretreatment of mouse aortas with the eNOS inhibitor NG-nitro-L-arginine methyl ester decreased ANG II-induced superoxide production in TgSMCnox1 mice compared with wild-type mice, indicating that uncoupled eNOS is also a significant source of increased superoxide in transgenic mice. Thus overexpression of Nox1 in vascular smooth muscle leading to enhanced production of reactive oxygen species in response to ANG II causes eNOS uncoupling and a decrease in nitric oxide bioavailability, resulting in impaired vasorelaxation. endothelial function; oxidative stress; angiotensin; nicotinamide adenine dinucleotide phosphate oxidase

There is some controversy about the source of the increased ROS production in hypertension, with evidence for involvement of NADPH oxidases (18), uncoupled endothelial nitric oxide synthase (eNOS) (19), xanthine oxidase (31), and mitochondria (7). Recent work suggests that substantial cross talk occurs among ROS-generating pathways. For example, in endothelial cells exposed to oscillatory shear stress, NADPH oxidase activity is required to maintain expression of xanthine oxidase, which then contributes to ROS production (24). Moreover, in ANG IIinduced hypertension, blockade of NADPH oxidases using apocynin prevents subsequent mitochondrial H2O2 production (7). Finally, NADPH oxidase-derived superoxide contributes to peroxynitrite formation, leading to oxidation of tetrahydrobiopterin (BH4) and uncoupling of eNOS (19). Most studies concerning cross talk among ROS pathways focus on the interaction of oxidant systems within endothelial cells. It is unclear whether ROS derived from medial smooth muscle cells (SMCs) can also influence endothelial sources of ROS and, indeed, if smooth muscle NADPH oxidases influence endothelial function. SMCs from large arteries express two NADPH oxidases, Nox1 and Nox4, but, of these, only Nox1 has been associated with hypertension (5, 9, 23, 34). Therefore, we investigated the possibility that ROS derived from Nox1 might uncouple eNOS, leading to endothelial dysfunction. To test this hypothesis, we used a transgenic mouse in which Nox1 is overexpressed specifically in SMCs (TgSMCnox1) and induced hypertension by ANG II infusion. We found that these mice do in fact exhibit eNOS uncoupling and impaired endothelium-dependent relaxation, emphasizing the importance of cross talk among cell types, as well as enzyme systems, within the vessel wall. MATERIALS AND METHODS

species (ROS) are present in many models of hypertension, including spontaneously hypertensive rats (30), DOCA-salt administration (1, 19), one- or two-kidney one-clip rats (6), and animals subjected to infusion of angiotensin II (ANG II) (27) or norepinephrine (22). Both superoxide and hydrogen peroxide (H2O2) are increased, whereas bioavailable nitric oxide (NO) is reduced (17, 19, 27). Increased tissue ROS levels mediate a number of pathologies associated with hypertension, including blood pressure regulation (18), endothelium-dependent control of vasomotor tone (26), renal cortical and medullary microvascular function (29), and end-organ damage (34). ELEVATIONS IN REACTIVE OXYGEN

Address for reprint requests and other correspondence: K. K. Griendling, Emory Univ., Division of Cardiology, 319 WMB, 1639 Pierce Dr., Atlanta, GA 30322 (e-mail: [email protected]). http://www.ajpheart.org

Animals. Mice used in this study were 6 –7 mo old. TgSMCnox1 mice were generated on a C57BL/6 background and have significantly increased Nox1 levels in vasculature (5). Animals were genotyped by polymerase chain reactions (PCR) using DNA prepared from earnotch biopsies. Procedures were approved by the Emory University Institutional Animal Care and Use Committee. Male TgSMCnox1 and C57BL/6 wild-type (WT) mice were divided into the following two groups: control (saline infusion) and ANG II infused. The mice were anesthetized, and micro-osmotic pumps were implanted subcutaneously in the midscapular region. Pumps delivered either 0.9% saline or ANG II at a rate of 0.7 mg·kg⫺1·day⫺1. After 14 days, the animals were killed by CO2 inhalation, and their aortas were harvested. For experiments involving BH4 administration, BH4 was compressed into rodent chow pellets, taking care to prevent oxidation of the compound by storing the pellets at ⫺20°C and providing fresh pellets daily. The concentration of BH4 in the pellets (1 mg/g) was calculated to provide a dose of 5 mg/day based on an average mouse

0363-6135/10 Copyright © 2010 the American Physiological Society

H673

H674

VSMC Nox1 PROMOTES eNOS UNCOUPLING

intake of 4 – 6 g of diet daily. BH4 was administered 48 h before ANG II treatment and continued for the entire period of ANG II infusion. Systolic blood pressure measurements. Systolic blood pressure was measured with the use of tail-cuff plethysmography (Visitech Systems). Blood pressure was measured two times before the implantation of osmotic pumps and on day 14 after pump placement. A set of 10 to 20 measurements was obtained for each animal, and the mean blood pressure was calculated. Western blotting. Aortas were harvested and cleaned of fat and connective tissue. Proteins were extracted and analyzed for total eNOS by Western blotting as described previously (15). With the use of nonboiled lysates and low-temperature SDS-PAGE, eNOS dimers/ monomers were immunoblotted (eNOS antibody 1:2,500; BD-Transduction Laboratory) as described elsewhere (15). Detection of intracellular superoxide with high-performance liquid chromatography. To evaluate intracellular production of superoxide, we measured the formation of 2-hydroxyethidium from dihydroethidium using high-performance liquid chromatography (HPLC) analysis (5). Hydroxyethidium was expressed per milligram protein. In some samples, polyethylene glycol (PEG)-superoxide dismutase (SOD, 100 U/ml) was added 1 h before addition of dihydroethidium. PEG-SOD inhibited the dihydroethidium signal by 60%. Measurement of NADPH oxidase activity. Aortas were dissected free of adventitia and then used to prepare membrane fractions, as described previously, with minor modifications (4). Protein aliquots (20 ␮g) were subjected to electron spin resonance spectroscopy (ESR) for quantitative measurement of NADPH (200 ␮mol/l)-dependent generation of superoxide radicals with 1 mmol/l 1-hydroxy-3-carboxypyrrolidine and 0.1 mmol/l diethylenetriamine pentaacetic acid in Chelex-treated phosphate-buffered saline. ESR spectra were recorded with an EMX ESR spectrometer (Bruker) and a super-high-Q microwave cavity exactly as described (4). SOD completely inhibited the NADPH-dependent signal. Measurement of H2O2. H2O2 was measured using a fluorometric horseradish peroxidase-linked assay (Amplex red assay; Molecular Probes) as described previously (35). H2O2 release was calculated using H2O2 standards and expressed as picomoles per milligram of tissue. The H2O2 signal was completely inhibited by catalase. Studies of vascular reactivity. Thoracic aortas were dissected free of adventitia, cut into 3-mm ring segments, and studied as previously described (12). Following contraction by PGF2␣ (3 ⫻ 10⫺6 mol/l), relaxations to cumulative concentrations of acetylcholine and nitroglycerin were examined. The degree of precontraction to PGF2␣ was chosen to approximate 80% of the maximal response to KCl (80 mmol/l). In some experiments, preconstricted isolated vessels were incubated in the organ chamber with apocynin (0.05 mmol/l) for 30 min before dose-response curves were performed. Determination of aortic NO production. Five 2-mm aortic rings were incubated for 60 min in 1.5 ml of Krebs/HEPES buffer containing 200 ␮mol/l iron diethyldithiocarbamate (Fe[DETC]2) and 10 ␮mol/l A-23187 at 37°C. The NO-Fe[DETC]2 complex was detected using ESR as described previously (3). Values were normalized to aortic dry weight. Measurement of aortic BH4 levels. Measurements of aortic biopterin content were performed using HPLC analysis and a differential oxidation method as described previously (8). The amount of BH4 was determined from the difference between total and alkaline-stable oxidized biopterin. A C-18 column (5 ⫻ 250 mm, 5 ␮m) was used with 5% methanol-95% water as a solvent at a flow rate of 1.0 ml/min. The fluorescence detector was set at 350 nm for excitation and 450 nm for emission. Real-time quantitative reverse transcriptase-PCR. Total RNA was purified from TgSMCnox1 and WT mouse aortas cleaned of fat tissue, adventitia, and blood with the use of the RNeasy kit (Qiagen). RNA from aortas and heterologous internal luciferase standards were reverse transcribed with Superscript II enzyme (Invitrogen) using random primers. Message expression was quantified on a Lightcycler AJP-Heart Circ Physiol • VOL

instrument (Roche) with SYBR green dye and specific mouse Nox2, Nox4, or p22phox primers and normalized to luciferase and 18S rRNA. Statistics. Results are expressed as means ⫾ SE. For multiple treatment groups, repeated-measures, two-way, or Latin-square design ANOVA followed by a Tukey-Kramer test was applied. For endothelium-dependent relaxation studies, one-way ANOVA with repeated measures followed by the Newman-Keuls test was used. RESULTS

NADPH-dependent superoxide generation and H2O2 production are elevated in aortas from TgSMCnox1 mice. We previously showed that aortic Nox1 expression in TgSMCnox1 mice is increased four- to fivefold (5), similar to that observed in hypertensive mice or in injured carotid arteries (25, 32). To confirm that overexpression of Nox1 in SMCs increased NADPH oxidase-dependent ROS production in the aorta, we measured NADPH-dependent superoxide generation and production of H2O2, the more stable ROS that is formed immediately from superoxide by SOD. Basal NADPH oxidase activity was significantly elevated in transgenic mice (50 ⫾ 10% over the level in WT mice) (Fig. 1A). Infusion of ANG II increased NADPH-dependent superoxide generation by twofold in WT mice and threefold in transgenic animals. Basal Nox1 and Nox4 expression were unchanged in TgSMCnox1 mice, whereas Nox4 was decreased following ANG II infusion in both sets of animals (Table 1). These findings agree well with our previous measurements of superoxide production in aortas from these animals (5). Consistent with these observations, H2O2 production was also enhanced in aortas from TgSMCnox1 mice following ANG II infusion (Fig. 1B). Nox1 overexpression impairs endothelium-dependent vasorelaxation: Improvement by apocynin. To determine if the increase in ROS production induced by Nox1 overexpression in smooth muscle impairs endothelium-dependent relaxation and increases blood pressure, we examined relaxation evoked by acetylcholine, which in the mouse thoracic aorta is entirely dependent on endothelial NO bioavailability (14), and measured blood pressure by tail cuff plethysmography. Consistent with our previous observations, a 2-wk infusion of ANG II increased blood pressure in WT mice from 101 ⫾ 3 to 148 ⫾ 4 mmHg (n ⫽ 20, P ⬍ 0.01), a response that was exacerbated in TgSMCnox1 (from 103 ⫾ 3 to 174 ⫾ 5 mmHg, n ⫽ 20, P ⬍ 0.01). Acetylcholine-induced endothelium-dependent relaxation in TgSMCnox1 mice was also impaired, with a maximal relaxation of 70 ⫾ 3% compared with 90 ⫾ 3% in WT mice (Fig. 2A). ANG II infusion inhibited endothelium-dependent vasodilatation in WT mice and worsened the impairment already present in TgSMCnox1 mice (P ⬍ 0.001, Fig. 2A). The NO donor, nitroglycerin, was used to test endothelium-independent relaxation. Endothelium-independent relaxations to nitroglycerin were similar in WT and TgSMCnox1 mice at baseline and were not altered by ANG II (Fig. 2D). The impairment in endothelium-dependent vasodilatation caused by Nox1 overexpression was completely prevented in aortas pretreated with the NADPH oxidase inhibitor apocynin, which inhibits assembly of the active NADPH oxidase complex. Aortic rings obtained from WT and TgSMCnox1 mice treated with apocynin showed virtually identical vascular reactivity to acetylcholine in the saline control and ANG II-treated groups (Fig. 2B). Preincubation with catalase also showed an improved relax-

299 • SEPTEMBER 2010 •

www.ajpheart.org

H675


Fig. 1. Production of aortic reactive oxygen species (ROS) in transgenic mice overexpressing NADPH oxidase (Nox) 1 in smooth muscle cells (TgSMCnox1) and wild-type (WT) mice. A: NADPH-dependent superoxide generation in aortas of TgSMCnox1 and C57BL/6 mice measured by electron spin oxidase microscopy (ESR) with 1-hydroxy-3-carboxypyrrolidine (CPH) as a spin probe. Mice were infused with saline or ANG II (0.7 mg·kg⫺1·day⫺1) for 14 days. Aortas were harvested, and membrane fractions were prepared by differential centrifugation. Superoxide production was measured using ESR after stimulation with 200 ␮mol/l of NADPH. Values were calculated as the difference of signals obtained from membranes in the presence and absence of NADPH. Values represent means ⫾ SE for 6 animals/group. ⫹Significant increase in superoxide level vs. saline-infused WT (P ⬍ 0.05); *significant increase in superoxide level vs. saline-infused WT mice (P ⬍ 0.001); #significant increase in superoxide level vs. saline-infused TgSMCnox1 mice (P ⬍ 0.001); ⬃significant increase in superoxide level vs. ANG II-infused WT mice (P ⬍ 0.001). B: aortic H2O2 release in TgSMCnox1 and C57BL/6 mice. Amplex Red was used to measure the release of H2O2 in C57BL/6 mice (open bars) and TgSMCnox1 mice (filled bars) following infusion with saline or ANG II for 14 days. Values represent means ⫾ SE for a minimum of 6 animals/group. *Significant increase in H2O2 level vs. saline-infused WT (P ⬍ 0.001); #significant increase vs. saline-infused TgSMCnox1 mice (P ⬍ 0.001); ⬃significant increase vs. ANG II-infused WT mice (P ⬍ 0.001).

ation response in TgSMCnox1 mice and after ANG II treatment in both genotypes (data not shown), suggesting that H2O2 normally contributes to vasoconstriction in the aortas of these animals. It should be noted that apocynin at concentrations 500 ␮mol/l and higher interfere with H2O2 measurements and may act as an antioxidant (13). For this reason, in these experiments, we used a low dose of apocynin (50 ␮mol/l), which had no nonspecific effects in the previous study. Effect of Nox1 overexpression on NO production and eNOS protein expression. One likely explanation for the impaired endothelium-dependent relaxation to acetylcholine in TgSMCnox1 is a reduction in bioavailable NO. Indeed, NO levels were decreased AJP-Heart Circ Physiol • VOL

by 20% in aortas of TgSMCnox1 mice under basal conditions (Fig. 3). ANG II decreased aortic NO levels in WT mice by 50 ⫾ 4%, a response that was exacerbated in TgSMCnox1 mice (63 ⫾ 5% decrease in bioavailable NO). Decreased NO bioavailability could be caused by either oxidative inactivation of NO, presumably by Nox1-derived superoxide, or uncoupling of eNOS. Based on previous data showing eNOS uncoupling in hypertension (19), we measured eNOS dimers to estimate eNOS uncoupling. As shown in Fig. 4, compared with WT mice, TgSMCnox1 mice have a significantly smaller amount of dimerized aortic eNOS, indicating possible uncoupling of eNOS in these mice. Total eNOS was unchanged. eNOS uncoupling can be caused by loss of BH4 due to oxidation (19), suggesting that TgSMCnox1 mice may have lower levels of BH4. To test this possibility, we measured reduced and oxidized aortic BH4 levels using HPLC. Indeed, reduced BH4 levels were decreased in TgSMCnox1 mice compared with WT mice, and ANG II decreased reduced BH4 to a greater extent in TgSMCnox1 mice than in WT mice (Fig. 5). Oxidized BH4 behaved in a reciprocal manner. Effect of BH4 supplementation and NG-nitro-L-arginine methyl ester on NO, superoxide production, eNOS uncoupling, and endothelium-dependent relaxation. Previous work has shown that oxidation of BH4 leads to eNOS uncoupling and generation of superoxide at the expense of NO. The loss of eNOS dimers in TgSMCnox1 mice thus suggests that superoxide derived from Nox1 may oxidize BH4 and uncouple eNOS, creating an additional source of superoxide in TgSMCnox1 mice. We tested this hypothesis by treating mice with oral BH4. BH4 administration beginning 2 days before ANG II had no effect on basal superoxide production but reduced superoxide in aortas of ANG II-infused TgSMCnox1 mice by 32 ⫾ 4%, and by a smaller but significant amount in WT hypertensive mice (17 ⫾ 5%, Fig. 6) without affecting Nox subunit expression (Table 1). As expected, this was accompanied by an increase in the amount of bioavailable NO (Fig. 3) and a partial reversal of the reduction in eNOS dimers caused by ANG II, indicative of improved eNOS coupling (Fig. 4). BH4 treatment restored NO levels in TgSMCnox1 mouse aortas to 97 ⫾ 4% of the values observed in WT untreated mice. It also partially restored bioavailable NO in aortas of both WT (33 ⫾ 5%) and TgSMCnox1 mice treated with ANG II (48 ⫾ 6%) and significantly improved endothelium-dependent relaxation in TgSMCnox1 mice both baTable 1. Expression of Nox subunit mRNA in WT and TgSMCnox1 mice treated with ANG II and tetrahydrobiopterin WT TgSMCnox1 WT ⫹ BH4 TgSMCnox1 ⫹ BH4 WT ⫹ ANG II TgSMCnox1 ⫹ ANG II WT ⫹ ANG II ⫹ BH4 TgSMCnox1 ⫹ ANG II ⫹ BH4

p22phox

Nox2

Nox4

200 ⫾ 20 190 ⫾ 10 200 ⫾ 10 210 ⫾ 20 380 ⫾ 50* 400 ⫾ 30* 360 ⫾ 30 410 ⫾ 20

2.0 ⫾ 0.2 2.0 ⫾ 0.2 2.1 ⫾ 0.2 2.3 ⫾ 0.3 4.1 ⫾ 0.3* 4.9 ⫾ 0.3* 3.6 ⫾ 0.3 3.8 ⫾ 0.3

390 ⫾ 20 370 ⫾ 20 410 ⫾ 20 370 ⫾ 20 150 ⫾ 20* 210 ⫾ 20*† 150 ⫾ 20* 240 ⫾ 20*†

Values are means ⫾ SE for 5 independent experiments. Units are copies/106 copies of 18S. Nox, NADPH oxidase; WT, wild type; TgSMCnox1, transgenic mice expressing Nox1 in smooth muscle cells; BH4, tetrahydrobiopterin. *P ⬍ 0.05 compared with corresponding control; †P ⬍ 0.05 compared with WT ANG II.


www.ajpheart.org

H676


Fig. 3. Nitric oxide (NO) levels in TgSMCnox1 mice after ANG II-induced hypertension: effect of BH4 supplementation. Calcium ionophore-stimulated NO production was estimated by ESR using the spin-trap iron diethyldithiocarbamate (Fe[DETC]2). Four to five 2-mm segments of thoracic aorta were incubated with Fe[DETC]2 and A-23187 (10 ␮mol/l) for 60 min, snap-frozen in liquid nitrogen, and subjected to ESR analysis. Mean data for 5–10 animals/group are shown. *Significant decrease in NO vs. saline-infused mice of the same genotype (P ⬍ 0.001); ⬃significant increase in NO vs. salineinfused TgSMCnox1 (P ⬍ 0.01); #P ⬍ 0.05 vs. WT mice with the same treatment; ⫹P ⬍ 0.05 vs. ANG II alone in mice of the same genotype.

sally and after ANG II treatment (Fig. 2C). Consistent with these observations, BH4 significantly attenuated hypertension in transgenic mice treated with ANG II (174 ⫾ 5 vs. 146 ⫾ 5 mmHg, n ⫽ 5, P ⬍ 0.05), and to a lesser extent in WT mice (148 ⫾ 3 to 129 ⫾ 4 mmHg, n ⫽ 5, P ⬍ 0.03). In additional studies, we treated aortic rings from TgSMCnox1 and WT mice infused with ANG II for 14 days with NG-nitroL-arginine methyl ester (L-NAME, 0.1 mmol/l) for 30 min and

Fig. 2. Nox1 overexpression impairs endothelium-dependent vasorelaxation in a NADPH oxidase-dependent manner in mice. After the infusion of vehicle or ANG II for 13 days, vascular reactivity to the endothelium-dependent agonist acetylcholine (A-C) or endothelium-independent NO donor nitroglycerin (D) was measured in aortic rings from TgSMCnox1 and C57BL/6 mice. [ACh], acetylcholine concentration. Rings were preconstricted with PGF2␣ (1 ␮mol/l). To study the effect of the NADPH oxidase inhibitor apocynin, rings from the same aortas were preincubated for 30 min with apocynin (0.05 mmol/l), which was added to the organ bath (B). In C, mice were treated with tetrahydrobiopterin (BH4) in the food during ANG II or saline infusion before vasodilator measurements. Data are expressed as means ⫾ SE. Lack of error bar indicates the error was smaller than the symbol; n ⫽ 4 –10 mice/group. A: *P ⬍ 0.001, TgSMCnox1 with ANG II vs. WT and WT with ANG II; #P ⬍ 0.05, TgSMCnox1 with ANG II vs. TgSMCnox1; †P ⬍ 0.001, TgSMCnox1 vs. WT; NS indicates not significant. C: #P ⬍ 0.05, TgSMCnox1 with ANG II vs. TgSMCnox1; *P ⬍ 0.0001, TgSMCnox1 with ANG II vs. TgSMCnox1 with ANG II plus BH4; †P ⬍ 0.0001, TgSMCnox1 vs. TgSMCnox1 treated with BH4.

AJP-Heart Circ Physiol • VOL

Fig. 4. Endothelial NO synthase (eNOS) protein expression. Representative Western blots (A) for dimer, monomer, and total eNOS using a low-temperature gel (3 different gels, but proteins from the same aortic homogenates). B: densitometry values for eNOS dimer/total eNOS ratio (n ⫽ 3). ⫹P ⬍ 0.05 vs. untreated WT mice; *P ⬍ 0.001 vs. untreated WT mice; **P ⬍ 0.05 vs. WT ANG II; ⬃P ⬍ 0.05 vs. WT ANG II; and #P ⬍ 0.01 vs. TgSMCnox1 mice with ANG II.


www.ajpheart.org


Fig. 5. Aortic BH4 levels in TgSMCnox1 and WT mice treated with ANG II. BH4 levels were quantified with the differential oxidation method and high-performance liquid chromatography. #P ⬍ 0.05 vs. BH4 level in WT mice; *P ⬍ 0.05 vs. BH4 level in untreated mice of the same genotype; ⫹P ⬍ 0.01 vs. BH4 level in WT ANG II; &P ⬍ 0.05 vs. oxidized BH4 level in WT mice; xP ⬍ 0.05 vs. oxidized BH4 level in TgSMCnox1 mice; ⬃P ⬍ 0.05 vs. oxidized BH4 level in WT ANG II mice.

measured superoxide production using dihydroethidium. LNAME treatment reduced superoxide production in response to ANG II by 20 ⫾ 5% in aortas from WT mice and by 68 ⫾ 9% in aortas from TgSMCnox1 mice (Fig. 7). These data suggest that superoxide production caused by overexpression of Nox1 is derived in part from subsequent uncoupling of eNOS. DISCUSSION

In this study, we provide evidence that ROS production by Nox1 in medial SMCs can uncouple eNOS, leading to a self-perpetuating cycle of superoxide production and impaired endothelium-dependent relaxation. This finding is similar to that reported in p47phox knockout mice but is the first to implicate smooth muscle sources of ROS as an initiating signal. Our results show that BH4 supplementation decreases aortic ROS production in aortas of TgSMCnox1 mice, in which the initial imbalance is NADPH oxidase overexpression. The kindling radical hypothesis originally proposed by Landmesser et al. (19) suggests that physiological levels of ROS can become pathological by creating a positive feedback loop in which ROS generation is enhanced by activation of another source of ROS. In addition to leading to eNOS uncoupling, NADPH oxidase activation is upstream of xanthine oxidase in endothe-

Fig. 6. Effect of BH4 treatment on superoxide production in TgSMCnox1 and WT mice infused with ANG II. Mice were infused with saline or ANG II (0.7 mg·kg⫺1·day⫺1) for 14 days. BH4 was administered in pellets starting 2 days before ANG II. Intracellular superoxide level in aortic rings was measured by HPLC after incubation with hydroxyethidium. Values represent means ⫾ SE for 5–10 animals/group. *P ⬍ 0.001 increase in superoxide level vs. salineinfused mice of the same genotype; #P ⬍ 0.01 vs. WT mice with the same treatment; ⫹P ⬍ 0.01 decrease in superoxide vs. ANG II alone in the same genotype. AJP-Heart Circ Physiol • VOL

H677

Fig. 7. Effect of eNOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on superoxide production in aortas from TgSMCnox1 and WT mice infused with ANG II. Mice were infused with saline or ANG II for 14 days. Aortic rings from the same mouse were incubated with or without L-NAME (0.1 mmol/l), and superoxide was measured by dihydroethidium-HPLC. Values represent means ⫾ SE for 5–10 animals/group. ⫹Significant change in superoxide in WT ⫹ L-NAME vs. WT (P ⬍ 0.05); *significant superoxide decrease in ANG II-infused WT ⫹ L-NAME vs. WT ⫹ ANG II (P ⬍ 0.05); #superoxide decrease in TgSMCnox1 infused with ANG II ⫹ L-NAME vs. TgSMCnox1 ⫹ ANG II (P ⬍ 0.005); ⬃superoxide increase in TgSMCnox1 ⫹ ANG II vs. WT ⫹ ANG II not treated with L-NAME (P ⬍ 0.001).

lial cells exposed to shear stress, as well as mitochondrial ROS generation in aortic endothelial cells stimulated with ANG II (7, 24). The fact that apocynin corrects the impairment in endothelium-dependent relaxation confirms that the primary defect in TgSMCnox1 mice is overactivation of Nox1, but the correction of superoxide production and endothelium-dependent relaxation, as well as the partial reversal of hypertension, by BH4 supplementation indicates that uncoupled eNOS, or possibly uncoupling of other NOS isoforms, also plays a role. Thus this system is an additional example of cross talk among oxidant systems and, uniquely, even across cell types. Nox1 is upregulated in a number of pathological conditions, including hypertension (25, 33) and neointimal formation after vascular injury (32). Increased expression and activity of this protein appears to contribute to blood pressure regulation. In mice in which Nox1 has been genetically deleted, ANG II infusion leads to an initial elevation in blood pressure similar to that observed in WT mice but does not cause sustained hypertension (9, 23). In contrast, TgSMCnox1 mice exhibit an enhanced blood pressure response (5). It is interesting to speculate that the failure to sustain blood pressure in Nox1 knockout mice may result from the secondary eNOS uncoupling demonstrated here. It should be noted, however, that, because BH4 treatment reduces blood pressure, this reduction in blood pressure itself could contribute to the observed responses. Nox1 also contributes to the increased medial thickness that is often observed in large arteries of hypertensive animals. ANG II-induced medial hypertrophy is impaired in Nox1 knockout mice but enhanced in TgSMCnox1 mice (5, 9). Some controversy exists over the mechanism by which Nox1 regulates medial hypertrophy, because effects on both extracellular matrix and smooth muscle proliferation are observed in Nox1deficient animals (9, 10, 21, 23). ROS regulate extracellular matrix integrity by upregulating a tissue inhibitor of metalloproteinases (10) and by inactivating matrix metalloproteinases (28). In the latter case, the extracellular ROS produced by uncoupled eNOS observed in this study is a likely source of oxidizing equivalents.


www.ajpheart.org

H678


Our observation that endothelium-dependent relaxation is impaired in TgSMCnox1 mice is consistent with similar findings in previous studies using hypertensive mice with altered NADPH oxidase activity (18, 20). However, the difference between previous studies and the model used here is that ROS produced by Nox1 in SMCs is largely intracellular and therefore might not be expected to reach the endothelium. One explanation may be that NO produced by eNOS is inactivated upon entering SMCs, thus preventing relaxation. This does not explain, however, the observation that eNOS itself becomes uncoupled. It is possible that the peroxynitrite formed upon NO interaction with Nox1-derived superoxide may exit SMCs and act to uncouple eNOS. Peroxynitrite has a low pKa, which allows the neutral/acid form of peroxynitrite to cross membranes (11). Indeed, extracellular supplementation of peroxynitrite leads to eNOS uncoupling (16). Alternatively, because H2O2 does not oxidize BH4 directly (16), Nox1-derived H2O2 may act on endothelial cells, or promote inflammatory cell infiltration, by initiating a ROS cascade that uncouples eNOS. This mechanism was recently demonstrated for H2O2 treatment of endothelial cells, which results in activation of NADPH oxidase and eNOS uncoupling (2). Another potential explanation is that the ROS produced by SMC Nox1 is either transported extracellularly, perhaps via activation of an ion channel, or stimulates the production of ROS by another source, such as xanthine oxidase. Finally, it has been suggested that H2O2 can induce eNOS protein expression (20), which conceivably contributes to enhanced uncoupling. However, we did not observe any change in eNOS expression in TgSMCnox1 mice. Based on these observations, we suggest that cross talk between oxidant systems occurs at multiple levels within the vessel wall. Not only can ROS produced within a cell type induce ROS formation by other enzymatic pathways, but ROS produced by cells in adjacent tissue can affect redox signaling as well. Such information may explain why in vitro studies of isolated cells do not always recapitulate in vivo responses. ROS generation in the vasculature regulates many aspects of cardiovascular diseases, and understanding the mechanisms controlling the generation of these important signaling molecules is of utmost importance in developing therapies that target pathophysiological mechanisms while keeping intact critical physiological signals.

5.

6.

7.

8. 9.

10.

11. 12.

13.

14.

15.

16.

17.

18.

19.

20.

ACKNOWLEDGMENTS This work was supported by National Heart, Lung, and Blood Institute Grants HL-38206, HL-05800, and HL-058863. DISCLOSURES

21.

K. K. Griendling and J. D. Lambeth share a patent on Nox1. REFERENCES 1. Beswick RA, Zhang H, Marable D, Catravas JD, Hill WD, Webb RC. Long-term antioxidant administration attenuates mineralocorticoid hypertension and renal inflammatory response. Hypertension 37: 781–786, 2001. 2. Boulden BM, Widder JD, Allen JC, Smith DA, Al-Baldawi RN, Harrison DG, Dikalov SI, Jo H, Dudley SC Jr. Early determinants of H2O2-induced endothelial dysfunction. Free Radic Biol Med 41: 810 – 817, 2006. 3. Dikalov S, Fink B. ESR techniques for the detection of nitric oxide in vivo and in tissues. Methods Enzymol 396: 597–610, 2005. 4. Dikalov SI, Dikalova AE, Bikineyeva AT, Schmidt HH, Harrison DG, Griendling KK. Distinct roles of Nox1 and Nox4 in basal and angiotensin AJP-Heart Circ Physiol • VOL

22.

23.

24.

II-stimulated superoxide and hydrogen peroxide production. Free Radic Biol Med 45: 1340 –1351, 2008. Dikalova A, Clempus R, Lassègue B, Cheng G, McCoy J, Dikalov S, San Martin A, Lyle A, Weber DS, Weiss D, Taylor WR, Schmidt HHW, Owens GK, Lambeth JD, Griendling KK. Nox1 overexpression potentiates angiotensin II-induced hypertension and vascular smooth muscle hypertrophy in transgenic mice. Circulation 112: 2668 –2676, 2005. Dobrian AD, Schriver SD, Prewitt RL. Role of angiotensin II and free radicals in blood pressure regulation in a rat model of renal hypertension. Hypertension 38: 361–366, 2001. Doughan AK, Harrison DG, Dikalov SI. Molecular mechanisms of angiotensin II-mediated mitochondrial dysfunction: linking mitochondrial oxidative damage and vascular endothelial dysfunction. Circ Res 102: 488 –496, 2008. Fukushima T, Nixon JC. Analysis of reduced forms of biopterin in biological tissues and fluids. Anal Biochem 102: 176 –188, 1980. Gavazzi G, Banfi B, Deffert C, Fiette L, Schappi M, Herrmann F, Krause KH. Decreased blood pressure in NOX1-deficient mice. FEBS Lett 580: 497–504, 2006. Gavazzi G, Deffert C, Trocme C, Schappi M, Herrmann FR, Krause KH. NOX1 deficiency protects from aortic dissection in response to angiotensin II. Hypertension 50: 189 –196, 2007. Goldstein S, Merenyi G. The chemistry of peroxynitrite: implications for biological activity. Methods Enzymol 436: 49 –61, 2008. Gongora MC, Qin Z, Laude K, Kim HW, McCann L, Folz JR, Dikalov S, Fukai T, Harrison DG. Role of extracellular superoxide dismutase in hypertension. Hypertension 48: 473–481, 2006. Heumuller S, Wind S, Barbosa-Sicard E, Schmidt HH, Busse R, Schroder K, Brandes RP. Apocynin is not an inhibitor of vascular NADPH oxidases but an antioxidant. Hypertension 51: 211–217, 2008. Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature 377: 239 –242, 1995. Klatt P, Schmidt K, Lehner D, Glatter O, Bachinger HP, Mayer B. Structural analysis of porcine brain nitric oxide synthase reveals a role for tetrahydrobiopterin and L-arginine in the formation of an SDS-resistant dimer. EMBO J 14: 3687–3695, 1995. Kuzkaya N, Weissmann N, Harrison DG, Dikalov S. Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase. J Biol Chem 278: 22546 –22554, 2003. Lacy F, O’Connor DT, Schmid-Schonbein GW. Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertension. J Hypertens 16: 291–303, 1998. Landmesser U, Cai H, Dikalov S, McCann L, Hwang J, Jo H, Holland SM, Harrison DG. Role of p47(phox) in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension 40: 511–515, 2002. Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, Harrison DG. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest 111: 1201–1209, 2003. Laude K, Cai H, Fink B, Hoch N, Weber DS, McCann L, Kojda G, Fukai T, Schmidt HH, Dikalov S, Ramasamy S, Gamez G, Griendling KK, Harrison DG. Hemodynamic and biochemical adaptations to vascular smooth muscle overexpression of p22phox in mice. Am J Physiol Heart Circ Physiol 288: H7–H12, 2005. Lee MY, San Martin A, Mehta PK, Dikalova AE, Garrido AM, Datla SR, Lyons E, Krause KH, Banfi B, Lambeth JD, Lassegue B, Griendling KK. Mechanisms of vascular smooth muscle NADPH oxidase 1 (Nox1) contribution to injury-induced neointimal formation. Arterioscler Thromb Vasc Biol 29: 480 –487, 2009. Lembo G, Vecchione C, Izzo R, Fratta L, Fontana D, Marino G, Pilato G, Trimarco B. Noradrenergic vascular hyper-responsiveness in human hypertension is dependent on oxygen free radical impairment of nitric oxide activity. Circulation 102: 552–557, 2000. Matsuno K, Yamada H, Iwata K, Jin D, Katsuyama M, Matsuki M, Takai S, Yamanishi K, Miyazaki M, Matsubara H, Yabe-Nishimura C. Nox1 is involved in angiotensin II-mediated hypertension: a study in Nox1-deficient mice. Circulation 112: 2677–2685, 2005. McNally JS, Davis ME, Giddens DP, Saha A, Hwang J, Dikalov S, Jo H, Harrison DG. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress. Am J Physiol Heart Circ Physiol 285: H2290 –H2297, 2003.


www.ajpheart.org

VSMC Nox1 PROMOTES eNOS UNCOUPLING 25. Mollnau H, Wendt M, Szocs K, Lassegue B, Schulz E, Oelze M, Li H, Bodenschatz M, August M, Kleschyov AL, Tsilimingas N, Walter U, Forstermann U, Meinertz T, Griendling K, Munzel T. Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling. Circ Res 90: E58 –E65, 2002. 26. Munzel T, Hink U, Heitzer T, Meinertz T. Role for NADPH/NADH oxidase in the modulation of vascular tone. Ann NY Acad Sci 874: 386 –400, 1999. 27. Rajagopalan S, Kurz S, Münzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG. Angiotensin II mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation: contribution to alterations of vasomotor tone. J Clin Invest 97: 1916 –1923, 1996. 28. Rajagopalan S, Meng XP, Ramasamy S, Harrison DG, Galis ZS. Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. J Clin Invest 98: 2572–2579, 1996. 29. Schnackenberg CG. Physiological and pathophysiological roles of oxygen radicals in the renal microvasculature. Am J Physiol Regul Integr Comp Physiol 282: R335–R342, 2002. 30. Schnackenberg CG, Welch WJ, Wilcox CS. Normalization of blood pressure and renal vascular resistance in SHR with a membrane-permeable

AJP-Heart Circ Physiol • VOL

31.

32.

33.

34.

35.

H679

superoxide dismutase mimetic: role of nitric oxide. Hypertension 32: 59 –64, 1998. Suzuki H, Swei A, Zweifach BW, Schmid-Schonbein GW. In vivo evidence for microvascular oxidative stress in spontaneously hypertensive rats Hydroethidine microfluorography. Hypertension 25: 1083–1089, 1995. Szöcs K, Lassègue B, Sorescu D, Hilenski LL, Valppu L, Couse TL, Wilcox JN, Quinn MT, Lambeth JD, Griendling KK. Upregulation of Nox-based NAD(P)H oxidases in restenosis after carotid injury. Arterioscler Thromb Vasc Biol 22: 21–27, 2002. Wingler K, Wunsch S, Kreutz R, Rothermund L, Paul M, Schmidt HH. Upregulation of the vascular NAD(P)H-oxidase isoforms Nox1 and Nox4 by the renin-angiotensin system in vitro and in vivo. Free Radic Biol Med 31: 1456 –1464, 2001. Yogi A, Mercure C, Touyz J, Callera GE, Montezano AC, Aranha AB, Tostes RC, Reudelhuber T, Touyz RM. Renal redox-sensitive signaling, but not blood pressure, is attenuated by Nox1 knockout in angiotensin II-dependent chronic hypertension. Hypertension 51: 500 –506, 2008. Zhang Y, Griendling KK, Dikalova A, Owens GK, Taylor WR. Vascular hypertrophy in angiotensin II-induced hypertension is mediated by vascular smooth muscle cell-derived H2O2. Hypertension 46: 732–737, 2005.


www.ajpheart.org