Vascular Oxidative Stress Precedes HighBlood Pressure in Spontaneously

Hypertensive Rats

LINDA NABHA, JESSICA C. GARBERN,*

CAROLYN L. BULLER, AND JOHN R. CHARPIEDivision of Pediatric Cardiology, Michigan Congenital Heart Center,

University of Michigan, Ann Arbor, Michigan, USA

This study examines whether longitudinal antioxidant treatment initiated in

prehypertensive spontaneously hypertensive rats (SHR) can attenuate vascular

oxidant stress and prevent blood pressure elevation during development. Male SHR

and age-matched Wistar-Kyoto rats (WKY) were treated from 6 to 11 weeks of age

with Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinoxyl) (1 mmol/l in drinking

water), a membrane-permeable superoxide dismutase mimetic. Mean systolic blood

pressures (SBPs) were measured by tail-cuff. Agonist-induced and basal O2�

production was measured in thoracic aortas of 6- and 11-week-old SHR and WKY by

lucigenin-derived chemiluminescence and oxidative fluorescent microscopy, respec-

tively. SBP of 6-week-old SHR (131 ± 5 mmHg) and WKY (130 ± 4 mmHg) were not

different; however, 11-week-old SHR SBP (171 ± 4 mmHg) was significantly greater

(p = .0001) than 11-week-old WKY SBP (143 ± 5 mmHg). Tempol treatment

completely, but reversibly, prevented this age-related rise in SHR SBP (SHR + Tem-

pol: 137 ± 4 mmHg; p < .0001 versus untreated SHR). Agonist-induced vascular O2�

was increased in 6- (p = .03) and 11-week-old SHR (p < .0001) and 11-week-old

WKY (p = .03) but not in 6-week-old WKY. Long-term Tempol treatment significantly

lowered O2� production in both strains. Basal O2

� measurements in both 6- and

11-week-old SHR were qualitatively increased compared with age-matched WKY; this

increase in SHR was inhibited with in vitro Tempol treatment. These data show that

antioxidant treatment to reduce oxidative stress prevents the age-related development

of high blood pressure in an animal model of genetic hypertension.

Keywords blood pressure, prehypertensive, spontaneously hypertensive rats,

superoxide anion, tempol

Clinical and Experimental Hypertension, 1:71–82, 2005

Copyright D Taylor & Francis, Inc.

ISSN: 1064-1963 print / 1525-6006 online

DOI: 10.1081/CEH-200044267

Received 15 April 2004; revised 10 August 2004; accepted 12 August 2004.*The first and second authors contributed equally to the research project and to preparation of

the manuscript.Address correspondence to Dr. John R. Charpie, Division of Pediatric Cardiology, Michigan

Congenital Heart Center, University of Michigan, Ann Arbor, MI 48109-0204, USA; Fax: (734)936-9470; E-mail: jcharpie@umich.edu

71

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IntroductionThe oxygen-derived free radical superoxide anion (O2

�) has been implicated in the

pathogenesis of multiple diseases including hypertension (1–3), diabetes (4), and hyper-

cholesterolemia (5). Several investigators have shown that O2� is produced in excess

in adult spontaneously hypertensive stroke-prone rats with established hypertension

compared with adult normotensive Wistar-Kyoto rats (WKY) (6, 7). Superoxide anion

normally is scavenged by superoxide dismutase (SOD) to form hydrogen peroxide and

eventually water. However, O2� may preferentially react with nitric oxide (NO) (8), an

important vasodilator, to form peroxynitrite (ONOO-), a vasoconstrictor that is toxic to

endothelial cells (9). Consequently, under conditions of excess O2� production such as

in hypertension, reduced NO bioavailability and increased oxidative stress may impair

endothelial function and elevate blood pressure (2, 10).

Because O2� appears to play a critical role in the pathogenesis of hypertension,

multiple researchers studied the use of antioxidants to lower blood pressure in both

human and experimental models of established hypertension (11, 12). Recently, Tempol

(4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl), a stable, membrane-permeable, met-

al-independent SOD mimetic, has been shown to decrease oxidative stress and normalize

blood pressure in adult rats with established hypertension (13–19). However, these

studies did not examine the longitudinal effect of Tempol in young, prehypertensive

rats; thus, we cannot necessarily conclude that increased O2� and high blood pressure

are mechanistically related during development. Therefore, it remains unclear whether

excess O2� precedes the developmental increase in blood pressure in genetic hyper-

tension or if excess O2� production is secondary to high blood pressure.

Previously, researchers described differences between prehypertensive spontaneous-

ly hypertensive rats (SHR) and age-matched WKY including upregulation of vascular

endothelial nitric oxide synthase (20) and increased angiotensin II-induced contraction

(21). Chabrashvili et al. (22) showed that NADPH oxidase, a possible enzymatic source

of O2�, is overexpressed in kidneys of young 4-week-old SHR prior to the develop-

ment of high blood pressure. Therefore, in our study we tested the hypothesis that

attenuation of oxidant stress by long-term Tempol treatment during the developmental

stage can prevent abnormal blood pressure elevation in SHR.

Methods

Chemicals

Angiotensin II (AII) (human; synthetic), Cu/Zn cytosolic SOD, dihydroethidium (DHE),

dimethyl sulfoxide (DMSO), Hanks balanced salt solution (HBSS), lucigenin (bis-N-

methylacridinium nitrate), No-Nitro-L-arginine (L-NNA), and Tempol were purchased

from Sigma Chemical Company (St. Louis, MO, USA). Sodium pentobarbital was

purchased from The Butler Company (Columbus, OH, USA) and Schering-Plough

Animal Health (Kenilworth, NJ, USA). Phosphate buffered saline (PBS) was obtained

from BioWhittaker (Walkersville, MD, USA). Tissue-Tek cryo-optimal cutting tem-

perature (OCT) compound was obtained from VWR Scientific Products (Torrance,

CA, USA).

Animal Preparation

All procedures were approved by the University of Michigan Committee on the Use and

Care of Animals. Six-week-old male SHR and age- and gender-matched WKY were

purchased from Harlan (Indianapolis, IN, USA). All rats were maintained on standard rat

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chow and housed in a continuous 12-hr light/dark cycle. Twenty SHR and 20 WKY was

randomly assigned to control and Tempol groups. SHRControl (n = 10) and WKYControl

(n = 10) received vehicle (tap water) while SHRTempol (n = 10) and WKYTempol (n = 10)

received 1 mmol/l Tempol in their drinking water for 5 consecutive weeks. Five weeks

was chosen because we found in preliminary studies (not shown) that developing SHR

reach a plateau in systolic blood pressure by 11 weeks of age. Water and Tempol volumes

were recorded at the time fresh drug was administered (every 2–3 days) to determine

individual oral intake. Body weights were measured weekly. Following 5 weeks of

Tempol treatment, a subset (n = 5) of the SHRTempol group was taken off of Tempol and

administered water (SHRTempol/Water) for an additional week.

Blood Pressure Measurements

SBP was measured weekly in conscious animals by the tail-cuff method as previously

described by Ikeda et al. (23) and modified by Kramer et al. (24). Briefly, rats were

placed in size-appropriate plexiglass (acrylic) restrainers and were whole-body heated to

32�C (Model 303, IITC Inc., Woodland Hills, CA, USA). After allowing rats to acclimate

for approximately 10 min, a minimum of three consecutive SBP measurements (Model

229 amplifier, IITC) were recorded and averaged for each animal. All measurements

were taken in the morning to minimize possible fluctuations due to circadian rhythms.

Weekly SBPs were expressed as mean ± standard error (SEM) for each treatment group.

In addition, after discontinuing Tempol at 11 weeks of age, SBPs in SHRTempol/Water were

measured daily or every other day for 1 week as described above.

Detection of O2������: Lucigenin-derived Chemiluminescence (LDCL)

Rats were anesthetized with sodium pentobarbital (50–75 mg/kg intraperitoneally).

Thoracic aortas were removed, cleaned of periadventitial tissue, and cut into 5-mm ring

segments. Aortic segments were incubated in serum-free media containing 10 mmol/l AII

for 12 hr at 37�C in an incubator (5% CO2, 95% room air). We chose 12 hr as the

incubation time based on preliminary data showing the optimum signal at 12 hr. Vascular

O2� was measured by LDCL in a luminometer (Minilumat LB 9506, PerkinElmer, Bad

Wildbad, Germany) essentially as described by Ohara et al. (5). Briefly, after incubation,

aortic segments were transferred to vials containing 2 ml of HBSS buffer (pH 7.4, 25�C)

and 5 mmol/l lucigenin. Immediately, vials were placed in the luminometer and photon

counts were recorded for 10 consecutive min. Minutes 1–5 were used as a period of dark

adaptation and were not included in the calculations. The area under the curve for

minutes 6–10 (AUC6 – 10) was calculated for each sample. AUC6 – 10 for blank counts,

obtained by measuring lucigenin alone, was subtracted from each sample. Raw data were

calculated as shown in Eq. 1.

Raw Data Sample ¼ AUC6 � 10; Sample � AUC6 � 10; Lucigenin

Wet tissue weightðmgÞ ð1Þ

AII-induced O2� production in segments was normalized to vessels from the same animal

incubated in serum-free media alone. Data are expressed as a fold-increase in O2�

production (Eq. 2).

Fold ChangeSample; All ¼ Raw Data Sample; All

Raw DataSample; SFM

ð2Þ

73Vascular Oxidative Stress

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Specificity of the chemiluminescence signal for O2� was monitored by co-incubation of

several segments with SOD (data not shown).

Experiments were completed in a tissue-free xanthine (0–400 nmol/l)/xanthine

oxidase (0.001 U/ml) system to assess the sensitivity of the LDCL assay. We found a

linear relationship between O2� production and xanthine concentration (Eq. 3).

AUC6 � 10; X=XO ¼ 6:0703 * ½ððxanthine concðnMÞÞ � 164:8Þ�; r2 ¼ 0:98 ð3Þ

Ohara et al. (5) describes a relationship to quantify O2� production as a function of

xanthine concentration.

Detection of O2������: Oxidative Fluorescent Microscopy

Basal O2� production was measured by oxidative fluorescent microscopy in a manner

similar to that previously described by Miller et al. (25). Arterial ring segments from

Table 1Oral intake and body weights

WKYControl WKYTempol SHRControl SHRTempol

Average water intake (mL/day) 35 ± 2 33 ± 2 36 ± 2 36 ± 2

Body weight, 6 weeks (g) 210 ± 6 177 ± 8 150 ± 7* 168 ± 8

Body weight, 11 weeks (g) 305 ± 4 283 ± 7 279 ± 4** 290 ± 8

SHR = spontaneously hypertensive rat, WKY = Wistar-Kyoto rat. Values are expressed asmean ± SEM (n = 10 rats per group).

*p < .0001 versus 6-week WKYControl.**p = .0004 versus WKYControl.

Figure 1. Systolic blood pressure (SBP) measurements. Weekly measurements in SBP in

WKYControl (open squares), WKYTempol (closed squares), SHRControl (open circles), and SHRTempol

(closed circles). Values are expressed as mean ± SEM. *p = .0006 versus WKYControl;yp < .0001

versus WKYControl;zp < .0001 versus SHRTempol;

xp = .02 versus WKYControl;kp = .001 versus

WKYControl. Tempol significantly attenuated the age-related rise in SBP by 11 weeks. *p < .0001

versus WKYControl;yp < .0001 versus SHRControl. SHR = spontaneously hypertensive rat;

WKY = Wistar Kyoto rat.

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6- and 11-week-old WKY and age-matched SHR were frozen in OCT embedding

medium, cut into 30 mm-thick sections, and placed on glass slides. DHE (a 20 mmol/l stock

solution prepared in DMSO and 40 mmol/l working solution prepared in PBS) was

topically applied to each tissue section, incubated for 30 min in the dark at 37�C in a

humidified chamber, and then coverslipped. In some cases, segments were treated acutely

with Tempol (1 mmol/l for 30 min at 37�C) prior to application of DHE. Images were

obtained with a laser scanning confocal microscope (Bio-Rad MRC-600, Hercules, CA,

Figure 2. Superoxide production in the aortic arteries of WKY (a) and SHR (b) as measured by

lucigenin-derived chemiluminescence. AII-induced O2� production in segments was normalized to

vessels incubated in serum-free media (SFM) alone. Data are expressed as a fold increase in O2�

production. Values are expressed as mean ± SEM. *p = .03 versus SFM of 11-week-old WKY;yp = .03 versus SFM of 6-week-old SHR; zp < .0001 versus SFM of 11-week-old SHRControl.

AII = angiotensin II; SHR = spontaneously hypertensive rat; WKY = Wistar Kyoto rat.

75Vascular Oxidative Stress

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USA). Once calibrated, the laser settings were not altered. Fluorescence was detected with

a 585 nm long-pass filter.

Statistical Analysis

SBP (mean ± SEM) was plotted for each group at every age. Differences among groups

over time were compared by ANOVA using Prism 3.0 (GraphPad Software, San Diego,

CA, USA). For determination of the effect of Tempol on SBP, unpaired t-tests were

performed with Bonferroni correction for multiple comparisons. Paired t-tests were used

to compare AII-induced O2� production to O2

� production in aortic segments incubated

in serum-free media alone. Values of p < .05 were considered statistically significant.

Results

Water Intake and Body Weight

Rats administered Tempol had similar overall fluid intake compared with controls

(Table 1). Tempol treatment had no effect on weight gain in either SHR or WKY. At both

6 and 11 weeks of age, SHR were significantly smaller than WKY (Table 1).

Figure 3. Superoxide production in the aortic arteries of 6-week-old WKY (a) and SHR (b) without

Tempol pretreatment and 6-week-old WKY (c) and SHR (d) with 30-min pretreatment of 1 mM

Tempol as measured by oxidative fluorescent microscopy. Superoxide production is increased in

6-week-old SHR compared with 6-week-old WKY that is inhibited by acute Tempol treatment.

E = endothelium; SM = smooth muscle cell layer.

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Blood Pressure Measurements

Figure 1 shows the weekly SBP measurements in all groups. At 6 weeks of age, SBP

measurements were not different among the groups. By age 9 weeks, SBP had

significantly increased in SHRControl compared with WKYControl (173 ± 2 versus 150 ±

5 mmHg, p = .002); at the conclusion of the study (age 11 weeks), SBP of SHRControl

remained significantly elevated compared with the other three groups. By 9 weeks of age,

Tempol treatment had significantly attenuated the increase in SBP in SHRControl

(SHRTempol = 133 ± 3 mmHg, p < .0001 versus SHRControl). At 11 weeks, Tempol

treatment significantly lowered SBP in SHR by 20% compared with SHRControl.

Mean SBP of SHRTempol was not statistically different from SBP of either WKYControl

(143 ± 5 mmHg) or WKYTempol at the conclusion of the study. SBP of WKYTempol was

significantly lower than WKYControl at 8, 9, and 10 weeks of age ( p = .02, .001, and

.02, respectively).

To confirm that SBP in adult SHRControl had in fact reached a plateau, SBP

measurements were repeated in a subset of this group (n = 5) at age 12 weeks. Similar to

Figure 4. Superoxide production in the aortic arteries of 11-week-old WKY (a) and SHR

(b) without Tempol pretreatment and 11-week-old WKY (c) and SHR (d) with 30-min pretreatment

of 1 mM Tempol as measured by oxidative fluorescent microscopy. Superoxide production is

increased in 11-week-old SHR compared with 11-week-old WKY that is inhibited by acute Tempol

treatment. E = endothelium; SM = smooth muscle cell layer.

77Vascular Oxidative Stress

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our previous findings, SBP in 12-week-old SHRControl (172 ± 3 mmHg) was not different

from 11-week-old SHRControl (171 ± 3 mmHg).

To determine whether prevention of the rise in SBP in SHR by Tempol is a

permanent or reversible effect, daily SBPs were measured after discontinuing Tempol

treatment in SHRTempol/Water. Initial SBP of SHRControl (171 ± 3 mmHg) was significantly

higher than both SHRTempol (140 ± 4 mmHg, p = .0005) and SHRTempol/Water

(135 ± 5 mmHg, p = .0006). However, after 7 days without Tempol treatment, mean

SBP of SHRTempol/Water (179 ± 3 mmHg) was not different than SHRControl (172 ±

3 mmHg), and both were significantly higher than SHRTempol (147 ± 6 mmHg, p = .005

and p = .007, respectively).

Detection of O2������: LDCL

Agonist-induced O2� production was significantly increased by 27.9 ± 0.2% in the aorta

of 6-week-old SHR. Similarly, there was a significant 39.1 ± 0.1% increase of agonist-

induced O2� production in the 11-week-old SHRControl (Figure 2b). In contrast, 6-week-

old WKY showed no significant change in agonist-induced O2� production; however

11-week-old WKYControl did show a modest increase in agonist-induced O2� production

(19.4 ± 0.1%) (Figure 2a). O2� production was completely attenuated in SHRTempol at

11 weeks of age (Figure 2b).

Detection of O2������: Oxidative Fluorescent Microscopy

Basal O2� production was increased in 6-week-old SHR (Figure 3b) compared with

6-week-old WKY (Figure 3a) as detected by oxidative fluorescent microscopy. Tempol

pretreatment of segments from 6-week-old rats had a slight inhibitory effect (Figure 3c

and 3d). Basal O2� production in 11-week-old SHR appeared to be increased compared

with 11-week-old WKY that was inhibited by Tempol pretreatment (Figure 4). Tempol

had no noticeable effect on O2� production in 11-week-old WKY.

DiscussionThis study demonstrates for the first time that longitudinal administration of Tempol to

prehypertensive SHR prevents the development of high blood pressure and oxidant

stress in the adult. Our data also confirm the observation that agonist-induced vascular

O2� production is increased in 6- (young) and 11-week-old (adult) SHR (but not in

WKY). Thus, these findings show that scavenging of increased O2� by an antioxidant

inhibits a critical pathway in the development of high blood pressure in genetic

hypertension and that increased vascular O2� may precede the development of high

blood pressure in SHR.

Previous investigators have shown that in experimental models of hypertension,

increased oxidative stress may contribute to endothelial dysfunction and increased blood

pressure (7, 26, 27). In our study, we also found that vascular O2� production is increased

in adult SHR with established hypertension. While the mechanism for the production of

excess O2� remains unclear, previous studies have found excess O2

� to be associated

with decreased NO bioavailability and endothelial dysfunction (2, 6, 10, 26). Excess O2�

reacts preferentially with NO to form ONOO� resulting in the inactivation of an

important molecule essential to endothelium-dependent vasodilation.

These observations have led to further investigation of various antioxidants as

antihypertensive treatments. In humans with essential hypertension, administration of

vitamin C has been shown to lower plasma isoprostanes, an index of oxidant stress, and

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attenuate abnormal endothelium-dependent vasodilation (12). A recent study in 20–

22-week-old SHR showed that administration of vitamins C and E was associated with

decreased activation of NADPH oxidase and enhanced NO production by endothelial

nitric oxide synthase (eNOS) in rats (28). However, these authors did not examine the

longitudinal effect of vitamins C and E on blood pressure. Another group demonstrated

that acetylsalicylic acid attenuates the age-related increase in SBP in SHR through its

antioxidant properties (29). Although vascular O2� production was completely blocked

by acetylsalicylic acid, hypertension was only partially inhibited suggesting a possible

alternative mechanism for the action of acetylsalicyclic acid and rendering it less ideal as

an antihypertensive agent. These data differ from our own results. Tempol, a SOD

mimetic, also has been proposed as a possible antihypertensive therapy (13, 14). In adult

rats with established hypertension, Tempol has been shown to lower mean arterial

pressure (13, 14, 19), reduce oxidative stress (14, 19), prevent vascular remodeling (19),

and improve endothelial function (30, 31). Our results suggest that O2� production may

be increased in the vasculature of young SHR prior to the development of hypertension.

Scavenging of O2� with Tempol can completely prevent the rise in SBP in SHR, unlike

previously studied treatments that exhibit an intermediary effect on blood pressure.

Our biochemical results are consistent with prior studies in young, prehypertensive

SHR showing that endothelial dysfunction correlates to production of O2� and precedes

the onset of high blood pressure (21). A study by Chabrashvili et al. (22) showed that

expression of NADPH oxidase, an enzyme suggested as a possible source of excess O2�

production in hypertension (32, 33), is increased in the kidneys of young, prehypertensive

SHR compared with age-matched WKY. Although this study did not directly examine the

vasculature per se, upregulation of this possible source of O2� is consistent with our

findings. Our work also is in agreement with Consentino et al. (34) who showed that

aortas from prehypertensive SHR exhibit significantly increased calcium ionophore-

induced production of O2� compared with age-matched WKY. However, our data are not

in complete agreement with the results described by Wu et al. (35) and Hong et al. (36)

who found that O2� was not significantly elevated in young SHR. Yet based on

differences in methodology, it remains unclear whether our results are truly inconsistent

with Wu et al. because these authors examined NADH-stimulated O2� production. In

contrast, AII may regulate O2� production by more than one enzymatic pathway, such

as the xanthine oxidase pathway as previously hypothesized (33, 37). Wu et al.

acknowledge that their method assesses extracellular production of O2� by an unknown

mechanism that may or may not have an actual physiological role. Furthermore, Hong et

al. did, in fact, observe a modest increase in O2� production in 5-week-old SHR

compared with WKY, although not statistically significant. However, Hong et al.

measured LDCL at 15-min intervals. By only recording measurements every 15 min,

these investigators may have missed the majority of the signal that, in our hands, often

occurs within the first 5 min. We used LDCL as a primary tool to assess the production

of O2� in vascular tissue, but we also confirmed our quantitative LDCL data with

qualitative data obtained by DHE staining. Both assays show that increased vascular O2�

is produced in the young and adult SHR that can be inhibited by Tempol. We have

attempted to perform a more thorough examination of O2� production by combining

LDCL with fluorescence microscopy in the young SHR.

AII has been shown to stimulate O2� production primarily through the NADH/

NADPH oxidase pathway (32, 38). In our study, using the LDCL technique, we were

unable to detect basal differences among any groups. Therefore, we chose to use AII to

stimulate O2� production because of previous data suggesting that AII plays a critical

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role in the development of high blood pressure. (17, 21, 33, 38, 39). Thus, our data

showing an increase in O2� due to AII-stimulation in both young and adult SHR suggest

that NADPH oxidase may play an important role in O2� generation during development.

Furthermore, the reduction in AII-induced O2� production by L-NNA suggests that nitric

oxide synthase plays an important role. However, additional studies are necessary to

precisely identify the specific enzymatic and cellular sources of O2�.

Another somewhat surprising finding was that Tempol completely prevented any

increase in SBP in the adult WKY. In fact, SBP in WKYTempol was statistically lower

than WKYControl beginning at 8 weeks of age. We also observed no significant increase in

agonist-induced vascular O2� production in the young WKY or WKYTempol. We did

however, observe a modest increase in O2� production in the adult WKYControl. These

findings are consistent with results of other investigators who have found a baseline level

of vascular O2� in normotensive rats that is significantly decreased by antioxidant

administration (29), and with the interpretation that Tempol inhibits the increase in O2�

production associated with normal aging in adult WKY. However, Tempol also may act,

at least in part, by a different mechanism to lower blood pressure in normotensive rats.

Data by Xu et al. (40) suggest that in normotensive rats, Tempol lowers blood pressure in

part by inhibiting the sympathetic nervous system. These investigators found that the

heart rate and blood pressure responses to Tempol were unaffected by the addition of a

nitric oxide synthase inhibitor, suggesting that Tempol acts by a NO-independent

mechanism in normotensive rats. However, a more recent study provides evidence that

augmented O2� contributes to blood pressure regulation through activation of renal

sympathetic nerve activity (41).

Finally, we observed that only 48 hr following Tempol withdrawal, the SBP of

SHRTempol/Water was indistinguishable from SHRControl. This finding may indicate that

despite the inhibitory effect of Tempol treatment on blood pressure and O2� production,

the genetic mechanisms that lead to an abnormal increase in SBP are still operational in

adult SHR and are not permanently altered by long-term Tempol treatment.

ConclusionThis study suggests that increased oxidative stress plays a critical role in the development

of high blood pressure in an animal model of genetic hypertension. Nonetheless, we cannot

rule out the possible contribution of high blood pressure to a further increase in vascular

oxidative stress in adult SHR. These results may have important therapeutic implica-

tions for the utility of antioxidants in attenuation of essential hypertension in humans.

AcknowledgmentThe authors gratefully acknowledge the technical assistance of Ankur Mehta, Vinod

Chadalavada, Simone Welch, and Collin Cowley, MD.

References1. Nakozono K, Watanabe N, Matsuno K, Sasaki J, Sato T, Inoue M. Does superoxide underlie

the pathogenesis of hypertension? Proc Natl Acad Sci U S A 1991; 88:10045–10048.

2. McIntyre M, Bohr DF, Dominiczak AF. Endothelial function in hypertension: the role of

superoxide anion. Hypertension 1999; 34:539–545.

3. Zalba G, San Jose G, Moreno MU, Fortuno MA, Fortuno A, Beaumont FJ, Diez J. Oxidative

stress in arterial hypertension: role of NAD(P)H oxidase. Hypertension 2001; 38:1395–1399.

4. Nassar T, Kadery B, Lotan C, Da’as N, Kleinman Y, Haj-Yehia A. Effects of the superoxide

L. Nabha et al.80

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y.

dismutase-mimetic compound tempol on endothelial dysfunction in streptozotocin-induced

diabetic rats. Eur J Pharmacol 2002; 436:111–118.

5. Ohara Y, Peterson TE, Harrison DG. Hypercholesterolemia increases endothelial superoxide

anion production. J Clin Invest 1993; 91:2546–2551.

6. Grunfeld S, Hamilton CA, Mesaros S, McClain SW, Dominiczak AF, Bohr DF, Malinski T.

Role of superoxide in the depressed nitric oxide production by the endothelium of genetically

hypertensive rats. Hypertension 1995; 26:854–857.

7. Kerr S, Brosnan MJ, McIntyre M, Reid JL, Dominiczak AF, Hamilton CA. Superoxide anion

production is increased in a model of genetic hypertension: role of the endothelium.

Hypertension 1999; 33:1353–1358.

8. Thomson L, Trujillo M, Telleri R, Radi R. Kinetics of cytochrome c2+ oxidation by

peroxynitrite: implications for superoxide measurements in nitric oxide-producing biological

systems. Arch Biochem Biophys 1995; 319:491–497.

9. Ballinger SW, Patterson C, Yan CN, Doan R, Burrow DL, Young CG, Yakes FM, Van Houten

B, Ballinger CA, Freeman BA, Runge MS. Hydrogen peroxide- and peroxynitrite-induced

mitochondrial DNA damage and dysfunction in vascular endothelial and smooth muscle cells.

Circ Res 2000; 86:960–966.

10. Spieker LE, Noll G, Ruschitzka FT, Maier W, Luscher TF. Working under pressure: the

vascular endothelium in arterial hypertension. J Hum Hypertens 2000; 14:617–630.

11. Akpaffiong MJ, Taylor AA. Antihypertensive and vasodilator actions of antioxidants in

spontaneously hypertensive rats. Am J Hypertens 1998; 11:1450–1460.

12. Taddei S, Virdis A, Ghiadoni L, Magagna A, Favilla S, Pompella A, Salvetti A. Restoration of

nitric oxide availability after calcium antagonist treatment in essential hypertension.

Hypertension 2001; 37:943–948.

13. Schnackenberg CG, Welch WJ, Wilcox CS. Normalization of blood pressure and renal

vascular resistance in SHR with a membrane-permeable superoxide dismutase mimetic: role

of nitric oxide. Hypertension 1998; 32:59–64.

14. Schnackenberg CG, Wilcox CS. Two-week administration of tempol attenuates both hyper-

tension and renal excretion of 8-iso prostaglandin F2a. Hypertension 1999; 33:424–428.

15. Nishiyama A, Fukui T, Fujisawa Y, Rahman M, Tian RX, Kimura S, Abe Y. Systemic and

regional hemodynamic responses to Tempol in angiotensin II-infused hypertensive rats.

Hypertension 2001; 37:77–83.

16. Beswick RA, Zhang H, Marable D, Catravas JD, Hill WD, Webb RC. Long-term antioxidant

administration attenuates mineralocorticoid hypertension and renal inflammatory response.

Hypertension 2001; 37:781–786.

17. Shastri S, Gopalakrishnan V, Poduri R, Wang HD. Tempol selectively attenuates angiotensin

II evoked vasoconstrictor responses in spontaneously hypertensive rats. J Hypertens 2002;20:1381–1391.

18. Sato M, Yanagisawa H, Nojima Y, Tamura J, Wada O. Zn deficiency aggravates hypertension

in spontaneously hypertensive rats: possible role of Cu/Zn-superoxide dismutase. Clin Exp

Hypertens 2002; 24:355–370.

19. Park JB, Touyz RM, Chen X, Schiffrin EL. Chronic treatment with a superoxide dismutase

mimetic prevents vascular remodeling and progression of hypertension in salt-loaded stroke-

prone spontaneously hypertensive rats. Am J Hypertens 2002; 15:78–84.

20. Vaziri ND, Ni Z, Oveisi F. Upregulation of renal and vascular nitric oxide synthase in young

spontaneously hypertensive rats. Hypertension 1998; 31:1248–1254.

21. Endemann D, Touyz RM, Li JS, Deng LY, Schiffrin EL. Altered antiogensin II-induced small

artery contraction during the development of hypertension in spontaneously hypertensive rats.

Am J Hypertens 1999; 12:716–723.

22. Chabrashvili T, Tojo A, Onozato ML, Kitiyakara C, Quinn MT, Fujita T, Welch WJ, Wilcox

CS. Expression and cellular localization of classic NADPH oxidase subunits in the

spontaneously hypertensive rat kidney. Hypertension 2002; 39:269–274.

23. Ikeda H, Davis G, Yamori Y. Indirect systolic and mean blood pressure determination by a

new tail cuff method in spontaneously hypertensive rats. Lab Anim 1991; 25:26–29.

81Vascular Oxidative Stress

Clin

Exp

Hyp

erte

ns D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Y

ork

Uni

vers

ity L

ibra

ries

on

08/1

3/14

For

pers

onal

use

onl

y.

24. Kramer JM, Beatty JA, Little HR, Plowey ED, Waldrop TG. Chronic exercise alters caudal

hypothalamic regulation of the cardiovascular system in hypertensive rats. Am J Physiol

2001; 280:R389–R397.

25. Miller FJ Jr, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in

vascular smooth muscle contributes to oxidative stress and impaired relaxation in

atherosclerosis. Circ Res 1998; 82:1298–1305.

26. McIntyre M, Hamilton CA, Rees DD, Reid JL, Dominiczak AF. Sex differences in the

abundance of endothelial nitric oxide in a model of genetic hypertension. Hypertension 1997;30:1517–1524.

27. Zalba G, Beaumont J, San Jose G, Fortuno A, Fortuno MA, Diez J. Vascular oxidant stress:

molecular mechanisms and pathophysiological implications. J Physiol Biochem 2000; 56:57–

64.

28. Ulker S, McKweon PP, Bayraktutan U. Vitamins reverse endothelial dysfunction through

regulation of eNOS and NAD(P)H oxidase activites. Hypertension 2003; 41:534–539.

29. Wu R, Lamontague D, de Champlain J. Antioxidative properties of acetylsalicylic acid on

vascular tissues from normotensive and spontaneously hypertensive rats. Circulation 2002;

105:387–392.

30. Haj-Yehia AI, Nassar T, Assaf P, Nassar G, Anggard EE. Effects of the superoxide dismutase-

mimetic compound TEMPOL on oxidant stress-mediated endothelial dysfunction. Antioxid

Redox Signal 1999; 1:221–232.

31. Schnackenberg CG, Wilcox CS. The SOD mimetic Tempol restores vasodilation in afferent

arterioles of experimental diabetes. Kidney Int 2001; 59:1859–1864.

32. Zhang H, Schmeisser A, Garlichs CD, Plotze K, Damme U, Mugge A, Daniel WG.

Angiotensin II-induced superoxide anion generation in human vascular endothelial cells: role

of membrane-bound NADH-/NADPH-oxidases. Cardiovasc Res 1999; 44:215–222.

33. Berry C, Hamilton CA, Brosnan MJ, Magill FG, Berg GA, McMurray JJV, Dominiczak AF.

Investigation into the sources of superoxide in human blood vessels: angiotensin II increases

superoxide production in human internal mammary arteries. Circulation 2000; 101:2206–

2212.

34. Cosentino F, Patton S, d’Uscio LV, Werner ER, Werner-Felmayer G, Moreau P, Malinski T,

Luscher TF. Tetrahydrobiopterin alters superoxide and nitric oxide release in prehypertensive

rats. J Clin Invest 1998; 101:1530–1537.

35. Wu R, Millette E, Wu L, de Champlain J. Enhanced superoxide anion formation in vascular

tissues from spontaneously hypertensive and desoxycorticosterone acetate-salt hypertensive

rats. J Hypertens 2001; 19:741–748.

36. Hong HJ, Loh SH, Yen MH. Suppression of the development of hypertension by the inhibitor

of inducible nitric oxide synthase. Br J Pharmacol 2000; 131:631–637.

37. Mervaala EMA, Cheng ZJ, Tikkanen I, Lapatto R, Nurminen K, Vapaatalo H, Muller DN,

Fiebeler A, Ganten U, Ganten D, Luft FC. Endothelial dysfunction and xanthine oxido-

reductase activity in rats with human renin and angiotensinogen genes. Hypertension 2001;37:414–418.

38. Rajagopalan S, Kurz S, Munzel 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 1996; 97:1916–1923.

39. Traub O, Webb RC. Angiotensin-converting enzyme inhibition during development alters

calcium regulation in adult hypertensive rats. J Pharmacol Exp Ther 1993; 267:1503–

1508.

40. Xu H, Fink GD, Chen A, Watts S, Galligan JJ. Nitric oxide-independent effects of tempol on

sympathetic nerve activity and blood pressure in normotensive rats. Am J Physiol 2001;281:H975–H980.

41. Shokoji T, Nishiyama A, Fujisawa Y, Hitomi H, Kiyomoto H, Takahashi N, Kimura S, Kohno

M, Abe Y. Renal sympathetic nerve responses to Tempol in spontaneously hypertensive rats.

Hypertension 2003; 41:266–273.

L. Nabha et al.82

Clin

Exp

Hyp

erte

ns D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Y

ork

Uni

vers

ity L

ibra

ries

on

08/1

3/14

For

pers

onal

use

onl

y.