This Article Abstract Full Text (PDF) All Versions of this Article: 91/10/938    most recent 01.RES.0000043280.65241.04v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Didion, S. P. Articles by Faraci, F. M. Search for Related Content PubMed PubMed Citation Articles by Didion, S. P. Articles by Faraci, F. M. Related Collections Genetically altered mice Brain Circulation and Metabolism Oxidant stress Endothelium/vascular type/nitric oxide

(Circulation Research. 2002;91:938.)
© 2002 American Heart Association, Inc.

Integrative Physiology

Increased Superoxide and Vascular Dysfunction in CuZnSOD-Deficient Mice

Sean P. Didion, Michael J. Ryan, Lisa A. Didion, Pamela E. Fegan, Curt D. Sigmund, Frank M. Faraci

From the Departments of Internal Medicine, Physiology, and Pharmacology, Cardiovascular Center, The University of Iowa College of Medicine, Iowa City, Iowa.

Correspondence to Frank M. Faraci, PhD, Department of Internal Medicine, E315-GH, University of Iowa College of Medicine, Iowa City, Iowa 52242-1081. E-mail frank-faraci{at}uiowa.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Increased superoxide is thought to play a major role in vascular dysfunction in a variety of disease states. Superoxide dismutase (SOD) limits increases in superoxide; however, the functional significance of selected isoforms of SOD within the vessel wall are unknown. We tested the hypothesis that selective loss of CuZnSOD results in increased superoxide and altered vascular responsiveness in CuZnSOD-deficient (CuZnSOD^-/-) mice compared with wild-type (CuZnSOD^+/+) littermates. Total SOD activity was reduced (P<0.05) by approximately 60% and CuZnSOD protein was absent in aorta from CuZnSOD^-/- as compared with wild-type mice. Vascular superoxide levels, measured using lucigenin (5 µmol/L)-enhanced chemiluminescence and hydroethidine (2 µmol/L)-based confocal microscopy, were increased (approximately 2-fold; P<0.05) in CuZnSOD^-/- mice as compared with wild-type mice. Relaxation of the carotid artery in response to acetylcholine and authentic nitric oxide was impaired (P<0.05) in CuZnSOD^-/- mice. For example, maximal relaxation to acetylcholine (100 µmol/L) was 50±6% and 69±5% in CuZnSOD^-/- and wild-type mice, respectively. Contractile responses of the carotid artery were enhanced (P<0.05) in CuZnSOD^-/- mice in response to phenylephrine and serotonin, but not to potassium chloride or U46619. In vivo, dilatation of cerebral arterioles (baseline diameter=31±1 µm) to acetylcholine was reduced by approximately 50% in CuZnSOD^-/- mice as compared with wild-type mice (P<0.05). These findings provide the first direct insight into the functional importance of CuZnSOD in blood vessels and indicate that this specific isoform of SOD limits increases in superoxide under basal conditions. CuZnSOD-deficiency results in altered responsiveness in both large arteries and microvessels.

Key Words: carotid artery • cerebral arterioles • genetically altered mice • nitric oxide • superoxide dismutase 1

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The bioactivity of nitric oxide depends, in part, on its interaction with reactive oxygen species, particularly superoxide.¹ Although there has been considerable effort to define the role of nitric oxide in the circulation, the role of superoxide within the vessel wall is poorly understood. Because the initial finding of Kontos and Wei that superoxide inactivates nitric oxide in vivo,² many studies have suggested that inactivation of nitric oxide by superoxide contributes to impaired vascular function.^3–7 Indeed, nitric oxide reacts with superoxide at a rate 3 times faster than dismutation of superoxide by superoxide dismutases (SODs).¹ Substances that generate superoxide (such as pyrogallol, tetrahydrobiopterin, amyloid ß peptide, and xanthine/xanthine oxidase) impair endothelium-dependent relaxation of blood vessels.^5,6,8,9 Under these conditions, impairment of endothelium-dependent relaxation is most likely due to interaction of superoxide with nitric oxide and reduction in nitric oxide bioavailability, because superoxide scavengers are effective in protecting vascular function.^5,6,8,9 Thus, local vascular levels of superoxide appear to have profound effects on endothelium-dependent relaxation.

Local vascular levels of superoxide reflect both the rate of superoxide formation and the rate of its removal by endogenous antioxidants (primarily SODs). In blood vessels, potential enzymatic sources of superoxide include cyclooxygenase, nitric oxide synthases, lipoxygenase, NAD(P)H oxidase, xanthine oxidase, and mitochondria.¹⁰ Much attention has been focused on sources of superoxide in relation to superoxide levels and vascular dysfunction. However, much less is known regarding the functional importance of alteration in expression or activity of SODs within the vascular wall.

SODs exist as three isoforms (each encoded by separate genes) localized within specific cellular compartments. Copper-zinc SOD (CuZnSOD; SOD1) is located predominately within the cytosol, as well as in the nucleus and is thought to be expressed in all mammalian cells. Manganese SOD (MnSOD; SOD2) is targeted to the mitochondrial matrix and is considered to be the primary SOD isoform in relation to oxidative stress in mitochondria. Extracellular-SOD (EC-SOD; SOD3), also a copper-zinc-containing SOD, is secreted extracellularly and is found primarily bound to heparan sulfate proteoglycan on cell surfaces. Within blood vessels, the predominant isoform of SOD (when expressed as percent of total SOD activity) is CuZnSOD.^11–14 For example, in normal mouse aorta, activity of CuZnSOD accounts for 53% to 80% of total SOD activity, MnSOD accounts for 2% to 7% of total vascular SOD, and EC-SOD accounts for the remainder.^11–14 A similar pattern of expression exists in human arteries.¹¹ It has been suggested that release of nitric oxide from endothelium is dependent on CuZnSOD, whereas EC-SOD activity is thought to be required for protection of nitric oxide as it diffuses through the vascular wall.^11,15,16 Although it is known that the three isoforms of SOD are expressed within the vessel wall, the functional importance of each SOD isoform is unclear.

To more directly examine the role of CuZnSOD and to distinguish it from EC-SOD and MnSOD, we tested the hypothesis that loss of CuZnSOD results in increased superoxide levels and impaired vascular function. To test this hypothesis, we used mice that were selectively deficient in CuZnSOD.¹⁷ Although, CuZnSOD-deficient mice have been used previously to examine the effects of cerebral and cardiac cell injury response to ischemia/reperfusion,^18–20 we are not aware of any studies that have examined superoxide levels and vascular responses. To this end, we measured superoxide levels and responses of carotid arteries and cerebral arterioles in wild-type mice and mice deficient in the expression of the gene for CuZnSOD.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Experimental Animals
Mice used for this study were derived from breeding pairs of heterozygous CuZnSOD-deficient (B6;129S-SOD1tm1Leb) mice obtained from Jackson Laboratory (Bar Harbor, Maine). Two groups of mice were studied: homozygous CuZnSOD-deficient (CuZnSOD^-/-) mice and wild-type (CuZnSOD^+/+) littermates. All breeding and genotyping was performed in a virus- and pathogen-free barrier-facility at the University of Iowa. The genotype of each mouse was ascertained by polymerase chain reaction of DNA isolated from tail biopsy samples as described on the Jackson Laboratory Web site.²¹ All experimental protocols were approved by the University of Iowa Animal Care and Use Committee.

SOD Activity
Total SOD activity of aortic homogenates from wild-type and CuZnSOD^-/- mice was determined as previously described.²² This assay is based on the competition of SOD and nitroblue-tetrazolium (NBT) for superoxide, thus the percent inhibition of NBT reduction is a measure of the amount of SOD present. The rate of NBT reduction to blue formazan was measured spectrophotometrically for 5 minutes at 560 nm. One unit of SOD activity is defined as the amount of protein that results in 50% inhibition of NBT reduction. SOD activity is expressed as U/mg protein. Total protein content was determined using a Bio-Rad protein assay kit.

Western Blotting for CuZnSOD
CuZnSOD protein expression in aorta of wild-type and CuZnSOD^-/- mice was examined by Western blotting as described previously.^23,24 Aorta (pool of 3) from wild-type and CuZnSOD^-/- mice was homogenized in extraction buffer (1% SDS, 10 mmol/L EDTA in water) and boiled, the protein extract being collected as supernatant after centrifugation. Protein concentrations were determined using a Bio-Rad protein assay kit, and then adjusted to 3 mg/mL with 4x Laemmli buffer. Protein samples were boiled and 75 µg of protein was loaded onto 4% to 20% gradient Tris-HCl gradient gel (BioRad) and electrophoresed. Proteins were blotted to a nitrocellulose membrane using a semi-wet cell. Proteins were blocked with PBS+0.05% Tween and 5% nonfat milk and then incubated with sheep polyclonal IgG Anti-CuZnSOD primary antibody (2 µL/mL; Upstate Biotechnology) at 4°C overnight. The membranes were washed with PBS+0.05% Tween and 5% nonfat milk and incubated for 1 hour with rabbit anti-sheep IgG-peroxidase-conjugated secondary antibody at a final concentration of 1:200 000 (Pierce). The membranes were developed with Supersignal (Pierce) for 1 to 3 minutes, exposed to x-ray film, and developed.

Measurement of Superoxide
Superoxide levels were measured using two approaches. First, basal superoxide levels were measured in aorta using lucigenin (5 µmol/L)-enhanced chemiluminescence as described previously.^3,4,25 In some experiments, vessel segments were preincubated for 30 minutes with either 4,5-dihydroxy-1,3-benzne disulfonic acid (Tiron; 10 mmol/L) or polyethylene-glycol-SOD (PEG-SOD) to quench the superoxide signal. Second, superoxide levels were also evaluated in carotid artery using hydroethidine (2 µmol/L)-based confocal microscopy as described previously.^3,26–28 Relative increases in ethidium bromide fluorescence were determined using SCION Image software for the PC (version 4.02; Scion Corporation) as previously described.³ Ethidium bromide fluorescence was normalized to the cross-sectional area of the vessel wall for each section.

Vascular Studies
Rings of carotid artery were studied in individual organ chambers as previously described.^3,29–31 After equilibration, vessels were contracted submaximally (50% to 60% of maximum) with the thromboxane analogue 9,11-dideoxy-11a,9a-epoxy-methanoprostaglandin F₂ (U46619). After reaching a stable contraction plateau, concentration-response curves were generated for the endothelium-dependent dilator acetylcholine (10 nmol/L to 100 µmol/L), for authentic nitric oxide (0.1 to 10 µmol/L), and for the non–nitric oxide dilator papaverine (0.1 to 100 µmol/L). We have shown previously using pharmacological and genetic approaches that responses of the carotid artery to acetylcholine are mediated by eNOS and nitric oxide.^30,31

Because contractile response to vasoconstrictors may be modulated by nitric oxide,^32–35 and because mice deficient in the gene that encodes for eNOS demonstrate enhanced sensitivity to serotonin,³⁰ we examined contractile responses in wild-type and CuZnSOD^-/- mice. Contractile responses to phenylephrine (10 nmol/L to 100 µmol/L), serotonin (10 nmol/L to 10 µmol/L), and potassium chloride (1 mmol/L to 100 mmol/L) were measured. At the end of each experiment, a concentration-response curve to U46619 (0.03 to 3.0 µg/mL) was generated in order to determine the maximal contractile response of each vessel.

Studies of Cerebral Arterioles
Wild-type and CuZnSOD^-/- mice were anesthetized with pentobarbital sodium (75 to 90 mg/kg IP), supplemented regularly at approximately 20 mg/kg per hour. Animals were ventilated mechanically, and arterial blood pressure and blood gasses were monitored as described previously.^36,37 A cranial window was made over the left parietal cortex, and a segment of a randomly selected pial arteriole was exposed. The diameter of cerebral arterioles was measured using a microscope equipped with a TV camera coupled to a video monitor and an image-shearing device. The diameter of one arteriole per animal was measured under control conditions and during topical application (cumulative administration) of acetylcholine (1 and 10 µmol/L) and nitroprusside (0.1 and 1 µmol/L). Arterial blood gasses were monitored and were similar in the two groups [wild type, PCO₂=36±2 mm Hg, PO₂=141±16 mm Hg, and pH=7.30±0.02; CuZnSOD^-/-, PCO₂=33±2 mm Hg, PO₂=116±11 mm Hg, and pH=7.36±0.02 (mean±SE)].

Blood Pressure Measurements
Because some forms of hypertension have been associated with increases in vascular superoxide and because we hypothesized that vascular superoxide levels would be increased in CuZnSOD-deficient mice. We measured blood pressure in wild-type and CuZnSOD^-/- mice using an automated tail-cuff device (BP-2000, Visitech Systems).^3,38 Because the tail-cuff method typically underestimates blood pressure, we also measured arterial pressure directly through the use of indwelling carotid catheters in freely moving mice as described previously.³⁸ Blood pressure measurements using both methods were performed in conscious mice because anesthesia in mice lowers blood pressure and tends to normalize differences in blood pressure as compared with the conscious state.

Drugs
Acetylcholine, papaverine, PEG-SOD, phenylephrine, serotonin, and Tiron were obtained from Sigma, and all were dissolved in saline. U46619 was obtained from Cayman Chemical and dissolved in 100% ethanol with subsequent dilution being made with saline. Hydroethidine was obtained from Molecular Probes and dissolved in DMSO at a concentration of 0.1 mol/L. Authentic nitric oxide was prepared as previously described.³⁹ All other reagents were of standard laboratory grade.

Statistical Analysis
All data are expressed as mean±SE. Relaxation to acetylcholine, nitric oxide, and papaverine is expressed as a percent relaxation to U46619-induced contraction. Comparisons of relaxation and contraction were made using analysis of variance followed by Bonferroni’s multiple comparison test. Comparison of blood pressure, SOD activity, and superoxide levels was made using paired t test. Statistical significance was accepted at P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Blood Pressure and Baseline Characteristics
Systolic blood pressure in conscious CuZnSOD^-/- mice (102±4 mm Hg; n=6), as measured using tail-cuff, was lower (P<0.05) as compared with that in wild-type mice (115±2 mm Hg; n=6). Measurements using carotid catheters confirmed tail-cuff measurements, in that blood pressure was lower (P<0.05) in CuZnSOD^-/- (114±3 mm Hg; n=4) as compared with wild-type littermates (134±5 mm Hg; n=5). Wild-type and CuZnSOD^-/- mice were of similar (P>0.05) age (5±1 and 5±1 months, respectively) and body weight (28±3 and 33±3 g, respectively).

SOD Activity
In wild-type mice, total aortic SOD activity was 155±22 U/mg, which is consistent to that reported previously in mouse aorta.¹¹ Total aortic SOD activity was reduced (P<0.05) in CuZnSOD^-/- mice by approximately 60% compared with activity levels in wild-type mice (Figure 1A). Western blotting confirmed that CuZnSOD protein was absent in vascular tissue in CuZnSOD^-/- mice (Figure 1B). These results indicate that loss of CuZnSOD through gene targeting results in an absence of CuZnSOD protein and a large reduction in total SOD activity in vessels, consistent with previous findings of CuZnSOD protein and activity levels in other tissues of CuZnSOD-deficient mice.^17–20

View larger version (19K): [in this window] [in a new window]   Figure 1. A, Total SOD activity expressed as U/mg total protein in aortic homogenates from CuZnSOD-/- (n=6) and wild-type (n=6) mice. B, Western blot for CuZnSOD protein expression in aorta from CuZnSOD-/- and wild-type mice. Aorta, represents protein pooled from 3 mice of each respective genotype. C, Superoxide levels, as detected by lucigenin-enhanced chemiluminescence, in aorta from CuZnSOD-/- (n=17) and wild-type (n=25) mice under basal conditions (open bars) and in the presence of 300 U/mL PEG-SOD (filled bars). WT indicates wild type; KO, CuZnSOD-/-. Values are mean±SE; *P<0.05 vs wild type; P<0.05 vs control.

Superoxide Levels
To determine whether loss of CuZnSOD is associated with increases in superoxide, basal superoxide levels were measured in wild-type and CuZnSOD^-/- mice. Basal superoxide levels, as measured using lucigenin (5 µmol/L)-enhanced chemiluminescence, were approximately 2-fold higher (P<0.05) in aorta of CuZnSOD^-/- mice compared with levels in aorta of wild-type mice (Figure 1C). Preincubation of aorta with Tiron (10 mmol/L; data not shown) or PEG-SOD (300 U/mL; Figure 1C) markedly reduced the lucigenin signal in both groups.

Consistent with results obtained with lucigenin, superoxide levels, as measured using hydroethidine, were higher in carotid artery of CuZnSOD^-/- mice compared with wild type (Figure 2A). Quantification of ethidium bromide signal (based on relative difference in fluorescent intensity), revealed higher (P<0.05) fluorescence in carotid artery of CuZnSOD^-/- mice as compared with wild-type CuZnSOD^+/+ mice (Figure 2B).

View larger version (18K): [in this window] [in a new window]   Figure 2. A, Representative confocal fluorescent sections of carotid arteries from wild-type (Left) and CuZnSOD-/- (Right) mice incubated with hydroethidine for detection of superoxide. In wild type, relatively equal levels of fluorescence were detected throughout the vessel wall. Similarly, in CuZnSOD-/- mice, fluorescence was noted to be equal throughout the vessel wall. However, the intensity of the fluorescent signal was higher than that observed in wild type, suggesting vascular superoxide levels are increased after deletion of the CuZnSOD gene. E indicates endothelium; A, adventitia. Scale Bar=100 µm. B, Relative superoxide levels, as detected by hydroethidine-based confocal microscopy in carotid artery from wild-type (n=13) and CuZnSOD-/- (n=8) mice. Relative superoxide levels are the mean of low-, medium-, and high-intensity fluorescence normalized to vessel area. WT indicates wild type. Values are mean±SE; *P<0.05 vs wild type.

Vascular Responses of Carotid Artery
Acetylcholine (Figures 3 and 4) and authentic nitric oxide (Figure 4) produced concentration-dependent relaxation in carotid artery from wild-type mice. In contrast, relaxation to acetylcholine (Figures 3 and 4) and nitric oxide (Figure 4) was impaired (P<0.05) in carotid artery from CuZnSOD^-/- mice compared with wild type. For example, maximal relaxation in response to acetylcholine was 50±6% and 69±5% in CuZnSOD^-/- and wild-type mice, respectively. To determine if these changes were selective, we examined relaxation responses to the non-endothelium-dependent, non-nitric oxide agonist, papaverine. Papaverine produced relaxation that was similar (P>0.05) in wild-type and CuZnSOD^-/- mice (Figure 4), indicating that the impaired response to acetylcholine and nitric oxide observed in CuZnSOD^-/- mice was selective.

View larger version (12K): [in this window] [in a new window]   Figure 3. Representative tracings of response of carotid arteries precontracted with U-44619 to acetylcholine from wild-type (Left) and CuZnSOD-/- (Right) mice.

View larger version (16K): [in this window] [in a new window]   Figure 4. Relaxation of carotid arteries from wild-type (open squares; n=8) and CuZnSOD-/- (filled circles; n=6) mice to the endothelium-dependent agonist acetylcholine (Left), to authentic nitric oxide (center), and to the endothelium-independent, non–nitric oxide agonist papaverine (Right). Values are mean±SE; *P<0.05 vs wild type.

Phenylephrine and serotonin produced concentration-dependent contraction in carotid artery from wild-type mice (Figures 5 and 6). Contractile responses to phenylephrine and serotonin were enhanced (P<0.05) in carotid arteries of CuZnSOD^-/- mice (Figures 5 and 6). Potassium chloride (Figure 6) and U46619 (data not shown) produced similar (P>0.05) levels of contraction in CuZnSOD^-/- and wild-type mice, suggesting that the enhanced response to phenylephrine and serotonin was selective.

View larger version (15K): [in this window] [in a new window]   Figure 5. Representative tracings of contractile response of carotid arteries to serotonin (top tracings) and phenylephrine (bottom tracings) from wild-type and CuZnSOD-/- mice.

View larger version (18K): [in this window] [in a new window]   Figure 6. Contraction of carotid arteries from wild-type (open squares; n=12) and CuZnSOD-/- (filled circles; n=12) mice to phenylephrine (Left), serotonin (Center), and potassium chloride (Right). Values are mean±SE; *P<0.05 vs wild type.

Effects of CuZnSOD-Deficiency on Dilator Responses of Cerebral Arterioles
Baseline diameter of cerebral arterioles was similar (P>0.05) in CuZnSOD^-/- (31±1 µm; n=9) and wild-type (33±2 µm; n=9) mice under control conditions. Mean arterial pressure (in anesthetized mice) was similar in both groups (76±4 mm Hg and 77±4 mm Hg in CuZnSOD^-/- and wild-type mice, respectively) under control conditions and was not affected by application of agonists into the cranial window.

Acetylcholine and nitroprusside produced concentration-dependent dilatation of cerebral arterioles (Figure 7). Dilatation of cerebral arterioles after topical application of acetylcholine was reduced by about 50% (P<0.05) in CuZnSOD^-/- mice compared with wild type (Figure 7). In contrast, dilatation of cerebral arterioles in response to nitroprusside was similar (P>0.05) in CuZnSOD^-/- and wild-type mice (Figure 7). These findings provide direct evidence that CuZnSOD deficiency impaired endothelial function in the microcirculation of a key organ in vivo.

View larger version (17K): [in this window] [in a new window]   Figure 7. Response of cerebral arterioles to acetylcholine (Left) and nitroprusside (Right) in CuZnSOD-/- (n=9) and wild-type (n=9) mice. Values are mean±SE; *P<0.05 vs wild type.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
There are several major new findings of the present study. First, CuZnSOD protein is absent and total SOD activity is markedly reduced in aorta from CuZnSOD^-/- as compared with wild-type mice. Second, basal superoxide levels in aorta and carotid artery, as measured using two independent methods of superoxide detection, were significantly higher in CuZnSOD^-/- mice under control conditions. These findings provide direct evidence that endogenous CuZnSOD limits increases in superoxide within the vessel wall under basal conditions. Third, in addition to increases in superoxide, relaxation of carotid arteries to acetylcholine and authentic nitric oxide and dilatation of cerebral arterioles in response to acetylcholine was significantly impaired in CuZnSOD^-/- mice as compared with wild-type mice. Fourth, contractile responses to phenylephrine and serotonin were augmented selectively in carotid artery of CuZnSOD^-/- mice. Taken together, these findings suggest that increases in basal superoxide in blood vessels due to deficiency in CuZnSOD produces impaired nitric oxide–mediated relaxation and enhanced contraction.

Selective Deficiency in CuZnSOD Decreases Total SOD Activity and Increases Superoxide Levels in Blood Vessels
It has been shown previously that deletion of the gene encoding CuZnSOD significantly reduces CuZnSOD protein and activity levels in nonvascular tissue from CuZnSOD-deficient mice.^{17–19,40–42} In the present study, we demonstrate for the first time that CuZnSOD protein is absent and total SOD activity is greatly reduced in aorta from CuZnSOD^-/- mice as compared with wild-type littermates. The percent reduction in total SOD activity (approximately 60%) in CuZnSOD^-/- mice is entirely consistent with what would be expected based on previous measurements, which indicated that 50% to 80% of total SOD activity in blood vessels is due to activity of CuZnSOD.^11–14

Consistent with our hypothesis, selective loss of CuZnSOD resulted in increased levels of superoxide in blood vessels. Superoxide was increased approximately 2-fold in both aorta and carotid artery in CuZnSOD^-/- mice, as measured using lucigenin-enhanced chemiluminescence and hydroethidine-based confocal microscopy. For both methods, the finding that the superoxide signal was markedly reduced by scavengers of superoxide suggests the assays are selective for superoxide. The present findings are consistent with previous reports, in which pharmacological inhibition of SOD activity with diethyldithiocarbamate (DDC), an inhibitor of copper-containing SODs (CuZnSOD and EC-SOD) results in increased superoxide in vessels.^4,43,44

Deficiency in CuZnSOD Alters Vascular Responses
Increased superoxide is thought to play a major role in vascular dysfunction (including endothelial dysfunction) in many disease states including atherosclerosis, diabetes, hypertension as well as aging, Alzheimer’s disease, subarachnoid hemorrhage, and ischemia/reperfusion.^16,45–47 This concept is based in part on the findings that scavengers of superoxide or gene transfer of antioxidant enzymes restores endothelium-dependent relaxation in several of these disease states.^3,7,26–28

Because of the association of superoxide levels with vascular dysfunction, many studies have focused on the identification of the source(s) of superoxide during pathological conditions. In addition to changes in the rate of superoxide production, local levels of superoxide are also dependent on the rate of metabolism of superoxide by SODs. Little is known, however, regarding the functional importance of decreased SOD expression and activity in blood vessels. Because basal superoxide levels were increased in CuZnSOD^-/- mice and because superoxide interacts readily with nitric oxide (thereby limiting its bioavailability), we examined endothelium-dependent and nitric oxide–mediated relaxation in CuZnSOD^-/- mice. Relaxation of carotid artery to the endothelium dependent-dilator acetylcholine and to authentic nitric oxide was reduced in CuZnSOD^-/- mice compared with wild type. These findings are consistent with previous studies in which superoxide levels were increased pharmacologically with DDC, resulting in impairment of endothelium-dependent relaxation in several different blood vessels, including cerebral arterioles.^4,43,44 However, pharmacological inhibition of endogenous SODs with DDC has at least three major limitations. First, DDC inhibits both CuZnSOD and EC-SOD activity. Thus, DDC does not provide any information regarding the importance of CuZnSOD versus EC-SOD within the vessel wall. This is of importance because CuZnSOD and EC-SOD together account for the majority (>90%) of the total SOD activity found in blood vessels.^11–14 Thus, DDC-induced increases in superoxide and impairment of vascular function, most likely reflects the loss of activity of both CuZnSOD and EC-SOD. Second, inhibition of SOD activity with DDC may not be complete, as there are uncertainties with regard to the cellular and subcellular access of DDC. Third, DDC may have nonspecific effects including inhibition of other copper-containing proteins. Thus, the use of CuZnSOD-deficient mice allowed us to examine the functional effects of selective CuZnSOD-deficiency on superoxide levels and vascular function.

In addition to impaired relaxation of carotid artery, we also observed impaired dilatation to acetylcholine in cerebral arterioles from CuZnSOD^-/- mice, suggesting that CuZnSOD limits increases in superoxide in cerebral blood vessels. These findings also indicate that the influence of CuZnSOD is not only limited to large conduit vessels but also extends to resistance vessels and the microcirculation of a key organ. The findings that relaxation to papaverine and nitroprusside was unaltered in carotid artery and cerebral arterioles in CuZnSOD^-/- mice, suggests that the impaired responses to acetylcholine were selective.

In addition to mediating relaxation, endothelium-derived nitric oxide can also exert a profound modulatory influence on the responsiveness of blood vessels to constrictor stimuli. For example, removal of endothelium or pharmacological inhibition of nitric oxide synthase has been shown to potentiate vascular responses to several constrictor stimuli including phenylephrine and serotonin.^32–35 We have shown previously contraction of the carotid artery to serotonin is selectively enhanced in eNOS-deficient mice.³⁰ Thus, nitric oxide produced by eNOS can exert a profound influence on vasoconstrictor responses. The present study indicates that responses of carotid artery to phenylephrine and serotonin are enhanced in CuZnSOD-deficient mice, presumably reflecting the loss of nitric oxide bioavailability. The enhanced contractile responses to phenylephrine and serotonin in CuZnSOD^-/- mice appear to be selective because contraction to potassium chloride and U46619 were not affected. Although a reduction in bioavailable nitric oxide seems the most likely explanation, we cannot rule out other effects of superoxide that may influence vasoconstrictor mechanisms.

In some experimental models of hypertension, administration of agents (ie, exogenous SODs) that reduce oxidative stress produce reductions in blood pressure.⁴⁸ The mechanism(s) that produces this effect has not been fully defined but may include vascular effects related to an increased nitric oxide bioavailability. Conversely, reductions in nitric oxide bioavailability, as in the case of eNOS-deficiency (ie, eNOS knockout mice), are associated with increases in blood pressure.⁴⁹ Thus, vascular levels of nitric oxide and superoxide affect vascular tone and appear to influence blood pressure. Based on this evidence, we hypothesized that increases in superoxide in vessels in CuZnSOD^-/- mice may be associated with an elevation in blood pressure. Although, superoxide levels in vessels were elevated, CuZnSOD^-/- mice were not hypertensive. Instead, measurements with two independent methods in conscious animals indicated that blood pressure in CuZnSOD^-/- mice was approximately 17 mm Hg less than that in wild-type littermates. Although this finding appears to be paradoxical, a recent report found that blood pressure in CuZnSOD transgenic mice was similar to that in nontransgenic mice, despite the fact that basal levels of superoxide were significantly lower in CuZnSOD transgenic mice.⁵⁰ It is also important to emphasize that increases in vascular superoxide levels, such as in diabetes and atherosclerosis or in response to lipopolysaccharide, are not always associated with increased arterial pressure. Collectively, these findings suggest that oxidative stress produced by CuZnSOD deficiency may influence blood pressure. At this time, we do not know if the mechanism that mediates the reduction in blood pressure in CuZnSOD^-/- mice is vascular, central, and/or renal.

Implications of Reduced CuZnSOD Activity to Vascular Dysfunction
The present study suggests that selective loss of CuZnSOD has profound effects on superoxide levels and vascular function. Thus, it seems likely that reduced CuZnSOD activity, due to a disease state or genetic mutation, would also have profound effects on vascular function. For example, impaired endothelial function in diabetic rats is associated with an approximately 50% reduction in total SOD activity.^51,52 Additionally, increases in superoxide and impaired vascular responses have been demonstrated in mice deficient in glutathione peroxidase-1, suggesting that other antioxidant enzymes, in addition to CuZnSOD, may also have an important role in regulation of vascular tone.⁵³

In summary, the results of the present study provide the first direct evidence that CuZnSOD protects nitric oxide–mediated vasorelaxation and counteracts vasoconstrictor responses. CuZnSOD normally maintains relatively low levels of vascular superoxide so that the selective loss of CuZnSOD activity results in enhanced superoxide and marked changes in vascular function. Functional effects of CuZnSOD deficiency were observed in vitro and in vivo in both large vessels and the microcirculation.

	Acknowledgments

 
This work was supported by National Institutes of Health grants NS-24621, HL-38901, HL-48058, HL-55006, HL-61446, HL-62984 (to F.M.F. and C.D.S.), and DK-25295 (to S.P.D.), Individual National Service Research Award HL-10425 (to M.J.R.), and National American Heart Association Scientist Development Award 0230327N (to S.P.D.). We thank Dale Kinzenbaw, Cynthia Lynch, and Pamela Tompkins for technical assistance. We also thank Jon Andresen for assistance with the SOD activity assay.

Received August 23, 2002; revision received October 14, 2002; accepted October 14, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Beckman JS, Koppenol WH. Nitric oxide, superoxide and peroxynitrite: the good, the bad, and the ugly. Am J Physiol. 1996; 271: C1424–C1437.[Medline] [Order article via Infotrieve]

2. Wei EP, Kontos HA, Christman CW, DeWitt DS, Povlishock JT. Superoxide generation and reversal of acetylcholine-induced cerebral arteriolar dilatation after acute hypertension. Circ Res. 1985; 57: 781–787.[Abstract/Free Full Text]

3. Didion SP, Ryan MJ, Baumbach GL, Sigmund CD, Faraci FM. Superoxide contributes to vascular dysfunction in mice that express human renin and human angiotensinogen. Am J Physiol Heart Circ Physiol. 2002; 283: H1569–H1576.[Abstract/Free Full Text]

4. Didion SP, Hathaway CA, Faraci FM. Superoxide levels and function of cerebral blood vessels after inhibition of CuZn-SOD. Am J Physiol Heart Circ Physiol. 2001; 281: H1697–H1703.[Abstract/Free Full Text]

5. Iadecola C, Zhang F, Niwa K, Eckman C, Turner SK, Fischer E, Younkin S, Borchelt DR, Hsiao KK, Carlson GA. SOD1 rescues cerebral endothelial dysfunction in mice overexpressing amyloid precursor protein. Nat Neurosci. 1999; 2: 157–161.[CrossRef][Medline] [Order article via Infotrieve]

6. Kinoshita H, Milstien S, Wambi C, Katusic ZS. Inhibition of tetrahydrobiopterin biosynthesis impairs endothelium-dependent relaxations in canine basilar artery. Am J Physiol. 1997; 273: H718–H724.[Medline] [Order article via Infotrieve]

7. Mayhan WG. Superoxide dismutase partially restores impaired dilatation of the basilar artery during diabetes mellitus. Brain Res. 1997; 760: 204–209.[CrossRef][Medline] [Order article via Infotrieve]

8. Ignarro LJ, Byrns RE, Buga GM, Wood KS, Chaudhuri G. Pharmacological evidence that endothelium-derived relaxing factor is nitric oxide: use of pyrogallol and superoxide dismutase to study endothelium-dependent and nitric oxide-elicited vascular smooth muscle relaxation. J Pharmacol Exp Ther. 1988; 244: 181–189.[Abstract/Free Full Text]

9. Dowell FJ, Hamilton CA, McMurray J, Reid JL. Effects of a xanthine oxidase/hypoxanthine free radical and reactive oxygen species generating system on endothelial function in New Zealand white rabbit aortic rings. J Cardiovasc Pharmacol. 1993; 22: 792–797.[Medline] [Order article via Infotrieve]

10. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000; 87: 840–844.[Abstract/Free Full Text]

11. Stralin P, Karlsson K, Johansson BO, Marklund SL. The interstitium of the human arterial wall contains very large amounts of extracellular superoxide dismutase. Arterioscler Thromb Vasc Biol. 1995; 15: 2032–2036.[Abstract/Free Full Text]

12. Fukai T, Siegfried MR, Ushio-Fukai M, Cheng Y, Kojda G, Harrison DG. Regulation of the vascular extracellular superoxide dismutase by nitric oxide and exercise training. J Clin Invest. 2000; 105: 1631–1639.[Medline] [Order article via Infotrieve]

13. Fukai T, Siegfried MR, Ushio-Fukai M, Griendling KK, Harrison DG. Modulation of extracellular superoxide dismutase expression by angiotensin II and hypertension. Circ Res. 1999; 85: 23–28.[Abstract/Free Full Text]

14. Fukai T, Galis ZS, Meng XP, Parthasarathy S, Harrison DG. Vascular expression of extracellular superoxide dismutase in atherosclerosis. J Clin Invest. 1998; 101: 2101–2111.[Medline] [Order article via Infotrieve]

15. Mugge A, Elwell JH, Peterson TE, Harrison DG. Release of intact endothelium derived relaxing factor depends on endothelial superoxide dismutase activity. Am J Physiol Cell Physiol. 1991; 260: C219–C225.[Abstract/Free Full Text]

16. Oury TD, Day BJ, Crapo JD. Extracellular superoxide dismutase: a regulator of nitric oxide bioavailability. Lab Invest. 1996; 75: 617–636.[Medline] [Order article via Infotrieve]

17. Matzuk MM, Dionne L, Guo Q, Kumar TR, Lebovitz RM. Ovarian function in superoxide dismutase 1 and 2 knockout mice. Endocrinology. 1998; 139: 4008–4011.[Abstract/Free Full Text]

18. Asimakis GK, Lick S, Patterson C. Postischemic recovery of contractile function is impaired in SOD2^+/- but not SOD1^+/- mouse hearts. Circulation. 2002; 105: 981–986.[Abstract/Free Full Text]

19. Kondo T, Reaume AG, Huang T-T, Carlson E, Murakami K, Chen SF, Hoffman EK, Scott RW, Epstein CJ, Chan PH. Reduction of CuZn-superoxide dismutase activity exacerbates neuronal cell injury and edema formation after transient focal cerebral ischemia. J Neurosci. 1997; 17: 4180–4189.[Abstract/Free Full Text]

20. Kawase M, Murakami K, Fujimura M, Morita-Fujimura Y, Gasche Y, Kondo T, Scott RW, Chan PH. Exacerbation of delayed cell injury after transient global ischemia in mutant mice with CuZn superoxide dismutase deficiency. Stroke. 1999; 30: 1962–1968.[Abstract/Free Full Text]

21. The Jackson Laboratory Web site. Available at: http://www.jax.org. Accessed October 14, 2002.

22. Spitz DR, Oberley LW. An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal Biochem. 1989; 179: 8–18.[CrossRef][Medline] [Order article via Infotrieve]

23. Fang X, Weintraub NL, Rios CD, Chappell DA, Zwacka RM, Engelhardt JF, Oberley LW, Yan T, Heistad DD, Spector AA. Overexpression of human superoxide dismutase inhibits oxidation of low-density lipoprotein by endothelial cells. Circ Res. 1998; 82: 1289–1297.[Abstract/Free Full Text]

24. Nakane H, Chu Y, Faraci FM, Oberley LW, Heistad DD. Gene transfer of extracellular superoxide dismutase increases superoxide dismutase activity in cerebral spinal fluid. Stroke. 2001; 32: 184–189.[Abstract/Free Full Text]

25. Didion SP, Faraci FM. Effects of NADH and NADPH on superoxide levels and cerebral vascular tone. Am J Physiol Heart Circ Physiol. 2002; 282: H688–H695.[Abstract/Free Full Text]

26. Lund DD, Faraci FM, Miller FJ Jr, Heistad DD. Gene transfer of endothelial nitric oxide synthase improves relaxation of carotid arteries from diabetic rabbits. Circulation. 2000; 101: 1027–1033.[Abstract/Free Full Text]

27. Nakane H, Miller FJ Jr, Faraci FM, Toyoda K, Heistad DD. Gene transfer of endothelial nitric oxide synthase reduces angiotensin II-induced endothelial dysfunction. Hypertension. 2000; 35: 595–601.[Abstract/Free Full Text]

28. 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.[Abstract/Free Full Text]

29. Bonthu S, Heistad DD, Chappell DA, Lamping KG, Faraci FM. Atherosclerosis, vascular remodeling, and impairment of endothelium-dependent relaxation in genetically altered hyperlipidemic mice. Arterioscler Thromb Vasc Biol. 1997; 17: 2333–2340.[Abstract/Free Full Text]

30. Lamping KG, Faraci FM. Role of sex differences and effects of endothelial NO synthase deficiency in responses of carotid arteries to serotonin. Arterioscler Thromb Vasc Biol. 2001; 21: 523–528.[Abstract/Free Full Text]

31. Faraci FM, Sigmund CD, Shesely EG, Maeda N, Heistad DD. Responses of carotid artery in mice deficient in expression of the gene for endothelial NO synthase. Am J Physiol. 1998; 274: H564–H570.[Medline] [Order article via Infotrieve]

32. Didion SP, Carmines PK, Ikenaga H, Mayhan WG. Enhanced constrictor responses of skeletal muscle arterioles during myocardial infarction. Am J Physiol. 1997; 273: H1502–H1508.[Medline] [Order article via Infotrieve]

33. Tabernero A, Giraldo J, Vila E. Effect of N^G-nitro-L-arginine methylester (L-NAME) on functional and biochemical 1-adrenoceptor-mediated responses in rat blood vessels. Br J Pharmacol. 1996; 117: 757–763.[Medline] [Order article via Infotrieve]

34. Lerman A, Sandok EK, Hilebrand FL Jr, Burnett JC Jr. Inhibition of endothelium-derived relaxing factor enhances endothelin-mediated vasoconstriction. Circulation. 1992; 85: 1894–1898.[Abstract/Free Full Text]

35. Zhang J, Van Meel JC, Pfaffendorf M, Zhang J, Van Zweiten PA. Endothelium-dependent, nitric oxide–mediated inhibition of angiotensin II-induced contractions in rabbit aorta. Eur J Pharmacol. 1994; 262: 247–253.[CrossRef][Medline] [Order article via Infotrieve]

36. Sobey CG, Faraci FM. Effects of a novel inhibitor of guanylyl cyclase on dilator responses of mouse cerebral arterioles. Stroke. 1997; 28: 837–842.[Abstract/Free Full Text]

37. Yamada M, Lamping KG, Duttaroy A, Zhang W, Cui Y, Bymaster FP, McKinzie DL, Felder CC, Deng C-X, Faraci FM, Wess J. Cholinergic dilation of cerebral blood vessels is abolished in M5 muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci U S A. 2001; 98: 14096–14101.[Abstract/Free Full Text]

38. Ryan MJ, Didion SP, Davis DR, Faraci FM, Sigmund CD. Endothelial dysfunction and blood pressure variability in selected inbred mouse strains. Arterioscler Thromb Vasc Biol. 2002; 22: 42–48.[Abstract/Free Full Text]

39. Didion SP, Heistad DD, Faraci FM. Mechanisms that produce nitric oxide-mediated relaxation of cerebral arteries during atherosclerosis. Stroke. 2001; 32: 761–766.[Abstract/Free Full Text]

40. Huang, T-T, Yasunami M, Carlson EJ, Gillespie AM, Reaume AG, Hoffman EK, Chan PH, Scott RW, Epstein CJ. Superoxide-mediated cytotoxicity in superoxide dismutase-deficient fetal fibroblasts. Arch Biochem Biophys. 1997; 344: 424–432.[CrossRef][Medline] [Order article via Infotrieve]

41. Reaume AG, Elliott JL, Hoffman EK, Kowall NW, Ferrante RJ, Siwek DF, Wilcox HM, Flood DG, Beal MF, Brown RH Jr, Scott RW, Snider WD. Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury. Nature Genetics. 1996; 13: 43–47.[CrossRef][Medline] [Order article via Infotrieve]

42. Ho YS, Gargano M, Cao J, Bronson RT, Heimler I, Hutz RJ. Reduced fertility in female mice lacking copper-zinc superoxide dismutase. J Biol Chem. 1998; 273: 7765–7769.[Abstract/Free Full Text]

43. Pagano PJ, Tornheim K, Cohen RA. Superoxide production by rabbit thoracic aorta: effect of endothelium-derived nitric oxide. Am J Physiol. 1993; 265: H707–H712.[Medline] [Order article via Infotrieve]

44. Wambi-Kiesse CO, Katusic ZS. Inhibition of copper/zinc superoxide dismutase impairs NO-mediated endothelium-dependent relaxation. Am J Physiol. 1999; 276: H1043–H1048.[Medline] [Order article via Infotrieve]

45. Sobey CG, Faraci FM. Subarachnoid haemorrhage: what happens to the cerebral arteries? Clin Exp Pharmacol Physiol. 1998; 25: 867–876.[Medline] [Order article via Infotrieve]

46. Kontos HA. Oxygen radicals in cerebral ischemia: the 2001 Willis lecture. Stroke. 2001; 32: 2712–2716.[Abstract/Free Full Text]

47. Droge W. Free radicals in the physiological control of cell function. Physiol Rev. 2002; 82: 47–95.[Abstract/Free Full Text]

48. Nakazono 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.[Abstract/Free Full Text]

49. Shesely EG, Maeda N, Kim HS, Desai KM, Krege JH, Laubach VE, Sherman PA, Sessa WC, Smithies O. Elevated blood pressures in mice lacking endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1996; 93: 13176–13181.[Abstract/Free Full Text]

50. Wang HD, Johns DG, Xu S, Cohen RA. Role of superoxide anion in regulating pressor and vascular hypertrophic response to angiotensin II. Am J Physiol Heart Circ Physiol. 2002; 282: H1697–H1702.[Abstract/Free Full Text]

51. Kobayashi T, Kamata K. Relationship among cholesterol, superoxide anion and endothelium-dependent relaxation in diabetic rats. Eur J Pharmacol. 1999; 367: 213–222.[CrossRef][Medline] [Order article via Infotrieve]

52. Kamata K, Kobayashi T. Changes in superoxide dismutase mRNA expression by streptozotocin-induce diabetes. Br J Pharmacol. 1996; 199: 583–589.

53. Forgione MA, Weiss N, Heydrick S, Cap A, Klings ES, Bierl C, Eberhardt RT, Farber HW, Loscalzo J. Cellular glutathione peroxidase deficiency and endothelial dysfunction. Am J Physiol Heart Circ Physiol. 2002; 282: H1255–H1261.[Abstract/Free Full Text]

This article has been cited by other articles:

				F. Jimenez-Altayo, L. Caracuel, F. J. Perez-Asensio, S. Martinez-Revelles, A. Messeguer, A. M. Planas, and E. Vila Participation of Oxidative Stress on Rat Middle Cerebral Artery Changes Induced by Focal Cerebral Ischemia: Beneficial Effects of 3,4-Dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6) J. Pharmacol. Exp. Ther., November 1, 2009; 331(2): 429 - 436. [Abstract] [Full Text] [PDF]

				S. P. Didion, D. A. Kinzenbaw, L. I. Schrader, Y. Chu, and F. M. Faraci Endogenous Interleukin-10 Inhibits Angiotensin II-Induced Vascular Dysfunction Hypertension, September 1, 2009; 54(3): 619 - 624. [Abstract] [Full Text] [PDF]

				M. Carlstrom, R. D. Brown, J. Sallstrom, E. Larsson, M. Zilmer, S. Zabihi, U. J. Eriksson, and A. E. G. Persson SOD1 deficiency causes salt sensitivity and aggravates hypertension in hydronephrosis Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2009; 297(1): R82 - R92. [Abstract] [Full Text] [PDF]

				J. R. Durrant, D. R. Seals, M. L. Connell, M. J. Russell, B. R. Lawson, B. J. Folian, A. J. Donato, and L. A. Lesniewski Voluntary wheel running restores endothelial function in conduit arteries of old mice: direct evidence for reduced oxidative stress, increased superoxide dismutase activity and down-regulation of NADPH oxidase J. Physiol., July 1, 2009; 587(13): 3271 - 3285. [Abstract] [Full Text] [PDF]

				C. Torrens, C. J. Kelsall, L. A. Hopkins, F. W. Anthony, N. P. Curzen, and M. A. Hanson Atorvastatin Restores Endothelial Function in Offspring of Protein-Restricted Rats in a Cholesterol-Independent Manner Hypertension, April 1, 2009; 53(4): 661 - 667. [Abstract] [Full Text] [PDF]

				N. Toda, K. Ayajiki, and T. Okamura Cerebral Blood Flow Regulation by Nitric Oxide: Recent Advances Pharmacol. Rev., March 1, 2009; 61(1): 62 - 97. [Abstract] [Full Text] [PDF]

				M. C. Gongora, H. E. Lob, U. Landmesser, T. J. Guzik, W. D. Martin, K. Ozumi, S. M. Wall, D. S. Wilson, N. Murthy, M. Gravanis, et al. Loss of Extracellular Superoxide Dismutase Leads to Acute Lung Damage in the Presence of Ambient Air: A Potential Mechanism Underlying Adult Respiratory Distress Syndrome Am. J. Pathol., October 1, 2008; 173(4): 915 - 926. [Abstract] [Full Text] [PDF]

				A. M. Beyer, W. J. de Lange, C. M. Halabi, M. L. Modrick, H. L. Keen, F. M. Faraci, and C. D. Sigmund Endothelium-Specific Interference With Peroxisome Proliferator Activated Receptor Gamma Causes Cerebral Vascular Dysfunction in Response to a High-Fat Diet Circ. Res., September 12, 2008; 103(6): 654 - 661. [Abstract] [Full Text] [PDF]

				S. Zabihi, P. Wentzel, and U. J. Eriksson Maternal Blood Glucose Levels Determine the Severity of Diabetic Embryopathy in Mice with Different Expression of Copper-Zinc Superoxide Dismutase (CuZnSOD) Toxicol. Sci., September 1, 2008; 105(1): 166 - 172. [Abstract] [Full Text] [PDF]

				L. Ai, M. Rouhanizadeh, J. C. Wu, W. Takabe, H. Yu, M. Alavi, R. Li, Y. Chu, J. Miller, D. D. Heistad, et al. Shear stress influences spatial variations in vascular Mn-SOD expression: implication for LDL nitration Am J Physiol Cell Physiol, June 1, 2008; 294(6): C1576 - C1585. [Abstract] [Full Text] [PDF]

				E. R. Kline, D. J. Kleinhenz, B. Liang, S. Dikalov, D. M. Guidot, C. M. Hart, D. P. Jones, and R. L. Sutliff Vascular oxidative stress and nitric oxide depletion in HIV-1 transgenic rats are reversed by glutathione restoration Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2792 - H2804. [Abstract] [Full Text] [PDF]

				C. A. Goodyear-Bruch, J. Jegathesan, R. L. Clancy, and J. D. Pierce Apoptotic-Related Protein Expression in the Diaphragm and the Effect of Dopamine During Inspiratory Resistance Loading Biol Res Nurs, April 1, 2008; 9(4): 293 - 300. [Abstract] [PDF]

				A. M. Beyer, G. L. Baumbach, C. M. Halabi, M. L. Modrick, C. M. Lynch, T. D. Gerhold, S. M. Ghoneim, W. J. de Lange, H. L. Keen, Y.-S. Tsai, et al. Interference With PPAR{gamma} Signaling Causes Cerebral Vascular Dysfunction, Hypertrophy, and Remodeling Hypertension, April 1, 2008; 51(4): 867 - 871. [Abstract] [Full Text] [PDF]

				H. Dayoub, R. N. Rodionov, C. Lynch, J. P. Cooke, E. Arning, T. Bottiglieri, S. R. Lentz, and F. M. Faraci Overexpression of Dimethylarginine Dimethylaminohydrolase Inhibits Asymmetric Dimethylarginine-Induced Endothelial Dysfunction in the Cerebral Circulation Stroke, January 1, 2008; 39(1): 180 - 184. [Abstract] [Full Text] [PDF]

				D. M. Arrick and W. G. Mayhan Acute infusion of nicotine impairs nNOS-dependent reactivity of cerebral arterioles via an increase in oxidative stress J Appl Physiol, December 1, 2007; 103(6): 2062 - 2067. [Abstract] [Full Text] [PDF]

				L. I. Schrader, D. A. Kinzenbaw, A. W. Johnson, F. M. Faraci, and S. P. Didion IL-6 Deficiency Protects Against Angiotensin II Induced Endothelial Dysfunction and Hypertrophy Arterioscler Thromb Vasc Biol, December 1, 2007; 27(12): 2576 - 2581. [Abstract] [Full Text] [PDF]

				K. A. Brown, S. P. Didion, J. J. Andresen, and F. M. Faraci Effect of Aging, MnSOD Deficiency, and Genetic Background on Endothelial Function: Evidence for MnSOD Haploinsufficiency Arterioscler Thromb Vasc Biol, September 1, 2007; 27(9): 1941 - 1946. [Abstract] [Full Text] [PDF]

				S. P. Didion, C. M. Lynch, and F. M. Faraci Cerebral vascular dysfunction in TallyHo mice: a new model of Type II diabetes Am J Physiol Heart Circ Physiol, March 1, 2007; 292(3): H1579 - H1583. [Abstract] [Full Text] [PDF]

				B. Rodriguez-Iturbe, L. Sepassi, Y. Quiroz, Z. Ni, and N. D. Vaziri Association of mitochondrial SOD deficiency with salt-sensitive hypertension and accelerated renal senescence J Appl Physiol, January 1, 2007; 102(1): 255 - 260. [Abstract] [Full Text] [PDF]

				S. P. Didion, D. A. Kinzenbaw, L. I. Schrader, and F. M. Faraci Heterozygous CuZn Superoxide Dismutase Deficiency Produces a Vascular Phenotype With Aging Hypertension, December 1, 2006; 48(6): 1072 - 1079. [Abstract] [Full Text] [PDF]

				D.-D. Chen and A. F. Chen CuZn Superoxide Dismutase Deficiency: Culprit of Accelerated Vascular Aging Process Hypertension, December 1, 2006; 48(6): 1026 - 1028. [Full Text] [PDF]

				K. Umeji, S. Umemoto, S. Itoh, M. Tanaka, S. Kawahara, T. Fukai, and M. Matsuzaki Comparative effects of pitavastatin and probucol on oxidative stress, Cu/Zn superoxide dismutase, PPAR-{gamma}, and aortic stiffness in hypercholesterolemia Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2522 - H2532. [Abstract] [Full Text] [PDF]

				J. Kitayama, C. Yi, F. M. Faraci, and D. D. Heistad Modulation of Dilator Responses of Cerebral Arterioles by Extracellular Superoxide Dismutase Stroke, November 1, 2006; 37(11): 2802 - 2806. [Abstract] [Full Text] [PDF]

				M. J. Ryan, G. R. McLemore Jr, and S. T. Hendrix Insulin Resistance and Obesity in a Mouse Model of Systemic Lupus Erythematosus Hypertension, November 1, 2006; 48(5): 988 - 993. [Abstract] [Full Text] [PDF]

				G. L. Baumbach, S. P. Didion, and F. M. Faraci Hypertrophy of Cerebral Arterioles in Mice Deficient in Expression of the Gene for CuZn Superoxide Dismutase Stroke, July 1, 2006; 37(7): 1850 - 1855. [Abstract] [Full Text] [PDF]

				F. M. Faraci, M. L. Modrick, C. M. Lynch, L. A. Didion, P. E. Fegan, and S. P. Didion Selective cerebral vascular dysfunction in Mn-SOD-deficient mice J Appl Physiol, June 1, 2006; 100(6): 2089 - 2093. [Abstract] [Full Text] [PDF]

				F. M. Faraci Reactive oxygen species: influence on cerebral vascular tone J Appl Physiol, February 1, 2006; 100(2): 739 - 743. [Abstract] [Full Text] [PDF]

				Q. Fang, H. Sun, D. M. Arrick, and W. G. Mayhan Inhibition of NADPH oxidase improves impaired reactivity of pial arterioles during chronic exposure to nicotine J Appl Physiol, February 1, 2006; 100(2): 631 - 636. [Abstract] [Full Text] [PDF]

				S. P. Didion, D. A. Kinzenbaw, and F. M. Faraci Critical Role for CuZn-Superoxide Dismutase in Preventing Angiotensin II-Induced Endothelial Dysfunction Hypertension, November 1, 2005; 46(5): 1147 - 1153. [Abstract] [Full Text] [PDF]

				A. Daiber, M. Oelze, S. Sulyok, M. Coldewey, E. Schulz, N. Treiber, U. Hink, A. Mulsch, K. Scharffetter-Kochanek, and T. Munzel Heterozygous Deficiency of Manganese Superoxide Dismutase in Mice (Mn-SOD+/-): A Novel Approach to Assess the Role of Oxidative Stress for the Development of Nitrate Tolerance Mol. Pharmacol., September 1, 2005; 68(3): 579 - 588. [Abstract] [Full Text] [PDF]

				S. Dayal, A. M. Devlin, R. B. McCaw, M.-L. Liu, E. Arning, T. Bottiglieri, B. Shane, F. M. Faraci, and S. R. Lentz Cerebral Vascular Dysfunction in Methionine Synthase-Deficient Mice Circulation, August 2, 2005; 112(5): 737 - 744. [Abstract] [Full Text] [PDF]

				I. A. Arenas, S. J. Armstrong, Y. Xu, and S. T. Davidge Chronic Tumor Necrosis Factor-{alpha} Inhibition Enhances NO Modulation of Vascular Function in Estrogen-Deficient Rats Hypertension, July 1, 2005; 46(1): 76 - 81. [Abstract] [Full Text] [PDF]

				J. I. Mendez, W. J. Nicholson, and W. R. Taylor SOD Isoforms and Signaling in Blood Vessels: Evidence for the Importance of ROS Compartmentalization Arterioscler Thromb Vasc Biol, May 1, 2005; 25(5): 887 - 888. [Full Text] [PDF]

				C. Yan, A. Huang, Z. Wu, P. M. Kaminski, M. S. Wolin, T. H. Hintze, G. Kaley, and D. Sun Increased superoxide leads to decreased flow-induced dilation in resistance arteries of Mn-SOD-deficient mice Am J Physiol Heart Circ Physiol, May 1, 2005; 288(5): H2225 - H2231. [Abstract] [Full Text] [PDF]

				S. P. Didion, C. M. Lynch, G. L. Baumbach, and F. M. Faraci Impaired Endothelium-Dependent Responses and Enhanced Influence of Rho-Kinase in Cerebral Arterioles in Type II Diabetes Stroke, February 1, 2005; 36(2): 342 - 347. [Abstract] [Full Text] [PDF]

				F. M. Faraci Oxidative Stress: The Curse That Underlies Cerebral Vascular Dysfunction? Stroke, February 1, 2005; 36(2): 186 - 188. [Full Text] [PDF]

				S. P. Didion and F. M. Faraci Ceramide-Induced Impairment of Endothelial Function Is Prevented by CuZn Superoxide Dismutase Overexpression Arterioscler Thromb Vasc Biol, January 1, 2005; 25(1): 90 - 95. [Abstract] [Full Text] [PDF]

				F. M. Faraci, C. Lynch, and K. G. Lamping Responses of cerebral arterioles to ADP: eNOS-dependent and eNOS-independent mechanisms Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2871 - H2876. [Abstract] [Full Text] [PDF]

				K. A. Blackwell, J. P. Sorenson, D. M. Richardson, L. A. Smith, O. Suda, K. Nath, and Z. S. Katusic Mechanisms of aging-induced impairment of endothelium-dependent relaxation: role of tetrahydrobiopterin Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2448 - H2453. [Abstract] [Full Text] [PDF]

				J.-M. Li and A. M Shah Endothelial cell superoxide generation: regulation and relevance for cardiovascular pathophysiology Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2004; 287(5): R1014 - R1030. [Abstract] [Full Text] [PDF]

				S. Adler and H. Huang Oxidant stress in kidneys of spontaneously hypertensive rats involves both oxidase overexpression and loss of extracellular superoxide dismutase Am J Physiol Renal Physiol, November 1, 2004; 287(5): F907 - F913. [Abstract] [Full Text] [PDF]

				R. Stocker and J. F. Keaney Jr. Role of Oxidative Modifications in Atherosclerosis Physiol Rev, October 1, 2004; 84(4): 1381 - 1478. [Abstract] [Full Text] [PDF]

				J. J. Andresen, F. M. Faraci, and D. D. Heistad Vasomotor responses in MnSOD-deficient mice Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1141 - H1148. [Abstract] [Full Text] [PDF]

				H. Shibata, T. Nabika, H. Moriyama, J. Masuda, and S. Kobayashi Correlation of NO Metabolites and 8-Iso-Prostaglandin F2a With Periventricular Hyperintensity Severity Arterioscler Thromb Vasc Biol, September 1, 2004; 24(9): 1659 - 1663. [Abstract] [Full Text] [PDF]

				F. M. Faraci and S. P. Didion Vascular Protection: Superoxide Dismutase Isoforms in the Vessel Wall Arterioscler Thromb Vasc Biol, August 1, 2004; 24(8): 1367 - 1373. [Abstract] [Full Text] [PDF]

				S. P. Didion, D. A. Kinzenbaw, P. E. Fegan, L. A. Didion, and F. M. Faraci Overexpression of CuZn-SOD Prevents Lipopolysaccharide-Induced Endothelial Dysfunction Stroke, August 1, 2004; 35(8): 1963 - 1967. [Abstract] [Full Text] [PDF]

				S. Veerareddy, C.-L. M. Cooke, P. N. Baker, and S. T. Davidge Gender differences in myogenic tone in superoxide dismutase knockout mouse: animal model of oxidative stress Am J Physiol Heart Circ Physiol, July 1, 2004; 287(1): H40 - H45. [Abstract] [Full Text] [PDF]

				J. Q. Liu and R. J. Folz Extracellular superoxide enhances 5-HT-induced murine pulmonary artery vasoconstriction Am J Physiol Lung Cell Mol Physiol, July 1, 2004; 287(1): L111 - L118. [Abstract] [Full Text] [PDF]

				D. Sun, A. Huang, E. H. Yan, Z. Wu, C. Yan, P. M. Kaminski, T. D. Oury, M. S. Wolin, and G. Kaley Reduced release of nitric oxide to shear stress in mesenteric arteries of aged rats Am J Physiol Heart Circ Physiol, June 1, 2004; 286(6): H2249 - H2256. [Abstract] [Full Text] [PDF]

				A. K. Nath, J. Enciso, M. Kuniyasu, X.-Y. Hao, J. A. Madri, and E. Pinter Nitric oxide modulates murine yolk sac vasculogenesis and rescues glucose induced vasculopathy Development, May 15, 2004; 131(10): 2485 - 2496. [Abstract] [Full Text] [PDF]

				M. J. Ryan, S. P. Didion, S. Mathur, F. M. Faraci, and C. D. Sigmund Angiotensin II-Induced Vascular Dysfunction Is Mediated by the AT1A Receptor in Mice Hypertension, May 1, 2004; 43(5): 1074 - 1079. [Abstract] [Full Text] [PDF]

				A. M. Devlin, E. Arning, T. Bottiglieri, F. M. Faraci, R. Rozen, and S. R. Lentz Effect of Mthfr genotype on diet-induced hyperhomocysteinemia and vascular function in mice Blood, April 1, 2004; 103(7): 2624 - 2629. [Abstract] [Full Text] [PDF]

				M. J. Ryan, S. P. Didion, S. Mathur, F. M. Faraci, and C. D. Sigmund PPAR{gamma} Agonist Rosiglitazone Improves Vascular Function and Lowers Blood Pressure in Hypertensive Transgenic Mice Hypertension, March 1, 2004; 43(3): 661 - 666. [Abstract] [Full Text] [PDF]

				C.-L. M Cooke and S. T Davidge Endothelial-dependent vasodilation is reduced in mesenteric arteries from superoxide dismutase knockout mice Cardiovasc Res, December 1, 2003; 60(3): 635 - 642. [Abstract] [Full Text] [PDF]

				M.-S. Zhou, A. G. Adam, E. A. Jaimes, and L. Raij In Salt-Sensitive Hypertension, Increased Superoxide Production Is Linked to Functional Upregulation of Angiotensin II Hypertension, November 1, 2003; 42(5): 945 - 951. [Abstract] [Full Text] [PDF]

				O. Jung, S. L. Marklund, H. Geiger, T. Pedrazzini, R. Busse, and R. P. Brandes Extracellular Superoxide Dismutase Is a Major Determinant of Nitric Oxide Bioavailability: In Vivo and Ex Vivo Evidence From ecSOD-Deficient Mice Circ. Res., October 3, 2003; 93(7): 622 - 629. [Abstract] [Full Text] [PDF]

				S. P. Didion and F. M. Faraci Angiotensin II Produces Superoxide-Mediated Impairment of Endothelial Function in Cerebral Arterioles Stroke, August 1, 2003; 34(8): 2038 - 2042. [Abstract] [Full Text] [PDF]

				F. M. Faraci Vascular Protection Stroke, February 1, 2003; 34(2): 327 - 329. [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 91/10/938    most recent 01.RES.0000043280.65241.04v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Didion, S. P. Articles by Faraci, F. M. Search for Related Content PubMed PubMed Citation Articles by Didion, S. P. Articles by Faraci, F. M. Related Collections Genetically altered mice Brain Circulation and Metabolism Oxidant stress Endothelium/vascular type/nitric oxide