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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1017.)
© 2001 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Mechanism of Endothelial Dysfunction in Apolipoprotein E–Deficient Mice

Livius V. d’Uscio; Timothy A. Baker; Carlos B. Mantilla; Leslie Smith; Deborah Weiler; Gary C. Sieck; Zvonimir S. Katusic

From the Departments of Anesthesiology and Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic and Foundation, Rochester, Minn.

Correspondence to Zvonimir S. Katusic, MD, PhD, Department of Anesthesiology and Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail katusic.zvonimir{at}mayo.edu

	Abstract

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Abstract—Endothelium-dependent relaxations mediated by NO are impaired in a mouse model of human atherosclerosis. Our objective was to characterize the mechanisms underlying endothelial dysfunction in aortas of apolipoprotein E (apoE)-deficient mice, treated for 26 to 29 weeks with a lipid-rich Western-type diet. Aortic rings from apoE-deficient mice showed impaired endothelium-dependent relaxations to acetylcholine (10^-9 to 10^-5 mol/L) and Ca²⁺ ionophore (10^-9 to 10^-6 mol/L) and endothelium-independent relaxations to diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10^-10 to 10^-5 mol/L) compared with aortic rings from C57BL/6J mice (P<0.05). By use of confocal microscopy of an oxidative fluorescent probe (dihydroethidium), increased superoxide anion (O₂^-) production was demonstrated throughout the aortic wall but mainly in smooth muscle cells of apoE-deficient mice. CuZn–superoxide dismutase (SOD) and Mn-SOD protein expressions were unaltered in the aorta exposed to hypercholesterolemia. A cell-permeable SOD mimetic, Mn(III) tetra(4-benzoic acid) porphyrin chloride (10^-5 mol/L), reduced O₂^- production and partially normalized relaxations to acetylcholine and DEA-NONOate in apoE-deficient mice (P<0.05). [¹⁴C]L-Citrulline assay showed a decrease of Ca²⁺-dependent NOS activity in aortas from apoE-deficient mice compared with C57BL/6J mice (P<0.05), whereas NO synthase protein expression was unchanged. In addition, cGMP levels were significantly reduced in the aortas of apoE-deficient mice (P<0.05). Our results demonstrate that in apoE-deficient mice on a Western-type fat diet, impairment of endothelial function is caused by increased production of O₂^- and reduced endothelial NO synthase enzyme activity. Thus, chemical inactivation of NO with O₂^- and reduced biosynthesis of NO are key mechanisms responsible for endothelial dysfunction in aortas of atherosclerotic apoE-deficient mice.

Key Words: endothelium • nitric oxide • superoxide anion • apolipoprotein E • atherosclerosis

	Introduction

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Atherosclerosis is a chronic process, which can be triggered by cardiovascular risk factors such as hypercholesterolemia, aging, hypertension, and diabetes mellitus.¹ Endothelium-derived vasoactive factors play an important regulatory role in vascular homeostasis and pathogenesis of atherosclerosis because of the strategic position of the endothelium between the vascular smooth muscle cells (VSMCs) and the circulating blood.^{2 3}

NO is a potent vasodilator that is formed in endothelial cells from L-arginine by endothelial NO synthase (eNOS), which is constitutively expressed.^{4 5 6} NO production is activated by the stimulation of cell surface receptors or by mechanical forces such as shear stress.^{7 8} Accumulating evidence suggests that alterations in the NO pathway play a central role in endothelial dysfunction induced by hypercholesterolemia. This may be of major importance inasmuch as NO can substantially inhibit several components of the atherogenic process, such as VSMC contraction and proliferation, platelet aggregation, and monocyte adhesion.^{9 10} Previous studies identified 3 mechanisms responsible for reduced bioavailability of NO in arteries exposed to hypercholesterolemia: (1) enhanced degradation of NO by superoxide anions (O₂^-),¹¹ (2) functional abnormalities of NO synthase (NOS) due to deficiency of substrate or cofactor,^{12 13} and (3) alteration in eNOS activity and/or protein expression.^{14 15 16}

Mice homozygous for the inactivated apoE gene provide a new model of human atherosclerosis. These mice develop spontaneous hypercholesterolemia and aortic atherosclerosis, which can be accelerated by A lipid-rich Western-type diet.^{17 18 19} Indeed, impaired endothelium-dependent relaxation in response to acetylcholine (ACh) has been observed in the aortas of apoE-deficient mice on a Western-type diet but not on a normal diet.^{20 21} However, the exact mechanisms of altered endothelial function (ie, role of O₂^-, eNOS expression, and eNOS activity) have not been determined in this animal model of human atherosclerosis.

	Methods

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Experimental Animals
Male C57BL/6J (control) mice and homozygous apoE-deficient mice (4 to 5 weeks old) were obtained from The Jackson Laboratories (Bar Harbor, Me). Housing facilities and all experimental protocols were approved by the Institutional Animal Care and Use Committee of Mayo Clinic. To accelerate the development of spontaneous atherosclerotic lesions in apoE-deficient mice, control C57BL/6J and apoE-deficient mice were fed a lipid-rich Western-type diet for 26 to 29 weeks (TD88137, Harlan Teklad).^{17 18} Total plasma cholesterol levels were 22.2±1.6 mmol/L in apoE-deficient mice and 6.4±0.7 mmol/L in C57BL/6J mice (P<0.05, n=10).

The mice were anesthetized (60 mg/kg body wt IP pentobarbital) and euthanized. The whole aorta was carefully removed and dissected free from connective tissue in cold (4°C) modified Krebs-Ringer bicarbonate solution (mmol/L: NaCl 118.6, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25.1, EDTA 0.026, and glucose 10.1). The aorta was cut into 4-mm rings (proximal, distal thoracic, and first part of abdominal aorta).

Experimental Setup
Isolated aortic rings from C57BL/6J and apoE-deficient mice were studied in parallel. Rings were connected to a force transducer for recording of isometric force and placed in organ baths filled with 25 mL Krebs’ solution (37°C, 94% O₂/6% CO₂, pH 7.4). After an equilibration period of 30 minutes, the rings were progressively stretched to their optimal passive tension as assessed by the response to 100 mmol/L KCl.

Concentration-dependent response curves to ACh (10^-9 to 10^-5 mol/L), Ca²⁺ ionophore (A23187, 10^-9 to 10^-6 mol/L), and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10^-10 to 10^-5 mol/L) were obtained. In a separate protocol, aortic rings were preincubated with or without the cyclooxygenase inhibitor indomethacin (10^-5 mol/L for 15 minutes) or superoxide dismutase (SOD, 75 U/mL for 5 minutes) or the NO-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME, 3x10^-4 mol/L for 20 minutes) or the cell-permeable SOD mimetic Mn(III) tetra(4-benzoic acid) porphyrin chloride (MnTBAP, 10^-5 mol/L for 15 minutes).

Oxidative Fluorescence Microscopy
The oxidative fluorescent dye dihydroethidium (Molecular Probes) was used as described previously.²² Unfixed frozen rings of distal thoracic aortic segments were cut into 30-µm-thick sections and placed on a glass slide. Samples were incubated with dihydroethidium (2x10^-6 mol/L) in a light-protected humidified chamber at 37°C for 30 minutes. Tissue sections were imaged by us of an Olympus Fluoview laser scanning confocal microscope.

Quantification of Vascular O₂^- Production
Lucigenin (5 µmol/L, Molecular Probes) was used to measure O₂^- levels in the aorta as described previously.²³ The results were expressed as counts per minute per microgram dry weight.

Measurement of Ca²⁺-Dependent NOS Enzyme Activity
[¹⁴C]L-Citrulline formation was measured by using a liquid scintillation counter (Beckman Instruments) as described.²⁴ Briefly, 4 whole aortas (n=1 experiment) were homogenized on ice in lysis buffer (Sigma Chemical Co). After centrifugation, equal amounts of total protein were added to the enzymatic reactions.

Western Blot Analysis
Mouse monoclonal anti-eNOS (Transduction), sheep polyclonal anti–von Willebrand factor (vWF, Cedarlane), and rabbit polyclonal anti–CuZn-SOD and anti–Mn-SOD (StressGen) were used. For actin, blots were rehybridized with monoclonal anti-actin (Sigma). Densitometry was carried out by using NIH Image, and the results were expressed as optical density (OD) per square millimeter aortic surface (eNOS and vWF) or relative to the respective intensity of the actin blot (CuZn-SOD and Mn-SOD).

Measurements of cGMP and cAMP
After homogenization, cGMP and cAMP radioimmunoassay kits (Amersham) were used as described.²⁵

Calculations and Statistical Analysis
Results are given as mean±SEM. The concentration-response curves of the different groups were compared by ANOVA for repeated measurements, followed by the Bonferroni correction. For simple comparison between 2 values, a paired or unpaired Student t test was used, where appropriate. A value of P<0.05 was considered significant.

An expanded Materials and Methods section can be found in an online data supplement, which can be accessed at http://atvb.ahajournals.org.

	Results

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Vascular Relaxations
In the aortas of apoE-deficient and C57BL/6J mice, endothelium-dependent relaxation in response to ACh was completely blocked by the NOS inhibitor L-NAME (0±1% and 1±1% for maximal relaxations, respectively; n=5). NO-mediated endothelium-dependent relaxations were reduced in apoE-deficient mice (P<0.0001 versus C57BL/6J for maximal relaxation, Figure 1A). The sensitivity (pD₂) to ACh was significantly shifted to the right in apoE mice (pD₂ 7.0±0.1) compared with C57BL/6J mice (pD₂ 7.4±0.1, P<0.05). Preincubation with SOD (75 U/mL) had no effect on relaxations to ACh in apoE-deficient and control mice (61±6% versus 59±3% and 91±2% versus 88±1% with and without SOD, respectively). In addition, indomethacin (10^-5 mol/L) did not affect ACh-induced relaxations in either group (64±4% for apoE-deficient mice and 93±2% for control mice, n=5 or 6).

View larger version (21K): [in this window] [in a new window]   Figure 1. Endothelium-dependent relaxations to ACh (A) and A23187 (B) and endothelium-independent relaxations to DEA-NONOate (C) in aortas of apoE and C57BL/6J mice after 26 weeks on Western-type diet. Relaxations to ACh, A23187, and DEA-NONOate were reduced (P<0.05, ANOVA+Bonferroni). Results are mean±SEM and expressed as percentage relaxation of the submaximal contraction to norepinephrine (1 to 5x10-8 mol/L).

A23187 also caused endothelium-dependent relaxations, which were impaired in the aortas of apoE-deficient mice compared with C57BL/6J mice (P<0.05, Figure 1B).

Endothelium-independent relaxations to the NO donor DEA-NONOate were reduced, and the concentration-response curve was shifted 3-fold to the right in apoE-deficient mice (P<0.05 versus C57BL/6J mice, Figure 1C; n=5). Maximal relaxations were unaltered.

Contractions of VSMCs
Contractions to 100 mmol/L KCl did not differ among control mice (1.41±0.05 mN/mm) and apoE-deficient mice (1.35±0.03 mN/mm).

Concentration-dependent contractions to phenylephrine were unchanged in apoE-deficient mice compared with C57BL/6J mice. Maximal contraction was 90±3% in apoE-deficient mice and 82±8% in control mice, and pD₂ was 6.9±0.1 and 7.0±0.1, respectively (n=6 to 8, P=NS).

Effect of SOD Mimetic
A novel cell-permeable SOD mimetic MnTBAP (10^-5 mol/L) significantly improved endothelium-dependent relaxation to ACh in aortas from apoE-deficient mice compared with untreated aortic rings (P<0.01, Figure 2A). However, maximal relaxations were still impaired compared with maximal relaxations in C57BL/6J mice in the presence or absence of MnTBAP (P<0.05). MnTBAP had no effect on sensitivity or maximal relaxations to ACh in C57BL/6J mice.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effects of the SOD mimetic MnTBAP on endothelium-dependent relaxations to ACh (A) and endothelium-independent relaxations to DEA-NONOate (B) in aortas of apoE and C57BL/6J mice after 26 weeks on a Western-type diet. MnTBAP (10-5 mol/L) significantly improved relaxations to ACh and to DEA-NONOate in aortas of apoE-deficient mice compared with untreated aortic rings (P<0.05, ANOVA+Bonferroni). Note that relaxations were still impaired compared with C57BL/6J mice (P<0.05, ANOVA+Bonferroni). Results are mean±SEM and expressed as percent relaxation of the submaximal contraction to phenylephrine (1 to 6x10-7 mol/L).

MnTBAP also significantly improved endothelium-independent relaxations to DEA-NONOate in aortas of apoE-deficient mice (pD₂ 7.7±0.1 versus 7.2±0.1, P<0.05 for aorta with MnTBAP versus aorta without MnTBAP; Figure 2B). MnTBAP did not affect relaxations to DEA-NONOate in C57BL/6J mice (pD₂ 8.1±0.1, P=NS).

Vascular O₂^- Production
After loading with the oxidation-sensitive dye dihydroethidium, a marked increase in ethidium bromide (EtBr) fluorescence was found throughout the vascular wall of apoE-deficient mouse aorta, which reflected an increase in O₂^- (Figure 3B) compared with O₂^- in C57BL/6J mice (Figure 3A). The increase in EtBr fluorescence was observed mainly in VSMCs but also in endothelial cells and atheromatous plaques (Figure 3B). MnTBAP reduced EtBr fluorescence (Figure 3D) in apoE-deficient mice compared with C57BL/6J mice (Figure 3C). Interestingly, MnTBAP reduced the increase in EtBr fluorescence, not only in endothelial cells but also in VSMCs and atheromatous plaques of aortas from apoE-deficient mice (Figure 3D).

View larger version (17K): [in this window] [in a new window]   Figure 3. Fluorescent photomicrographs showing in situ detection of O2- with use of confocal microscopic sections of distal thoracic aorta from C57BL/6J (left) and apoE-deficient (right) mice. Aortic sections were labeled with the oxidative dye dihydroethidium, which, in the presence of O2-, is oxidized to EtBr and gives red fluorescence (see Methods). High EtBr fluorescence was found in aortic walls of apoE-deficient mice (B) compared with C57BL/6J mice (A) and was localized mainly in VSMCs but also in endothelial cells and atheromatous plaques of apoE-deficient mice. Incubation with MnTBAP (10-5 mol/L) for 30 minutes before staining did not affect fluorescence of C57BL/6J mice (C) but reduced fluorescence in apoE-deficient mice (D).

When measured with lucigenin-enhanced chemiluminescence, O₂^- levels were 3-fold higher in aortas from apoE-deficient mice compared with control C57BL/6J mice (P<0.05, Figure 4A). Treatment of aortas with MnTBAP (10^-5 mol/L) reduced O₂^- levels in apoE-deficient mice (P<0.05).

View larger version (24K): [in this window] [in a new window]   Figure 4. A, Detection of superoxide anions in mouse aorta by lucigenin-enhanced chemiluminescence (CL). MnTBAP (10-5 mol/L) was added 15 minutes before recording. Photon counts were averaged over 8 minutes and were expressed as counts per minute per microgram dry weight. Results are mean±SEM (n=6 to 8). *P<0.05 vs control (C57BL/6J) mice; P<0.05 vs apoE-deficient mice without MnTBAP (ANOVA+Bonferroni). Representative Western Blot analysis of CuZn-SOD (B) and Mn-SOD (C) protein expression in aortas of C57BL/6J and apoE-deficient mice. Bar graphs indicate results of the relative densitometry compared with actin. Results are mean±SEM (n=3).

Western Blot Analysis
Expressions of cytosolic CuZn-SOD and Mn-SOD proteins were not different between apoE-deficient and C57BL/6J mice (n=3, Figure 4B and 4C). In addition, eNOS expression was not altered in apoE-deficient mice (n=3, Figure 5A). Interestingly, vWF protein expression was increased in aortas of apoE-deficient mice (3.9±0.2 versus 2.6±0.2 OD/mm² [P<0.05] for apoE-deficient mice versus control mice, respectively; n=3).

View larger version (18K): [in this window] [in a new window]   Figure 5. A, Representative Western Blot analysis of eNOS protein expression in aortas of C57BL/6J and apoE-deficient mice. Bar graph indicates results of relative densitometric analysis of eNOS protein expression as OD per square millimeter aortic surface. Results are mean±SEM (n=3). B, Bar graphs showing eNOS activity in aortas of C57BL/6J and apoE-deficient mice. [14 C]Citrulline formation was measured in aortic homogenates as described in Methods. Results are mean±SEM (n=7). *P<0.05 vs control (C57BL/6J) mice (unpaired Student t test).

Ca²⁺-Dependent NOS Activity
We measured Ca²⁺-dependent NOS activity in the aortas of apoE-deficient and C57BL/6J mice by assaying the conversion of [¹⁴ C]L-arginine to [¹⁴ C]L-citrulline in tissue homogenates. Aortas from apoE-deficient mice showed 2.3-fold less NOS enzyme activity than aortas from C57BL/6J mice (P<0.05, n=7; Figure 5B).

cGMP and cAMP Levels
Basal cGMP level was reduced in aortas from apoE-deficient mice compared with C57BL/6J mice (P<0.05, Figure 6A), whereas basal cAMP levels were not different (n=10, P=NS; Figure 6B).

View larger version (13K): [in this window] [in a new window]   Figure 6. Bar graphs showing basal cGMP (A) and cAMP (B) levels in aortas of C57BL/6J and apoE-deficient mice after 26 weeks on Western-type diet. Results are mean±SEM (n=10). *P<0.05 vs C57BL/6J mice (unpaired Student t test).

	Discussion

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There are several novel findings in the present study. The cell-permeable SOD mimetic MnTBAP reduced O₂^- formation and augmented endothelium-dependent and endothelium-independent relaxations mediated by NO. Increased O₂^- production was found in endothelial cells, VSMCs, and atheromatous plaques of apoE-deficient aorta. Increased breakdown of NO by O₂^- appears to be the key mechanism responsible for the decrease in biological activity of NO. This was reflected in selective impairment of cGMP production in aortic walls of apoE-deficient mice.

One of the hallmarks of atherosclerosis is impairment of endothelial function, which is present even before vascular structural changes occur.² Indeed, endothelial dysfunction is a common feature in subjects with cardiovascular risk factors, suggesting a role for the initiation of pathological changes in atherosclerosis.^{1 8 26} Consistent with previous results,^{20 27 28 29} the present study demonstrated that endothelium-dependent relaxations to ACh were impaired in the aorta of apoE-deficient mice on a Western-type diet. This is in line with the study in humans, which demonstrated a reduction of the initial rate of NO release in advanced atherosclerosis.¹⁵ Treatment with the SOD mimetic MnTBAP, but not SOD, normalized in part endothelium-dependent relaxations in the aorta of apoE-deficient mice, suggesting that excess production of free radicals throughout the aortic wall is responsible for the breakdown of NO.

It is known that superoxide radicals react with NO more rapidly to form peroxynitrite [k 6.7x10⁹ (mol/L)^-1 · s^-1] than with SOD [k 2x10⁹ (mol/L)^-1 · s^-1], which converts O₂^- to hydrogen peroxide.³⁰ MnTBAP is a cell-permeable Mn²⁺ (III)–containing metalloporphyrin that is able to catalyze the dismutation of O₂^- with a rate constant of 10⁷ (mol/L)^-1 · s^-1.³¹ Moreover, Mn²⁺ (III)–containing SOD mimetics have been reported to exhibit a catalase-like activity,³² converting hydrogen peroxide to water and oxygen, and they are also reported to be scavengers of peroxynitrite.³³ Importantly, in contrast to other metalloporphyrins, MnTBAP did not inhibit vascular relaxations in response to NO, suggesting that MnTBAP is not a scavenger of NO.³³ In the present study with apoE-deficient mouse aortas, MnTBAP was able to reduce the increased O₂^- production in endothelial cells, VSMCs, and atheromatous plaques as detected by lucigenin-enhanced chemiluminescence and EtBr fluorescence. Thus, the ability of MnTBAP to scavenge O₂^- may explain the improvement of endothelial function in apoE-deficient mice. Accordingly, treatment of hypercholesterolemic rabbits with polyethylene-glycolated SOD, but not native SOD, has been shown to restore, in part, endothelium-dependent relaxations in rabbit atherosclerotic aorta.³⁴ In addition, ex vivo gene transfer of CuZn-SOD and extracellular SOD reduced production of O₂^- in endothelium but not in VSMCs and failed to improve impaired relaxations to ACh in rabbit aortas.²²

Cytosolic CuZn-SOD and Mn-SOD protein levels were unaltered in the aortas of apoE-deficient mice compared with control mice despite increased O₂^- production. These findings are consistent with results reported by Fukai et al (1998).³⁵ They showed an increase of extracellular SOD activity in aortic macrophages, whereas the activities of cytosolic CuZn-SOD and Mn-SOD were unaltered in aortas of apoE-deficient mice.³⁵ These findings suggest that reduced SOD expression is not responsible for the increased formation of O₂^-, and they are in agreement with the most recent study.³⁶ In addition, the cyclooxygenase pathway is unlikely to be a source of O₂^- production because indomethacin did not affect endothelium-dependent relaxations in the aorta of apoE-deficient mice. The exact source of O₂^- in apoE-deficient mouse aortas remains to be determined.

Because endothelial function in the presence of MnTBAP was not completely normalized, we investigated whether other major mechanisms in the alteration of the NO pathway may be involved. Indeed, we found a reduction of Ca²⁺-dependent NOS enzyme activity in the aortas of apoE-deficient mice compared with aortas of control mice (see below), whereas Western blot analysis showed no change in eNOS protein expression. These findings were in contrast to studies on human arteries in which a reduction of immunoreactive eNOS in luminal endothelial cells was found.¹⁵ On the other hand, an increase of eNOS protein expression and mRNA in the atherosclerotic aortas of rabbits was found despite enhanced O₂^- production and impaired endothelium-dependent relaxations.^{11 37} The discrepancy may be related to the differential duration of the high cholesterol diet or to species differences or duration of the atherosclerotic process, ie, months in experimental animals versus decades in patients.

Deficiency of a substrate L-arginine may also lead to the reduced eNOS enzyme activity. However, chronic treatment with the NOS substrate L-arginine had no effect on impaired endothelial function in apoE-deficient mice,³⁸ suggesting that the endothelial dysfunction found in apoE-deficient mice is not due to the deficiency of the substrate L-arginine. Endothelium-dependent relaxations to ACh and A23187 were impaired, indicating that reduced Ca²⁺-dependent NOS activity rather than impairment of receptor-mediated signal transduction mechanisms is an important component responsible for endothelial dysfunction in apoE aortas. In addition, under in vivo conditions, several mechanisms may contribute to decreased eNOS activity. Chronic inhibition of the enzyme by free radicals^{11 39} or endogenous antagonists of NOS, such as asymmetric dimethyl arginine,⁴⁰ may reduce the activity of eNOS in atherosclerotic apoE-deficient mouse aortas.

The observed alteration in vascular responses may be dependent not only on the reduced availability of endothelium-derived relaxing factor(s) but also on the altered responsiveness of VSMCs to NO. Indeed, we found that basal cGMP levels were reduced in the aortas of apoE-deficient mice, although tissue cAMP levels were not different, indicating a selective loss of cGMP-dependent vascular function. Most importantly, relaxation of VSMCs to NO was impaired; the exact mechanism of VSMC dysfunction is unknown. This phenomenon may contribute to abnormal endothelium-dependent relaxation mediated by NO in atherosclerosis and is consistent with results of the previous studies.^{36 41}

In summary, an increased production of O₂^- throughout the aortic wall selectively impaired NO-mediated relaxations in apoE-deficient mice. Treatment with a SOD mimetic only in part improved endothelium-dependent relaxations. Increase of O₂^- production and the reduced activity of eNOS enzyme appear to be major mechanisms responsible for impaired endothelial function in the aortas of apoE-deficient mice.

	Acknowledgments

 
This work was supported by National Heart, Lung, and Blood Institute grant HL-53524, funds from the Bruce and Ruth Rappaport Program in Vascular Biology, and the Mayo Foundation. Livius V. d’Uscio is a recipient of a stipend from the Swiss National Sciences Foundation and a postdoctoral fellowship from the American Heart Association, Northland Affiliate. The authors would like to thank Janet Beckman for typing the manuscript.

Received February 9, 2001; accepted March 9, 2001.

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