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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1477.)
© 2001 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Extracellular Superoxide Dismutase Deficiency and Atherosclerosis in Mice

Marie-Louise Sentman; Thomas Brännström; Sanna Westerlund; Mikko O. Laukkanen; Seppo Ylä-Herttuala; Samar Basu; Stefan L. Marklund

From the Department of Medical Biosciences, Clinical Chemistry (M.-L.S., S.L.M.), and the Department of Medical Biosciences, Pathology (T.B.), Umeå University Hospital, Umeå, Sweden; the A.I. Virtanen Institute and Department of Medicine (S.W., M.O.L., S.Y.-H.), University of Kuopio, Kuopio, Finland; and the Department of Geriatrics, Faculty of Medicine (S.B.), Uppsala University, Uppsala, Sweden.

Correspondence to Prof Stefan L. Marklund, Department of Medical Biosciences, Clinical Chemistry, Umeå University Hospital, SE-901 85 Umeå, Sweden. E-mail stefan.marklund{at}klinkemi.umu.se

	Abstract

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Abstract—— Lipoprotein peroxidation in the arterial wall has been implicated in atherogenesis. The superoxide radical is formed in arteries and can induce such oxidation. Extracellular superoxide dismutase (EC-SOD) occurs in high concentration in the vascular wall interstitium, and in this study, we examined the importance of the enzyme in atherogenesis. On an apolipoprotein E–null background, the limited aortic lesions induced by a 1-month atherogenic diet were larger in EC-SOD wild-type mice than in EC-SOD–null mice, whereas there were no differences between the EC-SOD genotypes in the larger lesions seen after 3 months on the diet or after 8 months on normal chow. Despite smaller or equal lesions in the EC-SOD–null mice, their cholesterol levels were somewhat higher. Also, on a wild-type background, there were no effects produced by the absence or presence of EC-SOD on atherogenic diet–induced aortic root lesions. The urinary excretion of the lipid peroxidation biomarker 8-isoprostaglandin F₂ was related to the rates of atherogenesis in the mice but was not influenced by the EC-SOD genotype. Likewise, the EC-SOD status had no effect on the staining for oxidized low density lipoprotein epitopes in aortic root sections. Our findings suggest that EC-SOD has little influence on atherogenesis in mice.

Key Words: aortic lesions • isoprostanes • LDL • oxidation • superoxide anion radical

	Introduction

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Atherosclerosis has been suggested to be linked to the oxidation of lipoproteins, primarily LDL, in the vascular wall.¹ However, the mechanisms by which LDL may be oxidized in vivo have not been fully revealed. Evidence for the involvement of the superoxide anion radical has been found in a variety of in vitro cell culture systems.² The mechanisms by which superoxide might modify LDL could involve direct oxidation by the protonated form HO₂^·3,4 or catalysis by transition metal ions such as copper and iron⁵ or ceruloplasmin⁶ or by the reactive peroxynitrite formed through the reaction between NO and O₂^·-.⁷ There are numerous sources of O₂^·- in the vascular wall, including NAD(P)H oxidases in endothelial cells,⁸ smooth muscle cells,⁹ macrophages,¹⁰ and adventitial fibroblasts.¹¹ Other potential sources of O₂^·- are lipoxygenases,¹² xanthine oxidase,¹³ and NO synthases.¹⁴ Furthermore, the production of O₂^·- is markedly increased in a variety of vascular pathologies.^15–17

See p 1387

The major defenses against the superoxide anion radical are the superoxide dismutases (SODs). CuZn-SOD exists in the cytosol,¹⁸ Mn-SOD exists in the mitochondrial matrix,¹⁹ and extracellular SOD (EC-SOD) is secreted to the extracellular space.²⁰ Because the superoxide anion radical crosses membranes poorly, the SOD isoenzymes exert separate protective roles in their respective compartments. A distinguishing property of EC-SOD is its high affinity for heparin and heparan sulfate, and the enzyme exists primarily anchored to heparan sulfate proteoglycans on cell surfaces and in the connective tissue matrix.^21,22 EC-SOD occurs in particularly high concentrations in the arterial wall in humans and other mammalian species and is evenly distributed with a high concentration in the intima.²³ Furthermore, a marked upregulation has been demonstrated in atherosclerotic mouse aorta.²⁴ Thus, the enzyme is present in abundance where the oxidation of LDL presumably takes place. EC-SOD occurs also in the blood and forms an equilibrium between the plasma phase and the endothelial cell surfaces.²⁵

Mice lacking EC-SOD have been generated,²⁶ and these mice would be expected to have an increased level of superoxide radical in the interstitium of the vessels. The goal of the present study was to determine the influence of EC-SOD and superoxide radicals in the interstitium on the development of atherosclerosis. Mice are naturally resistant to atherosclerosis, and diets containing high fat and cholesterol are required to produce even minor lesions.²⁷ ApoE-null mutant mice, on the other hand, are hypercholesteremic and develop lesions spontaneously on normal chow and also on high fat atherogenic diets.^28,29 In the present study, we exposed EC-SOD–null mice, apoE-null mice, and mice with various combined genotypes to an atherogenic diet or standard mouse chow and examined the development of atherosclerosis.

	Methods

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Animals and Treatments
EC-SOD–null mutant mice (background, crossed 5 times with C57BL/6) were obtained from a breeding colony established at Umeå University, Umeå, Sweden.²⁶ The apoE-deficient mouse strain,³⁰ C57BL/6J-Apoc3^tm1Unc (background, crossed 10 times with C57BL/6), was purchased from The Jackson Laboratory (Bar Harbor, Me). The EC-SOD–deficient mice were bred to the apoE-deficient mice, and the resulting offspring were bred again to create double- and single-knockout mice of all combinations. The genotypes of the mice were determined by polymerase chain reaction with gene-specific primers³¹ and according to suggestions from The Jackson Laboratory.

The pups were weaned at 3 weeks of age and then maintained on regular chow. At 6 weeks of age, some groups of mice were put on an "atherogenic" diet containing 15% fat, 1.5% cholesterol, and 0.5% cholic acid (TD 88051, Teklad Premier). Diet and water were provided ad libitum. The present study followed the principles of laboratory animal care of the National Institutes of Health (NIH publication No. 86-23, revised 1985) and was approved by the local animal ethics committee. All mice were weighed at 6 weeks of age and then once a month throughout the study, when blood was collected from the tail artery. At euthanasia, mice were anesthetized by metoxyphan, and blood and urine were collected.

Analysis of Atherosclerotic Lesions
The degree of atherosclerosis was determined either by quantifying sudanophilic en face lesions in pinned out aortas³² or by quantifying oil red O–stained lesions in cross sections from the aortic root.³³ Briefly, the mice were perfused, first with PBS and then with a fixative solution (4% paraformaldehyde, 5% sucrose, 20 mmol/L EDTA, and 20 µmol/L butylated hydroxytoluene, pH 7.4). The aorta was opened longitudinally from the aortic root to the iliac branch; all branching vessels were removed; and the aorta from the iliac bifurcation to a point equidistant between the aortic valve and the brachiocephalic artery was removed, pinned out flat on a black wax surface, and stained with Sudan IV. The aortas were then photographed, and the total surface and the entire lesion areas were measured by planimetry. For the study of the aortic root, the heart and 1 mm of the thoracic aorta were removed. The aortic root was dissected from surrounding tissue, embedded in OTC (Miles Laboratories), and frozen at -20°C. A total of one hundred 10-µm-thick cross sections were collected, stained with oil red O, and counterstained with hematoxylin. The lesions were quantified in 1 cross section per 50 µm from the start of the aortic root spanning 1000 µm of the ascending aorta. Sections were viewed by a light microscope, images captured were with a video camera (Hamamatsu color chilled 3CCD camera, Openlab), and the total lesion areas were quantified with the assistance of an image analysis software package (Openlab 3).

Serum Lipid Pattern Analysis
Sera from 5 or 6 animals fed the atherogenic diet for 1 month were pooled, 350 µL was analyzed by separation on a 10x300-mm Superose 6 column (Amersham Pharmacia Biotech), and the cholesterol levels were determined in the collected 300-µL fractions.

Immunohistochemistry for Oxidized LDL
Aortic roots, kept for 30 minutes in the fixative solution (see above), were paraffin-embedded, and 5- to 6-µm-thick sections were cut. The sections were stained for macrophages and malondialdehyde-modified lysines, epitopes characterizing oxidized LDL.³⁴ As controls for the staining, the primary antibodies were replaced with class- and species-matched immunoglobulins.

Blood and Urine Chemical Analyses
Serum cholesterol and triglyceride levels were analyzed in a Vitros DT60 apparatus. The level of thiobarbituric acid–reactive substances (TBARs) was determined in mouse plasma samples by a fluorometric method.³⁵ Urine creatinine was determined in a Vitros 950 apparatus. Unextracted urinary samples (50 µL) were analyzed for 8-isoprostaglandin F₂ (8-iso-PGF₂) by a highly specific and validated radioimmunoassay.³⁶

Statistical Analysis
Because the data mostly were skewed from the normal distribution, the differences versus EC-SOD wild-type mice in the various treatment groups were evaluated by the nonparametric Mann-Whitney U test. A value of P<0.05 was regarded as significant. Univariate correlations between the en face lesion areas and serum cholesterol levels were assessed by Spearman correlation coefficient analysis.

	Results

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Development of Atherosclerotic Lesions
ApoE-null mutant mice develop atherosclerotic lesions rapidly when kept on an atherogenic diet and also when kept on normal chow. To test whether EC-SOD influences the process, EC-SOD/apo-E double-knockout mice and, for comparison, apoE-null mice were exposed to the diets, and the formation of aortic en face lesions was determined.

When mice were fed the atherogenic diet, small lesions were present in mice from all groups after only 1 month. The EC-SOD/apoE-null double-mutant mice were significantly less affected by lesions than were the apoE-deficient mice (P<0.05). After 3 months on the atherogenic diet, the lesions were more advanced, and no difference between the genotypes was detected. Likewise, when the mice were kept on normal chow for 8 months, there was no influence by the presence or absence of EC-SOD on the development of atherosclerotic lesions in the apoE-null mice (Table, Figure 1).

View this table: [in this window] [in a new window]   Table 1. Atherosclerotic Lesions and Blood and Urine Analyses in the Various Groups

View larger version (21K): [in this window] [in a new window]   Figure 1. Atherosclerotic lesion development in mice with and without EC-SOD. Mice were kept on the atherogenic (athero.) diet or normal diet for different times, as indicated. Open circles indicate males; solid triangles, females. S/s indicates wild-type (S) and EC-SOD–null (s) alleles; similarly, E/e indicates wild-type (E) and apoE-null (e) alleles. Bars indicate medians.

We also determined atherogenesis in mice with a normal apoE background by subjecting EC-SOD–null and wild-type control mice to the atherogenic diet for 5 months. In these mice, there were no aortic en face lesions; therefore, they were examined by histology of the aortic roots. The lesion areas of the EC-SOD–deficient mice did not differ significantly from those of the wild-type mice (Table, Figure 1).

To test whether EC-SOD influences the growth of the aorta, we determined the total en face and aortic root section areas for the various genotypes. There were no significant differences between the EC-SOD–deficient mice and EC-SOD–replete mice (data not shown).

Lipids
Despite smaller or equal lesions in EC-SOD–null mice on an apoE-null background, their serum cholesterol levels were higher in most groups (Table). On the other hand, there were no differences in cholesterol between EC-SOD–null and wild-type mice on a normal apoE background kept on the atherogenic diet for 5 months (Table).

To further analyze this phenomenon, we plotted lesion areas versus serum cholesterol levels in the various groups but found no significant correlations between the parameters in any of the groups (data not shown).

We also examined lipid patterns in serum from mice subjected to the atherogenic diet for 1 month by separation on a Superose 6 column. No notable differences were found in the patterns between apoE-null mice and apoE/EC-SOD double-knockout mice (Figure 2).

View larger version (11K): [in this window] [in a new window]   Figure 2. Separation of serum on Superose 6. Pools of serum from apoE-knockout mice (n=5, solid circles) and EC-SOD/apoE double-knockout mice (n=6, open triangles) kept on the atherogenic diet for 1 month were separated on Superose 6, and cholesterol was analyzed in collected fractions.

ApoE-null mice deposit lipids in the skin and a variety of internal organs.²⁸ We examined esophagus, liver, and skin from the mice by histology but did not find any differences related to the absence or presence of EC-SOD in the extent of the lipid deposition (data not shown).

Serum TBARs and Urinary Excretion of Isoprostanes
To assess the extent of systemic lipid peroxidation in the mice, serum levels of TBARs and urinary excretion of 8-iso-PGF₂, an F₂-isoprostane, were determined. The TBAR levels were not significantly influenced by either diets or genotypes. On the other hand, the 8-iso-PGF₂ excretion was markedly increased in the mice with high cholesterol levels in the apoE-knockout group. However, the presence or absence of EC-SOD did not significantly influence the levels (Table).

Oxidatively Modified Lipoproteins Present in the Aortic Root
To evaluate whether the EC-SOD/apoE double-mutant mice suffered from increased levels of oxidatively modified lipoproteins compared with the apoE mutant mice, the aortic roots of mice fed the atherogenic diet for 1 month were investigated (Figure 3). There was a correlation between the extent of staining for macrophages and oxidized LDL, but neither parameter differed between EC-SOD/apoE double-knockout and apoE-knockout mice.

View larger version (10K): [in this window] [in a new window]   Figure 3. Staining for oxidized LDL (oxLDL) and macrophages in aortic root sections. The sections were derived from apoE-knockout mice (n=8, solid circles) and EC-SOD/apoE double-knockout mice (n=12, open triangles) kept on the atherogenic diet for 1 month, as described in Methods. The intensities of staining were semiquantitatively graded in blinded sections as 0, 1, 2, and 3. In wild-type mice kept on the diet, no macrophage staining was detected, and only 1 mouse showed a grade 1 staining for oxLDL (n=6, data not shown). Aortas from EC-SOD heterozygous/apoE-knockout mice showed staining similar to those from the double-knockout mice and the apoE-null mutant mice (data not shown).

	Discussion

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Contrary to expectation, the early small lesions that had developed after 1 month on the atherogenic diet were smaller in the double-mutant mice than in the apoE-null mice. However, the results for the EC-SOD heterozygous mice on an apoE-null background did not differ from the results for the double-knockout mice (Figure 1). After mice were fed an atherogenic diet for 3 months, the lesions were more advanced, and the differences between the genotypes were lost. We also kept mice with an apoE-null genetic background on regular chow for 8 months. The resulting lesions were relatively large and were not influenced by the absence or presence of EC-SOD. The mechanisms behind the enhanced development of atherosclerosis in the apoE-null mice are very complex.³⁷ Therefore, we subjected EC-SOD–null and littermate wild-type control mice to the atherogenic diet for 5 months. No aortic en face lesions were seen, but aortic root lesions developed, and they did not differ in extent between the genotypes (Figure 1). In aggregate, the results suggest that the high vascular wall EC-SOD activity in the wild-type mice and, by inference, low O₂^·- concentration in the interstitium enhance or have little effect on the development of atherosclerotic lesions in mice. Potential problems resulting from genetic heterogeneity were minimized by the fact that both groups of animals had been bred on the C57BL/6 background; furthermore, littermate mice were used for the study, making it unlikely that other genetic differences can account for our findings.

Throughout the study, the cholesterol levels were somewhat higher in the EC-SOD–null mice, despite the smaller lesions in the early phase. However, in accord with previous findings,²⁹ there was no correlation between cholesterol levels and lesion sizes. Also, we did not find any major differences in lipoprotein profiles between EC-SOD–null and EC-SOD–replete mice on an apoE-null background after 1 month on the atherogenic diet (Figure 2). Furthermore, the lipid deposition in the skin and internal organs of apoE-null mice was not influenced by the absence or presence of EC-SOD. Finally, there were no obvious differences in general health status of the different genotypes as indicated by the similar weight developments (Table).

The present findings suggest that extracellular superoxide radicals and derived reactive products are of minor importance for atherogenesis in vivo in mice and may even offer protection in the early phase. The results are in agreement with recent studies reporting that disruption of the gp91^phox38 or p47^phox,39 components of the neutrophil-type superoxide radical–producing NADPH oxidases, did not significantly alter the development of atherosclerosis in apoE-null mice. These components exist in the endothelium⁸ and in adventitial fibroblasts.¹¹ However, in smooth muscle cells, another terminal oxidase, Mox1, substitutes gp91^phox, and it is unclear whether p47^phox is necessary for its oxidase activity.⁴⁰ Significant superoxide radical formation from NADH oxidase may thus still exist in the vascular wall in these models, supplemented by xanthine oxidase¹³ and formation of the radical as a byproduct from lipoxygenase¹² and NO synthase.¹⁴ The presence of the high wild-type EC-SOD activity should decrease the O₂^·- concentration in the vascular extracellular space irrespective of the source. The effect of overexpression of the intracellular CuZn-SOD has also been examined and was found not to alter the atherogenesis in C57BL/6 mice.⁴¹ Peroxynitrite, the product of NO and O₂^·-, has been implicated in atherogenesis in vitro⁷ and in vivo.⁴² Yet, if the blood pressure is controlled, disruption of endothelial NO synthase or inducible NO synthase does not alter atherogenesis in apoE-null background mice.⁴³ These and our present findings are in accord in suggesting that ONOO^- is of minor importance in atherogenesis, at least in apoE-null background mice. Regarding mechanisms of LDL oxidation and atherogenesis, it has been shown that disruption of the 12/15-lipoxygenase gene reduces lesion formation on an apoE-null background.⁴⁴ However, lipoxygenases can also produce superoxide radicals leaking to the extracellular space,^12,45 but our present findings suggest that loss of this activity did not contribute to the phenotype. Finally, it should be noted that the present results do not contradict the notion that lipoprotein oxidation is involved in atherogenesis; they merely suggest that superoxide radical is not essential for the process. We found high rates of F₂-isoprostane excretion that were broadly related to the rates of atherogenesis and high intensities of oxidized LDL and macrophage staining in the aortic roots, which, however, were not influenced by the absence or presence of EC-SOD.

What could be the explanation for the differences in early lesion formation between the different EC-SOD genotypes (Table, Figure 1)? There are numerous reports showing that production of superoxide may have signaling effects and that it stimulates cellular proliferation and migration, for instance.⁴⁶ In most cases studied, the superoxide is produced intracellularly, and the actual signaling species is poorly defined but seems mostly to be hydrogen peroxide or other derived species. In some cases, however, there is evidence of a more direct involvement of superoxide radicals.⁴⁷ One might speculate that a high EC-SOD activity interferes with a superoxide signal that suppresses mechanisms that promote the early phase of atherogenesis. It has been suggested that the superoxide radical can react with the lipid peroxy radical and alkoxy radical formed during lipid peroxidation and that at least the latter reaction might lead to chain termination.⁴⁸ Such a reaction might balance other pro-oxidant reactions, and it has been found that protective effects of SOD have a dose optimum in several pathophysiological models.⁴⁸ The high wild-type EC-SOD level might reduce superoxide below a putative optimum in the apoE-null mice, and it was found that the lesions were smaller in the EC-SOD heterozygous mice (Figure 1, Table). Although the superoxide radical rapidly forms hydrogen peroxide by spontaneous disproportionation, the presence of (EC-)SOD may increase that formation by diverting the superoxide radical from other reaction routes. Furthermore, CuZn-SOD can, in the presence of hydrogen peroxide, initiate lipid peroxidation,⁴⁹ and the active sites of CuZn-SOD and EC-SOD are very similar.⁵⁰ Thus, in the vessel wall, EC-SOD may increase the hydrogen peroxide formation and also initiate lipid peroxidation. However, the efficiency of this putative reaction is unknown. Our main conclusion from the present study is that EC-SOD and probably extracellular superoxide radicals are not major participants in atherogenesis.

	Acknowledgments

 
This study was supported by the Swedish Medical Research Council (grant 12566). We wish to thank Eva Bern, Agneta Öberg, Lena Hedman, Karin Wallgren, and Inger Bodin for skillful technical assistance and Jan Borén for technical advice and material.

Received February 19, 2001; accepted May 29, 2001.

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