Published online before print June 26, 2003, doi: 10.1161/01.RES.0000082978.92494.B1
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© 2003 American Heart Association, Inc.
Integrative Physiology |
Aorta of ApoE-Deficient Mice Responds to Atherogenic Stimuli by a Prelesional Increase and Subsequent Decrease in the Expression of Antioxidant Enzymes
From the Division of Biopharmaceutics of the Leiden/Amsterdam Center for Drug Research, Leiden, The Netherlands. Present address for P.A.C.’t.H. is the Center for Human and Clinical Genetics, LUMC, Leiden, The Netherlands. Present address for J.T. is Amsterdam Medical Therapeutics, Amsterdam, The Netherlands.
Correspondence to Theo J.C. Van Berkel, LACDR, Division of Biopharmaceutics, PO Box 9502, 2300 RA Leiden, The Netherlands. E-mail t.berkel{at}lacdr.leidenuniv.nl
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Abstract |
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Oxidative stress has been implicated in the development of atherosclerotic lesions. We evaluated the relationship between extent of atherosclerotic lesion formation and vascular expression of pro- and antioxidant enzymes in apoE-deficient mice. On normal chow, these mice showed elevated serum cholesterol levels (7.5- to 9.5-fold), and age-dependent, spontaneous development of all stages of atherosclerotic lesions, starting at the age of 12 weeks. RNA was extracted from the aortic arch and descending aorta, and mRNA expression of pro- and antioxidant enzymes was measured with real-time PCR. Local infiltration of monocytes/macrophages, reflected by increased vascular expression of CD68 mRNA (>10-fold), indicated that the arch was more susceptible than the descending aorta. The expression of catalase-1 and various isoforms of superoxide dismutase, glutathione peroxidase, and glutathione S-transferase alpha was significantly increased in the aortic arch, but not in the descending aorta, in the period preceding lesion formation (age 6 to 12 weeks). These expression levels were 1.5 to 5 times higher than in age-matched wild-type animals. Remarkably, there was an inverse relationship between extent of lesion formation and the mRNA levels of antioxidant enzymes, most of which started to decline after 12 weeks, as lesions developed. In contrast, inducible nitric oxide synthase expression increased 4-fold in the aortic arch over the course of the disease. Our results suggest that the arterial wall responds to increased serum levels of atherogenic lipoproteins by stimulating expression of antioxidant enzymes. The observed co-ordinate decline in expression of many of these protective systems may greatly accelerate the development of atherosclerosis.
Key Words: oxidative stress • vascular gene expression • real-time PCR • atherosclerosis • redox balance
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Introduction |
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Oxidative stress–induced endothelial dysfunction probably represents one of the first stages in the development of atherosclerotic lesions.1–3 Accordingly, the atherosclerotic vessel wall contains increased levels of reactive oxygen species (ROS) including hydroxyl radicals (OH.), superoxide anions (O2-·), hydrogen peroxide (H2O2), and lipid peroxides (LOOH).4–6 ROS affect several redox-sensitive pathways in vascular cells, which result in a markedly altered cellular composition of the tissue. Migration and proliferation of vascular smooth muscle cells in the area is induced, as well as expression of adhesion molecules and chemotactic factors by the endothelium.7,8 Consequently, increased arterial adhesiveness at predisposed sites provides an exquisite environment for local infiltration of circulating immune cells, resulting in chronic inflammation.9 ROS affect vascular function directly by scavenging available nitric oxide (NO·), thereby modulating vessel contractility.4,6
ROS are continuously produced in all cells, for example during mitochondrial respiration and by enzyme systems that utilize molecular oxygen. Oxidative stress, resulting from pathological levels of ROS, has been observed in both endothelial- and smooth muscle cells in the presence of increased levels of atherogenic lipoproteins such as oxidized low-density lipoproteins (oxLDL),1,10 or on increased interaction of circulating monocytes with the arterial wall.3,11 In a later stage of atherosclerosis, oxidative stress may be aggravated through increased ROS production by NAD(P)H oxidase, inducible NO· synthase (iNOS), and myeloperoxidase produced by infiltrating monocytes/macrophages.3
In light of the postulated role of oxidative stress in atherogenesis, we quantitatively analyzed the expression of enzymes involved in antioxidant defense and production of ROS in the aorta. Figure 1 provides an overview of the studied enzymes that convert O2-·, H2O2, or LOOH. In addition, we measured expression levels of heme oxygenase (HO-1), because, although being a possible source of ROS, its role in iron homeostasis may be cytoprotective, as was observed in atherosclerotic endothelium, lesional macrophages, and foam-cells.12–14 Furthermore, we studied the expression of eNOS and iNOS. Although a source for cytoprotective levels of NO·, pro-, and anti-atherogenic roles within the context of atherosclerosis have been reported for both enzymes.15–17 We used apoE-deficient mice on normal chow that spontaneously display the entire spectrum of lesions observed during atherogenesis in man.18,19 Changes in expression levels were correlated with progression of lesion formation. We show that the expression of several antioxidant enzymes was increased in the period preceding lesion formation, and that their expression decreased at the time when lesion formation became apparent.
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Materials and Methods |
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Materials
Mayer’s hematoxylin stain, Oil-Red-O, and diethylpyrocarbonate were from Sigma. DNAse I (RNAse-free) and glycogen were from Invitrogen. Ribogreen was from Molecular Probes. All real-time PCR supplies were from Applied Biosystems. RevertAid M-MuLV Reverse Transcriptase was from MBI Fermentas. PCR primers were synthesized by Eurogentec or Isogen B.V.
Animals
Female apoE-deficient mice (back-crossed for 9 generations on a C57bl/6 background) and C57bl/6 mice were obtained from Jackson Labs (Bar Harbor, Mass) and Broekman (Someren, The Netherlands), respectively. The mice were kept on a regular dark/light cycle, and received water and regular chow ad libitum. Mice were anesthetized by subcutaneous injection of ketamine (75 mg/kg; Eurovet), droperidol (1 mg/kg), fluanisone (0.75 mg/kg), and fentanyl (0.04 mg/kg; all from Janssen-Cilag). Blood was drawn from the inferior caval vein for analysis of serum lipids. Subsequently, a 10-minute whole-body perfusion was performed using ice-cold phosphate-buffered saline, containing 1 mmol/L EDTA, administered by heart puncture (left ventricle). After perfusion, organs were excised, frozen in liquid N2, and kept at -80°C until RNA isolation. The aorta was prepared free of peri-adventitial fat in situ. The aortic arch was isolated from the base (most proximal to the heart) and severed from the descending aorta just behind the left subclavian artery. The descending aorta was harvested from the arch down to the bifurcation.
Assessment of Lesion Formation
Serial sections (10 µm thick) of the hearts were cut using a Leica CM3050S cryostat, starting at the apex and through the aortic valve area into the proximal part of the aorta. Ten consecutive sections per mouse, clearly showing the tricuspid valves, were immersed 10 times in 60% isopropanol, incubated for 15 minutes with Oil-Red-O (0.06% [wt/vol] in 60% isopropanol), washed, and counterstained for 3 minutes with Mayer’s hematoxylin stain. Sections were visualized using a Leica microscope, and lesion area was quantified with Leica Qwin imaging software. Macrophage infiltration in the atherosclerotic lesions was determined by immunolocalization of CD68 (see expanded Materials and Methods section in the online data supplement available at http://www.circresaha.org).
Determination of Lipid Levels
Blood was allowed to clot, and spun for 10 minutes at 2000g to obtain serum. Sera were kept at 4°C and analyzed for total cholesterol, free cholesterol, and triglyceride levels using enzymatic kits (Roche, Mannheim, Germany). The distribution of cholesterol over the different lipoprotein fractions was evaluated by loading of 30 µL serum onto a Superose 6 column (3.2x30 mm, Smart-system, Pharmacia).
RNA Isolation and cDNA Synthesis
RNA was isolated essentially as described20 (for details see online data supplement), treated with DNAse I, and reverse-transcribed with RevertAid M-MuLV Reverse Transcriptase using oligo(dT) primers.
Determination of mRNA Levels
mRNA levels were quantitatively determined on an ABI Prism 7700 Sequence Detection system (Applied Biosystems) using SYBR-green technology. PCR primers (online Table S1, in the online data supplement) were designed using Primer Express 1.5 Software with the manufacturer’s default settings. After PCR amplification, dissociation curves were constructed to confirm formation of the intended PCR products. Expression levels of target genes were related to the averaged expression levels of three housekeeping genes: HPRT, GAPDH, and 36B4. Relative expression levels were calculated with the 
Ct rule (Applied Biosystems, User Bulletin No. 2, and Winer et al21). For details, see the online data supplement.
Statistical Analysis
An unpaired Student t test and a two-tailed Spearman rank-correlation test were performed to determine statistical significance. The software package SPSS version 7.5 (SPSS Inc) was used for statistical analyses.
An expanded Materials and Methods section is available in the online data supplement at http://www.circresaha.org.
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Results |
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Development of Atherosclerotic Lesions in ApoE-Deficient Mice
The aim of our study was to quantitatively monitor changes in vascular gene expression of proteins potentially associated with atherogenesis. To match these changes to the extent of disease, lesion formation at the base of the aortic tree was determined in apoE-deficient mice and wild-type mice of various ages, on normal chow. Minimal fatty-streaks were found in 6- to 12-week-old apoE-deficient animals, with a rapid increase in lesion size and complexity in the older mice (Figures 2A through 2F). Infiltration of monocytes/macrophages was evident from the age of 12 weeks, as measured through immunohistochemical staining of CD68 (Figures 2G through 2J). Figure 3A shows the increase in lesion area with age. Based on previous studies by others,22 we estimate that after 12 weeks approximately 5% of the aortic tree is covered with lesions, and approximately 30% and 50% after 21 and 34 weeks, respectively.
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) and descending aorta (
) of apoE-deficient mice of different age (mean of 5 to 6 animals per group ±SD) were quantified by real-time PCR. mRNA levels are expressed relative to the expression of 3 different housekeeping genes and related to the levels in 6-week-old animals. An unpaired Student t test was applied to test if mRNA levels were significantly different from the mRNA levels in 6-week-old animals (*P<0.05).
Serum total cholesterol (TC), free cholesterol (FC), and triglyceride (TG) levels were also measured in apoE-deficient mice to investigate a possible relationship between gene expression and serum lipid levels. No gross changes in serum lipid levels were observed in mice of different age and extent of lesion development (Figure 4). Both TC and FC levels were up to 35% higher in 12- to 21-week-old mice compared with 6-week-old mice (P<0.05); however, TC levels were 7.5- to 9.5-fold higher in apoE-deficient mice than in wild-type animals, irrespective of age, and the same held true for free cholesterol levels. We did not observe a difference in TG levels between apoE-deficient and wild-type mice. On fractionation of the sera by size-exclusion chromatography, serum cholesterol was mainly contained within the very low-density lipoprotein fraction (
50%), with a second peak in the intermediate- to low-density lipoprotein fraction (25% to 30%).
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) and wild-type (
Changes in Gene Expression During Atherosclerosis Development
It appears that apoE-deficient mice do not develop visible atherosclerotic lesions before the age of 12 weeks, despite prolonged exposure to high serum levels of atherogenic lipids. Although we did not measure serum lipid levels before the age of 6 weeks, we infer (from Figure 4) that they are already elevated at birth. In addition, prenatal exposure to maternal hypercholesterolemia has been reported.23 As the development of atherosclerosis may be associated with changed expression of vascular anti- and pro-oxidant enzymes, we measured the mRNA levels of 16 candidate genes in the aorta (online Table S1).
Whereas the base of the aortic tree of each animal was used to assess the extent of lesion formation, the arterial tree of the same animal was used to determine changes in gene expression. Although lesion area at the base of the aortic tree shows a strong, linear correlation with the lesion area covering the entire aorta, lesions develop more readily at the base of the aortic tree and aortic arch than in the descending aorta.22 Therefore, the aortic arch and the descending aorta were analyzed separately. The expression level of CD68 was again used as a measure of influx of monocytes/macrophages, and was more pronounced, and emerged at an earlier stage, in the aortic arch than in the descending aorta (Figure 3B). mRNA levels for 
-actin and PECAM-1 were determined as a marker for VSMCs and ECs, respectively. The observed gradual decline in the expression of the SMC marker -actin (Figure 3C) probably reflects remodeling of the vascular wall, hallmarked by a change from contractile to synthetic phenotype.24 Whereas ECs contribute only very little to the RNA pool analyzed for each time point, the technique was sensitive enough to detect expression of PECAM-1. PECAM-1 mRNA levels remained constant during the course of lesion formation (Figure 3D).
Gene expression was measured using real-time PCR technology. The threshold cycle (Ct) at which the fluorescent signal reaches a certain threshold value, was used as a measure for gene expression. Online Table S1 gives threshold cycles for the analyzed genes in the descending aorta and the aortic arch. The Ct values are approximate indications of the abundance of a particular mRNA within the tissues, but also depend on amplicon size and, possibly, amplicon sequence. In 6-week-old apoE-deficient animals, expression of most of the evaluated genes in the aortic arch was approximately 2-fold lower (difference of 1 Ct) than in the descending aorta (online Table S1). This may be due to slight differences in composition and/or embryonic origin of the tissues. However, there were some notable exceptions: GPx-1 and GSTA4, which have been shown to be upregulated by low levels of oxidative stress,25 were expressed at a higher level in the aortic arch than in the descending aorta.
In Figure 5, the relative mRNA levels of the examined genes in the aortic arch and the descending aorta of apoE-deficient animals are shown in relation to age. To distinguish atherosclerosis-induced changes in gene expression from age-dependent variation in expression levels, we compared mRNA levels in the aortic arch of 6- to 34-week-old apoE-deficient mice with those in age-matched wild-type animals (Table). The expression levels of several genes coding for proteins associated with the combat of oxidative stress was increased in the period preceding manifestation of atherosclerotic lesions. The mRNA levels of GPx-1, GPx-4, Cat-1, SOD-1, SOD-2, and HO-1 were all elevated in the aortic arch of apoE-deficient mice in the age of 6 to 12 weeks, compared with wild-type animals (Table). Furthermore, in apoE-deficient mice the mRNA levels of GPx-3 and Cat-1 were higher at age 9 to 12 weeks than at 6 weeks (Figure 5). Also, the expression of GSTA3 and GSTA4, which reduce lipid peroxidation products,26,27 were increased 2- to 25-fold in the aortic arch of many, but not all, mice in the 9- and 12-week age groups, compared with 6-week-old mice. In contrast, the mRNA levels of aforementioned antioxidant enzymes in the descending aorta were not induced.
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Remarkably, from the age of 12 weeks onwards, when lesion formation initiates both at the base and arch of the aortic tree (Figures 2 and 3 
) in apoE-deficient mice, the expression of most antioxidant enzymes in the aortic arch, ie, GPx-1, GPx-3, GPx-4, Cat-1, SOD-1, SOD-2, GR, GSTA3, and GSTA4, declined (Spearman’s rank-correlation test, P<0.05) (Figure 5). The expression of many antioxidant enzymes in the descending aorta also declined and reached levels comparable to those in the aortic arch. However, the expression of GPx-1, GPx-4, and GSTA4 was much lower in the aortic arch than in the descending aorta in these old apoE-deficient animals, suggesting that at this stage of disease these enzymes are hardly expressed in atherosclerotic tissue. In contrast, the expression of HO-1 remained high in apoE-deficient animals (Table).
The expression of eNOS was determined as a marker for endothelial integrity.28 eNOS mRNA levels were significantly lower in the descending aorta of 12- to 21-week-old apoE-deficient mice compared with 6-week-old animals. The expression of eNOS in the aortic arch was not significantly altered. iNOS mRNA levels gradually increased in the aortic arch and were 4-fold higher in mice of 34 weeks, compared with mice of 6 weeks. In contrast, the iNOS mRNA levels were 5-fold lower in the descending aorta of 12- to 21-week-old animals, again reflecting major differences in the response of the two parts of the aorta to the hyperlipidemic status.
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Discussion |
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Oxidative stress plays a major role in the development of atherosclerosis.1–3 Accordingly, increased levels of ROS have been found in the atherosclerotic vessel wall.4–6 In the present study, we investigated a possible relationship between expression of pro- and antioxidant enzymes, and the age-dependent development of atherosclerotic lesions in apoE-deficient mice. Homogenates of aorta segments (aortic arch versus descending aorta) were used, rather than isolated vascular cells. As accumulation of proatherogenic lipid affects all cell types present within the vascular wall, the response of the entire tissue to the hyperlipidemic state is relevant as an indication of vascular defense as a whole. Nevertheless, the measured expression levels in the present study could be influenced by changes in the cellular composition of the vessel wall. We have demonstrated influx of macrophages, and possible changes in the VSMC phenotype, whereas EC content was shown to be relatively constant. Laser-capture is needed to indicate whether there are differences between cell-types in the response to lipid-induced stress.
Analysis of mRNA levels with real-time PCR technology is more sensitive and isoform-specific than analysis at the protein level. However, whereas decreases in mRNA levels are likely to be accompanied by loss of protein expression, increased mRNA levels do not always reflect increases in protein expression and enzyme activities.29 For many of the antioxidant enzymes analyzed in this study, mRNA levels were shown to correlate well with protein levels and enzyme activities.30–34 We found a good correlation between CD68 mRNA levels and immunohistochemical staining of the protein. Analysis of other expression patterns (GPx, SOD, and catalase) at the protein (activity) level was complicated by low sensitivity of the immunohistochemical stainings, cross-reactivity of antibodies, and lack of isoform-specific enzyme substrates.
In the period preceding visible lesion formation (6 to 12 weeks after birth), mRNA levels of many antioxidant enzymes in the aortic arch of apoE-deficient mice were higher (1.5- to 5-fold) than those in age-matched wild-type mice. The increase in expression levels may have started at a younger age than studied here and/or may be (partly) caused by prenatal exposure to high cholesterol levels, because it was recently demonstrated in an expression profiling study that extracellular SOD was even enhanced in the offspring of hypercholesterolemic mice that were otherwise normocholesterolemic.23 However, the expression of several antioxidant enzymes (GPx-3, Cat-1, GSTA3, and GSTA4) was still rising at the age of 9 to 12 weeks, compared with 6-weeks. Coordinated induction of antioxidant enzymes may be highly relevant. Induction of SOD-1 and SOD-2, which convert O2-· into the less reactive H2O2, alone may not be sufficient to prevent atherosclerotic lesion formation.5 However, coordinate induction of SODs with GPx-1, GPx-3, GPx-4, Cat-1, GSTA3, and GSTA4 may be protective for the endothelium, because the latter enzymes will diminish levels of H2O2 and lipid peroxides. An indication for coregulation of enzymes with a primary antioxidant function (GPx, Cat, SOD, and GSTA isoforms) is derived from the Pearson correlation coefficients of their expression levels in individual 9- and 12-week-old mice (online Table S2). The coordinated induction of antioxidant enzymes is probably mediated by ROS and lipid peroxides, as has been demonstrated for SOD-1, SOD-2, GPx-1, catalase, and GSTA3.7,31,33,35,36 Possibly, the presence of binding sites for redox-active transcription factors in their promoter regions, such as the antioxidant responsive element,26 governs this coordinated response. Interestingly, at the age of 9 weeks, the expression of several antioxidant enzymes appear to be positively correlated to CD68 expression levels, whereas at 12 weeks, they tend to be negatively correlated (online Table S2). This may indicate that macrophage infiltration is partly responsible for the initial induction and subsequent decrease of antioxidant enzyme expression levels.
In the period of lesion formation (at the age of 15 to 34 weeks), a decrease in the mRNA expression was observed for many antioxidant enzymes, ie, GPx-1, GPx-3, GPx-4, Cat-1, SOD-1, SOD-2, GR, GSTA3, and GSTA4. The results correlate well with the observed absence of GPx-1, and decrease in GSTA3 and GR enzyme activities in human atherosclerotic lesions,37 indicative of a disturbed, GSH-dependent antioxidant defense. In earlier reports, high levels of antioxidant activities have been linked, both in human and mouse studies, to a resistance toward atherosclerotic plaque development.23,38 Our results support the notion that induction of antioxidant activities (partially) prevents progression of atherogenesis. Age-dependent changes in the expression levels of pro- and antioxidant enzymes in apoE-deficient mice were often more prominent in the aortic arch than in the descending aorta. The lower laminar shear stress in regions of high blood flow turbidity, such as found at the level of the tricuspid valves and the lesser curvatures of branch-points, possibly contributes to the differential sensitivity toward atherogenic stimuli, reflected in gene expression patterns (this study), and at the level of progression of lesion formation.39
As iNOS is highly expressed by macrophages, and as the expression profile overlaps with that of CD68, the increased iNOS mRNA levels in the aortic arch of older apoE-deficient mice are probably derived from infiltrating macrophages. Also in a microarray analysis, iNOS was identified as an upregulated gene during progression of atherosclerosis in apoE-deficient mice.40 In the microarray study, the difference in iNOS expression between apoE-deficient and wild-type mice was much greater than observed in our study. This may be due to the Western-type diet used, greatly accelerating the rate of lesion development in apoE-deficient mice. Increased iNOS expression may lead to increased generation of ROS and reactive nitrogen species41 and aggravate atherogenesis, as demonstrated in iNOS/apoE double knockout mice.16 However, absence of iNOS per se has also been shown to induce lesion formation.15 Similar paradoxical findings have been reported for eNOS.17
Although highly variable, the mRNA levels of HO-1 seem to be higher in apoE-deficient animals than in wild-type animals. HO-1 expression is most likely an important, early indicator of inflammation in the vessel wall.13 Induction of HO-1 expression attenuated diet-induced lesion formation in LDL receptor–deficient mice.14 Also in young apoE-deficient mice, elevated HO-1 expression may delay the progression of atherosclerosis.
In summary, we have surveyed the expression of pro- and antioxidant enzymes in the aorta of apoE-deficient mice by real-time PCR. Many antioxidant enzymes show an initial increase in mRNA levels in apoE-deficient mice at 6 to 12 weeks after birth, which is mainly apparent in the aortic arch. The increase is followed by a decrease in the expression at later time-points, accompanied by a massive increase in atherosclerotic lesions. We hypothesize that in young apoE-deficient animals, the aorta compensates for the oxidative stress induced by atherogenic stimuli by stimulating the expression of antioxidant enzymes. This may delay atherosclerotic lesion formation in these young mice. From the age of 12 weeks, the antioxidant capacity of the arterial wall collapses, which may explain the appearance of atherosclerotic lesions.
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Acknowledgments |
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J.T. was funded by Unyphar, a collaboration between Yamanouchi and the Universities of Groningen, Leiden, and Utrecht. This article is dedicated to the memory of Martin Bijsterbosch, an active and respected member of the scientific community, who passed away on May 9, 2003, at the age of 49.
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Footnotes |
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Deceased. 
Original received April 9, 2002; resubmission received March 4, 2003; revised resubmission received June 12, 2003; accepted June 12, 2003.
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