Published online before print January 26, 2006, doi: 10.1161/01.ATV.0000205850.49390.3b
© 2006 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Glucose-6-Phosphate Dehydrogenase Deficiency Decreases Vascular Superoxide and Atherosclerotic Lesions in Apolipoprotein E–/– Mice
From the Vascular Biology Unit (R.M., S.X., K.A.M., R.M., R.A.C.), Whitaker Cardiovascular Institute (J.A.L., D.E.H., J.L.), Evans Department of Medicine Boston University School of Medicine, Boston, Mass.
Correspondence to Reiko Matsui, MD, Vascular Biology Unit, Boston University School of Medicine, X707, 650 Albany St, Boston, MA 02118. E-mail rmatsui{at}bu.edu
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Abstract |
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Objective— Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway that is a major source of cellular NADPH. The purpose of this study was to examine whether G6PD deficiency affects vascular oxidants and atherosclerosis in high-fat fed apolipoprotein (apo) E–/– mice.
Methods and Results— G6PD-mutant mice whose G6PD activity was 20% of normal were crossbred with apoE–/– mice. Among male apoE–/– mice that were fed a western-type diet for 11 weeks, G6PD wild-type (E-WT), and G6PD hemizygous (E-Hemi) mice were compared. Basal blood pressure was significantly higher in E-Hemi. However, superoxide anion release, nitrotyrosine, vascular cell adhesion molecule (VCAM)-1, and inducible nitric oxide synthase immunohistochemical staining were less in E-Hemi compared with E-WT aorta. Serum cholesterol level was lower in E-Hemi, but aortic lesion area was decreased in E-Hemi even after adjusting for serum cholesterol.
Conclusions— Lower NADPH production in G6PD deficiency may result in lower NADPH oxidase-derived superoxide anion, and thus lower aortic lesion growth. The association of higher blood pressure with lower serum cholesterol levels in this mouse model is indicative of the complex effects that G6PD deficiency may have on vascular disease.
Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the pentose phosphate pathway that is a major source of cellular NADPH. The purpose of this study was to examine whether G6PD deficiency affects vascular oxidants and atherosclerosis in high-fat fed apolipoprotein (apo) E–/– mice. Lower NADPH production in G6PD deficiency may result in lower NADPH oxidase-derived superoxide anion, and thus lower aortic lesion growth. The association of higher blood pressure with lower serum cholesterol levels in this mouse model is indicative of the complex effects that G6PD deficiency may have on vascular disease.
Key Words: atherosclerosis • genetically altered mice • reactive oxygen species • NADPH
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Introduction |
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Enhanced vascular superoxide anion production is associated with hypercholesterolemia and may contribute to the initiation and progression of atherosclerosis.1 Vascular cell-derived superoxide anion mediates oxidative modification of low-density lipoprotein (LDL)2,3 and oxidized LDL further promotes superoxide production and foam cell formation.4 Also, reactive oxygen species promote various processes including endothelial dysfunction, smooth muscle cell growth and migration, and induction of adhesion molecules.5 A major source of superoxide anion in vascular cells is NADPH oxidase, the expression of which is enhanced in atherosclerotic lesions.5–7 Pharmacological inhibition of NADPH oxidase decreased aortic superoxide anion production and atherosclerotic lesions in apoE–/– mice,8 and genetic deficiency in the p47phox subunit of NADPH oxidase attenuates the inflammatory response to hypercholesterolemia9 and reduces aortic lesions in apoE–/– mice.10
Glucose-6-phosphate dehydrogenase (G6PD), a key enzyme in the pentose phosphate pathway, provides NADPH for various cellular reactions including glutathione (GSH) recycling, superoxide anion production via NADPH oxidase, NO synthesis, and cholesterol synthesis. Inhibition of G6PD results in decreased production of superoxide and/or NO in granulocytes11,12 and other cell types including endothelial cells.13–15 In addition, recent studies suggest that the level of pentose phosphate pathway-derived NADPH may regulate vascular superoxide production.16 Consistent with these studies, we found that G6PD-deficient mice had lower aortic superoxide production and less hypertrophy in response to angiotensin II infusion.17 This is a rather paradoxical result, because G6PD is generally considered to be an antioxidant enzyme. G6PD null embryonic stem cells are extremely sensitive to oxidative stress.18 In various conditions G6PD activity is rapidly upregulated in response to oxidative stress, presumably to maintain GSH in its reduced form.19–22 Human G6PD deficiency, the most common genetic enzymopathy, is reported to either enhance or decrease the risk of cardiovascular disease,23,24 but the mechanisms by which risk might be affected are not known.
In the present study, we examined whether G6PD deficiency affects the development of atherosclerosis by modification of oxidant production because of an altered supply of NADPH. We crossbred mice whose G6PD activity was &20% of normal with apoE–/– mice. We found that lower activity of G6PD is associated with higher basal blood pressure, but lower superoxide, serum cholesterol, and atherosclerotic aortic lesions.
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Materials and Methods |
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Animal Model
The G6PD-deficient mouse model in the C3H strain was bred at our institution from frozen embryos obtained from the Medical Research Council (Harwell, UK).25 The G6PD deficient mouse was originally created by Pretsch and Charles26 and showed decreased translation of the protein caused by a single mutation in the untranslated region in the splice site of the X-linked G6PD gene.27 Male hemizygotes (XmY) with a C3H background were bred with female apoE–/– mice with C57BL/6J background obtained Jackson Laboratories (Bar Harbor, Me), and female offspring (F1) (apoE+/–, G6PD XmX) were bred with male apoE–/– mice. Among offspring (F1', F2) of this breeding, male apo E–/– mice littermates were selected with X or Xm G6PD genotype which was ascertained by polymerase chain reaction (PCR) as previously described.22 Therefore, both apoE–/– control (E-WT) and apoE–/– G6PD mutant (E-Hemi) mice used in this study were littermates and had &25% C3H and 75% C57BL/6 genetic background (Figure I, available online at http://atvb.ahajournals.org). Further analysis of their genetic background was done based on analyzing Ath-1, the principal genetic locus that determines atherosclerosis susceptibility in the C57BL/6 strain28 and tnfsf4 the gene within that locus to which that susceptibility has been attributed29 (Supplement, please see http://atvb.ahajournals.org.). Male E-WT and E-Hemi mice were fed a western diet (21% fat, 0.15% cholesterol w/w, TD 88137; Harlan Teklad, Madison, Wis) between age 10 and 21 weeks. Systolic blood pressure was determined by tail cuff plethysmography (Visitech Systems, Apex, NC) as described previously.30,31 This method includes training sessions, a quiet, warm, and dark environment, and provides values similar to those obtaining by intra-arterial measurements in conscious mice.29 The protocol was approved by the Boston University Medical Center Institutional Animal Care and Use Committee.
Measurement of Serum Cholesterol
At 21 weeks of age, mice were anesthetized with isoflurane and blood, aorta, heart, and liver were removed. Serum cholesterol was measured enzymatically using a kit from Sigma Diagnostics.8
Detection of Aortic Superoxide Anion by Lucigenin Chemiluminescence
Measurement of superoxide anion from intact mouse aorta was performed according to the method published previously.8,30 Briefly, the aorta was isolated under a dissecting microscope and incubated in a tube containing 1 mL of physiological buffer with lucigenin (5 µmol/L). This lower concentration of lucigenin was demonstrated not to be involved in redox cycling.32 The tube was placed in a luminometer (model 20e; Turner Design, Mountainview, Calif) in which the light chamber was maintained at 37°C. The luminometer was set to report arbitrary units of emitted light; after a 15-minute equilibration, repeated measurements were integrated every 30 seconds, and an average value was obtained over a 5- to 10-minute period. Tiron (10 mmol/L), a cell-permeable nonenzymatic scavenger of superoxide anion, was then added to quench all superoxide anion-dependent chemiluminescence. Tiron-quenchable chemiluminescence was normalized to aortic wet weight.
Determination of G6PD Activity
Freshly isolated aorta was homogenized in 20 mmol/L Tris buffer with 0.35 mol/L sucrose and centrifuged at 12 000g for 5 minutes. The supernatant was analyzed for protein concentration, and enzymatic activity of G6PD was assayed according to the method described elsewhere.17,33
Quantification of Aortic Atherosclerotic Lesion Area
Atherosclerotic lesions were quantified by planimetry of Sudan IV-stained lesions on the aortic intima as described previously.8 The entire thoracic and abdominal aorta was cut open longitudinally through its ventral side under a dissecting microscope, and immersed in Sudan IV (Fisher). Quantification of stained lesion area was performed on the digitized images using Scion Image and NIH Image software.
Immunohistochemistry of Aortic Sections
The aortic arch was placed in 4% formalin overnight, dehydrated, and embedded in paraffin. Tissue sections (5 µm) were obtained from the descending thoracic aorta, 3 mm distal to the left subclavian artery, and processed as previously described in detail.17,30 Specificity of anti-3-nitrotyrosine antibody was confirmed as previously described.17 Polyclonal anti-VCAM-1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Polyclonal anti-iNOS antibody was from Biomol (Plymouth Meeting, Pa). To assure specific staining, staining with all antibodies was routinely performed with comparisons made to a nonspecific IgG control antibody as shown in Figure 4a.
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Semi-Quantitative Analysis of Immunohistochemistry
Scoring of nitrotyrosine and iNOS in aorta was performed based on the method previously used in our laboratory.30 Photographs of immuno-stained mouse aorta were taken under microscope (x100 magnification) and randomly shown to observers without identification of samples. Score (grade 0 to 4) was given to aortic endothelium, media, and adventitia, respectively, according to the intensity of staining. The average of scores from 3 observers for each element was taken as scores for the sample.
Western Blot
A part of the thoracic aorta was homogenized in lysis buffer (1% NP-40, 0.25% deoxycholic acid, 50 mmol/L Tris, pH 7.4, 1 mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L NaF, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl floride, 10 µg/mL leupeptin). Protein was analyzed by immunoblot as previously described.17 Polyclonal anti-G6PD antibody was obtained from Bethyl Laboratories (Montgomery, Tex). Monoclonal anti-
-actin antibody was from Sigma (St. Louis, Mo), and polyclonal mouse VCAM-1 antibody was from R&D systems (Minneapolis, Minn).
Measurement of Tissue Glutathione
A piece of frozen tissue (heart, aorta) was homogenized in 0.35 N perchloric acid and centrifuged at 3000g for 10 minutes. The supernatant was used to assay glutathione (GSH) according to the colorimetric method provided by a kit (GSH-400; Oxis, Portland, Ore). The precipitate was re-suspended in 0.2 N NaOH and used for protein assay (Bio-Rad). Tissue GSH content was expressed as nmol/mg protein.
Data Analysis
Data are expressed as mean±SE. Statistical comparisons were performed by ANOVA and Student t test. Significance was accepted when P<0.05.
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Genetic Characterization of Mice in the Study
Among mice used in the study, the percentage of C57BL/6 background was tested by analysis of microsatellites on the Ath-1 locus on chromosome-1 that determines susceptibility to atherosclerosis.28,29 Of the 10 mice analyzed in each group 5 of the mice were homozygous C57BL/6 at this locus in the E-WT group and 5 were homozygous in the E-Hemi group (Figure II, available online at http://atvb.ahajournals.org). The remaining 5 mice in each group analyzed were heterozygous C57BL/6 at this locus, indicating that the genetic background of Ath-1 locus was similar between E-WT and E-Hemi. In addition, the cardiac mRNA expression of tnfsf4 that is the critical gene within Ath-1 locus that determines susceptibility of the C57BL/6 strain to atherosclerosis29 was not significantly different between E-WT and E-Hemi mice (Figure III, available online at http://atvb.ahajournals.org). Thus, differences in the 2 groups could not be ascribed to differences in genetic background of Ath-1 locus.
G6PD-Deficient apoE –/– Mice (E-Hemi) Demonstrate Higher Blood Pressure and Lower Serum Cholesterol Than apoE–/– Mice (E-WT)
After administration of western diet, blood pressure measured by tail-cuff was significantly higher in E-Hemi mice (E-WT 92±2 versus E-Hemi 100±3, mm Hg, P<0.05; Figure 1). When mice were euthanized at 21 weeks of age, E-Hemi mice demonstrated the same body weight, heart weight, and liver weight as those of E-WT mice. However, serum cholesterol level was significantly lower (E-WT 1756±82 versus E-Hemi 1567±57, mg/dL; P<0.05; Table I, available online at http://atvb.ahajournals.org). After administration of western diet, G6PD activity in E-Hemi mouse aorta was 23% of that in E-WT mouse aorta. Immuno-blot also confirmed that G6PD protein expression was lower in E-Hemi aorta compared with E-WT (Figure IV, available online at http://atvb.ahajournals.org).
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Aortic Lesion Area Is Significantly Less in E-Hemi Compared With E-WT
Sudan IV-stained lesions were concentrated in the aortic arch and spinal artery branches in both E-WT and E-Hemi mice (Figure 2). The average atherosclerotic lesion area was significantly less in E-Hemi mice (E-WT 1653±214 versus E-Hemi 938±131, x103 µm2, n=15 to 16, P<0.01; Figure 2). When mice in the 2 groups were selected to match serum cholesterol levels within the same range (E-WT 1478±87 versus E-Hemi 1533±58, mg/dL, n=12 to 13, not significant), the lesion area was still significantly less in E-Hemi mice (E-WT 1715±260 versus E-Hemi 1025±133, x103 µm2, P<0.05). The significant difference in lesion area also was found in mice that were homozygous C57BL/6 at the Ath-1 locus (P<0.01, n=5, Figure II).
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Less Aortic Lesion in E-Hemi Mice Was Associated With Lower Expression of VCAM-1
VCAM-1 expression as a marker of vascular inflammation was studied by immunohistochemistry and immunoblot. VCAM-1 staining was localized on atheromatous plaques but also observed in nonlesion endothelium in E-WT mouse aorta. E-Hemi mouse aorta showed less staining compared with E-WT mouse aorta (Figure 4a). Immunoblot of aortic homogenate also showed lower expression of VCAM-1 in E-Hemi mice (Figure 3).
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G6PD-Deficient Mice Generate Lower Superoxide Anion and Demonstrate Lower 3-o-Nitrotyrosine Staining in the Aorta
Aortic NADPH content was decreased &50% in the G6PD mutant mice,17 and therefore superoxide anion generation was measured in aorta of apoE–/– mice to examine whether lower NADPH associated with G6PD deficiency might contribute to lower superoxide production via vascular NADPH oxidase. Superoxide anion production detected by lucigenin was significantly lower in E-Hemi mouse aorta (E-WT 9.3±1.1 versus E-Hemi 6.2±0.9 mU/min/mg aorta, n=12 to 15, P<0.05). In addition, nitrotyrosine staining was less in E-Hemi mice (Figure 4a). Semi-quantitative analysis in 7 to 8 mice per group showed significantly less staining in E-Hemi mice aorta, consistent with lower production of superoxide anion (Figure 4b). Nitrotyrosine staining was less in lesions (Figure 4a), and significantly less in the media and adventitia that are not involved with lesions (Figure 4b).
Inducible Nitric Oxide Synthase Is Less in E-Hemi Compared With E-WT Mouse Aorta
Inducible NO synthase (iNOS) is induced by inflammatory cytokines in atherosclerotic lesions and may be responsible for producing reactive oxygen/nitrogen species including nitric oxide and peroxynitrite. Immunohistochemistry demonstrated iNOS expression in endothelium, media, and adventitia of E-WT mouse aortas, but significantly less expression was observed in all aortic cell layers in E-Hemi mice (Figure 5).
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GSH Content Was Not Decreased in E-Hemi Mice Fed Western Diet
Because GSH can be decreased by acute oxidative stress in G6PD-deficient tissues,22 GSH content was measured in heart and aorta of E-WT and E-Hemi mice. Although G6PD activity of E-Hemi heart was 20% of E-WT heart (data not shown), GSH in the heart (E-WT 7.3+0.2 versus E-Hemi 7.3+0.5 nmol/mg protein, n=4) and in the aorta (E-WT 39+12 versus E-Hemi 33+13 nmol/mg protein, n=4 to 6) were not significantly different.
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Discussion |
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In this study, apoE–/– mice with G6PD deficiency demonstrated less atherosclerotic lesions associated with lower superoxide anion production and nitrotyrosine in the aorta. The aorta of the G6PD mutant mouse has 10% to 20% of normal G6PD activity and 50% of normal NADPH content compared with WT mice.17 The data presented here suggest that a lower supply of NADPH is associated with lower superoxide anion production via NADPH oxidase, which contributes to decreased lesion formation in apoE–/– mice. These results are compatible with NADPH oxidase having a relatively high Michaelis constant for NADPH,34 which therefore makes its activity likely to be influenced by an insufficient supply of its substrate. A role of NADPH oxidase in regulating atherogenesis is also consistent with reports showing less atherosclerosis in p47phox-deficient mice10 or with pharmacological inhibition of NADPH oxidase.8
There are several other factors that may have influenced atherogenesis in the apoE–/– mice in this study. First, we found that systolic blood pressure was significantly higher in E-Hemi mice. Although apoE+/+, G6PD mutant mice showed a trend toward higher basal blood pressure in our earlier study,17 the statistically significant difference observed in apoE–/– mice in this study may be because of the effect that additional factors including hypercholesterolemia have on blood pressure. The higher blood pressure in E-Hemi mice is consistent with a clinical report showing higher blood pressure in G6PD-deficient men.23 Higher basal blood pressure in E-Hemi mice might be attributed to less endothelial nitric oxide (NO) production, which may also be because of a lower supply of NADPH. In agreement with this interpretation, NO bioavailability is decreased by inhibiting G6PD activity in endothelial cells in culture.14 Decreased NO bioavailability would be expected to result in enhanced atherosclerosis in E-Hemi mice because eNOS deficiency enhances atherosclerosis in apoE–/– mice.35 The fact that this was not observed indicates that other factors such as the decrease in NADPH oxidase-derived superoxide anion overcame that of NO in the development of atherosclerosis. Inhibiting NADPH oxidase may reduce vascular inflammation without changing vascular tone.36
Second, serum cholesterol was significantly lower in G6PD deficient apoE–/– mice. 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase, as well as several other enzymes downstream of it that are involved in cholesterol biosynthesis, requires NADPH as a cofactor and has a relatively high Km for the cofactor (0.08 mmol/L37). Therefore, endogenous cholesterol synthesis is NADPH-dependent and is likely impaired in G6PD-deficient mice with diminished generation of NADPH. When E-Hemi mice were fed regular diet, serum cholesterol was also 20% lower than E-WT mice (data not shown). We speculate that endogenous cholesterol synthesis is lower in G6PD-deficient animals. In fact, an epidemiological study reported that the serum levels of total cholesterol, LDL cholesterol, and high-density lipoprotein cholesterol were significantly lower in G6PD-deficient men.38 However, in our study, the lower serum cholesterol did not likely contribute to the decreased atherosclerosis, because the lesion area was still significantly less when comparing groups of mice with similar serum cholesterol values.
Third, the G6PD mutant mouse used in this study to breed with the apoE–/– was of the C3H strain that is atherosclerosis-resistant compared with the C57BL/6 strain.39 In this study after breeding with apoE–/– mice of a C57BL/6 background, approximately one-quarter of the genomic background of both E-WT and E-Hemi littermate mice were of the C3H background. Although they were littermates, a small deference in genes could have influenced the extent of atherosclerosis in mice used in this study.40 The Ath-1 locus on mouse chromosome-1 has been reported to render C57BL/6 mice more susceptible and C3H mice more resistant to diet-induced atherosclerosis.28 Recently, tnfsf4 was identified within the Ath-1 locus as the major gene that influences susceptibility to atherosclerosis.29 Therefore, we tested microsatellite markers on the Ath-1 locus by PCR and tnfsf4 expression in mouse hearts by quantitative PCR (Figures II, III). These data confirmed that lesser lesion in E-Hemi did not result from any difference in genetic background of Ath-1 locus or from different expression of tnfsf4.
Fourth, lower levels of iNOS were observed in E-Hemi atherosclerotic mouse aorta. Previous studies have shown that inhibition of G6PD decreased NO production in some cells,11,13,15 and iNOS gene expression was decreased by inhibition of G6PD in glial cells.41 If reactive oxygen species contribute to activation of transcription factors such as NF-
B to induce the iNOS gene, decreased superoxide anion may result in less iNOS induction. Further studies are required to elucidate the mechanism by which iNOS is decreased in E-Hemi aorta. NO itself is thought to be an anti-atherogenic factor, reducing VCAM-1 expression in cultured cells,42 and NOS inhibitors increase atherogenesis.43 However, peroxynitrite, a reaction product of NO and superoxide anion, is a strong oxidant that may promote LDL oxidation.3 Also, in oxidative stress, uncoupled iNOS can produce superoxide.44 These have been suggested as reasons why genetic deficiency45,46 or pharmacological inhibition of iNOS reduces atherosclerosis.47 Thus, less iNOS in G6PD deficient mouse aorta may have contributed to decreased superoxide anion, reactive nitrogen species, nitrotyrosine, and atherosclerotic lesions. It is also possible that the effect of the decreased iNOS derived oxidants on atherosclerosis in this model overcame any potential effect of decreased eNOS function. The decreased expression of iNOS is also consistent with that of VCAM-1, another NF-B–dependent gene involved in atherosclerosis. It is clear that the alterations in iNOS and nitrotyrosine in E-Hemi mice occurred in all layers of the aortic wall, not just in atherosclerotic lesions, consistent with the decrease in reactive species being a cause of the decreased lesions, rather than the decrease in reactive species being a result of the decreased lesions.
Acute oxidative stress is often accompanied by depletion of GSH and induction of G6PD activity. In such cases inhibiting G6PD may limit GSH reductase activity which regenerates GSH to its reduced form.19,20 However, we did not find significant decreases in tissue GSH levels in the heart or aorta of E-Hemi mice, suggesting that other mechanisms to maintain GSH levels are effective in chronic states of oxidant stress. GSH synthesis is induced by oxidized-LDL in macrophages48 and known to be upregulated by oxidative stress in the lung,49 so that it is possible that GSH level in the E-Hemi mice are compensated by increased de novo synthesis.
This study together with our previous study17 implicates an important role of NADPH derived from the pentose phosphate pathway acting as substrate for vascular superoxide generation via NADPH oxidase. Also, our results support a role for vascular superoxide production in contributing to the progression of atherosclerosis. Potentially because of the conflicting effects of G6PD deficiency cited, and the genetic complexity of humans with G6PD deficiency, the clinical cardiovascular manifestations of G6PD deficiency may have remained undetected. Our results are consistent with clinical studies suggesting that G6PD deficiency contributes to a higher blood pressure,23 but a lower serum cholesterol38 and cardiovascular mortality associated with atherosclerosis.24 In addition to resistance to malaria,50 this report may indicate protective aspects of G6PD deficiency for atherosclerosis for a large population with the most prevalent enzymopathy in the world.
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Acknowledgments |
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The studies were supported by National Institutes of Health grants SCOR HL55993, and R01 HL55620, R01 AG 27080, and R03 AG19078.
Received July 15, 2005; accepted January 13, 2006.
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References |
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