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【摘要】
Background- We have recently demonstrated that activity of red blood cell glutathione peroxidase-1 is inversely associated with the risk of cardiovascular events in patients with coronary artery disease. The present study analyzed the effect of glutathione peroxidase-1 deficiency on atherogenesis in the apolipoprotein E-deficient mouse.
Methods and Results- Female apolipoprotein E-deficient mice with and without glutathione peroxidase-1 deficiency were placed on a Western-type diet for another 6, 12, or 24 weeks. After 24 weeks on Western-type diet, double-knockout mice (GPx-1 -/- ApoE -/- ) developed significantly more atherosclerosis than control apolipoprotein E-deficient mice. Moreover, glutathione peroxidase-1 deficiency led to modified atherosclerotic lesions with increased cellularity. Functional experiments revealed that glutathione peroxidase-1 deficiency leads to increased reactive oxygen species concentration in the aortic wall as well as increased overall oxidative stress. Peritoneal macrophages from double-knockout mice showed increased in vitro proliferation in response to macrophage-colony-stimulating factor. Also, we found lower levels of bioactive nitric oxide as well as increased tyrosine nitration as a marker of peroxynitrite production.
Conclusions- Deficiency of an antioxidative enzyme accelerates and modifies atherosclerotic lesion progression in apolipoprotein E-deficient mice.
The present study analyzed the effect of glutathione peroxidase-1 (GPx-1) deficiency on atherogenesis in the apolipoprotein E-deficient mouse (ApoE -/- ) on the C57BL/6J background. Our data demonstrate that deficiency of an antioxidative enzyme accelerates and modifies atherosclerotic lesion progression in ApoE -/- mice.
【关键词】 antioxidants atherosclerosis nitric oxide
Introduction
Oxidative stress is defined as an imbalance between the production and degradation of reactive oxygen species (ROS). Enzymatic inactivation of ROS is achieved mainly by superoxide dismutases, catalase and the glutathione peroxidases. 1 Indeed, glutathione and the glutathione peroxidases constitute the principal antioxidant defense system in mammalian cells. 2-4 Glutathione peroxidase-1 (GPx-1), the ubiquitous intracellular form and key antioxidant enzyme within many cells, including the endothelium, consumes reduced glutathione to convert hydrogen peroxide to water and lipid peroxides to their respective alcohols. 5 It also acts as a peroxynitrite reductase. 6 Because of its major role in the prevention of oxidative stress, GPx-1 may be an important antiatherogenic enzyme. 7 In fact, we have recently shown in patients with coronary artery disease that a low activity of red blood cell GPx-1 is associated with an increased risk of cardiovascular events independently from traditional risk factors or atherosclerosis. 8 A mouse model of GPx-1 deficiency is available. These animals appear healthy and are fertile. 9 However, a recent in vitro study showed increased cell-mediated oxidation of low-density lipoprotein (LDL) in this model. 10 Furthermore, GPx-1 deficiency causes endothelial dysfunction in mice 11 that is aggravated by hyperhomocysteinemia. 12 GPx-1 deficiency is accompanied by increased periadventitial inflammation, neointima formation, and collagen deposition surrounding the coronary arteries. 13 GPx-1 activity is decreased or absent in carotid atherosclerotic plaques, and the absence of GPx-1 activity in atherosclerotic lesions has been linked to the development of more severe lesions in humans. 14 Recently, t?Hoen et al 15 reported that the expression of several antioxidant enzymes including GPx-1 was increased in the aortic arch of apolipoprotein E-deficient (ApoE -/- ) mice in the period preceding lesion formation, and that their expression decreased at the time when lesion formation became apparent, whereas Biswas et al suggested that decreased glutathione synthesis as well as reduced transcription and activity of GPx-1 precede lipid peroxidation and detectable atheroma in these mice. 16 Despite this plethora of both experimental and clinical data linking GPx-1 deficiency to cardiovascular pathology, no direct evidence has yet established a role of GPx-1 during atherogenesis. Recently, de Haan et al 17 reported that in C57BL/J6 mice GPx-1 deficiency did not accelerate atherosclerotic lesion development after 12 and 20 weeks on a high-fat diet. Since we obtained the same result in GPx-1-deficient (GPx-1 -/- ) C57BL/J6 mice even after 1 year on a high-fat diet supplemented with cholate (data not shown), we extended our analysis to the commonly used ApoE -/- mouse model by crossbreeding GPx-1 -/- mice with this strain.
Methods
Mice
GPx-1 -/- ApoE -/- mice (generously provided by Ye-Shi Ho, Department of Biochemistry, Wayne State University, Detroit, Mich) were bred by generating F2 hybrids from the ApoE -/- and GPx-1 -/- parental strains. The GPx-1 -/- ApoE -/- strain could then be propagated successfully by incrossing. Genotype determination was performed as described (please see http://atvb.ahajournals.org). 13
Induction of Atherosclerosis
At 7 to 8 weeks of age, female ApoE -/- as well as GPx-1 -/- ApoE -/- mice were placed on a Western-type diet (WTD) for another 6 weeks (5 GPx-1 -/- ApoE -/- and 5 control ApoE -/- mice), 12 weeks (16 GPx-1 -/- ApoE -/- and 16 control ApoE -/- mice), or 24 weeks (12 GPx-1 -/- ApoE -/- and 15 control ApoE -/- mice). The WTD contained 21% (wt/wt) fat and 0.15% (wt/wt) cholesterol (ssniff; Spezialdiäten GmbH, Soest, Germany). Mice were kept in accordance with standard animal care requirements, housed 4 to 5 per cage, and maintained on a 12-hour light-dark cycle. Water and food were given ad libitum.
Lipoprotein Analysis and Blood Pressure Measurements
For detailed description of quantitative cholesterol and triglyceride analyses, fast protein liquid chromatography gel filtration of pooled plasma samples as well as blood pressure measurements with a computerized tail-cuff system (please see http://atvb.ahajournals.org) were performed.
GPx-1 Activity
GPx-1 activity was determined in washed red blood cells obtained immediately after sampling from whole blood anticoagulated with EDTA. Hemolyzed cells were stored frozen for up to 1 week. Enzyme activity was measured as described, with minor modifications (Ransel; Randox, Crumlin, UK). 18
Tissue Preparation and Quantitative Morphometry of Atherosclerotic Lesion Development
Mice were euthanized by exposure to carbon dioxide. Peritoneal cavities were opened and the cadavers fixed in 4% buffered formaldehyde. Hearts and aortas were resected en bloc down to the iliac bifurcation and carefully cleaned of perivascular adipose tissue under a dissection microscope (Leica MZ6; Leica, Bensheim, Germany). The aortic arch and the rest of the aorta from the arch to the iliac bifurcation were separated. Longitudinal sections of the aortic arch were stained with trichrome and computer-assisted (Image Pro Discovery; Media Cybernetics, Silver Spring, Md) measurement of plaque size was performed as described previously 19 (supplemental Figure I, please see http://atvb.ahajournals.org).
Immunohistochemical and Histochemical Analyses
Immunostaining of murine tissues with the murine MAbs was performed using the Vector M.O.M. immunodetection kit (Vector Laboratories, Burlingham, Calif; please see http://atvb.ahajournals.org). The following antibodies and dilutions were used: murine monoclonal IgM EO6 (1:500) binding the phosphorylcholine headgroup of oxidized but not native phospholipids, rabbit anti-murine JE (MCP-1, 1:100), murine monoclonal IgG NA59 (1:100) binding 4-hydroxynonenal lysine-epitopes, rabbit anti-nitrotyrosine (1:500; Upstate, Dundee, UK), and murine anti-smooth muscle -actin (1A4, 1:100; Sigma, St Louis, Mo). Collagen content was analyzed by picrosirius red and polarized light microscopic imaging. Percent-positive area for immunohistochemical or picrosirius red staining of the inner aortic arch intima (lesser curvature) was quantified by Photoshop-based image analysis as described. 20,21 Briefly, pixels with similar chromogen characteristics were selected with the "magic wand" tool and the "select similar" command, and the ratio of the positively stained area to the total lesion area studied was calculated with the "histogram" command in Photoshop. For details of the procedure for determination of MCP-1 immunostaining intensity please see http://atvb.ahajournals.org. All quantitative morphometric and immunohistochemical data were collected independently by 2 experienced operators blinded to the mice genotypes.
Determination of ApoB IgG and IgM Immune Complexes (IgG and IgM IC/apoB) and Autoantibodies to Oxidized LDL
The IC/apoB in mouse plasma were assessed after 12 and 24 weeks on the WTD with a "sandwich" chemiluminescence immunoassay as previously described. 22,23
The levels of IgG and IgM autoantibodies binding to malondialdehyde (MDA)-LDL and copper-oxidized LDL (low-density lipoprotein) were determined by chemiluminescence-based enzyme-linked immunosorbent assay as described. 24 MDA-LDL and copper-oxidized low-density lipoprotein was generated as previously described. 25,26 Mice sera were diluted 1:200.
Oxidative Fluorescent Microtopography and Detection of ROS Formation Using Diogenes-Enhanced Chemiluminescence
Superoxide was detected in situ using dihydroethidium fluorescence as described recently. 27 For superoxide and hydrogen peroxide detection, please see http://atvb.ahajournals.org.
Isolation of Peritoneal Macrophages
Mouse peritoneal macrophages were prepared from GPx-1 -/- ApoE -/-, and ApoE -/- mice were prepared by intraperitoneal injection of 1 mL 3% thioglycollate (Merck, Darmstadt, Germany). After 4 days, cells were harvested by intraperitoneal lavage with 7 mL DMEM and centrifuged for 5 minutes at 1250 rpm. The pellet was resuspended in DMEM with 10% fetal calf serum and plated in bacterial dishes. After incubation for 4 hours, nonadherent cells were removed.
Proliferation Assay
The thioglycollate-elicited macrophages were incubated for 4 days with macrophage-colony stimulating factor (10 ng/mL; PeproTech, London, UK). Cells were detached with Accutase (PAA Laboratories, Pasching, Austria) and plated in a 96-well plate (2.5 x 10 4 cells/well). Cells were incubated again with macrophage-colony stimulating factor and BrdU for another 16 hours, and the proliferation assay (Roche, Mannheim, Germany) was performed according to the manufacturer?s instruction. Briefly, after fixation and permeabilization of the cells, the incorporated BrdU was detected by an anti-BrdU-POD antibody followed by incubation with Luminol. The amount of bound antibody was quantified by determination of relative light units with a chemiluminescence plate reader (Fluoroscan; Thermo Labsystems, Waltham, Mass).
TUNEL Assay
Thioglycollate-elicited peritoneal macrophages were plated in 16-well chamber slides (1 x 10 5 cells/well) in DMEM supplemented with 10% fetal calf serum. After 24 hours, nonadherent cells were removed and the resident macrophages were incubated for another 7 days without additional stimuli. Staining of apoptotic cells was performed with the in situ cell death staining kit (Roche, Mannheim, Germany) according to the manufacturer?s instructions. Percentage of apoptotic cells was evaluated by counting the stained cells/1 x 10 5 cells seeded.
Reporter Cell Assay for Determination of Aortic Nitric Oxide Production
Aortic nitric oxide production was bioassayed by determination of the cGMP content in RFL-6 rat lung fibroblasts as reporter cells (please see supplemental materials).
Statistical Analyses
Data were analyzed with SPSS 12.0 for Windows (SPSS Inc). Most of the outcome parameters used in this study did not follow a normal distribution as judged by Shapiro-Wilk tests, so statistical analyses were performed with Mann-Whitney U tests. Except for plasma lipid contents and blood pressure measurements (mean±SD), data in the text are presented as median and interquartile range. Data in the Figures are presented as boxplots with median, interquartile range, minimum, and maximum. Differences were considered significant when P <0.05.
Results
Generation of GPx-1 -/- ApoE -/- Mice
To study the role of GPx-1 in atherogenesis, we generated double-knockout (GPx-1 -/- ApoE -/- ) mice by crossing GPx-1 -/- with ApoE -/- mice, both on the C57/BL6 background (please see supplemental materials).
GPx-1 Deficiency Does Not Affect Serum Lipoproteins or Systolic Blood Pressure
The WTD significantly increased plasma total cholesterol but not triglyceride levels in both GPx-1 -/- ApoE -/- mice and control ApoE -/- mice compared with baseline values with no significant differences between the 2 strains after 6, 12, and 24 weeks, respectively (supplemental Figure IIA). Furthermore, fast protein liquid chromatography analysis of lipoproteins showed that disruption of GPx-1 gene function did not affect lipoprotein distribution in mice consuming the atherogenic diet (supplemental Figure IIB).
Also systolic blood pressure was similar after 6 weeks on a WTD (118±4.4 mm Hg in GPx-1 -/- ApoE -/- mice [n=4] versus 114.6±2.7 mm Hg in ApoE -/- mice [n=3]).
Atherosclerosis Lesion Progression
Compared with control ApoE -/- mice (n=15), double-knockout mice (GPx1 -/- ApoE -/-, n=12) developed significantly more atherosclerosis after 24 weeks on the WTD as indicated by plaque area en face of the aorta from the arch down to the iliac bifurcation ( Figure 1A and 1 B). This difference was mainly caused by significantly more atherosclerosis in the distal part of the aorta from the diaphragm down to the iliac bifurcation ( P <0.01, data not shown). A similar, albeit statistically not significant, trend could already be observed as early as after 12 weeks on the WTD ( Figure 1 A), but not yet after 6 weeks on the WTD (% plaque area, median and interquartile range: GPx-1 -/- ApoE -/-, 0.7/0.65; ApoE -/-, 1.1/1.0). Taken together, these data indicate that GPx-1 deficiency accelerates atherosclerotic lesion development in ApoE -/- mice.
Figure 1. Atherosclerosis lesion progression. A, After consuming the WTD for 12 or 24 weeks, both GPx-1 -/- ApoE -/- and ApoE -/- mice aortae from the arch down to the iliac bifurcation were isolated and stained for lipid deposition with Sudan IV. Sudan stained atherosclerotic lesions en face were then quantified using Photoshop-based image analysis. Data are presented as boxplots with median, interquartile range, minimum, and maximum. B, Representative specimens of GPx-1 -/- ApoE -/- and ApoE -/- mice aortae used to calculate the percentages of Sudan stained areas shown in the boxplots in (A).
Phenotypic Analysis of Atherosclerotic Lesions
Quantification of the maximal area and thickness (median/interquartile range) of the inner aortic arch intima (lesser curvature) revealed no significant differences in control mice compared with double-knockout mice after 6 weeks on the WTD (area, 446.372/575.210 µm 2 versus 184.386/185.062 µm 2; thickness, 298/380 µm versus 203/167 µm. The relatively high medians and interquartile ranges are attributable to extreme outliers and high interindividual variations at this early time point). In contrast, consistently, albeit not significantly, less atherosclerosis was observed in control mice compared with double-knockout mice after 12 weeks (area, 115.962/114.594 µm 2 versus 128.633/87.096 µm 2; thickness, 215/151 µm versus 237/173 µm) or 24 weeks on the WTD (area, 382.551/182.114 µm 2 versus 416.530/71.370 µm 2; thickness, 328/201 µm versus 334/100 µm). Furthermore, the histomorphological aspect of lesions in double-knockout mice differed markedly from lesions in control mice in terms of plaque composition. At 12 weeks on the WTD, atherosclerotic lesions in ApoE -/- mice contained significantly less macrophages (area covered by macrophages; Figures 2A and 3 A, left panels) and significantly more collagen (area covered by collagen; Figures 2C and 3 C, left panels) than lesions in double-knockout mice. Furthermore, a slight but yet not significant increase in the amount of smooth muscle cells could be observed (area covered by smooth muscle cells; Figures 2B and 3 B, left panels). At 24 weeks on the WTD, the difference in the amount of smooth muscle cells in double-knockout mice was highly significant ( Figures 2B and 3 B, right panels). Macrophages and collagen content were not significantly different at this time point ( Figures 2A, 2C, 3A, 3 C, right panels). Collectively, these data indicate that GPx-1 deficiency leads to increased cellularity with a higher relative number of macrophages in early and a higher relative number of smooth muscle cells in advanced atherosclerotic lesions when compared with control ApoE -/- mice.
Figure 2. Phenotypic analysis of atherosclerotic lesions after 12 (left panels) and 24 weeks (right panels) on the WTD. Atherosclerotic lesions of the inner aortic arch intima (lesser curvature) of GPx-1 -/- ApoE -/- and ApoE -/- mice were quantified for macrophages (A), smooth muscle cells (B), and collagen (C). Percent-positive area for macrophages, smooth muscle cells, and collagen was quantified by Photoshop-based image analysis (see also Figure 3 ). Data are presented as boxplots with median, interquartile range, minimum, and maximum.
Figure 3. Representative examples of atherosclerotic lesion composition after 12 (left panels) and 24 weeks (right panels) on the WTD. Atherosclerotic lesions of the inner aortic arch intima (lesser curvature) of GPx-1 -/- ApoE -/- and ApoE -/- mice were stained with trichrome (A) (for quantification of macrophages), mouse anti-smooth muscle -actin (1A4) (B) (for quantification of smooth muscle cells), and picrosirius red with subsequent polarization (C) (for quantification of collagen). Percent-positive area for macrophages (*), smooth muscle cells (brown), and collagen (yellow, green, orange, and red polarized color) were quantified by Photoshop-based image analysis (see also Figure 4 ). The lumen is to the upper left corner. The demarcation between intima and media is indicated by an arrowhead. C, Note that the adventitial tissue (*) also polarizes after picrosirius red staining (internal positive control). Magnification x 64.
Figure 4. In situ detection of superoxide in the aorta from GPx-1 -/- ApoE -/- and ApoE -/- mice after 6 weeks on the WTD. Left, Fluorescent photomicrographs of microscopic sections of aortic rings. Vessels were labeled with dihydroethidium dye, which produces red fluorescence when oxidized to ethidine by superoxide. The green autofluorescence corresponds to the basal laminae. E indicates endothelium; M, media; A, adventitia. Right, Data presented as boxplots with median, interquartile range, minimum, and maximum.
Superoxide Formation in the Aortic Wall and Detection of ROS Formation in Aortic Vessel Segments, Heart Mitochondria, and Membrane Fractions
We assessed superoxide formation in cryosectioned aortic rings after 6 weeks on a WTD by means of dihydroethidium-derived fluorescence. Staining of aortic sections with dihydroethidium revealed a marked (50%) increase in vascular superoxide in GPx-1 -/- ApoE -/- mice compared with control ApoE -/- mice. This increase was observed primarily in the endothelium, but less pronounced in the intima and adventitia ( Figure 4 ). Furthermore, detection of ROS formation in aortic vessel segments, heart mitochondria, and membrane fractions point toward increased oxidative stress in GPx-1 -/- ApoE -/- mice because of activation of mitochondria and NADPH oxidases or because of impaired antioxidative defense mechanism in the GPx-1 -/- animals (supplemental Figure III).
Influence of GPx-1 Deficiency on Lipoprotein Oxidation
We did not find evidence for an increased lipoprotein oxidation in GPx-1 -/- ApoE -/- mice as determined by both immunohistochemical quantification of oxidized LDL and measurement of ApoB IgG and IgM immune complexes and autoantibodies to oxidized LDL (supplemental Figure IV).
Proliferation, Apoptosis, and Recruitment of Monocytes
Next, we investigated whether proliferative activity and/or less apoptosis of monocyte-derived macrophages might account for the increased cellularity of early atherosclerotic lesions in GPx-1 -/- ApoE -/- mice. The proliferation rate of mouse peritoneal macrophages was investigated with a BrdU-based chemiluminescence assay. After stimulation with macrophage-colony-stimulating factor macrophages from GPx-1 -/- ApoE -/- mice showed significantly more BrdU incorporation than macrophages from ApoE -/- control mice ( Figure 5 A). Apoptosis of mouse peritoneal macrophages was investigated with a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. After cultivation for 7 days without additional stimuli, percentage of apoptotic cells was not significantly different in control mice compared with double-knockout mice ( Figure 5 B).
Figure 5. Proliferative activity and apoptosis. A, After differentiation of thioglycollate-elicited mouse peritoneal monocytes for 4 days in macrophage-colony stimulating factor (10 ng/mL), cells were incubated with BrdU for another 16 hours and the proliferation rate was investigated with a BrdU-based chemiluminescence assay (n=8). B, After cultivation of thioglycollate-elicited mouse peritoneal monocytes for 7 days without additional stimuli, staining of apoptotic cells was performed with an in situ cell death staining kit. Percentage of apoptotic cells was evaluated by counting the stained cells/1 x 10 5 cells (n=8). Data are presented as boxplots with median, interquartile range, minimum, and maximum.
These results indicate that increased proliferative activity rather than diminished apoptotic rates of macrophages contribute to the increased cellularity of early atherosclerotic lesions in GPx-1 -/- ApoE -/- mice. Furthermore, we did not find evidence for increased expression of MCP-1 in lesions of GPx-1 -/- ApoE -/- mice, that might account for increased recruitment of monocytes/macrophages into the lesions (supplemental Figure V).
Vascular Nitric Oxide Production and Protein Nitration
The bioassay of aortic nitric oxide production indicated that less bioactive nitric oxide is generated in response to acetylcholine in aortae of double-knockout mice compared with control mice. Likewise, the observation of extensive protein nitration in atherosclerotic lesions of double-knockout mice suggests that peroxynitrite is produced and may be involved in the different evolution of the lesions in control and double-knockout mice (supplemental Figure VI).
Discussion
The present study on atherosclerosis development in GPx-1 -/- ApoE -/- mice has provided several key results. Atherosclerotic lesion development is more rapid in GPx-1 -/- ApoE -/- double-knockout mice with larger lesions after 12 (not significant) and 24 weeks on a WTD compared with ApoE -/- mice. The lesions are more cellular with an increase in macrophage content in early lesions and an increase in smooth muscle cell content in advanced lesions. As expected, arterial walls of double-knockout mice contain more ROS than tissue from controls. Consistent with the previously reported endothelial dysfunction in GPx-1 -/- mice, 11-13 aortic tissue from double-knockout mice produced less bioactive nitric oxide in response to acetylcholine than that of ApoE -/- control mice. Somewhat unexpectedly, we could not find consistent evidence of increased lipoprotein oxidation in GPx-1 -/- ApoE -/- mice by immunohistochemistry as well as determination of apoB-immune complexes and autoantibodies against oxidized LDL. Taking into account the inverse association of GPx-1 activity and the risk for cardiovascular events in humans with coronary artery disease, 8 these results provide further strong evidence that GPx-1 plays an important antiatherogenic role in the arterial wall.
The accelerated development of atherosclerosis in the present study is in contrast to recent observations in simple GPx-1 -/- mice, which have no enhanced atherosclerotic lesion development. 17 One should keep in mind though, that lesion development in fat fed C57BL/6 mice is very discrete and confined to the aortic root. Furthermore, this model is associated with severe nonvascular pathology (eg, hepatic) caused by the extreme diet modification. Therefore, it has been generally abandoned for atherosclerosis research after the availability of hyperlipidemic ApoE -/- and LDL-receptor -/- mice.
Besides the increased lesion size in double-knockout mice, another striking difference between the 2 genotypes in atherosclerotic lesion development relates to histomorphological composition in the aortic arch lesion. At 12 weeks on the WTD, lesions in the aortic arch were particularly rich in macrophages. GPx-1 -/- ApoE -/- mice showed a lesional macrophage content that was approximately twice that of corresponding lesions in ApoE -/- mice ( Figure 4 A). This increase in macrophage cellularity was paralleled by a comparative decrease in extracellular matrix as determined by the collagen content ( Figure 4 C). At 24 weeks on the WTD, lesions in the aortic arch were much more advanced with smooth muscle cells being the predominant cell type. At this time point, the relative number of smooth muscle cells was significantly higher in GPx-1 -/- ApoE -/- mice when compared with ApoE -/- mice with no concomitant increase in collagen content, which is surprising at first glance ( Figure 4 B). However, this latter observation is in line with recent data demonstrating that increased oxidative stress activates matrix metalloproteinases and decreases fibrillar collagen synthesis in rat cardiac fibroblasts 28 and, vice versa, overexpression of GPx attenuates matrix metalloproteinase-9 zymographic and protein levels in mouse myocardium. 29
This propensity toward more cellular atherosclerotic lesions of GPx-1 -/- ApoE -/- mice was accompanied by severely impaired disposal of ROS in mitochondria. Whereas severe oxidative stress is cytotoxic, mild oxidative stress may stimulate cell proliferation, as shown recently in various cell types including vascular smooth muscle cells. 30-33 Proliferation of vascular smooth muscle cells is a hallmark of atherosclerosis development. Furthermore, many data support the concept that also macrophages in early lesions may derive, at least in part, from local proliferation, especially under circumstances of enhanced oxidative stress. 34 The combination of hypertension and hyperlipidemia in LDL-receptor -/- mice with hyperglycemia and further oxidative stress by glucose-oxidized LDL has recently been shown to induce macrophage proliferation in vascular lesions. 35 We could show that GPx-1 deficiency increases proliferation of macrophages in vitro in response to macrophage-colony-stimulating factor. In contrast, GPx-1 deficiency obviously does not influence the extent of apoptosis of macrophages in vitro or MCP-1 protein expression in vivo. Therefore, we propose that increased cellularity of early atherosclerotic lesions in GPx-1 -/- ApoE -/- mice is mainly caused by increased proliferative activity rather than diminished apoptotic rates or increased recruitment of monocytes/macrophages into the lesions.
The data obtained in GPx-1 -/- ApoE -/- mice add further information to the role of antioxidant defenses in atherosclerosis. Antioxidant enzymes have been the topic of many investigations in experimental animals and epidemiologic studies in humans. In mouse models of atherosclerosis, data are available on the 3 superoxide dismutases (SOD), catalase, and glutathione peroxidase. It is remarkable that neither overexpression of copper, zinc-SOD, or of endothelial cell-SOD protects against atherosclerosis in ApoE -/- or LDL-receptor -/- mice. 36-38 Accordingly, deletion of endothelial cell-SOD is not proatherogenic. 39 In contrast to these models of atherosclerosis, neointima formation in balloon injury models has been reported to be inhibited by overexpression of endothelial cell-SOD. 40 Because homozygous Mn-SOD deficiency leads to severe pathology and death soon after birth, only mice with heterozygous Mn-SOD deficiency have been analyzed for atherosclerosis development. In these mice, there was an increase in branchpoint lesions observed in the aorta, interpreted as interaction of high shear-stress with the genotype. However, only 4 animals per group were analyzed and no data on the overall size of lesions were presented. 41 In summary, the SODs with the possible exception of Mn-SOD apparently do not protect hyperlipidemic mice against atherosclerosis. Similarly, also, inactivation of NADPH oxidase (with a significant reduction in vascular superoxide generation) had no protective effect. 42,43 Finally, no association of red blood cell copper-zinc-SOD activity with cardiovascular risk was observed by us in the same study that revealed the inverse association of GPx-1 activity and cardiovascular risk. 8
While the role of SODs in atherogenesis has been surprisingly minor, it has been shown that overexpression of catalase is protective in a hyperlipidemic mouse model. 38 Like glutathione peroxidases, catalase inactivates H 2 O 2. Even though definitive experimental evidence is missing to date, these data implicate that H 2 O 2 may be relatively more important in atherogenesis than superoxide anion. This may be related to the fact that the latter is very short-lived (also in the absence of SOD) and contributes to the generation of H 2 O 2. It is conceivable that as long as H 2 O 2 is effectively converted to water, minor changes in the production and half-life of superoxide outside mitochondria may not be critical for atherogenesis. Aside from these considerations, it is well-established that superoxide and H 2 O 2 induce different cellular responses, eg, by different effector mechanisms in transcriptional regulation. 33,44
An intriguing aspect of the increased lesion size relates to the reduction of antioxidant enzymes including GPx-1 in the aortic arch of ApoE -/- mice before or at least when lesions develop. 15,16 Even though these 2 studies come to slightly different results, this may be interpreted as indicating that GPx-1 and other antioxidant enzymes cannot be critical for atherosclerotic lesion development, because they are no longer expressed at high levels when lesions developed. It should be noted, though, that there is definitely remaining GPx-1 activity at all stages of atherosclerotic lesions in the aortic arch. This could provide some protection in the ApoE -/- mouse, which is lost in the double-knockouts. Furthermore, in the early phase of lesion development, when GPx-1 expression has been reported to be normal in one study, there is a significant difference in lesion composition toward a more macrophage-rich atheroma in GPx-1 -/- ApoE -/- mice. Previous work has already demonstrated that GPx-1 was expressed at higher level in the descending aorta than in the aortic arch in Apo E -/- mice from the age of 12 weeks onwards. 15 The substantially higher residual expression of GPx-1 in atherosclerotic lesions of the descending aorta would indicate that the difference in enzyme activity between ApoE -/- and GPx-1 -/- ApoE -/- is more pronounced in this part of the aorta. This observation might explain the larger difference in lesion development observed in the abdominal aorta after 24 weeks.
Our knowledge concerning the contributions of different ROS and their reaction products to atherogenesis is still incomplete. This fact is impressively underscored by the surprising but notorious lack of any true preventive effects of different antioxidant treatments in large clinical trials. 45,46 However, the results from different genetically modified mouse models show that modification of antioxidant defense systems may indeed add important clues to our understanding of oxidative stress in atherogenesis and may eventually redirect clinical interest toward the development of effective preventive interventions in patients at risk for cardiovascular disease.
Acknowledgments
The authors thank Ye-Shi Ho, Department of Biochemistry, Wayne State University, Detroit, Mich, for generously providing GPx-1 -/- mice backcrossed to C57BL/6. The authors are indebted to Antje Canisius, Adriana Degreif, Elizabeth Miller, and Carolin Orning for their excellent technical assistance.
Sources of Funding
This work was supported by the Mainzer Forschungsföderungsprogramm (MAIFOR 2004 to M.T.), the Deutsche Forschungsgemeinschaft (DFG LA 499/3-1 and 499/3-2 to K.J.L. and M.T.), SFB 553 (TP1 to H.L. and U.F., TP17 to T.M.), and the Center of Preventive Medicine of the Medical Faculty (University of Mainz).
Disclosure
None.
M.T. and V.O. contributed equally to this work.
Original received April 26, 2006; final version accepted December 21, 2006.
【参考文献】
Forsberg L, de Faire U, Morgenstern R. Oxidative stress, human genetic variation, and disease. Arch Biochem Biophys. 2001; 389: 84-93.
Raes M, Michiels C, Remacle J. Comparative study of the enzymatic defense systems against oxygen-derived free radicals: the key role of glutathione peroxidase. Free Radic Biol Med. 1987; 3: 3-7.
Ursini F, Maiorino M, Brigelius-Flohe R, Aumann KD, Roveri A, Schomburg D, Flohe L. Diversity of glutathione peroxidases. Methods Enzymol. 1995; 252: 38-53.
Arthur JR. The glutathione peroxidases. Cell Mol Life Sci. 2000; 57: 1825-1835.
Flohe L. Glutathione peroxidase. Basic Life Sci. 1988; 49: 663-668.
Sies H. Glutathione and its role in cellular functions. Free Radic Biol Med. 1999; 27: 916-921.
Glass CK, Witztum JL. Atherosclerosis. the road ahead. Cell. 2001; 104: 503-516.
Blankenberg S, Rupprecht HJ, Bickel C, Torzewski M, Hafner G, Tiret L, Smieja M, Cambien F, Meyer J, Lackner KJ. Glutathione peroxidase 1 activity and cardiovascular events in patients with coronary artery disease. N Engl J Med. 2003; 349: 1605-1613.
Ho YS, Magnenat JL, Bronson RT, Cao J, Gargano M, Sugawara M, Funk CD. Mice deficient in cellular glutathione peroxidase develop normally and show no increased sensitivity to hyperoxia. J Biol Chem. 1997; 272: 16644-16651.
Guo Z, Van Remmen H, Yang H, Chen X, Mele J, Vijg J, Epstein CJ, Ho YS, Richardson A. Changes in expression of antioxidant enzymes affect cell-mediated LDL oxidation and oxidized LDL-induced apoptosis in mouse aortic cells. Arterioscler Thromb Vasc Biol. 2001; 21: 1131-1138.
Forgione MA, Weiss N, Heydrick S, Cap A, Klings ES, Bierl C, Eberhardt RT, Farber HW, Loscalzo J. Cellular glutathione peroxidase deficiency and endothelial dysfunction. Am J Physiol Heart Circ Physiol. 2002; 282: H1255-H1261.
Dayal S, Brown KL, Weydert CJ, Oberley LW, Arning E, Bottiglieri T, Faraci FM, Lentz SR. Deficiency of glutathione peroxidase-1 sensitizes hyperhomocysteinemic mice to endothelial dysfunction. Arterioscler Thromb Vasc Biol. 2002; 22: 1996-2002.
Forgione MA, Cap A, Liao R, Moldovan NI, Eberhardt RT, Lim CC, Jones J, Goldschmidt-Clermont PJ, Loscalzo J. Heterozygous cellular glutathione peroxidase deficiency in the mouse: abnormalities in vascular and cardiac function and structure. Circulation. 2002; 106: 1154-1158.
Lapenna D, de Gioia S, Ciofani G, Mezzetti A, Ucchino S, Calafiore AM, Napolitano AM, Di Ilio C, Cuccurullo F. Glutathione-related antioxidant defenses in human atherosclerotic plaques. Circulation. 1998; 97: 1930-1934.
t?Hoen PA, Van der Lans CA, Van Eck M, Bijsterbosch MK, Van Berkel TJ, Twisk J. Aorta of ApoE-deficient mice responds to atherogenic stimuli by a prelesional increase and subsequent decrease in the expression of antioxidant enzymes. Circ Res. 2003; 93: 262-269.
Biswas SK, Newby DE, Rahman I, Megson IL Depressed glutathione synthesis precedes oxidative stress and atherogenesis in Apo-E(-/-) mice. Biochem Biophys Res Commun. 2005; 338: 1368-1373;Epub 2005 Oct: 1326.
de Haan JB, Witting PK, Stefanovic N, Pete J, Daskalakis M, Kola I, Stocker R, Smolich JJ. Deficiency in the antioxidant enzyme glutathione peroxidase-1 (Gpx1) does not increase atherosclerosis in C57BL/J6 mice fed a high fat diet. J Lipid Res. 2006; 28: 28.
Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med. 1967; 70: 158-169.
Mach F, Schonbeck U, Sukhova GK, Atkinson E, Libby P. Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature. 1998; 394: 200-203.
Lehr HA, Mankoff DA, Corwin D, Santeusanio G, Gown AM. Application of photoshop-based image analysis to quantification of hormone receptor expression in breast cancer. J Histochem Cytochem. 1997; 45: 1559-1565.
Lehr HA, van der Loos CM, Teeling P, Gown AM. Complete chromogen separation and analysis in double immunohistochemical stains using Photoshop-based image analysis. J Histochem Cytochem. 1999; 47: 119-126.
Svenungsson E, Jensen-Urstad K, Heimburger M, Silveira A, Hamsten A, de Faire U, Witztum JL, Frostegard J. Risk factors for cardiovascular disease in systemic lupus erythematosus. Circulation. 2001; 104: 1887-1893.
Schneider M, Witztum JL, Young SG, Ludwig EH, Miller ER, Tsimikas S, Curtiss LK, Marcovina SM, Taylor JM, Lawn RM, Innerarity TL, Pitas RE High-level lipoprotein expression in transgenic mice: evidence for oxidized phospholipids in lipoprotein but not in low density lipoproteins. J Lipid Res. 2005; 46: 769-778;Epub 2005 Jan: 2016.
Freigang S, Horkko S, Miller E, Witztum JL, Palinski W. Immunization of LDL receptor-deficient mice with homologous malondialdehyde-modified and native LDL reduces progression of atherosclerosis by mechanisms other than induction of high titers of antibodies to oxidative neoepitopes. Arterioscler Thromb Vasc Biol. 1998; 18: 1972-1982.
Horkko S, Bird DA, Miller E, Itabe H, Leitinger N, Subbanagounder G, Berliner JA, Friedman P, Dennis EA, Curtiss LK, Palinski W, Witztum JL. Monoclonal autoantibodies specific for oxidized phospholipids or oxidized phospholipid-protein adducts inhibit macrophage uptake of oxidized low-density lipoproteins. J Clin Invest. 1999; 103: 117-128.
Tsimikas S, Palinski W, Witztum JL. Circulating autoantibodies to oxidized LDL correlate with arterial accumulation and depletion of oxidized LDL in LDL receptor-deficient mice. Arterioscler Thromb Vasc Biol. 2001; 21: 95-100.
Munzel T, Afanas?ev IB, Kleschyov AL, Harrison DG. Detection of superoxide in vascular tissue. Arterioscler Thromb Vasc Biol. 2002; 22: 1761-1768.
Siwik DA, Pagano PJ, Colucci WS. Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts. Am J Physiol Cell Physiol. 2001; 280: C53-C60.
Shiomi T, Tsutsui H, Matsusaka H, Murakami K, Hayashidani S, Ikeuchi M, Wen J, Kubota T, Utsumi H, Takeshita A Overexpression of glutathione peroxidase prevents left ventricular remodeling and failure after myocardial infarction in mice. Circulation. 2004; 109: 544-549;Epub 2004 Jan: 2026.
Martindale JL, Holbrook NJ. Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol. 2002; 192: 1-15.
Matsuoka T, Wada J, Hashimoto I, Zhang Y, Eguchi J, Ogawa N, Shikata K, Kanwar YS, Makino H. Gene delivery of Tim44 reduces mitochondrial superoxide production and ameliorates neointimal proliferation of injured carotid artery in diabetic rats. Diabetes. 2005; 54: 2882-2890.
Hu Y, Rosen DG, Zhou Y, Feng L, Yang G, Liu J, Huang P Mitochondrial manganese-superoxide dismutase expression in ovarian cancer: role in cell proliferation and response to oxidative stress. J Biol Chem. 2005; 280: 39485-39492;Epub 32005 Sep: 39422.
Stocker R, Keaney JF, Jr. Role of oxidative modifications in atherosclerosis. Physiol Rev. 2004; 84: 1381-1478.
Sakai M, Kobori S, Miyazaki A, Horiuchi S. Macrophage proliferation in atherosclerosis. Curr Opin Lipidol. 2000; 11: 503-509.
Lamharzi N, Renard CB, Kramer F, Pennathur S, Heinecke JW, Chait A, Bornfeldt KE. Hyperlipidemia in concert with hyperglycemia stimulates the proliferation of macrophages in atherosclerotic lesions: potential role of glucose-oxidized LDL. Diabetes. 2004; 53: 3217-3225.
Tribble DL, Gong EL, Leeuwenburgh C, Heinecke JW, Carlson EL, Verstuyft JG, Epstein CJ. Fatty streak formation in fat-fed mice expressing human copper-zinc superoxide dismutase. Arterioscler Thromb Vasc Biol. 1997; 17: 1734-1740.
Laukkanen MO, Leppanen P, Turunen P, Porkkala-Sarataho E, Salonen JT, Yla-Herttuala S. Gene transfer of extracellular superoxide dismutase to atherosclerotic mice. Antioxid Redox Signal. 2001; 3: 397-402.
Yang H, Roberts LJ, Shi MJ, Zhou LC, Ballard BR, Richardson A, Guo ZM Retardation of atherosclerosis by overexpression of catalase or both Cu/Zn-superoxide dismutase and catalase in mice lacking apolipoprotein E. Circ Res. 2004; 95: 1075-1081;Epub 2004 Nov: 1074.
Sentman ML, Brannstrom T, Westerlund S, Laukkanen MO, Yla-Herttuala S, Basu S, Marklund SL. Extracellular superoxide dismutase deficiency and atherosclerosis in mice. Arterioscler Thromb Vasc Biol. 2001; 21: 1477-1482.
Laukkanen MO, Kivela A, Rissanen T, Rutanen J, Karkkainen MK, Leppanen O, Brasen JH, Yla-Herttuala S. Adenovirus-mediated extracellular superoxide dismutase gene therapy reduces neointima formation in balloon-denuded rabbit aorta. Circulation. 2002; 106: 1999-2003.
Ballinger SW, Patterson C, Knight-Lozano CA, Burow DL, Conklin CA, Hu Z, Reuf J, Horaist C, Lebovitz R, Hunter GC, McIntyre K, Runge MS. Mitochondrial integrity and function in atherogenesis. Circulation. 2002; 106: 544-549.
Kirk EA, Dinauer MC, Rosen H, Chait A, Heinecke JW, LeBoeuf RC. Impaired superoxide production due to a deficiency in phagocyte NADPH oxidase fails to inhibit atherosclerosis in mice. Arterioscler Thromb Vasc Biol. 2000; 20: 1529-1535.
Hsich E, Segal BH, Pagano PJ, Rey FE, Paigen B, Deleonardis J, Hoyt RF, Holland SM, Finkel T. Vascular effects following homozygous disruption of p47(phox) : An essential component of NADPH oxidase. Circulation. 2000; 101: 1234-1236.
Liu H, Colavitti R, Rovira II, Finkel T. Redox-dependent transcriptional regulation. Circ Res. 2005; 97: 967-974.
Brown BG, Cheung MC, Lee AC, Zhao XQ, Chait A. Antioxidant vitamins and lipid therapy: end of a long romance? Arterioscler Thromb Vasc Biol. 2002; 22: 1535-1546.
Lonn E, Bosch J, Yusuf S, Sheridan P, Pogue J, Arnold JM, Ross C, Arnold A, Sleight P, Probstfield J, Dagenais GR. Effects of long-term vitamin E supplementation on cardiovascular events and cancer: a randomized controlled trial. Jama. 2005; 293: 1338-1347.
作者单位：Institute of Clinical Chemistry and Laboratory Medicine (M.T., V.O., F.C., H.R., K.J.L.), Department of Medicine II (A.L.K., M.O., A.D., S.B., T.M.), Department of Pharmacology (H.L., U.F.), Central Laboratory Animal Facility (K.R.), Johannes Gutenberg University, Mainz, Germany; Division of Cardiol