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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:1996.)
© 2002 American Heart Association, Inc.

Vascular Biology

Deficiency of Glutathione Peroxidase-1 Sensitizes Hyperhomocysteinemic Mice to Endothelial Dysfunction

Sanjana Dayal; Kara L. Brown; Christine J. Weydert; Larry W. Oberley; Erland Arning; Teodoro Bottiglieri; Frank M. Faraci; Steven R. Lentz

From the Departments of Internal Medicine (S.D., K.L.B., F.M.F., S.R.L.), Radiation Oncology (C.J.W., L.W.O.), and Pharmacology (F.M.F.), University of Iowa College of Medicine, Iowa City; Baylor Institute of Metabolic Disease (E.A., T.B.), Dallas, Tex; and Veterans Affairs Medical Center (S.R.L.), Iowa City, Iowa.

Correspondence to Steven R. Lentz, MD, PhD, Department of Internal Medicine, C303 GH, The University of Iowa, Iowa City, IA 52242. E-mail steven-lentz{at}uiowa.edu

	Abstract

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Objective— We tested the hypothesis that deficiency of cellular glutathione peroxidase (GPx-1) enhances susceptibility to endothelial dysfunction in mice with moderate hyperhomocysteinemia.

Methods and Results— Mice that were wild type (Gpx1^+/+), heterozygous (Gpx1^+/-), or homozygous (Gpx1^-/-) for the mutated Gpx1 allele were fed a control diet or a high-methionine diet for 17 weeks. Plasma total homocysteine was elevated in mice on the high-methionine diet compared with mice on the control diet (23±3 versus 6±0.3 µmol/L, respectively; P<0.001) and was not influenced by Gpx1 genotype. In mice fed the control diet, maximal relaxation of the aorta in response to the endothelium-dependent dilator acetylcholine (10^-5 mol/L) was similar in Gpx1^+/+, Gpx1^+/-, and Gpx1^-/- mice, but relaxation to lower concentrations of acetylcholine was selectively impaired in Gpx1^-/- mice (P<0.05 versus Gpx1^+/+ mice). In mice fed the high-methionine diet, relaxation to low and high concentrations of acetylcholine was impaired in Gpx1^-/- mice (maximal relaxation 73±6% in Gpx1^-/- mice versus 90±2% in Gpx1^+/+ mice, P<0.05). No differences in vasorelaxation to nitroprusside or papaverine were observed between Gpx1^+/+ and Gpx1^-/- mice fed either diet. Dihydroethidium fluorescence, a marker of superoxide, was elevated in Gpx1^-/- mice fed the high-methionine diet (P<0.05 versus Gpx1^+/+ mice fed the control diet).

Conclusions— These findings demonstrate that deficiency of GPx-1 exacerbates endothelial dysfunction in hyperhomocysteinemic mice and provide support for the hypothesis that hyperhomocysteinemia contributes to endothelial dysfunction through a peroxide-dependent oxidative mechanism.

Key Words: endothelium • homocysteine • nitric oxide • peroxide

	Introduction

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Hyperhomocysteinemia is an emerging risk factor for cardiovascular events and venous thrombosis,^1,2 but the mechanisms responsible for the vascular pathology of hyperhomocysteinemia are still incompletely understood. Like many other cardiovascular risk factors, hyperhomocysteinemia produces endothelial dysfunction, which is possibly due to oxidative inactivation of endothelium-derived NO.^3,4 The oxidative stress hypothesis⁴ is supported by the observation that auto-oxidation of homocysteine in vitro generates reactive oxygen species (ROS), including hydrogen peroxide and superoxide, and promotes oxidation of LDL.^5–7 Treatment of cultured endothelial cells with homocysteine decreases bioavailable NO^8,9 and produces cytotoxicity mediated by hydrogen peroxide.¹⁰ Homocysteine also indirectly contributes to oxidative stress by inhibiting the expression of antioxidant enzymes, such as cellular glutathione peroxidase (GPx-1), an effect that may sensitize one to the toxic effects of homocysteine-derived hydrogen peroxides and lipid peroxides.^8,11

However, the role of oxidative stress in the vascular dysfunction of hyperhomocysteinemia in vivo is less clear, and its clinical importance has been questioned.¹² Attempts to demonstrate elevated levels of oxidation products in humans with hyperhomocysteinemia have produced conflicting results.^13–16 Recent studies with human subjects have demonstrated that administration of antioxidant vitamins such as vitamin C or vitamin E can attenuate impairment of endothelium-dependent vasodilatation during acute hyperhomocysteinemia produced by oral methionine loading.^17–19 Perhaps the strongest evidence of an oxidative mechanism for endothelial dysfunction in hyperhomocysteinemia has been obtained from studies in hyperhomocysteinemic mice. Mild to moderate hyperhomocysteinemia (plasma total homocysteine [tHcy] levels of 10 to 30 µmol/L) can be produced in mice by genetic and/or dietary approaches, and it is associated with impairment of endothelium-dependent relaxation.^20–24 Impaired endothelium-dependent vasodilatory responses in hyperhomocysteinemic mice can be restored toward normal by administration of the thiol antioxidant l-2-oxothiazolidine-4-carboxylic acid (OTC)²³ or by transgenic overexpression of GPx-1.²⁴

To further explore the role of oxidative stress in the endothelial dysfunction of hyperhomocysteinemia in vivo, we tested the hypothesis that deficiency of GPx-1 increases susceptibility to endothelial dysfunction in mice with moderate hyperhomocysteinemia. Our results show that deficiency of GPx-1 exacerbates endothelial dysfunction in hyperhomocysteinemic mice, which suggests that hyperhomocysteinemia contributes to vascular dysfunction through an oxidative mechanism that involves peroxides.

	Methods

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Mice
GPx-1–deficient mice containing a targeted disruption of the Gpx1 gene²⁵ were generously provided by Dr Y.S. Ho (Wayne State University, Detroit, Mich). To minimize the potential influence of differences in genetic background, GPx-1–deficient mice were crossbred to C57BL6 mice (The Jackson Laboratory, Bar Harbor, Me) for at least 7 generations and then interbred to generate littermates that were wild type (Gpx1^+/+), heterozygous (Gpx1^+/-), or homozygous (Gpx1^-/-) for the mutated Gpx1 allele. Genotyping for the wild-type and mutated Gpx1 alleles was performed by polymerase chain reaction as described by Forgione et al.²⁶ At the time of weaning, mice were placed on either a control diet (LM-485 chow, Harlan Teklad) or a high methionine diet (LM-485 chow and drinking water supplemented with 0.5% L-methionine). After 17 weeks of the experimental diet, mice were euthanized with sodium pentobarbital (75 mg IP). Blood was collected by cardiac puncture into EDTA (final concentration 5 mmol/L) for measurement of plasma tHcy and methionine, and the thoracic aorta and liver were removed for ex vivo studies. The experimental protocol was approved by the University of Iowa and Veterans Affairs Animal Care and Use Committees.

Plasma tHcy and Methionine
Plasma tHcy, defined as the total concentration of homocysteine after quantitative reductive cleavage of all disulfide bonds,²⁷ was measured by high-performance liquid chromatography and electrochemical detection.²⁸ Plasma methionine was measured by high-performance liquid chromatography coupled to coulometric electrochemical detection. The method was modified from that described by Martin et al²⁸ by increasing the electrochemical potential to 1100 mV to detect methionine.

GPx-1 Activity
Hepatic GPx-1 activity was measured with hydrogen peroxide as the substrate, as described previously.^29,30 Liver samples were kept frozen at -80°C until analysis, and then they were homogenized in 0.05 mol/L potassium phosphate buffer, pH 7.8. Homogenates were assayed in 0.1 mol/L potassium phosphate, pH 7.0, containing 1.0 mmol/L EDTA and 1.0 mmol/L NaN₃ (to inhibit catalase), 1.0 mmol/L glutathione, 0.35 U/mL glutathione reductase, 0.15 mmol/L NADPH, and 0.15 mmol/L hydrogen peroxide. GPx-1 activity was determined from the rate of oxidation of NADPH and was measured spectrophotometrically at 340 nm. One unit of GPx-1 activity was defined as the amount of protein required to oxidize 1.0 µmol NADPH per minute. The protein concentration of the homogenates was determined by using a modified Bradford assay (Bio-Rad).

Other Antioxidant Enzyme Assays
Catalase activity of liver homogenates was determined by monitoring the disappearance of hydrogen peroxide spectrophotometrically at 240 nm³¹ and was expressed in k units per milligram protein, as described previously.³² Total superoxide dismutase (SOD) activity of liver homogenates was measured by a modification of the nitro blue tetrazolium method, as described previously.^32,33 One unit of SOD activity was defined as the amount of protein required to inhibit the reduction of nitro blue tetrazolium by 50%.³²

Vasomotor Responses
Relaxation of precontracted aortic rings was measured in vitro as described previously.^21,22 After removal of loose connective tissue, the thoracic aorta was cut into multiple 3- to 4-mm rings that were suspended in an organ chamber containing oxygenated Krebs buffer maintained at 37°C. Rings were contracted submaximally by using the thromboxane analogue U46619, and relaxation dose-response curves were generated by cumulative addition of the endothelium-dependent vasodilator acetylcholine (10^-8 to 10^-5 mol/L) or the endothelium-independent vasodilators sodium nitroprusside (10^-8 to 10^-5 mol/L) or papaverine (10^-8 to 10^-5 mol/L). We have demonstrated previously that responses to acetylcholine in mouse aortas are mediated by NO.^34,35

Dihydroethidium Fluorescence
Dihydroethidium, an oxidative fluorescent dye, was used to detect superoxide in segments of carotid artery as described previously.^36,37 Briefly, fresh unfixed segments of the common carotid artery were frozen in OCT compound, and transverse sections (10 µm) were generated with a cryostat and placed on glass slides. Sections were then incubated in a light-protected chamber at room temperature for 30 minutes with 10 µmol/L dihydroethidium (Molecular Probes). Images were obtained with the use of a Bio-Rad MRC-1024 laser scanning confocal microscope equipped with a krypton/argon laser. The excitation wavelength was 488 nm, and emission fluorescence was detected with the use of a 585 nm long-pass filter. Identical laser settings were used for acquisition of images from different groups of mice. Fluorescent images were acquired as 8 bit (256 intensity levels) and were subsequently analyzed with NIH Image software (available online at http://rsb.info.nih.gov/nih-image). Data are reported as the percentage of surface area of carotid sections within the upper 20% of fluorescence intensity.

Statistical Analysis
Comparisons between genotypes (Gpx1^+/+, Gpx1^+/-, and GPx1^-/- mice) were performed by unpaired 2-tailed Student t tests. Responses to vasodilators were analyzed by 2-way repeated-measures ANOVA, with the Tukey post hoc test used for multiple comparisons. Correlation coefficients were calculated by the Pearson method. A value of P<0.05 was used to define statistical significance. Values are reported as mean±SE.

	Results

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Plasma Levels of tHcy and Methionine
Mice were studied after 17 weeks on a control or high-methionine diet (ie, at 20 weeks of age). Body weight did not differ between diets or between Gpx1 genotypes (data not shown). Plasma tHcy was elevated on the high-methionine diet (23.3±3.1 µmol/L) compared with the control diet (6.1±0.3 µmol/L, P<0.001) and was not influenced by Gpx1 genotype (Figure 1A). Plasma methionine was elevated to a similar extent in Gpx1^+/+, Gpx1^+/-, andGpx1^-/- mice that were fed the high-methionine diet (P<0.05 versus control diet, Figure 1B). The overall correlation between plasma tHcy and plasma methionine was strongly positive (r=0.72, P<0.0001).

View larger version (23K): [in this window] [in a new window]   Figure 1. Effect of diet and Gpx1 genotype on plasma levels of tHcy and methionine. Plasma concentrations of tHcy (A) or methionine (B) were measured in Gpx1+/+ (open bars), Gpx1+/- (hatched bars), and Gpx1-/- (filled bars) mice fed either the control diet (n=13, 10, and 9 for Gpx1+/+,Gpx1+/-, andGpx1-/- mice, respectively) or the high-methionine (high Met) diet (n=6, 15, and 7 for Gpx1+/+,Gpx1+/-, andGpx1-/- mice, respectively). *P<0.05 vs control diet.

GPx-1 Activity
Hepatic GPx-1 activity in Gpx1^+/+ mice tended to be slightly lower for the high-methionine diet (299±63 mU/mg) than the control diet (347±47 mU/mg), although this difference did not reach statistical significance (P>0.05, Figure 2). Compared with Gpx1^+/+ mice, Gpx1^+/- mice had hepatic GPx-1 activity that was decreased by 50% on either the control (177±42 mU/mg) or high-methionine (186±49 mU/mg) diet. Hepatic GPx-1 activity was undetectable in Gpx1^-/- mice fed either diet.

View larger version (18K): [in this window] [in a new window]   Figure 2. Effect of diet and Gpx1 genotype on hepatic GPx-1 activity. GPx-1 activity was measured in liver samples from Gpx1+/+ (open bars), Gpx1+/- (hatched bars), and Gpx1-/- (filled bars) mice fed either the control diet (n=10, 10, and 6 for Gpx1+/+,Gpx1+/-, andGpx1-/- mice, respectively) or the high Met diet (n=8, 11, and 7 for Gpx1+/+,Gpx1+/-, andGpx1-/- mice, respectively). *P<0.05 vs Gpx1+/+ mice.

Activity of Catalase and SOD
Hepatic catalase activity and SOD activity were similar in mice fed control or high-methionine diets and were not influenced by Gpx1 genotype (Table).

View this table: [in this window] [in a new window]   Table 1. Hepatic Levels of Catalase Activity and SOD Activity in Gpx1+/+ and Gpx1-/- Mice

Vasomotor Responses
Aortic rings from all groups of mice contracted similarly to the thromboxane analogue U46619 (data not shown). For mice fed the control diet, maximal relaxation to the endothelium-dependent vasodilator acetylcholine (10^-5 mol/L) was similar in Gpx1^+/+, Gpx1^+/-, and Gpx1^-/- mice, but relaxation to lower concentrations of acetylcholine was impaired in Gpx1^-/- mice (P<0.05 versus Gpx1^+/+ mice, Figure 3A). No significant differences in relaxation to the endothelium-independent vasodilators nitroprusside or papaverine were observed between Gpx1^+/+, Gpx1^+/-, and Gpx1^-/- mice fed the control diet (Figure 3B and 3C).

View larger version (18K): [in this window] [in a new window]   Figure 3. Relaxation of aortic rings from mice fed the control diet. Relaxation responses to acetylcholine (A), nitroprusside (B), or papaverine (C) were measured in Gpx1+/+ (filled circles, n=13), Gpx1+/- (open squares, n=10), and Gpx1-/- (open triangles, n=9) mice fed the control diet. *P<0.05 vs Gpx1+/+ mice.

For mice fed the high-methionine diet, impairment of relaxation to acetylcholine was evident in Gpx1^-/- mice at both high and low concentrations of acetylcholine (Figure 4A). Maximal relaxation to 10^-5 mol/L acetylcholine was significantly decreased in Gpx1^-/- mice (73±6%) compared with Gpx1^+/+ mice (90±2%) (P<0.05). No differences in relaxation to nitroprusside or papaverine were observed between Gpx1^+/+, Gpx1^+/-, and Gpx1^-/- mice fed the high-methionine diet (Figure 4B and 4C).

View larger version (18K): [in this window] [in a new window]   Figure 4. Relaxation of aortic rings from mice fed the high Met diet. Relaxation responses to acetylcholine (A), nitroprusside (B), or papaverine (C) were measured in Gpx1+/+ (filled circles, n=8), Gpx1+/- (open squares, n=15), and Gpx1-/- (open triangles, n=7) mice fed the high Met diet. *P<0.05 vs Gpx1+/+ mice.

Dihydroethidium Fluorescence
Dihydroethidium, an oxidative fluorescent dye, was used to detect superoxide in segments of carotid artery, as described previously.^36,37 Dihydroethidium is freely permeable to cells and is oxidized to the fluorescent dye ethidium bromide in the presence of superoxide.³⁶ Dihydroethidium fluorescence was detected at similar levels in Gpx1^+/+ and Gpx1^-/- mice fed the control diet (Figure 5A and 5B) and in Gpx1^+/+ mice fed the high-methionine diet (Figure 5C) but was elevated 3-fold in Gpx1^-/- mice fed the high-methionine diet (P<0.05 versus Gpx1^+/+ mice fed control diet, Figure 5D and 5E).

View larger version (26K): [in this window] [in a new window]   Figure 5. In situ detection of superoxide by dihydroethidium fluorescence. A through D, Representative laser-scanning fluorescent confocal micrographs of carotid artery from Gpx1+/+ mice fed the control diet (A), Gp1x-/- mice fed the control diet (B), Gpx1+/+ mice fed the high Met diet (C), and Gpx1-/- mice fed the high Met diet (D). E, Summary data for dihydroethidium (DHE) fluorescence, presented as the percentage of surface area of carotid sections within the upper 20% of fluorescence intensity, for Gpx1+/+ mice (open bars) and Gpx1-/- mice (filled bars) fed either the control diet or high Met diet (n=6 for Gpx1+/+ mice fed the control diet and n=4 for Gpx1-/- mice fed the control diet, Gpx1+/+ mice fed the high Met diet, and Gpx1-/- mice fed the high Met diet). *P<0.05 vs Gpx1+/+ mice fed the control diet.

	Discussion

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The major finding of the present study is that deficiency of GPx-1 produces impairment of endothelium-dependent relaxation of the aorta in mice with moderate hyperhomocysteinemia. GPx-1–deficient mice also had impaired relaxation responses to low concentrations of acetylcholine in the absence of hyperhomocysteinemia, but maximal relaxation to acetylcholine was impaired only when GPx-1–deficient mice were fed the hyperhomocysteinemic diet. Deficiency of GPx-1 did not affect vascular responses to the endothelium-independent vasodilators nitroprusside and papaverine, either in the absence or presence of hyperhomocysteinemia. These findings suggest that hyperhomocysteinemia contributes to endothelial dysfunction through a peroxide-dependent oxidative mechanism.

At least 4 isoforms of glutathione peroxidase have been described, but GPx-1 is the major cellular isoform in most tissues.³⁸ GPx-1 reduces hydrogen peroxide to water and can also reduce lipid peroxides to their corresponding alcohols.³⁸ The GPx-1–deficient mice used in the present study were generated by targeted homologous recombination of the Gpx1 gene.²⁵Gpx1^-/- mice are overtly healthy, but they have enhanced susceptibility to several forms of oxidative injury, including myocardial reperfusion injury,³⁹ virus-induced myocarditis,⁴⁰ peroxide-induced DNA strand breakage,⁴¹ and neutrophil-mediated hepatic injury during endotoxemia.⁴² Our present results indicate that they also have increased sensitivity to endothelial dysfunction, especially when they are fed a hyperhomocysteinemic diet. These findings, together with a recent study in which transgenic overexpression of Gpx-1 was found to rescue heterozygous cystathionine ß-synthase (CBS) mice (CBS^+/- mice) from endothelial dysfunction,²⁴ establish a critical role for GPx-1 in protecting endothelium from oxidative damage during moderate hyperhomocysteinemia in vivo.

Normal relaxation responses to acetylcholine in mouse aortas are dependent on endothelium-derived NO.^34,35 Expression of endothelial NO synthase (eNOS) appears to be unaffected in hyperhomocysteinemia, but the bioavailability of endothelium-derived NO is decreased.²⁰ Hyperhomocysteinemia may promote the oxidative inactivation of NO and thereby provide sensitization to endothelial dysfunction through several different mechanisms (Figure 6). Auto-oxidation of homocysteine in vitro produces hydrogen peroxide and, in the presence of transition metals such as copper or iron, can also generate more highly reactive ROS, such as superoxide and hydroxyl radical.^5–7 If auto-oxidation of homocysteine occurs during hyperhomocysteinemia in vivo, excessive production of hydroxyl radicals may cause lipid peroxidation, and both superoxide and lipid peroxyl radicals may react rapidly with endothelium-derived NO to produce peroxynitrite and lipid peroxynitrites. In support of this mechanism, elevated levels of superoxide and 3-nitrotyrosine, a product of protein modification by peroxynitrites, have been detected in the aortas of hyperhomocysteinemic mice.²⁰

View larger version (20K): [in this window] [in a new window]   Figure 6. Possible mechanisms for protection from hyperhomocysteinemia-induced endothelial dysfunction by GPx-1. Auto-oxidation of homocysteine may produce hydrogen peroxide (H2O2). Indirect oxidative effects of hyperhomocysteinemia may include generation of superoxide from uncoupled eNOS, xanthine oxidase, or NAD(P)H oxidase, downregulation of antioxidant enzymes, and depletion of intracellular glutathione. In the presence of transition metals such as copper or iron, H2O2 and superoxide (O2· -) may react to form a hydroxyl radical (·OH).6,7 The hydroxyl radical may promote the generation of lipid peroxyl radicals (LOO·), and both superoxide and lipid peroxyl radicals may react rapidly with endothelium-derived NO (·NO) to produce peroxynitrite (ONOO-) or lipid peroxynitrite (LOONO), respectively. The protective role of GPx-1 may be mediated by reduction of hydrogen peroxide, lipid peroxides (LOOH), and peroxynitrite. ONO- indicates nitrite.

Whether or not homocysteine auto-oxidation is a major mechanism for the generation of ROS in vivo is uncertain.¹² Conversion of homocysteine to its disulfide forms in plasma is mediated mainly by thiol-disulfide exchange reactions rather than by copper-dependent oxidation,⁴³ which suggests that homocysteine is unlikely to be a major source of hydrogen peroxide in vivo. Therefore, it is perhaps more likely that indirect mechanisms are responsible for the oxidative stress of hyperhomocysteinemia (Figure 6). Indirect oxidative effects of hyperhomocysteinemia may include generation of superoxide from xanthine oxidase⁴⁴ or uncoupled eNOS,⁴⁵ downregulation of antioxidant enzymes,^8,11,46 and depletion of intracellular glutathione.²³ Another potential source of superoxide is vascular NAD(P)H oxidase, which appears to contribute to endothelial dysfunction in hypertension and diabetes⁴⁷ but may be less important in hyperhomocysteinemia.⁴⁴ Each of these indirect effects is consistent with increased dihydroethidium fluorescence in Gpx1^-/- mice fed the high-methionine diet (Figure 5). Regardless of whether hyperhomocysteinemia promotes oxidative stress through direct or indirect mechanisms, the protective role of GPx-1 is presumably mediated by the reduction of hydrogen peroxide and lipid peroxides (Figure 6). GPx-1 may also provide protection from the toxic effects of peroxynitrite through its peroxynitrite reductase activity.⁴⁸

Our results confirm the findings of Forgione et al²⁶ that deficiency of GPx-1 leads to impairment of endothelium-dependent vasodilator function in mice with normal plasma levels of tHcy. Forgione et al observed endothelial dysfunction with paradoxical vasoconstriction to endothelium-dependent agonists in mesenteric arterioles of Gpx1^-/- mice fed standard chow. In contrast, the degree of impairment of endothelium-dependent relaxation of the aortas of Gpx1^-/- mice that we observed in the absence of hyperhomocysteinemia was relatively mild and was apparent only with submaximal concentrations of acetylcholine (Figure 3A). Similar differences in the magnitude of endothelial dysfunction between the aorta and mesenteric arterioles²⁰ or between the aorta and cerebral arterioles (S.R. Lentz, F.M. Faraci, unpublished data, 2002) have been observed in CBS-deficient mice with mild hyperhomocysteinemia. Further work will be required to determine whether these observations reflect differences in experimental methods, genetic background, or inherent differences between large arteries and small arterioles in susceptibility to oxidative stress.

We did not detect significant endothelial dysfunction in the aortas of Gpx1^+/+ or Gpx1^+/- mice that were fed the high-methionine diet despite the fact that these mice had moderately elevated plasma levels of tHcy. This result is consistent with a previous study in which the high-methionine diet produced endothelial dysfunction in CBS^+/- mice but not in their CBS^+/+ littermates.²² Plasma tHcy levels obtained in mice fed the high-methionine diet in the present study (23±3 µmol/L) were somewhat higher than those obtained in the previous study (13±2 µmol/L),²² which probably reflects differences in the timing of sample collection. Plasma samples in the present study were drawn from nonfasted mice, whereas they were drawn after 4 to 6 hours of fasting in the previous study.²² This interpretation is consistent with the higher plasma levels of methionine in the present study (57±8 µmol/L) compared with mice fed the same diet in the previous study (28±4 µmol/L).²² Plasma levels of tHcy exhibit a marked diurnal variation in mice, especially when they are fed a methionine-enriched diet (Jonathan Smith, oral communication, April 2002). Therefore, it is likely that mice fed the high-methionine diet were exposed to a similar degree of chronic hyperhomocysteinemia in both studies. These levels are within the pathophysiological range of plasma tHcy seen in most patients with moderate hyperhomocysteinemia.¹

In summary, we have demonstrated that that deficiency of GPx-1 exacerbates endothelial dysfunction in mice with moderate hyperhomocysteinemia. These in vivo findings provide support for the hypothesis that vascular damage in hyperhomocysteinemia is mediated through a peroxide-dependent oxidative mechanism. Although the present findings do not exclude the possibility that hyperhomocysteinemia also may produce endothelial dysfunction through additional mechanisms, they do establish a critical role for GPx-1 in providing protection from endothelial dysfunction in the face of diet-induced hyperhomocysteinemia.

	Acknowledgments

 
This work was supported in part by the Office of Research and Development, Department of Veterans Affairs and National Institutes of Health grants HL-63943, DK-25295, NS-24621, and HL-62984, an American Heart Association-Bugher Foundation Award, and the Cardiovascular Interdisciplinary Research Fellowship (HL-07121) funded by the National Heart, Lung, and Blood Institute. We thank Katina Wilson, Jessica Hook, and Ryan McCaw for technical assistance, Dr Douglas Spitz for advice regarding antioxidant enzyme assays, and Dr Francis J. Miller, Jr, for advice regarding dihydroethidium fluorescence.

	Footnotes

 
Consulting Editor for this article was Alan M. Fogelman, MD, Professor of Medicine and Executive Chair, Departments of Medicine and Cardiology, UCLA School of Medicine, Los Angeles, Calif.

Received April 29, 2002; accepted September 30, 2002.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Eikelboom JW, Lonn E, Genest JJ, Hankey G, Yusuf S. Homocyst(e)ine and cardiovascular disease: a critical review of the epidemiologic evidence. Ann Intern Med. 1999; 131: 363–375.[Abstract/Free Full Text]
2. den Heijer M, Rosendaal FR, Blom H, Gerrits WBJ, Bos GMJ. Hyperhomocysteinemia and venous thrombosis: a meta-analysis. Thromb Haemost. 1998; 80: 874–877.[Medline] [Order article via Infotrieve]
3. Lentz SR. Homocysteine and cardiovascular physiology. In: Carmel R, Jacobsen DW, eds. Homocysteine in Health and Disease. Cambridge, UK: Cambridge University Press; 2001: 441–450.
4. Loscalzo J. The oxidant stress of hyperhomocyst(e)inemia. J Clin Invest. 1996; 98: 5–7.[Medline] [Order article via Infotrieve]
5. Parthasarathy S. Oxidation of low density lipoprotein by thiol compounds leads to its recognition by the acetyl LDL receptor. Biochim Biophys Acta. 1987; 917: 337–340.[Medline] [Order article via Infotrieve]
6. Heinecke JW, Rosen H, Suzuki LA, Chait A. The role of sulfur-containing amino acids in superoxide production and modification of low density lipoprotein by arterial smooth muscle cells. J Biol Chem. 1987; 262: 10098–10103.[Abstract/Free Full Text]
7. Jones BG, Rose FA, Tudball N. Lipid peroxidation and homocysteine induced toxicity. Atherosclerosis. 1994; 105: 165–170.[CrossRef][Medline] [Order article via Infotrieve]
8. Upchurch GR, Welch GN, Fabian AJ, Freedman JE, Johnson JL, Keaney JF, Loscalzo J. Homocyst(e)ine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase. J Biol Chem. 1997; 272: 17012–17017.[Abstract/Free Full Text]
9. Zhang X, Li H, Jin H, Ebin Z, Brodsky S, Goligorsky MS. Effects of homocysteine on endothelial nitric oxide production. Am J Physiol. 2000; 279: F671–F678.
10. Starkebaum G, Harlan JM. Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest. 1986; 77: 1370–1376.[Medline] [Order article via Infotrieve]
11. Outinen PA, Sood SK, Pfeifer SI, Pamidi S, Podor TJ, Li J, Weitz JI, Austin RC. Homocysteine-induced endoplasmic reticulum stress and growth arrest leads to specific changes in gene expression in human vascular endothelial cells. Blood. 1999; 94: 959–967.[Abstract/Free Full Text]
12. Jacobsen DW. Hyperhomocysteinemia and oxidative stress: time for a reality check? Arterioscler Thromb Vasc Biol. 2000; 20: 1182–1184.[Free Full Text]
13. Blom HJ, Engelen DP, Boers GH, Stadhouders AM, Sengers RC, de Abreu R, TePoele-Pothoff MT, Trijbels JM. Lipid peroxidation in homocysteinaemia. J Inherit Metabol Dis. 1992; 15: 419–422.[CrossRef][Medline] [Order article via Infotrieve]
14. Nightingale AK, James PP, Morris-Thurgood J, Harrold F, Tong R, Jackson SK, Cockcroft JR, Frenneaux MP. Evidence against oxidative stress as mechanism of endothelial dysfunction in methionine loading model. Am J Physiol. 2001; 280: H1334–H1339.
15. Dudman NPB, Wilcken DEL, Stocker R. Circulating lipid hydroperoxide levels in human hyperhomocysteinemia: relevance to development of arteriosclerosis. Arterioscler Thromb. 1993; 13: 512–516.[Abstract/Free Full Text]
16. Domagala TB, Libura M, Szczeklik A. Hyperhomocysteinemia following oral methionine load is associated with increased lipid peroxidation. Thromb Res. 1997; 87: 411–416.[CrossRef][Medline] [Order article via Infotrieve]
17. Chambers JC, McGregor A, Jean-Marie J, Obeid OA, Kooner JS. Demonstration of rapid onset vascular endothelial dysfunction after hyperhomocysteinemia: an effect reversible with vitamin C therapy. Circulation. 1999; 99: 1156–1160.[Abstract/Free Full Text]
18. Kanani PM, Sinkey CA, Browning RL, Allaman M, Knapp HR, Haynes WG. Role of oxidant stress in endothelial dysfunction produced by experimental hyperhomocyst(e)inemia in humans. Circulation. 1999; 100: 1161–1168.[Abstract/Free Full Text]
19. Raghuveer G, Sinkey CA, Chenard C, Stumbo P, Haynes WG. Effect of vitamin E on resistance vessel endothelial dysfunction induced by methionine. Am J Cardiol. 2001; 88: 285–290.[CrossRef][Medline] [Order article via Infotrieve]
20. Eberhardt RT, Forgione MA, Cap A, Leopold JA, Rudd MA, Tolliet M, Heyrick S, Stark R, Klings ES, Moldovan NI, Yaghoubi M, Goldschmidt-Clermont PJ, Farber HW, Cohen R, Loscalzo J. Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia. J Clin Invest. 2000; 106: 483–491.[Medline] [Order article via Infotrieve]
21. Lentz SR, Erger RA, Dayal S, Maeda N, Malinow MR, Heistad DD, Faraci FM. Folate dependence of hyperhomocysteinemia and endothelial dysfunction in cystathionine ß-synthase-deficient mice. Am J Physiol. 2000; 278: H970–H975.
22. Dayal S, Bottiglieri T, Arning E, Maeda N, Malinow MR, Sigmund CD, Heistad DD, Faraci FM, Lentz SR. Endothelial dysfunction and elevation of S-adenosylhomocysteine in cystathionine ß-synthase–deficient mice. Circ Res. 2001; 88: 1203–1209.[Abstract/Free Full Text]
23. Weiss N, Heydrick S, Zhang YY, Bierl C, Cap A, Loscalzo J. Cellular redox state and endothelial dysfunction in mildly hyperhomocysteinemic cystathionine beta-synthase-deficient mice. Arterioscler Thromb Vasc Biol. 2002; 22: 34–41.[Abstract/Free Full Text]
24. Weiss N, Zhang YY, Heydrick S, Bierl C, Loscalzo J. Overexpression of cellular glutathione peroxidase rescues homocyst(e)ine-induced endothelial dysfunction. Proc Natl Acad Sci U S A. 2001; 98: 12503–12508.[Abstract/Free Full Text]
25. 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.[Abstract/Free Full Text]
26. 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. 2002; 282: H1255–H1261.
27. Mudd SH, Finkelstein JD, Refsum H, Ueland PM, Malinow MR, Lentz SR, Jacobsen DW, Brattström L, Wilcken B, Wilcken DEL, Blom HJ, Stabler SP, Allen RH, Selhub J, Rosenberg IH. Homocysteine and its disulfide derivatives: a suggested consensus terminology. Arterioscler Thromb Vasc Biol. 2000; 20: 1704–1706.[Free Full Text]
28. Martin SC, Hilton AC, Bartlett WA, Jones AF. Plasma total homocysteine measurement by ion-paired reversed-phase HPLC with electrochemical detection. Biomed Chromatogr. 1999; 13: 81–82.[CrossRef][Medline] [Order article via Infotrieve]
29. Paglia PE, Valentine WN. Studies on the quantitation and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med. 1967; 70: 158–169.[Medline] [Order article via Infotrieve]
30. Zhong W, Oberley LW, Oberley TD, Yan T, Domann FE, St. Clair DK. Inhibition of cell growth and sensitization to oxidative damage by overexpression of manganese superoxide dismutase in rat glioma cells. Cell Growth Differ. 1996; 7: 1175–1186.[Abstract]
31. Beers RF, Jr, Sizer IW. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem. 1952; 195: 133–140.[Free Full Text]
32. Spitz DR, Elwell JH, Sun Y, Oberley LW, Oberley TD, Sullivan SJ, Roberts RJ. Oxygen toxicity in control and H₂O₂ resistant Chinese hamster fibroblasts. Arch Biochem Biophys. 1990; 279: 249–260.[CrossRef][Medline] [Order article via Infotrieve]
33. Spitz DR, Oberley LW. An assay for superoxide dismutase in mammalian tissue homogenates. Anal Biochem. 1989; 179: 8–18.[CrossRef][Medline] [Order article via Infotrieve]
34. Faraci FM, Sigmund CD, Shesely EG, Maeda N, Heistad DD. Responses of carotid artery in mice deficient in expression of the gene for endothelial NO synthase. Am J Physiol. 1998; 274: H564–H570.[Medline] [Order article via Infotrieve]
35. Lake-Bruse KD, Faraci FM, Shesely EG, Meada N, Heistad DD. Gene transfer of endothelial nitric oxide synthase (eNOS) in eNOS-deficient mice. Am J Physiol. 1999; 46: H770–H776.
36. Miller FJ Jr, Gutterman DD, Rios CD, Heistad DD, Davidson BL. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res. 1998; 82: 1298–1305.[Abstract/Free Full Text]
37. Hathaway CA, Heistad DD, Piegors DJ, Miller FJ Jr. Regression of atherosclerosis in monkeys reduces vascular superoxide levels. Circ Res. 2002; 90: 277–283.[Abstract/Free Full Text]
38. Arthur JR. The glutathione peroxidases. Cell Mol Life Sci. 2000; 57: 1825–1835.[CrossRef][Medline] [Order article via Infotrieve]
39. Yoshida T, Maulik N, Engelman RM, Ho YS, Magnenat JL, Rousou JA, Flack JE, Deaton D, Das DK. Glutathione peroxidase knockout mice are susceptible to myocardial ischemia reperfusion injury. Circulation. 1997; 96 (suppl II): II-216–II-220.
40. Beck MA, Esworthy RS, Ho YS, Chu FF. Glutathione peroxidase protects mice from viral-induced myocarditis. FASEB J. 1998; 12: 1143–1149.[Abstract/Free Full Text]
41. Reddy VN, Lin LR, Ho YS, Magnenat JL, Ibaraki N, Giblin FJ, Dang L. Peroxide-induced damage in lenses of transgenic mice with deficient and elevated levels of glutathione peroxidase. Ophthalmologica. 1997; 211: 192–200.[Medline] [Order article via Infotrieve]
42. Jaeschke H, Ho YS, Fisher MA, Lawson JA, Farhood A. Glutathione peroxidase-deficient mice are more susceptible to neutrophil-mediated hepatic parenchymal cell injury during endotoxemia: importance of an intracellular oxidant stress. Hepatology. 1999; 29: 443–450.[CrossRef][Medline] [Order article via Infotrieve]
43. Sengupta S, Wehbe C, Majors AK, Ketterer ME, DiBello PM, Jacobsen DW. Relative roles of albumin and ceruloplasmin in the formation of homocysteine, homocysteine-cysteine mixed disulfide and cystine in circulation. J Biol Chem. 2001; 276: 46896–46904.[Abstract/Free Full Text]
44. Bagi Z, Ungvari Z, Koller A. Xanthine oxidase-derived reactive oxygen species convert flow-induced arteriolar dilation to constriction in hyperhomocysteinemia: possible role of peroxynitrite. Arterioscler Thromb Vasc Biol. 2002; 22: 28–33.[Abstract/Free Full Text]
45. Doshi SN, Mcdowell IFW, Moat SJ, Payne N, Durrant HJ, Lewis MJ, Goodfellow J. Folic acid improves endothelial function in coronary artery disease via mechanisms largely independent of homocysteine lowering. Circulation. 2002; 105: 22–26.[Abstract/Free Full Text]
46. Huang RF, Hsu YC, Lin HL, Yang FL. Folate depletion and elevated plasma homocysteine promote oxidative stress in rat livers. J Nutr. 2001; 131: 33–38.[Abstract/Free Full Text]
47. Cai H, Harrison DG, Drummond GR, Cai H, Davis ME, Ramasamy S, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress: transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide. Circ Res. 2000; 87: 840–844.[Abstract/Free Full Text]
48. Sies H, Sharov VS, Klotz LO, Briviba K. Glutathione peroxidase protects against peroxynitrite-mediated oxidations: a new function for selenoproteins as peroxynitrite reductase. J Biol Chem. 1997; 272: 27812–27817.[Abstract/Free Full Text]

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