Vascular Biology |
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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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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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.1316 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.1719 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.2024 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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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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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.
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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).
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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).
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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).
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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).
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| Discussion |
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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-1deficient 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.57 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
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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 |
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| Footnotes |
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Received April 29, 2002; accepted September 30, 2002.
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L. Qiu, E. O. List, and J. J. Kopchick Differentially Expressed Proteins in the Pancreas of Diet-induced Diabetic Mice Mol. Cell. Proteomics, September 1, 2005; 4(9): 1311 - 1318. [Abstract] [Full Text] [PDF] |
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S. Dayal and S. R Lentz ADMA and hyperhomocysteinemia Vascular Medicine, July 1, 2005; 10(1_suppl): S27 - S33. [Abstract] [PDF] |
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J. A. Leopold and J. Loscalzo Oxidative Enzymopathies and Vascular Disease Arterioscler Thromb Vasc Biol, July 1, 2005; 25(7): 1332 - 1340. [Abstract] [Full Text] [PDF] |
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R. Schnabel, K. J. Lackner, H. J. Rupprecht, C. Espinola-Klein, M. Torzewski, E. Lubos, C. Bickel, F. Cambien, L. Tiret, T. Munzel, et al. Glutathione Peroxidase-1 and Homocysteine for Cardiovascular Risk Prediction: Results from the AtheroGene Study J. Am. Coll. Cardiol., May 17, 2005; 45(10): 1631 - 1637. [Abstract] [Full Text] [PDF] |
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S. Dayal and S. R Lentz ADMA and hyperhomocysteinemia Vascular Medicine, May 1, 2005; 10(2_suppl): S27 - S33. [Abstract] [PDF] |
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R. W. Powers, R. E. Gandley, D. L. Lykins, and J. M. Roberts Moderate Hyperhomocysteinemia Decreases Endothelial-Dependent Vasorelaxation in Pregnant But Not Nonpregnant Mice Hypertension, September 1, 2004; 44(3): 327 - 333. [Abstract] [Full Text] [PDF] |
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S. Dayal, E. Arning, T. Bottiglieri, R. H. Boger, C. D. Sigmund, F. M. Faraci, and S. R. Lentz Cerebral Vascular Dysfunction Mediated by Superoxide in Hyperhomocysteinemic Mice Stroke, August 1, 2004; 35(8): 1957 - 1962. [Abstract] [Full Text] [PDF] |
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J. Zhou, G. H. Werstuck, S. Lhotak, A. B. L. de Koning, S. K. Sood, G. S. Hossain, J. Moller, M. Ritskes-Hoitinga, E. Falk, S. Dayal, et al. Association of Multiple Cellular Stress Pathways With Accelerated Atherosclerosis in Hyperhomocysteinemic Apolipoprotein E-Deficient Mice Circulation, July 13, 2004; 110(2): 207 - 213. [Abstract] [Full Text] [PDF] |
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V. Vitvitsky, S. Dayal, S. Stabler, Y. Zhou, H. Wang, S. R. Lentz, and R. Banerjee Perturbations in homocysteine-linked redox homeostasis in a murine model for hyperhomocysteinemia Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2004; 287(1): R39 - R46. [Abstract] [Full Text] [PDF] |
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D. J. Kurz, S. Decary, Y. Hong, E. Trivier, A. Akhmedov, and J. D. Erusalimsky Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells J. Cell Sci., May 1, 2004; 117(11): 2417 - 2426. [Abstract] [Full Text] [PDF] |
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S. Blankenberg, H. J. Rupprecht, C. Bickel, M. Torzewski, G. Hafner, L. Tiret, M. Smieja, F. Cambien, J. Meyer, K. J. Lackner, et al. Glutathione Peroxidase 1 Activity and Cardiovascular Events in Patients with Coronary Artery Disease N. Engl. J. Med., October 23, 2003; 349(17): 1605 - 1613. [Abstract] [Full Text] [PDF] |
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E. Cediel, D. Sanz-Rosa, M. P. Oubina, N. de las Heras, F. R. G. Pacheco, O. Vegazo, J. Jimenez, V. Cachofeiro, and V. Lahera Effect of AT1 receptor blockade on hepatic redox status in SHR: possible relevance for endothelial function? Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2003; 285(3): R674 - R681. [Abstract] [Full Text] [PDF] |
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F. M. Faraci Hyperhomocysteinemia: A Million Ways to Lose Control Arterioscler Thromb Vasc Biol, March 1, 2003; 23(3): 371 - 373. [Full Text] [PDF] |
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