(Circulation. 2005;112:737-744.)
© 2005 American Heart Association, Inc.
Stroke |
From the Departments of Internal Medicine (S.D., A.M.D., R.B.M., F.M.F., S.R.L.) and Pharmacology (F.M.F.), University of Iowa, Carver College of Medicine, Iowa City; Baylor Institute of Metabolic Disease, Dallas, Tex (E.A., T.B.); Department of Nutritional Sciences and Toxicology, University of CaliforniaBerkeley (M.-L.L., B.S.); and Veterans Affairs Medical Center, Iowa City, Iowa (S.R.L.).
Correspondence to Steven R. Lentz, MD, PhD, Department of Internal Medicine, C32 GH, University of Iowa, Iowa City, IA 52242. E-mail steven-lentz{at}uiowa.edu
Received December 12, 2004; revision received April 28, 2005; accepted May 6, 2005.
| Abstract |
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Methods and Results Plasma total homocysteine was similar in wild-type (Mtr+/+) and heterozygous (Mtr+/) mice fed a control diet (4.5±0.3 and 5.3±0.4 µmol/L, respectively) and mildly elevated in Mtr+/+ and Mtr+/ mice fed a low-folate (LF) diet (7.5±0.7 and 9.6±1.2 µmol/L, respectively; P<0.001 versus control diet). Dilatation of cerebral arterioles to the endothelium-dependent dilator, acetylcholine (10 µmol/L) was blunted in Mtr+/ mice compared with Mtr+/+ mice fed the control diet (21±4 versus 32±4%; P<0.05). Both Mtr+/+ and Mtr+/ mice exhibited impaired dilatation of cerebral arterioles to acetylcholine when they were fed the LF diet (12±2 and 14±2%, respectively; P<0.01 versus Mtr+/+ mice fed the control diet). Elevated levels of superoxide and hydrogen peroxide were detected by confocal microscopy in cerebral arterioles of Mtr+/ mice fed the control diet and in both Mtr+/+ and Mtr+/ mice fed the LF diet.
Conclusions These findings demonstrate that defective homocysteine remethylation caused by deficiency of either MS or folate produces oxidative stress and endothelial dysfunction in the cerebral microcirculation of mice.
Key Words: cerebrovascular circulation endothelium homocysteine superoxides
| Introduction |
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Studies in animals and humans have established that hyperhomocysteinemia causes endothelial dysfunction, with impaired endothelium-dependent dilatation of the aorta and other peripheral vessels.9 The cerebral microcirculation appears to be particularly susceptible to endothelial dysfunction during hyperhomocysteinemia.10,11 A major mechanism of hyperhomocysteinemia-induced endothelial dysfunction, in both cerebral and peripheral blood vessels, is related to decreased bioavailability of endothelium-derived nitric oxide (NO).12 There is strong evidence that elevation of homocysteine is linked to oxidative inactivation of NO, but alternative mechanisms of decreased NO bioavailability during hyperhomocysteinemia also have been proposed.13,14 It is not known whether endothelial dysfunction is caused by homocysteine itself or a related metabolite.
Homocysteine is formed during methyl transfer reactions that use S-adenosylmethionine (SAM) as a methyl donor.15 The metabolic fate of homocysteine in most cells depends on the relative activity of 2 cytoplasmic enzymes: (1) cystathionine ß-synthase (CBS), which catalyzes the first step in the transsulfuration of homocysteine to cysteine, and (2) methionine synthase (MS), a cobalamin (vitamin B12)-dependent enzyme that remethylates homocysteine to form methionine. During the MS reaction, a methyl group from 5-methyltetrahydrofolate (5-MTHF) is first transferred to cobalamin, which serves as an intermediate methyl carrier. The methyl group is subsequently transferred to homocysteine, generating methionine. The tissue distribution of CBS is limited largely to liver, kidney, intestine, pancreas, and brain, whereas MS is widely expressed in most tissues.15 The availability of 5-MTHF for homocysteine remethylation depends on the action of methylenetetrahydrofolate reductase (MTHFR). A common polymorphism in the MTHFR gene, 677C
T, produces impaired MTHFR activity and is associated with hyperhomocysteinemia and increased risk of coronary heart disease and stroke, especially under conditions of low-folate (LF) status.16
Using a murine model of CBS deficiency, we11,17,18 and others19 have found that defective transsulfuration is associated with enhanced susceptibility to diet-induced hyperhomocysteinemia and endothelial dysfunction. Endothelial dysfunction also has been observed in MTHFR-deficient mice, which have an indirect defect in homocysteine remethylation resulting from a relative lack of 5-MTHF.10,20 It is not known, however, whether endothelial dysfunction can be caused by a specific genetic defect in MS-catalyzed homocysteine remethylation.
In the present study, we tested the hypothesis that endothelial dysfunction and oxidative stress occur in mice with a direct defect in homocysteine remethylation caused by a targeted disruption of the Mtr gene, which encodes MS.21 Because homozygous disruption of the Mtr gene produces embryonic lethality,22 we chose to study heterozygous (Mtr+/) mice fed either a control diet or an LF diet. This approach allowed us to examine the vascular effects of an isolated defect in the remethylation pathway in mice with either normal or deficient folate intake. Our findings indicate that impaired homocysteine remethylation resulting from a deficiency of either MS or folate causes oxidative stress and endothelial dysfunction in the cerebral microcirculation.
| Methods |
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Vasomotor Responses
Relaxation of aortic rings was measured as described previously.17,18 After removal of loose connective tissue, the proximal aorta was cut into multiple 3- to 4-mm rings and suspended in an organ chamber containing oxygenated Krebs buffer maintained at 37°C. Rings were precontracted submaximally using the thromboxane analog U46619, and relaxation dose-response curves were generated by cumulative addition of the endothelium-dependent vasodilator acetylcholine (108 to 105 mol/L) or the endothelium-independent vasodilator nitroprusside (108 to 105 mol/L).
Dilatation of cerebral arterioles was measured in vivo as described previously.10,11,24 Briefly, mice were anesthetized with sodium pentobarbital and ventilated mechanically with room air and supplemental oxygen. A cranial window was made over the left parietal cortex, and a segment of a randomly selected pial arteriole (
30 µm in diameter) was exposed. The diameter of the arteriole was measured with a video microscope coupled to an image-shearing device under control conditions and during superfusion with acetylcholine (1 and 10 µmol/L) and nitroprusside (0.1 and 1 µmol/L).
Detection of Vascular ROS
The oxidative fluorescent dyes dihydroethidium (DHE) and 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (DCF) (both from Molecular Probes, Inc) were used to detect superoxide and peroxide, respectively, in sections of cerebral arterioles as described previously.11,25 Frozen transverse sections (10 µm) of cerebral arterioles were preincubated with PBS, 250 U/mL polyethylene glycol-superoxide dismutase (PEG-SOD, Sigma Chemical Co), or 500 U/mL PEG-catalase (Sigma Chemical Co) and then incubated with 10 µmol/L of either DHE or DCF for 30 minutes at room temperature in a dark chamber. Fluorescence was detected by laserscanning confocal microscopy.11
Metabolite Assays
Plasma tHcy, defined as the total concentration of homocysteine after quantitative reductive cleavage of all disulfide bonds,26 was measured by high-performance liquid chromatography (HPLC) with SBDF (ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate) fluorescence detection.27 Plasma methionine was measured by HPLC coupled to fluorescence detection after precolumn derivatization with o-phthaldialdehyde as described previously.28 Plasma and tissue folate was measured with the Quantaphase II folate radioimmunoassay (Bio-Rad Laboratories). SAM and SAH concentrations in liver and brain were determined by HPLC using UV detection as described previously.29 Plasma and tissue levels of ADMA, symmetric dimethyarginine (SDMA), and L-arginine were determined by HPLC using precolumn derivatization with o-phthaldialdehyde.30
Statistical Analysis
ANOVA, followed by Tukeys post-hoc test, was used to compare the effects of Mtr genotype, sex, and diet on tHcy, folate, methionine, SAM, SAH, ADMA, SDMA, and L-arginine. Unpaired 2-tailed Student t test was used to compare cerebral vasomotor responses. Responses to vasodilators in aorta were analyzed by 2-way repeated-measures ANOVA with Tukeys post hoc test for multiple comparisons. Correlation analysis was performed by linear regression, followed by 1-way ANOVA. A value of P=0.05 was used to define statistical significance. Values are reported as mean±SE.
| Results |
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Tissue Levels of Folate, SAM, and SAH
Levels of folate in the liver were significantly decreased in both Mtr+/+ and Mtr+/ mice fed the LF diet (P<0.001) (Table 1). Methylation capacity in the liver also was significantly influenced by diet, with mice fed the LF diet having lower levels of SAM (P<0.01) and lower SAM-to-SAH ratios (P<0.05) than mice fed the control diet. By Tukeys multiple comparison analysis, these effects of diet reached significance for Mtr+/ mice but not for Mtr+/+ mice (Table 1). Levels of SAH in liver did not differ significantly between Mtr+/+ and Mtr+/ mice or between the control and LF diets. No significant effects of Mtr genotype or diet were found on folate, SAM, SAH, or SAM-to-SAH ratio in brain, although there was a trend toward lower folate levels in mice fed the LF diet (P=0.08) (Table 2). As observed previously in CBS-deficient and MTHFR-deficient mice,10,18 the SAM-to-SAH ratio was higher in brain (Table 2) than in liver (Table 1).
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Vasomotor Responses
We observed concentration-dependent relaxation of aortic rings to the endothelium-dependent vasodilator acetylcholine and the endothelium-independent vasodilator nitroprusside in all groups of mice (Figure 2). Relaxation responses to acetylcholine were similar in Mtr+/+ and Mtr+/ mice regardless of diet (Figure 2A and 2B). Similarly, no differences in relaxation responses to nitroprusside were observed between Mtr+/+ and Mtr+/ mice or between diets (Figure 2C and 2D).
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To determine the effects of Mtr genotype and diet on vasomotor responses in the cerebral circulation, dilatation of pial arterioles was measured in anesthetized mice in vivo (Figure 3). When mice were fed the control diet, dose-dependent dilatation to 1.0 and 10 µmol/L acetylcholine was observed in both Mtr+/+ and Mtr+/ mice, but dilatation to 10 µmol/L acetylcholine was blunted in Mtr+/ mice compared with Mtr+/+ mice (P<0.05; Figure 3A and 3B). When mice were fed the LF diet, both Mtr+/+ and Mtr+/ mice exhibited impaired dilatation responses to 10 µmol/L acetylcholine (P<0.05; Figure 3A and 3B). Dose-dependent dilatation of cerebral arterioles in response to nitroprusside was not affected by either diet or Mtr genotype (Figure 3C and 3D).
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Vascular ROS
Superoxide was detected by laserscanning confocal microscopy in sections of cerebral arterioles using the oxidative fluorescent dye DHE. As expected, DHE fluorescence was localized to nuclei because of the high affinity of oxidized DHE for nucleic acids (Figure 4).31 Compared with Mtr+/+ mice fed the control diet (Figure 4A), higher levels of microvascular DHE fluorescence were observed in Mtr+/ mice fed the control diet (Figure 4B) and in both Mtr+/+ and Mtr+/ mice fed the LF diet (Figure 4C and 4D). DHE fluorescence in Mtr+/ mice fed the LF diet was strongly inhibited by PEG-SOD (Figure 4E), which suggests that superoxide was the major cause of enhanced DHE fluorescence in this model.
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Staining with DCF, which detects peroxides and other ROS,31 was elevated in Mtr+/ mice (Figure 5B and 5D) compared with Mtr+/+ mice (Figure 5A and 5C) fed either the control or LF diets. Most of the enhanced DCF fluorescence in Mtr+/ mice fed the LF diet was inhibited by PEG-catalase (Figure 5E), which suggests that enhanced DCF fluorescence in these mice was mediated largely by hydrogen peroxide. A background signal caused by autofluorescence of the elastic lamina was observed in all sections.
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ADMA
Previous studies have suggested that hyperhomocysteinemia-induced endothelial dysfunction may be mediated by elevation of ADMA, an endogenous inhibitor of NO synthases.32,33 We found that plasma levels ADMA were similar in Mtr+/+ and Mtr+/ mice fed either the control diet or the LF diet (Table 3), although there was a nonsignificant trend toward higher levels of ADMA in mice fed the LF diet compared with mice fed the control diet (P=0.06). There were no significant effects of Mtr genotype or diet on plasma SDMA, L-arginine, or ratio of L-arginine to ADMA (Table 3). Levels of ADMA in the liver did not differ between Mtr+/+ and Mtr+/ mice fed either the control diet (44.9±8.3 versus 50.0±1.0 nmol/g; P=0.51) or the LF diet (34.5±3.4 versus 47.2±7.3 nmol/g; P=0.13). A positive correlation was found between plasma ADMA and liver ADMA (R=0.48, P=0.05).
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| Discussion |
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In agreement with previous studies,10,17 we found that the LF diet produced a highly significant (P<0.001) elevation of plasma tHcy in both Mtr+/+and Mtr+/ mice. The influence of Mtr genotype on plasma tHcy was less ±substantial, with minimal elevation of plasma tHcy seen in Mtr+/ mice fed the control diet (P=0.05) (Figure 1). The modest effect of the Mtr+/ genotype on plasma tHcy is consistent with previous findings in MS-deficient mice.22 The concentration of tHcy in plasma is strongly determined by its metabolism in the liver,18 which is capable of remethylating homocysteine to methionine through an alternative pathway catalyzed by betaine:homocysteine methyltransferase (BHMT). Therefore, we speculate that increased hepatic BHMT-dependent remethylation may maintain plasma tHcy at relatively normal levels in Mtr+/ mice fed the control diet.
Previous studies have suggested that hyperhomocysteinemia produces endothelial dysfunction through oxidative inactivation of endothelium-derived NO.14,34 Our present results are consistent with these prior studies, because we observed endothelial dysfunction with increased superoxide and/or hydrogen peroxide in cerebral arterioles of Mtr+/+ and Mtr+/ mice fed the LF diet (Figures 4 and 5
). An interesting new finding from the present study is that, despite the absence of hyperhomocysteinemia, Mtr+/ mice fed the control diet also exhibited endothelial dysfunction and elevated levels of both superoxide and peroxide in cerebral arterioles. We have considered several possible mechanisms to account for these findings.
One possibility is that homocysteine itself is responsible for producing oxidative stress and endothelial dysfunction. Homocysteine can undergo auto-oxidation through its free thiol group, potentially leading to direct inactivation of endothelium-derived NO.34 Homocysteine also may increase oxidative stress indirectly by depleting intracellular glutathione or downregulating antioxidant enzymes such as glutathione peroxidase or by upregulating vascular NAD(P)H oxidase.35 Unlike Mtr+/+ and Mtr+/ mice fed the LF diet, Mtr+/ mice fed the control diet did not have a significant elevation of tHcy in plasma, but we cannot exclude the possibility that homocysteine levels may have been elevated in vascular tissue, which lacks compensatory BHMT activity. It remains possible, therefore, that high levels of homocysteine in cerebral microvessels produce local oxidative stress and endothelial dysfunction in Mtr+/ mice.
Another possibility is that it is not homocysteine but another factor related to folate metabolism that is responsible for cerebral vascular dysfunction. Folates are known to have antioxidant properties, and several clinical intervention studies have demonstrated that treatment with folic acid can improve endothelial function in subjects with hypercholesterolemia or atherosclerosis.36 In some of these studies, the improvement in endothelial function was associated with a decrease in markers of systemic oxidative stress.37 Treatment with high-dose folic acid also improves endothelial function in hyperhomocysteinemic subjects,36 an effect that may be independent of plasma tHcy.38,39 In agreement with these findings in humans, folate deficiency has been shown to cause increased lipid peroxidation and decreased cellular antioxidant activity in rats.40,41
Although Mtr+/+ and Mtr+/ mice have similar plasma levels of total folate (Figure 1), it is conceivable that differences in tissue levels of folate or folate pool distribution may contribute to decreased antioxidant activity in Mtr+/ mice. Deficient MS activity in Mtr+/ mice might be expected to cause buildup of 5-MTHF and depletion of other folates, including those required for thymidylate and purine synthesis. An analogous "methyl folate trap" is responsible for megaloblastic anemia in humans with deficient MS activity resulting from pernicious anemia.42 5-MTHF itself has been shown to be a weak scavenger of superoxide and to enhance NO production from endothelial NO synthase, possibly by improving the redox state of the endothelial NO synthase cofactor tetrahydrobiopterin.43 It is not known, however, whether other forms of folate are more or less potent antioxidants compared with 5-MTHF. Clearly, more work is needed to define the redox effects of various folate metabolites.
To begin to address role of tissue folates in the phenotype of MS-deficient mice, we measured levels of total folate in liver and brain, and we did not detect any significant differences between Mtr+/+ and Mtr+/ mice (Tables 1 and 2
). Interestingly, however, levels of folate in brain were >10-fold lower than in liver, and the relative influence of the LF diet on folate level was attenuated in brain compared with liver. These findings suggest that tissue-specific differences in folate uptake and metabolism may modulate the phenotypic response to altered homocysteine remethylation. Because of technical limitations, we were not able to measure the distribution of individual folate metabolites in these tissues, but this remains an important goal of future studies.
We also considered the possibility that altered cellular methylation capacity may have contributed to endothelial dysfunction and oxidative stress. Compared with mice fed the control diet, Mtr+/+ and Mtr+/ mice fed the LF diet had significantly lower SAM-to-SAH ratios in the liver (Table 1). The diminished hepatic methylation capacity of these mice might be expected to alter DNA methylation or the generation of other methylated products. No changes in SAM-to-SAH ratio were observed in the brains of mice fed the LF diet, however, and Mtr+/ mice fed the control diet did not exhibit altered methylation capacity in either liver or brain. We have not yet been able to reliably measure SAM or SAH in vascular tissue, so the potential importance of altered methylation to the vascular phenotype of Mtr+/ mice remains to be investigated.
Another methylation product that may influence vascular function during hyperhomocysteinemia is ADMA.44 ADMA is derived from protein methylation reactions that produce homocysteine as a byproduct, and plasma ADMA is elevated during hyperhomocysteinemia in monkeys and humans.44 The effects of hyperhomocysteinemia on ADMA metabolism may be complex, however. A homocysteine-induced decrease in methylation capacity might result in decreased production of ADMA, but homocysteine also may inhibit the hydrolysis of ADMA by dimethylarginine dimethylaminohydrolase.45 We observed only a small trend toward elevated plasma ADMA in Mtr+/+ and Mtr+/ mice fed the LF diet (Table 3), and hepatic levels of ADMA were not elevated in Mtr+/+ or Mtr+/ mice fed the LF diet.
The observation that responses to acetylcholine in aortic rings were unaffected by Mtr genotype or the LF diet is consistent with previous findings in murine models of mild hyperhomocysteinemia. In CBS-deficient mice, for example, endothelial function in the aorta was relatively normal when the concentration of plasma tHcy was <20 µmol/L but became impaired with higher concentrations of plasma tHcy.17,18 In comparison, endothelial dysfunction in the cerebral microcirculation of CBS-deficient mice occurred with very mild elevation of plasma tHcy (8 to 15 µmol/L).11 A similar relative insensitivity of the aorta compared with smaller vessels such as cerebral arterioles or mesenteric arterioles has been observed in MTHFR-deficient mice.10,20 Our present findings in MS-deficient mice provide additional support for the hypothesis that, compared with large conduit vessels such as the aorta, microvessels are hypersensitive to hyperhomocysteinemia-induced endothelial dysfunction.
In summary, we have demonstrated oxidative stress and endothelial dysfunction in cerebral microvessels of mice with genetic or diet-induced defects in homocysteine remethylation. The occurrence of cerebrovascular dysfunction without hyperhomocysteinemia in Mtr+/ mice fed the control diet may have important implications with regard to the mechanisms of stroke, dementia, and other cerebrovascular diseases that are associated with altered homocysteine metabolism. Our findings also may have implications for the ongoing clinical trials of folic acid supplementation to prevent cardiovascular disease and stroke.46
| Acknowledgments |
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A. M. Devlin, R. Singh, R. E. Wade, S. M. Innis, T. Bottiglieri, and S. R. Lentz Hypermethylation of Fads2 and Altered Hepatic Fatty Acid and Phospholipid Metabolism in Mice with Hyperhomocysteinemia J. Biol. Chem., December 21, 2007; 282(51): 37082 - 37090. [Abstract] [Full Text] [PDF] |
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A. Narayanasamy, B. Subramaniam, C. Karunakaran, P. Ranganathan, R. Sivaramakrishnan, T. Sharma, V. S. Badrinath, and J. Roy Hyperhomocysteinemia and Low Methionine Stress Are Risk Factors for Central Retinal Venous Occlusion in an Indian Population Invest. Ophthalmol. Vis. Sci., April 1, 2007; 48(4): 1441 - 1446. [Abstract] [Full Text] [PDF] |
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S. M. Colgan and R. C. Austin Homocysteinylation of Metallothionein Impairs Intracellular Redox Homeostasis: The Enemy Within! Arterioscler Thromb Vasc Biol, January 1, 2007; 27(1): 8 - 11. [Full Text] [PDF] |
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S. Dayal, K. M. Wilson, L. Leo, E. Arning, T. Bottiglieri, and S. R. Lentz Enhanced susceptibility to arterial thrombosis in a murine model of hyperhomocysteinemia Blood, October 1, 2006; 108(7): 2237 - 2243. [Abstract] [Full Text] [PDF] |
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F. M. Faraci Hydrogen peroxide: watery fuel for change in vascular biology. Arterioscler Thromb Vasc Biol, September 1, 2006; 26(9): 1931 - 1933. [Full Text] [PDF] |
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