Published online before print May 24, 2001, doi: 10.1161/hh1101.092180
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© 2001 American Heart Association, Inc.
Integrative Physiology |
Endothelial Dysfunction and Elevation of S-Adenosylhomocysteine in Cystathionine ß-Synthase–Deficient Mice
From the Departments of Internal Medicine (S.D., C.D.S., D.D.H., F.M.F., S.R.L.), Pharmacology (D.D.H., F.M.F.), and Physiology and Biophysics (C.D.S.), University of Iowa College of Medicine, Iowa City, Iowa; Veterans Affairs Medical Center (D.D.H., S.R.L.), Iowa City, Iowa; Baylor Institute of Metabolic Disease (T.B., E.A.), Dallas, Tex; Department of Pathology (N.M.), University of North Carolina, Chapel Hill, NC; Oregon Regional Primate Research Center (M.R.M.), Beaverton, Oreg.
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
Abstract—Hyperhomocysteinemia
is associated with increased risk for cardiovascular
events, but it is not certain whether it is a mediator of vascular
dysfunction or a marker for another risk factor. Homocysteine levels
are regulated by folate bioavailability and also by the methyl donor
S-adenosylmethionine (SAM) and
its metabolite
S-adenosylhomocysteine (SAH).
We tested the hypotheses that endothelial dysfunction
occurs in hyperhomocysteinemic mice in the absence of folate deficiency
and that levels of SAM and SAH are altered in mice with dysfunction.
Heterozygous cystathionine ß-synthase–deficient
(CBS+/–) and wild-type
(CBS+/+) mice were fed a folate-replete,
methionine-enriched diet. Plasma levels of total homocysteine were
elevated in CBS+/– mice compared with
CBS+/+ mice after 7 weeks (27.1±5.2 versus
8.8±1.1 µmol/L; P<0.001)
and 15 weeks (23.9±3.0 versus 13.0±2.3 µmol/L;
P<0.01). After 15 weeks, but
not 7 weeks, relaxation of aortic rings to acetylcholine was
selectively impaired by 35%
(P<0.05) and thrombomodulin
anticoagulant activity was decreased by 20%
(P<0.05) in
CBS+/– mice. Plasma levels of folate did
not differ between groups. Levels of SAH were elevated 
2-fold in
liver and brain of CBS+/– mice, and
correlations were observed between plasma total homocysteine and SAH in
liver (r=0.54;
P<0.001) and brain
(r=0.67;
P<0.001). These results
indicate that endothelial dysfunction occurs in
hyperhomocysteinemic mice even in the absence of folate deficiency.
Endothelial dysfunction in
CBS+/– mice was associated with increased
tissue levels of SAH, which suggests that altered SAM-dependent
methylation may contribute to vascular dysfunction in
hyperhomocysteinemia.
Key Words: acetylcholine • endothelium • homocysteine • methylation • thrombomodulin
Elevation of plasma concentration of total homocysteine (tHcy) is considered to be a clinical risk factor for cardiovascular disease.1 2 3 Mild or moderate hyperhomocysteinemia (plasma tHcy concentration of 15 to 50 µmol/L) is found in up to 40% of patients with myocardial infarction, stroke, or venous thrombosis.1 2 3 Although strong associations between hyperhomocysteinemia and cardiovascular events have been observed in many retrospective and some prospective studies, a few prospective studies have failed to confirm this association.2 One potential explanation for the inconsistent results of the prospective studies is that hyperhomocysteinemia itself may not be the causative agent in vascular dysfunction, but instead may be a marker for another risk factor.4 5 6
One factor that is often associated with
hyperhomocysteinemia is deficiency of folate, because folate is
required for efficient remethylation of homocysteine to methionine
(Figure 1
). In addition to causing hyperhomocysteinemia,
folate deficiency may impair endothelial function
through effects on endothelial nitric oxide synthase
(eNOS), a major mediator of endothelium-dependent
relaxation.7 In a previous
study,8 we examined the
influence of folate on endothelial function in mice
with heterozygous deficiency of the cystathionine ß-synthase (CBS)
gene. We found that both the plasma concentration of tHcy and the
severity of endothelial dysfunction in aorta were
strongly influenced by the folate content of the
diet.8 Whether
endothelial dysfunction in CBS-deficient mice was
caused by hyperhomocysteinemia, folate deficiency, or another factor
related to homocysteine metabolism was not
determined.
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Another proposed mechanism for endothelial
dysfunction in hyperhomocysteinemia is that hyperhomocysteinemia may be
a marker for altered methylation of cellular substrates that utilize
S-adenosylmethionine (SAM) as a
methyl donor.9 SAM serves as
a donor for methyl transfer reactions that produce a variety of
methylated products, including proteins, nucleic acids,
phospholipids, and other substrates
(Figure 1
). The major byproduct of these
methyltransferase reactions is
S-adenosylhomocysteine (SAH),
which undergoes hydrolysis to form homocysteine. SAH is a potent
inhibitor of
methyltransferases,10 which
suggests that elevation of SAH in hyperhomocysteinemia could lead to
inhibition of methylation reactions in endothelial
cells. In support of this hypothesis, incubation of cultured
endothelial cells with exogenous homocysteine was found
to increase intracellular levels of SAH and inhibit methylation of
p21ras, resulting in decreased cellular
proliferation.11 The
influence of hyperhomocysteinemia on tissue levels of SAH has not been
well characterized, however, and it is not known whether elevated
levels of SAH are associated with endothelial
dysfunction.
In the present study, we have utilized a folate-replete, methionine-enriched diet with CBS-deficient mice to ask two related questions. First, does endothelial dysfunction occur in hyperhomocysteinemic mice in the absence of folate deficiency? Second, are tissue levels of SAM and SAH altered in hyperhomocysteinemic mice with endothelial dysfunction? Our results indicate that endothelial dysfunction does occur during chronic hyperhomocysteinemia in mice with normal folate status. Our findings also demonstrate that hyperhomocysteinemic mice with endothelial dysfunction have elevated levels of SAH and a decreased SAM/SAH ratio. These findings are compatible with the hypothesis that altered SAM-dependent methylation may contribute to endothelial dysfunction in hyperhomocysteinemia.
Materials and Methods
Mice and Experimental Protocol
To minimize the potential influence of differences in
genetic background, CBS-deficient
mice12 were crossbred to
C57BL/6J mice (The Jackson Laboratory, Bar
Harbor, Maine) for at least eight generations, and comparisons
were performed between heterozygous CBS-deficient
(CBS+/–) mice and wild-type
(CBS+/+) littermates. Homozygous
CBS-deficient mice were not studied because they exhibit growth
retardation, hepatic dysfunction, and shortened survival. Genotyping
for the targeted CBS allele was performed by polymerase chain
reaction.12 At the time of
weaning, mice were fed LM-485 chow (Harlan
Teklad), which contains 7.5 mg/kg folic acid and 4.0 g/kg
L-methionine, and were
provided with drinking water that was either unsupplemented (control)
or supplemented with 0.5%
L-methionine (high
methionine). The terms "control diet" and "high-methionine
diet" refer to the combination of solid chow and drinking water
consumed by each group. After either 7 or 15 weeks of experimental
diet, mice were euthanized with sodium pentobarbital (75 mg IP). The
breeding schedule was designed to allow for the study of two mice
(littermates) of the appropriate age on each study day. For the 7-week
time point, the ages of the mice ranged from 9 to 10 weeks, with mean
values of 6.7 to 6.8 weeks on experimental diet. For the 15-week time
point, the ages of the mice ranged from 17 to 20 weeks, with mean
values of 14.8 to 15.1 weeks on experimental diet. Blood was collected
in EDTA (final concentration 5 mmol/L) for measurement of plasma
levels of tHcy, methionine, and folate, and the thoracic aorta, lung,
liver, and brain were removed for ex vivo studies. The experimental
protocol was approved by the University of Iowa and Veterans Affairs
Animal Care and Use Committees.
Vasomotor Responses
After removal of loose connective tissue, the
proximal 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 using
the thromboxane analogue U46619, and relaxation
dose-response curves were generated by cumulative addition of the
endothelium-dependent vasodilator acetylcholine or the
endothelium-independent vasodilators sodium
nitroprusside or papaverine, as described
previously.8 We have used
these methods previously and demonstrated that responses to
acetylcholine in mouse aorta are mediated by
eNOS.13 14 15
Thrombomodulin Activity
Thrombomodulin activity (thrombomodulin-dependent
activation of protein C) was measured using a two-stage assay described
previously.8 16
Activity was measured in rings of proximal thoracic aorta 1.0 mm
in length or in lysates of lung tissue prepared by
homogenization in 0.02 mol/L Tris-HCl and 0.1 mol/L
NaCl (pH 8). Reference curves were generated using rabbit lung
thrombomodulin (American
Diagnostica). One unit of activity was defined
as the amount of activated protein C generated in the presence
of 1.0 nmol/L rabbit thrombomodulin. The thrombomodulin activity in
lung homogenates was expressed as units/mg of total
protein.
Other Assays
Plasma tHcy, defined as the total concentration of
homocysteine after quantitative reductive cleavage of all disulfide
bonds,17 was measured by
high-performance liquid chromatography and
electrochemical detection as described
previously.18 19
Plasma levels of methionine were measured by high-performance
liquid chromatography coupled to coulometric
electrochemical detection. The method was modified from that described
by Martin et al20 by
increasing the electrochemical potential to +1100 mV to detect
methionine. Plasma levels of folate were measured by an automated
chemiluminescence immunoassay (Chiron
Diagnostics).
Levels of SAM and SAH in liver and brain tissue were measured by high-performance liquid chromatography coupled to a UV detector as described previously.21 Tissues were collected in ice-cold perchloric acid (0.4 mol/L) immediately after the mice were euthanized, and samples were deproteinized by homogenization and centrifugation within 1 hour. The clear supernatant fraction was stored at -80°C until the time of analysis.
Statistical Analysis
Comparisons between genotypes
(CBS+/+ versus
CBS+/– mice) or between diets (control diet
versus high-methionine diet) were performed within each age group using
the unpaired, two-tailed Student’s
t test. Responses to
vasodilators in aorta were analyzed using two-way
repeated-measures ANOVA with Bonferroni multiple-comparison
analysis at specific concentrations of vasodilator. Correlation
coefficients were calculated using the Pearson method. A value of
P<0.05 was used to define
statistical significance. Values are reported as
mean±SE.
Results
In a previous study, moderate hyperhomocysteinemia
(plasma tHcy 25 µmol/L) was produced by feeding heterozygous
CBS-deficient mice a diet that was deficient in
folate.8 To produce
hyperhomocysteinemia without folate deficiency, mice in the present
study were fed a folate-replete diet that contained supplemental
methionine in drinking water. Beginning at the time of weaning (3
weeks of age), CBS+/– and
CBS+/+ littermates were fed either the
control diet or the high-methionine diet for up to 15 weeks. All mice
appeared to be healthy, and body weight did not differ between
CBS+/– and
CBS+/+ mice or between mice fed the control
and high-methionine diets (not shown).
Plasma Levels of tHcy, Methionine, and
Folate
After 7 weeks, plasma tHcy was mildly elevated in
CBS+/– mice fed the control diet
(P<0.01 versus
CBS+/+ mice) and markedly elevated in
CBS+/– mice fed the high-methionine diet
(P<0.001 versus
CBS+/+ mice)
(Table 1). Similar differences in levels of plasma tHcy
between CBS+/– and
CBS+/+ mice were observed after 15 weeks
(Table 1). Plasma methionine concentration tended to be
higher in mice fed the high-methionine diet than in mice fed the
control diet and higher in CBS+/– mice than
in CBS+/+ mice, but these differences did
not reach statistical significance
(Table 1). Plasma folate did not differ between
groups.
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Vasomotor Responses in Aorta
Acetylcholine, nitroprusside, and papaverine each
produced dose-dependent relaxation of aortic rings. After 7 weeks,
despite marked differences in plasma tHcy, no differences in relaxation
to acetylcholine or nitroprusside were observed between
CBS+/+ and
CBS+/– mice fed either diet (not shown).
After 15 weeks, relaxation to acetylcholine did not differ
significantly between CBS+/– and
CBS+/+ mice fed the control diet
(Figure 2A) but was impaired in
CBS+/– mice compared with
CBS+/+ mice fed the high-methionine diet
(Figure 2B). Maximal relaxation to the highest dose of
acetylcholine was 90±3% in CBS+/+ mice and
73±8% in CBS+/– mice
(P<0.05). No differences in
relaxation to nitroprusside
(Figures 2C and 2D) or papaverine (not shown) were observed
between CBS+/+ and
CBS+/– mice fed either diet. Vessels from
all groups of mice contracted similarly to the thromboxane
analogue U46619 (not shown).
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, CBS+/– mice (n=10 to 11). *P<0.05 vs CBS+/+ mice.
Thrombomodulin Activity
Thrombomodulin anticoagulant activity was measured in
aortic arch and lung. After 7 weeks, no differences in thrombomodulin
activity were observed between CBS+/+ and
CBS+/– mice fed either diet (not shown).
After 15 weeks, thrombomodulin activity in aortic arch was 20%
lower in CBS+/– mice compared with
CBS+/+ mice fed the control diet
(Figure 3A). Similar decreases (20% to 25%) in
thrombomodulin activity in aortic arch also were observed in
CBS+/– or CBS+/+
mice fed the high-methionine diet compared with
CBS+/+ mice fed the control diet
(Figure 3A). No differences in thrombomodulin activity in
lung were observed between CBS+/+ and
CBS+/– mice fed either diet
(Figure 3B).
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Tissue Levels of SAM and SAH
After 7 weeks, levels of SAM in liver did not differ
significantly between groups, but levels of SAH in liver were elevated
2-fold in CBS+/– mice fed the
high-methionine diet compared with CBS+/+
mice fed the control diet
(P<0.001)
(Table 2). CBS+/– mice fed the
high-methionine diet also had a lower SAM/SAH ratio in liver compared
with CBS+/+ mice fed the control diet
(P<0.05). After 15 weeks,
levels of SAH in liver were elevated and SAM/SAH ratio was decreased in
all three groups with elevated plasma tHcy
(CBS+/+ mice fed the high-methionine diet,
CBS+/– mice fed the control diet, and
CBS+/– mice fed the high-methionine diet)
(Table 2).
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Levels of SAM and SAH also were measured in brain. Compared
with levels in liver, levels of SAM in brain were 2- to 4-fold lower,
levels of SAH were 10- to 15-fold lower, and SAM/SAH ratio was 3- to
5-fold higher
(Table 3). After 7 weeks, levels of SAH in brain were
elevated in CBS+/– mice fed the
high-methionine diet (P<0.05).
These mice also had elevated levels of SAM in brain
(P<0.05). After 15 weeks,
levels of SAM in brain no longer differed between groups, but levels of
SAH remained elevated in CBS+/– mice fed
the high-methionine diet compared with
CBS+/+ mice fed the control diet
(P<0.05). Like SAM/SAH ratio
in liver, SAM/SAH ratio in brain was decreased in all three groups with
elevated plasma tHcy.
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When data from all mice were analyzed, strong
correlations were observed between plasma tHcy and SAH in liver
(r=0.54;
P<0.001) and plasma tHcy and
SAH in brain (r=0.67;
P<0.001)
(Figure 4). Plasma tHcy correlated inversely with SAM/SAH
ratio in liver (r=-0.27;
P<0.05) and SAM/SAH ratio in
brain (r=-0.31;
P<0.01).
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, CBS+/+ mice fed the high-methionine diet; 
, CBS+/– mice fed the high-methionine diet.Discussion
By providing supplemental methionine to
CBS+/– mice, we have produced moderate
hyperhomocysteinemia (plasma tHcy 25 µmol/L) in the absence of
folate deficiency. Hyperhomocysteinemic mice had normal vascular
responses after 7 weeks but developed impaired
endothelium-dependent relaxation and decreased
thrombomodulin anticoagulant activity in aorta after 15 weeks. This
finding demonstrates that endothelial dysfunction can
occur during hyperhomocysteinemia in mice, even in the absence of
folate deficiency. Another major finding of this study is that
endothelial dysfunction in
CBS+/– mice was associated not only with
elevation of plasma tHcy but also with elevation of SAH and decreased
SAM/SAH ratio in liver and brain. This finding suggests a possible role
for altered SAM-dependent methylation reactions in the vascular
pathology of hyperhomocysteinemia.
Endothelial dysfunction has been observed in
animal models of experimental hyperhomocysteinemia in several species,
including nonhuman
primates16 and
rats.22 23 24
In a previous study, we found that
endothelium-dependent relaxation in aorta was preserved
in CBS+/– mice with normal folate levels
and very mild hyperhomocysteinemia (plasma tHcy 6 µmol/L) but
impaired in folate-deficient CBS+/– mice
with higher levels of plasma tHcy (25
mmol/L).8 Abnormal
endothelial function in heterozygous CBS-deficient mice
also was reported independently by Eberhardt et
al,25 who detected evidence
for endothelial dysfunction in aorta and mesenteric
microvessels in CBS+/– mice with mild
hyperhomocysteinemia (plasma tHcy 9 to 10 µmol/L). One potential
explanation for the greater impairment of responses in the studies of
Eberhardt et al than in our studies is that the diets may have
contained different amounts of folate, methionine, or other
constituents that influence tHcy levels. Plasma levels of folate were
not reported by Eberhardt et al.
The precise mechanisms by which hyperhomocysteinemia produces endothelial dysfunction are incompletely defined. Impairment of endothelium-dependent relaxation in hyperhomocysteinemia appears to be mediated in part by decreased bioavailability of endothelium-derived nitric oxide, which may be caused either by decreased production or increased degradation of nitric oxide.26 Homocysteine-induced oxidative inactivation of nitric oxide has been observed in studies of cultured endothelial cells,27 and indirect evidence for increased oxidative inactivation of nitric oxide during hyperhomocysteinemia has been obtained in some studies in animals.23 In support of this mechanism, CBS-deficient mice have been reported to exhibit increased generation of superoxide anion in aorta.25 In our study, vasodilator responses to nitroprusside were normal in CBS+/– mice fed the high-methionine diet, which suggests that the sensitivity of soluble guanylyl cyclase in vascular smooth muscle to nitric oxide was relatively normal. Normal responses to nitroprusside also were observed in previous studies of CBS-deficient mice.8 25
Expression of eNOS does not appear to be decreased during hyperhomocysteinemia,25 but production of nitric oxide by eNOS may be limited by endogenous inhibitors such as asymmetric dimethylarginine (ADMA).28 29 ADMA is derived from the hydrolysis of proteins that are methylated on arginine residues by SAM-dependent protein arginine methyl transferases.30 It has been proposed, therefore, that hyperhomocysteinemia due to methionine loading may increase production of ADMA through increased SAM-dependent arginine methylation.29 Alternatively, hyperhomocysteinemia may produce elevation of ADMA by inhibiting its metabolism by the enzyme dimethylarginine dimethylaminohydrolase.30 31 Our observations in hyperhomocysteinemic mice are consistent with the latter mechanism, because we observed elevation of SAH without elevation of SAM, and chronically elevated levels of SAH would be expected to inhibit most methyltransferases.32 The effect of hyperhomocysteinemia on the specific protein arginine methyltransferase that produces ADMA is not known, however, so this question will require further study.
In comparison with our previous findings in CBS+/– mice fed a low-folate diet,8 CBS+/– mice fed high-methionine had almost identical levels of plasma tHcy but differed in the duration of hyperhomocysteinemia required to produce endothelial dysfunction. When fed a low-folate diet, CBS+/– mice developed marked impairment to endothelium-dependent vasodilators in aorta after 6 to 7 weeks.8 In the present study, when CBS+/– mice were fed a high-methionine diet, aortic responses to endothelium-dependent vasodilators were preserved after 7 weeks but became impaired after 15 weeks. These findings suggest that the vascular phenotype of hyperhomocysteinemic mice is dependent on both the duration of hyperhomocysteinemia and the underlying defect in homocysteine metabolism.
The more rapid onset of endothelial dysfunction in CBS+/– mice with folate deficiency compared with CBS+/– mice given supplemental methionine suggests that the homocysteine remethylation pathway may be particularly important for maintaining normal vasomotor responses during hyperhomocysteinemia. Endothelial cells appear to lack both CBS activity and betaine-homocysteine methyltransferase and therefore must rely on the folate-dependent enzyme methionine synthase for homocysteine remethylation.6 For this reason, endothelial cells may be particularly sensitive to folate deficiency, and intracellular levels of homocysteine may increase disproportionately in endothelial cells compared with liver when mice are fed a folate-deficient diet. This effect may be exacerbated in CBS-deficient mice, because increased plasma tHcy (derived from liver and other tissues) may lead to increased uptake of Hcy by endothelial cells. Alternatively, effects of folate deficiency unrelated to hyperhomocysteinemia, such as direct impairment of endothelial nitric oxide production7 may have contributed to endothelial dysfunction in CBS+/– mice fed the low-folate diet.
Another potential mechanism by which vascular function may be impaired in hyperhomocysteinemia is through altered methylation of cellular substrates that utilize SAM as a methyl donor.9 An inverse correlation between hepatic SAM concentration and plasma tHcy has been observed in folate-deficient rats33 and in mice deficient in methylenetetrahydrofolate reductase.32 Incubation of cultured endothelial cells with exogenous homocysteine increases intracellular levels of SAH and inhibits carboxymethylation of p21ras, resulting in decreased cellular proliferation.11 In uremic patients, hyperhomocysteinemia is associated with increased levels of SAH and decreased protein methylation in erythrocytes.34 A recent study in healthy young women demonstrated that elevation of plasma tHcy was associated with elevation of SAH in plasma and lymphocytes and with hypomethylation of lymphocyte DNA.35
Our findings in CBS-deficient mice are concordant with these clinical observations, as we found that hyperhomocysteinemic mice with endothelial dysfunction had increased tissue levels of SAH in liver and brain. For technical reasons, we were unable to measure levels of SAH in aorta in mice. Nevertheless, our findings are compatible with the hypothesis that altered SAM-dependent methylation may contribute to vascular dysfunction in hyperhomocysteinemia. For example, hypomethylation of DNA may alter the expression of genes encoding antioxidant enzymes such as catalase.36 Interestingly, in a recent case-control study, plasma SAH was found to be a more sensitive marker of cardiovascular disease than plasma tHcy.37 This observation raises the question of whether elevation of SAH, rather than elevation of tHcy, is a risk factor for cardiovascular events. If confirmed in larger studies, this finding would lend further credence to the hypothesis9 that inhibition of methyltransferases by SAH may be a causative factor in vascular disease. It is important to note, however, that neither SAH nor tHcy correlated perfectly with the presence of endothelial dysfunction in CBS+/– mice, and additional studies will be necessary to directly test the hypothesis that elevation of tHcy, through its effects on SAH, is a marker of altered methylation reactions in endothelial cells.
CBS+/– mice fed the high-methionine diet for 15 weeks not only had impaired endothelium-dependent vasomotor function but also had decreased thrombomodulin anticoagulant activity. Thrombomodulin activity was decreased in aorta but not in lung. Thrombomodulin activity can be inhibited by exogenous homocysteine in cultured endothelial cells,38 39 40 and we have observed decreased thrombomodulin activity in carotid artery and aorta of monkeys with moderate hyperhomocysteinemia.16 Clinical evidence for impairment of thrombomodulin-dependent activation of protein C in hyperhomocysteinemia has been unconvincing, however.41 Our observations in monkeys and mice suggest that thrombomodulin may be more susceptible to hyperhomocysteinemia in large conduit vessels such as aorta than in small vessels such as those found in the pulmonary microvasculature. One potential mechanism for the differential effects of hyperhomocysteinemia on thrombomodulin activity in large and small vessels is that oxidative stress may be more prominent in large vessels. Thrombomodulin activity is possibly inhibited through oxidative mechanisms in vivo, because thrombomodulin contains a critical oxidation-sensitive methionine residue near its thrombin binding domain.42 Another possible mechanism is that hyperhomocysteinemia may alter the expression or activity of the endothelial cell protein C receptor, which is expressed mainly on large vessels.43
In summary, moderate hyperhomocysteinemia was produced in CBS-deficient mice with normal folate status. In comparison with previous findings in CBS+/– mice with folate deficiency,8 a longer duration of hyperhomocysteinemia was needed to produce endothelial dysfunction in aorta in CBS+/– mice without folate deficiency. The development of endothelial dysfunction in mice was associated with increased levels of SAH and decreased SAM/SAH ratio in liver and brain. These findings provide support for the hypothesis that, in addition to hyperhomocysteinemia itself, related factors such as folate deficiency and altered SAM-dependent methylation reactions may contribute to vascular dysfunction. Additional studies will be needed to identify specific methylation products that may be altered in hyperhomocysteinemia.>
Acknowledgments
This work was supported by National Institutes of Health Grants HL63943, HL07344, DK25295, and NS24621, the Office of Research and Development, Department of Veterans Affairs, and an American Heart Association-Bugher Foundation Award. Genotyping was performed by Lucinda Robbins, Norma Sinclair, and Patricia Lovell in the University of Iowa Transgenic Facility under the direction of Curt D. Sigmund. We thank Kara Brown for technical assistance.
Footnotes
Original received December 6, 2000; revision received April 30, 2001; accepted April 30, 2001.
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