Circulation Research. 2001
Published online before print May 24, 2001,
doi: 10.1161/hh1101.092180
A more recent version of this article appeared on June 8, 2001
(Circulation Research. 2001;0:hh1101.092180.)
© 2001 American Heart Association, Inc.
Endothelial Dysfunction and Elevation of S-Adenosylhomocysteine in Cystathionine ß-Synthase–Deficient Mice
Sanjana Dayal,
Teodoro Bottiglieri,
Erland Arning,
Nobuyo Maeda,
M. René Malinow,
Curt D. Sigmund,
Donald D. Heistad,
Frank M. Faraci
Steven R. Lentz
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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Figure 1. Homocysteine metabolism in CBS-deficient mice. Homocysteine is produced from dietary methionine through the intermediates SAM and SAH. Genetic deficiency of CBS decreases conversion of homocysteine to cystathionine. Remethylation of homocysteine to methionine requires 5-methyltetrahydrofolate (5-methyl THF). SAM serves as a methyl donor for methyl transfer reactions. In liver and kidney, some homocysteine is remethylated to methionine through an alternative pathway that utilizes betaine (not shown).
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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.
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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Figure 2. Relaxation of aorta to acetylcholine or nitroprusside in mice fed a control diet (A and C) or a high-methionine diet (High Met; B and D) for 15 weeks. •, CBS+/+ mice (n=8 to 14);  , CBS+/– mice (n=10 to 11). *P<0.05 vs CBS+/+ mice.
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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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Figure 3. Thrombomodulin activity in aorta (A) or lung (B) of mice fed a control diet or a high-methionine diet (High Met) for 15 weeks. White bars, CBS+/+ mice (n=8 to 14); black bars, CBS+/– mice (n=10 to 11). *P<0.05 vs CBS+/+ mice fed control diet.
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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).
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.
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).
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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S. J. James, S. Melnyk, M. Pogribna, I. P. Pogribny, and M. A. Caudill
Elevation in S-Adenosylhomocysteine and DNA Hypomethylation: Potential Epigenetic Mechanism for Homocysteine-Related Pathology
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[Abstract]
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J. D. Symons, A. E. Mullick, J. L. Ensunsa, A. A. Ma, and J. C. Rutledge
Hyperhomocysteinemia Evoked by Folate Depletion: Effects on Coronary and Carotid Arterial Function
Arterioscler Thromb Vasc Biol,
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[Abstract]
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Z. Bagi, Z. Ungvari, and A. Koller
Xanthine Oxidase-Derived Reactive Oxygen Species Convert Flow-Induced Arteriolar Dilation to Constriction in Hyperhomocysteinemia: Possible Role of Peroxynitrite
Arterioscler Thromb Vasc Biol,
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[Abstract]
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, CBS+/+ mice fed the high-methionine diet; 
, CBS+/– mice fed the high-methionine diet.
