Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi 39216
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ABSTRACT |
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To test the hypothesis that
endothelial dysfunction in hyperhomocysteinemia was due to increased
levels of nitrotyrosine and matrix metalloproteinase (MMP) activity in
response to antagonism of peroxisome proliferator-activated
receptor- (PPAR-
), cystathionine
-synthase (CBS)
/+
mice were bred, tail tissue was analyzed for genotype by PCR, and tail
vein blood was analyzed for homocysteine (Hcy) by spectrofluorometry.
To induce PPAR-
, mice were administered 8 µg/ml of ciprofibrate
(CF) and grouped: 1) wild type (WT), 2) WT + CF, 3) CBS, 4) CBS + CF (n = 6 in each
group). In these four groups of mice, plasma Hcy was 3.0 ± 0.2, 2.5 ± 1.2, 15.2 ± 2.6 (P < 0.05 compared
with WT), 11.0 ± 2.9 µmol/l. Mouse urinary protein was 110 ± 11, 86 ± 6, 179 ± 13, 127 ± 9
µg · day
1 · kg
1
by Bio-Rad dye binding assay. Aortic nitrotyrosine was 0.099 ± 0.012, 0.024 ± 0.004, 0.132 ± 0.024 (P < 0.01 compared with WT), 0.05 ± 0.01 (scan unit) by Western
analysis. MMP-2 activity was 0.053 ± 0.010, 0.024 ± 0.002, 0.039 ± 0.009, 0.017 ± 0.006 (scan unit) by zymography.
MMP-9 was specifically induced in CBS
/+ mice and inhibited by CF
treatment. Systolic blood pressure (SPB) was 90 ± 2, 88 ± 16, 104 ± 8 (P < 0.05 compared with WT), 96 ± 3 mmHg. Aortic wall stress
[(SPB · radius2/wall
thickness)/2(radius + wall thickness)] was 10.2 ± 1.9, 9.7 ± 0.2, 16.6 ± 0.8 (P < 0.05 compared
with WT), 13.1 ± 2.1 dyn/cm2. The results suggest
that Hcy increased aortic wall stress by increasing nitrotyrosine and
MMP-9 activity.
extracellular matrix; matrix metalloproteinase; tissue inhibitor of
metalloproteinase; collagen; elastin; cystathionine -synthase; nitric oxide; arteriosclerosis; fibrate
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INTRODUCTION |
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HOMOCYSTEINE (Hcy) is
involved in nucleic acid methylation (50). There are four
ways by which hyperhomocysteinemia (HHcy) is developed: 1)
by methionine-rich protein diet, 2) vitamin
B12/folate deficiency, 3)
heterozygous/homozygous in cystathione -synthase (CBS) activity and
B6 deficiency, and 4) renovascular stenosis and
volume retention. Although the treatment of vitamin
B12/folate reduced the level of plasma Hcy and ameliorated
vascular dysfunction, in part, by conversion to methionine
(37), the mechanism of other causes of HHcy is unclear. In
human, heterozygosity (
/+) in CBS activity was associated with HHcy
and increased oxidative stress (8). In CBS
/+ mice, Hcy
induced endothelial dysfunction (11). Hcy caused vascular
disease (20), including arteriosclerosis (43), endothelial cell desquamation (40),
thromboresistance (23), smooth muscle cell proliferation
(44), collagen synthesis (44), oxidation of
low-density lipoprotein (17), increased monocyte adhesion
to the vessel wall (21), platelet aggregation (7), coagulation (33), blood rheology
(25), and activation of plasminogen and matrix
metalloproteinase (MMP) (16). Hcy decreased endothelial
nitric oxide (NO) concentration and promoted the formation of
nitrotyrosine in the vessel wall (29). Hcy induced
oxidative stress (2) and activated NF-
B
(5). A negative correlation between high Hcy level and
peroxisome proliferator-activated receptor (PPAR) expression has been
suggested (6). The agonists of PPAR attenuated oxidative
stress-mediated vascular dysfunction (12) and hypertension
(34). In addition, PPAR promoted the synthesis of
superoxide dismutase (SOD) and catalase (18) and decreased
NADPH oxidase (18). Although Hcy induced constrictive vascular remodeling and PPAR agonists ameliorated remodeling in vitro
(30), the increased oxidative stress was associated with increased MMP activity (45), and the agonists of PPAR
decreased oxidative stress and MMP activity in macrophages
(24). It was unclear whether PPAR ameliorated Hcy-mediated
MMP activation in vivo. We hypothesized that Hcy downregulated
PPAR-
, thereby causing oxidation of NO to nitrotyrosine with
consequent increased activation of MMP. MMP-induced changes in vascular
wall structure, in turn, may alter wall stress and hemodynamics.
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MATERIALS AND METHODS |
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Animal model.
A breeding pair of mice heterozygous for the CBS gene
(C57BL/6J-Cbs tm1Unc) was obtained from Jackson Laboratories
and bred at the mice breeding facility of the University of Mississippi Medical Center. CBS /+ was created by inactivating CBS gene using homologous recombination in C57BL/6J mouse embryonic stem cells by
disrupting the coding sequence of the CBS gene with the Neo gene as
described (49). Homozygous
/
CBS mice do not survive past 2-3 wk. However, the heterozygous (
/+) CBS-deficient mice and their wild-type (+/+) littermate controls survive normally. At this
age, CBS
/+ mice develop homocysteinemia. To minimize sex
differences, we performed all experiments in male mice. Animal room
temperature is maintained between 22 and 24°C. A 12-h light-dark cycle was maintained by artificial illumination. In accordance with the
National Institutes of Health Guidelines for animal research, all
animal procedures are reviewed and approved by the Institutional Animal
Care and Use Committee of the University of Mississippi Medical Center,
Jackson. To induce PPAR-
, we administered ciprofibrate (CF; Sigma)
to CBS
/+ and wild-type +/+ mice at 40 µg/day in drinking water.
The mice were grouped as follows: 1) wild type, 2) wild type plus CF, 3) CBS, and 4)
CBS plus CF. To determine selectivity of CF in the absence of Hcy, we
administered CF to wild-type mice. In humans, 100 mg/day of CF has a
potent effect (12). On the basis of the fact that the
binding constant between CF and PPAR is in the micromolar range
(31), 8 µg/ml of CF were administered to mice in
drinking water. Because mice continuously drink and excrete ~5 ml of
water/day, each ingests 2 mg · kg
1 · day
1
CF. This produced a blood concentration of ~32 µmol/l, enough to
saturate most binding sites on PPAR. The animals were fed standard chow
and water ad libitum. To determine whether treatment with CF caused any
change in food and water intake, we measured food and water every 2 days during the treatment period. There was no difference in food and
water intake in either group. Because previous studies have
demonstrated significant vascular dysfunction at 12 wk of
homocysteinemia (26), CF was administered for 12 wk.
Genotype and phenotype determination.
The tail vein blood and tissue from offspring at the age of ~8 wk
weighing ~20 ± 3 g were collected and analyzed for
1) genomic DNA by PCR using specific CBS primers
(49) and 2) the levels of plasma Hcy. The DNA
was extracted and amplified by PCR for sequences in intron 3 and Neo
insert in CBS /+ mice (49). The PCR primers used were:
5'-GCCTCTGTCTGCTAACCTA-3' and 5'-GAGGTCGACGGTATCGATA-3' (49). On the basis of the genotype and the levels of
plasma Hcy, the mice were categorized as wild type (+/+) and CBS (
/+) (49). This produced wild-type littermates from the same
breed of mice.
Aortic morphology and in situ MMP activity.
The aorta was stained with trichrome and van Gieson for collagen and
elastin as described (46). The intima-media thickness was
measured by a digital micrometer. To determine total MMP activity in
the aortas of CBS /+ and +/+ mice, we performed in situ zymography as
described (46). Briefly, freshly isolated aortic segments were laid onto gelatin-gel preequilibrated with Triton X-100 and incubation buffer. The gels after 18 h were stained for lytic activity with Coomassie blue.
Plasma Hcy and urinary protein. Hcy was measured by a modification of a procedure by Frantzen et al. (13). Briefly, the plasma was reduced by trace amounts of reduced glutathione. The Hcy was converted to S-adenosyl-L-homocysteine (SAH) by incubating the plasma with SAH hydrolase (Sigma) and adenosine. The fluorescence of the incubate SAH was measured 422 nm when excited at 320 nm. The free nucleotides do not fluoresce. The adenosine and hydrolase were used in reference buffer. The standards of SAH (Sigma) were prepared. Because mouse urine had large quantities of proteins [primary mouse major urinary protein (MUP)] and males had much higher urinary MUP than females, to collect the urine, we caged male mice in 24-h metabolic cages for several days of acclimatization to reduce separation effects before hemodynamic measurements. MUP was measured by Bio-Rad dye binding assay.
Hemodynamic measurements. Mice were anesthetized with tribromoethanol (100 mg/kg ip). This drug had minimal effect on cardiovascular function in mice (32). The aortic blood pressure (BP), heart rate, and systolic (SBP) and diastolic blood pressure were measured by a PE-10 catheter in the aorta through right common carotid artery (27). The catheter was connected to a pressure transducer (Micro-Med) positioned at the level of heart. Pulsatile arterial pressure signal was analyzed by a computer using customized software (Micro-Med). To determine plasma Hcy and to ensure mouse-to-mouse variation, we collected 0.5 ml of blood from each mouse by the same catheter.
Tissue processing. Under deep anesthesia, the mice were euthanized by arresting the heart in diastole with injections of 0.2 ml/100 g body wt (iv) of a 20% KCl solution. The thoracic aorta was dissected. Aortic tissue homogenates were prepared (48). For PPAR measurements, aortic nuclear extracts were prepared (47). Bio-Rad dye binding assay was applied to estimate total protein concentration in the tissue extracts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with and without the reduction.
PPAR, nitrotyrosine, and -actin.
The levels of nitrotyrosine and PPAR were measured by Western blot
analysis using mouse monoclonal anti-nitrotyrosine antibody (Upstate
Biotechnology) and mouse polyclonal anti-PPAR antibody (Calbiochem).
The 10% SDS-PAGE at 20-mA/gel was carried out. The samples were
prepared in reducing buffer. PPAR antibody recognized both
- and
-isoforms. We established the specificity of antibodies by
immunoprecipitating the antigen before loading them onto the gel, by
antibody conjugated-agarose beads (Upstate Biotechnology). To determine
whether total protein loaded onto the gel was identical, we performed
-actin Western blots using anti-
-actin antibody (Sigma). Alkaline
phosphatase-conjugated secondary antibody was used as the detection
system. Bands on blots were scanned by a Bio-Rad GS-700 densitometer.
Zymographic analysis of MMP activity.
To determine MMP-2 and -9 activity, we performed gelatin substrate gel
zymography containing 1% gelatin in 8% SDS-PAGE (46). The aortic tissue homogenates were loaded onto the gel under identical condition of total protein. The scanned band intensity was normalized by -actin.
Preparation of Hcy, acetylcholine, nitroprusside, and CF
solutions.
The concentration of Hcy was determined by spectrophometric titration
with dithio-bis-nitrobenzoate (absorption measured at 412 nm) using
412 nm of 13,600 M
1cm
1
(48). Concentrations of acetylcholine, nitroprusside, and
CF were based on weight measurements. All dilutions from stock
solutions were made before the experiment. Buffer was used as vehicle control.
Aortic ring preparation and vascular contractility. The aortic function was measured as described (29). The lumen diameter was measured by a micrometer. The wall thickness was measured by putting micrometer edges on both sides of the aortic wall. The wall stress was measured as follows: (SBP · radius2/wall thickness)/2(radius + wall thickness). The ring was aerated with 95% O2 and 5% CO2 (pH 7.4) and equilibrated at 37°C continuously. Aortic rings were perfused with physiological salt solution containing (in mM) 131.5 NaCl, 0.2 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 0.5 CaCl2, 23.5 NaHCO3, and 11.2 glucose. The rings were mounted in between two stainless steel wires, one connected to a force transducer and the other connected to a micrometer, in an isometric myograph (World Precision Instrument). The signal from the ring under experimentation was digitalized by on-line analysis using CVMS software (World Precision Instrument). To evaluate the viability of aortic ring, we contracted the ring three times by inducing active muscle tone using 20 mM CaCl2, rinsed, and reequilibrated before experiment. To accommodate for different sizes and lengths of different rings, we normalized the generated tension in grams with the weight of the tissue in grams (38). Precontracted rings with CaCl2 were relaxed by endothelium-dependent acetylcholine and endothelium-independent nitroprusside. The optimum dose, an effective concentration (EC50), was measured by a dose-dependence curve between amplitude of contraction and added concentrations of vasoactive agents. We measured the levels of PPAR, nitrotyrosine, and MMP activity in aortic segments after function measurements and observed similar results before and after.
Statistical analysis.
Values are given as means ± SE from n = 6 in each
group. Differences between groups were evaluated by using ANOVA,
followed by the Bonferroni post hoc test (42), focusing on
the effects of /+ CBS (+/+ mice to
/+ mice) and treatment (
/+
mice treated with CF compared with
/+ mice). P < 0.05 was considered significant.
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RESULTS |
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Genotype/phenotype of CBS mice.
PCR analysis of genomic DNA revealed one PCR product of 1.5 kb of a
normal allele and two products of normal and homologous disrupted
alleles with different electrophoretic mobility in /+ alleles. There
were two groups of mice: one with Hcy levels between 3 and 6 µM and
the other with Hcy levels between 8 and 15 µM. On the basis of
genotype and the levels of Hcy, mice were divided into wild-type (+/+)
littermates and heterozygous (
/+) CBS mice, respectively.
Morphology and in situ MMP activity.
Histological analysis revealed increased collagen and decreased
elastin in aortic media of CBS /+ mice compared with wild-type mice
(Fig. 1). There was significant MMP
activity in the aortas of
/+ compared with +/+ mice. In +/+
mice, only adventitia regions of the aortas showed high MMP activity,
whereas in
/+ mice, the adventitia, media, and lumen showed high MMP
activity (Fig. 2).
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|
BP in CBS /+ mice.
SBP was 104 ± 8 mmHg in CBS
/+ mice and 90 ± 2 mmHg in
wild-type +/+ mice (Table 1). CF
treatment in CBS
/+ mice for 12 wk decreased the SBP by 10 mmHg
(Table 1).
|
Proteinuresis.
CBS /+ mice demonstrated an increase in protein excretion in
urine compared with wild-type +/+ mice. Treatment with CF decreased the
levels of MUP in CBS
/+ mice to control levels (Table 1).
Plasma Hcy.
The levels of plasma Hcy in CBS /+ mice did not decrease to the
control levels after CF treatment (Table 1).
Levels of PPAR.
To determine whether CF had classical biological effect, such as liver
proliferation, we measured liver wt/body wt ratios. The results
suggested reduced liver size in CBS /+ mice. Treatment with CF
increased liver wt-to-body wt ratios (Table 1). CF tended to induce
both PPAR-
and -
(Fig. 3).
|
CF inhibits nitrotyrosine generation.
The levels of nitrotyrosine were increased in CBS /+ mice
compared with wild-type +/+ littermates (P < 0.01). Treatment with CF inhibited the generation of nitrotyrosine in
both the
/+ as well as wild-type +/+ controls (Fig.
4).
|
Activation of MMP-2 and -9 in
/+ CBS mice.
Zymographic analysis revealed increased MMP-9 activity in CBS mice.
Treatment with CF completely inhibited the MMP-9 and decreased MMP-2
activity by 50% in
/+ as well as wild-type +/+ control mice (Fig.
5).
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Improvement of aortic function by CF.
Aortic response to acetylcholine was attenuated in CBS /+ mice
compared with wild-type +/+ mice. The dose-response curves were shifted
to the left in CBS
/+ mice after CF treatment compared with CBS
/+
mice (Fig. 6). These results suggest that
treatment with CF improved vascular function in CBS
/+ mice.
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DISCUSSION |
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A linear relationship between SBP and plasma levels of Hcy has
been suggested in elderly patients (41). Some studies have also linked PPAR to hypertension (1, 10) and suggested
that peroxisome proliferator fibrates may improve hemodynamic
(34) and renal parameters (14, 15). We
suggest that in the absence of PPAR response, reactive oxygen species
generated by Hcy, in part, induced hypertension by increasing vessel
wall stress in CBS /+ mice. The administration of Hcy in rats
increased BP (26). The changes in BP by Hcy were
independent of amount of Hcy but dependent on the duration, suggesting
structural changes by Hcy (26). Histological analysis
revealed that aortic intima-media was thickened in the CBS
/+ mice
compared with controls (Table 1 and Fig. 1). Similar results were
obtained by others (9). The thickening of aortas was
associated with increased MMP activity (Fig. 2). Others have shown that
CF inhibits the synthesis of the ECM component fibrinogen
(19) and that the agonist of PPAR causes regression of
intima-media thickness in humans (28). The ECM,
particularly elastin (50% of the normal vessel wall), surrounding the
vascular smooth muscle cell enhances muscle cell compliance with load.
Elastin is a good substrate for MMP-2 and -9 (38). To
reduce vessel wall stress, especially in the absence of endothelial NO
and HHcy, the MMP is activated to dilate the vessel by remodeling and
degrading elastin. Because elastin turnover is remarkably lower than
collagen (36), the degraded elastin is replaced by stiffer
collagen, and the Hcy induces collagen synthesis (44).
Therefore, vessel wall stress is increased (Table 1). Previously we
quantified the decreased elastin and increased collagen in
homocysteinemic aortas (29). Here we demonstrated increased collagen and decreased elastin in aortas of HHcy mice.
Treatment with peroxisome proliferator decreased lipid profile
(35). A number of studies have demonstrated that the
levels of Hcy in fasting and loading conditions are not changed after fibrate therapy. In fact, some studies reported a slight increase (3). Here is the paradox: drugs that repair vascular
endothelial function may increase Hcy accumulation. Our results suggest
that plasma Hcy levels were not altered by fibrate treatment in CBS /+ mice (Table 1). In addition, the levels of PPAR were increased after fibrate treatment (Fig. 3), suggesting that CF induced PPAR expression.
Hcy induced multiorgan damage (26) and increased the MUP
in CBS /+ mice (Table 1). CF treatment reduced the injury response by
Hcy (Table 1). Others have shown that treatment with fibrate decreases
renovascular resistance and hypertension (15). The mouse
model of abrogation of endothelial NO generation has suggested an
increase in BP (39). Hcy decreased bioavailability of
endothelial NO. The increase in Hcy in CBS
/+ mice was associated
with the increase in SBP (Table 1). The treatment with Hcy induced both nitrotyrosine and MMP-2 and -9 activity (29). Treatment
with nicotinamide, an inhibitor of poly(ADP-ribose)synthetase, an
enzyme that can be activated by oxidants and peroxynitrite
(4), inhibited the Hcy-mediated nitrotyrosine generation
and MMP activation (29). Nicotinamide also ameliorated
Hcy-mediated vascular dysfunction (29). Here we report
that the activation of MMP and treatment with CF decreased
nitrotyrosine levels and MMP activity, respectively (Figs. 4 and 5).
Numerous studies have demonstrated impairment of vasodilatory response
of aortas after acute treatment with Hcy. In CBS /+ mice, the
response to acetylcholine was attenuated (9). Folic acid
regressed the Hcy-mediated vascular dysfunction in CBS
/+ mice
(22). CF has been shown to improve the oxidative
stress-mediated vascular dysfunction (12). Our results
suggest that CF reversed Hcy-mediated vascular dysfunction in CBS
/+
mice (Fig. 6), in part by decreasing nitrotyrosine and MMP activity.
Under normal conditions, NO binds to the metal ion and keeps the MMP in
latent form. However, in HHcy, NO favors peroxynitrite formation and nitrates the neighboring tyrosine residues. Consequently, the levels of
peroxynitrite were increased and generated nitrotyrosine. Also it is
known that Hcy induced endothelial nitric oxide synthase as well as
superoxide (2). It is a paradox that Hcy may increase both
NO and nitrotyrosine. However, this can be explained by the scenario
that, during HHcy, Hcy may promote NO generation. The increased
oxidative stress may favor nitration and promote nitrotyrosine. The
decrease in nitrotyrosine in CF-treated mice may be due, in part, to
the fact that PPAR increased antioxidant enzymes such as SOD/catalase
and decreases NADPH oxidase (18). Collectively, these
studies suggest a role of both the superoxide and NO in generation of
nitrotyrosine. Although we have not established a cause and
effect relationship between PPAR expression and MMP activity, previous
studies by us and others have suggested decreased MMP activity after CF
treatment (24, 30). The results of this study suggest that
Hcy downregulated PPAR-
, thereby causing oxidation of NO to
nitrotyrosine with consequent increased activation of MMP. MMP-induced
changes in vascular wall structure, in turn, may alter wall stress and hemodynamics.
Limitations.
The goal of this study was to test whether the high MMP and
nitrotyrosine arterial levels in HHcy animals are secondary to Hcy-induced downregulation of PPAR-. We have shown that CF, a PPAR-
agonist, increased PPAR expression in HHcy mouse aorta while
reducing aortic nitrotyrosine and MMP levels. However, MMP and
nitrotyrosine levels were also downregulated by CF in wild-type mice, a
phenotype in which CF did not upregulate PPAR expression. We speculated
that this phenotype may be due to a threshold level of PPAR in normal
mice. It is possible that CF had other effects on vascular muscle,
perhaps inducing transcription of other enzymes. Indeed, other effects
of CF are likely another possible explanation for the observation that
CF downregulated MMP activity and nitrotyrosine levels but not PPAR
expression, especially in wild-type mice. There was a basal level of
nitrotyrosine in the aorta of wild-type +/+ mice. However, it was lower
than in
/+ mice (Fig. 4). Similar levels have been reported by others
(9). Treatment with CF decreased nitrotyrosine levels in
CBS
/+ mice. Collectively, these studies suggest that Hcy interferes
with NO signaling via the cGMP pathway, which may impact remodeling,
structure, and function in the vessel wall.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institutes of Health Grants GM-48595 and HL-71010 and by the Kidney Care Foundation.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. C. Tyagi, Dept. of Physiology and Biophysics, Univ. of Mississippi Medical Center, 2500 No. State St., Jackson, MS 39216-4505 (E-mail: styagi{at}physiology.umsmed.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published October 25, 2002;10.1152/ajplung.00183.2002
Received 7 June 2002; accepted in final form 4 October 2002.
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