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Atherosclerosis and Lipoproteins |
From the Division of Internal Medicine and Cardiovascular Diseases (S.H.W., R.D.S., P.J.M.B., T.E.P., D.R.H., A.L.) and the Division of Hypertension (L.O.L., K.A.N.), Mayo Clinic and Foundation, Rochester, Minn, and the Lipid Research Laboratory (M.A.), Rappaport Family Institute for Research in the Medical Sciences, Haifa, Israel.
Correspondence to Amir Lerman, MD, Division of Cardiovascular Diseases, Mayo Clinic, 200 First St SW, Rochester, MN 55905. E-mail lerman.amir{at}mayo.edu
| Abstract |
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Key Words: hypercholesterolemia nitric oxide vasorelaxation endothelial NO synthase
| Introduction |
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Recent evidence has suggested that the HRIs may have important therapeutic effects in hypercholesterolemia (HC), in the absence of cholesterol lowering. First, in vitro studies have shown that the HRIs directly upregulate endothelial NO synthase (eNOS), the constitutive enzyme catalyzing the synthesis of NO in the vasculature.6 Furthermore, a lipid-independent decrease in superoxide formation by macrophages has been demonstrated, consistent with an antioxidant effect.7 However, there is a paucity of data regarding the effects of the HRIs in hypercholesterolemia, in the absence of lipid lowering, in vivo.
Experimental porcine HC is characterized by a decrease in NO bioavailability, with downregulation of eNOS in association with increased production of oxygen-derived free radicals that may inactivate NO.8 9 Thus, a decrease in NO production and an increase in its breakdown may contribute to abnormal endothelium-dependent vasorelaxation. We hypothesized that the HRIs may have direct beneficial effects on NO bioavailability and oxidative stress in vivo. Thus, we designed the present study to determine the effects of simvastatin, an HRI, on large- and small-vessel coronary endothelial function, coronary eNOS, cGMP production, and oxidative stress in the setting of experimental HC in the absence of cholesterol lowering.
| Methods |
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Lipid Parameters
Plasma total, LDL, and HDL cholesterol
levels were measured after completion of 12 weeks of therapy. Plasma
total and LDL cholesterol levels were determined by
applying the technique of Allain et
al,13 with use of a
commercial reagent (Roche).
In Vitro Determination of Vascular
Reactivity
Epicardial Vessels
The method has been previously described in
detail.14 In brief, segments
of the left circumflex artery 2 to 3 mm long were dissected. To
determine the endothelium-dependent vasorelaxation
properties of epicardial arteries, the arteries were first contracted
with 10-7 mol/L endothelin-1 (Phoenix) and
then relaxed with cumulative concentrations of bradykinin
(10-11 to 10-6
mol/L, Sigma Chemical Co), substance P
(10-11 to 10-8
mol/L, Sigma), and calcium ionophore (10-11
to 10-8 mol/L, Sigma). At the end of these
experiments, 10-3.5 mol/L papaverine
(Sigma) was added to determine the maximal vasodilatory capacity of the
vessel.
Arterioles
Coronary vasomotor tone in the arterioles was
determined by using a previously described
method.15 In brief, segments
(2 to 3 mm long) of the secondary branch of the left circumflex
artery (<500 µm in diameter) were dissected with use of a dissecting
microscope. The vessels were precontracted with
10-8 mol/L endothelin-1, and then the
response to 10-11 to
10-6 mol/L bradykinin was
recorded.
In addition, epicardial vessels and arterioles were preincubated with NG-monomethyl-L-arginine (L-NMMA, 10-4 mol/L, Sigma), the NO synthase inhibitor, for 20 minutes before the addition of endothelium-dependent agents to assess the contribution of endogenous NO release in vasorelaxation.
The contraction attained with endothelin-1 for each vessel at baseline was considered as 0% relaxation. Subsequent measurements of coronary artery relaxation were expressed as a percent reduction in contraction (with the maximal relaxation attained with papaverine being 100% relaxation). At least 5 rings were used in each group of experiments, and no more than 2 rings from each animal were used per group of experiments.
Western Blotting for eNOS
Arteries from 3 pigs in each experimental group were
snap-frozen in liquid nitrogen and subsequently homogenized
in lysis buffer (50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl,
0.02% sodium azide, 0.1% SDS, 100 µg/mL phenylmethylsulfonyl
fluoride, 1 µg/mL aprotinin, 1% NP-40, and 0.5% sodium
deoxycholate) by use of a tissue homogenizer. The
lysate was analyzed for protein content by a Bradford assay
(Bio-Rad). Equal amounts of protein were resolved under reducing
conditions on an 8% SDS-polyacrylamide gel.
Immunoblotting was performed with use of a monoclonal
antibody to eNOS (Signal Transduction Laboratories) at a dilution of
1:1000 in a nonfat milk/Tris buffer. The membrane was subsequently
probed with a secondary anti-mouse antibody conjugated to horseradish
peroxidase (Amersham Life Sciences) at a dilution of 1:5000 and
developed with chemiluminescence (Pierce). The membrane was then
exposed to x-ray film (Kodak), which was subsequently developed.
Densitometry was performed by use of NIH image.
Immunohistochemistry for eNOS
One section taken from the proximal segment of the
circumflex artery was analyzed from 8 animals in each group, as
previously described.8 The
sections were mounted, dried, and fixed. Endogenous
peroxidase activity was blocked by incubating slides in 1.5% hydrogen
peroxide/50% absolute methanol for 10 minutes. The slides were then
placed in 0.25% SDS for 10 minutes at room temperature. Nonspecific
protein binding sites were blocked with 5% goat serum diluted in
PBS/0.05% Tween 20 at room temperature for 10 minutes. A monoclonal
antibody to eNOS was then used (Signal Transduction Laboratories). This
antibody was diluted to 1:500 in 1% goat serum and PBS/0.05% Tween 20
and applied to the slides overnight at 4°C. A biotinylated secondary
antibody was then applied (mouse IgG) at a 1:400 dilution for 30
minutes with 1% normal goat serum and 2% normal swine serum. The
slides were subsequently incubated for 30 minutes with
streptavidinhorseradish peroxidase (Dako, 1:500 dilution). After a
further washing, color development was performed with the use of
3-amino-9-ethylcarbazole substrate solution for 15 minutes at room
temperature. A counterstain was then performed with the use of
hematoxylin for 30 seconds. The slides were then rinsed for 5 minutes
in running tap water and mounted in aqueous glycerol gelatin
media.
cGMP Production
Vascular rings were collected from 10 animals in each
experimental group, and the endothelium was manually
removed by rolling the vessel with wire. The rings were then placed in
an organ chamber filled with Krebs solution. After 1 hour of
incubation, 146 µL of 3-isobutyl-1-methylxanthine
(10-4 mol/L) and 100 µL of
indomethacin (10-5 mol/L)
were added to the solution for 30 minutes. Samples were then randomized
to either control or the NO donor, diethylamine NONOate (DEA)
(10-6 mol/L), for 1 minute and then
shock-frozen.
Homogenization was carried out on dry ice with the addition of 2 mL of absolute alcohol. After being allowed to sit for 5 minutes, the samples were centrifuged for 10 minutes at 4000 rpm at 10°C. Samples were dried, and 500 µL of Tris buffer was added, after which they were centrifuged as before and placed on ice. After the preparation of standards, 50 µL of radioactive dye and 50 µL of antiserum were subsequently added to both samples and standards. After vortex mixing, tubes were placed on ice and refrigerated for 90 minutes, ammonium sulfate was added, and samples were allowed to sit for 5 minutes. The samples were subsequently centrifuged for 10 minutes at 4000 rpm at 10°C. Supernatant was discarded, 1.1 mL of water added, and the samples were vortexed again. Finally, samples were placed in a scintillation counter.
Plasma Lipid Peroxidation
Plasma
F2-Isoprostanes
Blood samples were taken from each group after 12
weeks of the experiment. Samples were collected in EDTA tubes, and the
plasma was stored at -80°C until the time of the assay. The total
levels of 8-isoprostaglandin F2
were measured with an enzyme immunoassay kit (EIA, Cayman). Before the
enzyme immunoassay, an alkaline hydrolysis was
used.16 Plasma samples were
purified by Sep-Pak C-18 columns (Milford) before analysis. The
samples, tracer, and antiserum were added to wells precoated with mouse
monoclonal antibody. The plates were washed to remove all unbound
reagents. EllmanÕs reagent (containing the substrate to
acetylcholinesterase) was added to the wells. Spectrophotometric
analysis was performed at 405 nm.
TBARS Assay
Samples were taken from 6 pigs in each group. The
extent of plasma lipid peroxidation was measured directly in the medium
by the thiobarbituric acidreactive substances (TBARS) assay, with the
use of malondialdehyde (MDA) for preparation of a standard
curve.17
Catalase and Glutathione Peroxidase
Assays
For measurement of glutathione peroxidase, a
previously described method18
was used. In brief, coronary artery samples from each
experimental group (n=6) were homogenized in 5 mL of 0.25
mol/L sucrose by using a Polytron homogenizer
(Brinkmann) for 15 seconds. The homogenates were
centrifuged for 1 hour at
105 000g, and the supernatant
was diluted 1:10. The diluted supernatant (0.1 mL) was added to 0.8 mL
of mixture consisting of 50 mmol/L potassium phosphate (pH 7.0),
1 mmol/L EDTA, 1 mmol/L NaN3, 0.2
mmol/L NADPH, 1 enzyme unit per milliliter of glutathione disulfide
reductase, and 1 mmol/L glutathione, and the reaction mixture was
allowed to incubate for 5 minutes. Hydrogen peroxide (0.1 mL of
0.25 mmol/L) was added to 0.9 mL of the incubated sample. The
absorbance at 340 nm was followed for 5 minutes, and the activity of
glutathione peroxidase was calculated by subtracting the activity of
the blank from the activity of each sample. The millimolar absorptivity
for NADPH at 340 nm is 6.22. All measurements were performed in
duplicate. For measurement of catalase activity, the method of
Aebi et al was used as previously
described.19
Statistical Analysis
Data are expressed as mean±SEM. In vitro vascular
reactivity was analyzed by a 2-way ANOVA, followed by a
Bonferroni t test. TBARS,
F2-isoprostanes, catalase, glutathione
peroxidase, cGMP, and eNOS protein levels were analyzed by a
1-way ANOVA, followed by a Bonferroni
t test. Statistical
significance was inferred at a value of
P<0.05.
| Results |
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Plasma HMGCoA Reductase Activity
HMGCoA reductase activity was measured in 6 pigs in the
HC and HC+S groups. There was no detectable activity in any of the HC
group, but there was detectable activity in all of the 6 HC+S pigs
measured (median 0.7, interquartile range 0.5 to 1.0;
P<0.005 between
groups).
In Vitro Determination of Vascular
Reactivity
Epicardial Vessels
The mean vasorelaxation to bradykinin was significantly
attenuated in the HC group compared with the N group (32.3±1.2%
versus 42.9±1.6%, P<0.0001;
Figure 1
). There was increased vasorelaxation to bradykinin
in the HC+S group compared with the HC group (38.7±1.5%,
P<0.005 versus HC group;
Figure 1
). There was no difference in the precontraction to
endothelin-1 between the groups. The vasorelaxation response to
bradykinin in all groups was significantly attenuated by preincubation
of the vessels with the NO synthase inhibitor L-NMMA
(P<0.0001 in all cases). In
addition, the enhanced vasorelaxation response to bradykinin in the N
and HC+S groups compared with the HC group was abolished by
preincubation of the vessels with L-NMMA.
|
The maximal vasorelaxation to substance P was significantly
attenuated in the HC group compared with the N group (50.5±11.9%
versus 79.3±5.3%, respectively;
P<0.05). This attenuated
response was normalized in the HC+S group (74.9±4.0%,
P<0.05 versus HC group;
Figure 2
). In addition, although there was no significant
attenuation of vasorelaxation to another
endothelium-dependent agent, calcium ionophore, in the
HC group compared with the N group, there was enhanced vasorelaxation
in the HC+S group (40.0±1.3%) compared with the HC group
(31.8±1.3%, P<0.05). There
was no attenuation of the vasorelaxation to sodium nitroprusside, an
endothelium-independent agent, in the HC compared with
the N group.
|
Arterioles
The maximal arteriolar vasorelaxation to bradykinin was
also significantly attenuated in the HC pigs compared with the N pigs
(71.9±4.9% versus 96.8±1.34%, respectively;
P<0.005). This was completely
reversed in the HC+S group (98.4±0.6%,
P<0.0001 versus HC group;
Figure 3
). The difference between the HC+S and HC groups was
abolished by preincubation of the HC+S vessels with
L-NMMA.
|
Immunohistochemistry for eNOS
Immunoreactivity for eNOS was present in an intact
endothelial cell layer in the N, HC, and HC+S groups,
by use of labeling with the monoclonal antibody to eNOS
(Figure 4
). Western blotting was used to quantify the amount
of eNOS present in the 3 groups.
|
Western Blotting
Western blotting is shown in
Figure 4
. eNOS protein was present in the vessel wall in
all 3 homogenates from the N group and was significantly
decreased in the HC group
(P<0.05). eNOS protein was
also present in all 3 of the coronary tissue
homogenates from the HC+S group and was significantly
increased compared with that in the HC group
(P<0.05). Ponceau staining
confirmed equal protein loading between lanes.
cGMP Levels
Baseline generation of cGMP was the same in all 3
groups (P=NS). However, there
was significantly greater generation of cGMP in response to DEA in the
HC group compared with the N group (99.2±10.9 versus 67.1±3.7 pmol/mg
protein, P<0.05). This was
completely reversed after treatment with simvastatin
(60.2±4.5 pmol/mg protein,
P<0.05).
Plasma Lipid Peroxidation
Plasma
F2-Isoprostanes
There was a significant increase in the plasma
F2-isoprostane levels in the HC group (n=14)
compared with the N group (n=10; 203.4±12.1 versus 124.3±16.0 pg/mL,
respectively; P<0.005). There
was a significant decrease in the levels in the HC+S group (n=9)
compared with the HC group (150.7±12.1 pg/mL,
P<0.05;
Table
).
There was no significant difference in levels between the HC+S and N
groups.
TBARS Assay
With use of the TBARS assay, the HC group had a
significantly higher level of aldehydes compared with the N group
(6.2±0.2 versus 4.5±0.3 nmol MDA equivalent per milliliter,
P<0.005; both n=6). This high
plasma lipid peroxidation in the HC group was significantly decreased
by treatment with simvastatin (3.9±0.1 nmol MDA equivalent
per milliliter, P<0.0001; n=6;
Table
).
There was no significant difference in levels between the HC+S group
and the N group.
Catalase and Glutathione Peroxidase
Assay
There was a decrease in the catalase levels in the HC
group compared with the N group (6.0±0.4 versus 7.3±0.8 µmol
H2O2 consumption per
minute per milliliter), which was reversed in the HC+S group (7.5±0.5
µmol H2O2 consumption
per minute per milliliter). However these changes did not reach
statistical significance
(P=0.14). There was no
significant difference between the glutathione peroxidase levels in the
3 groups (see
Table
).
| Discussion |
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One of the beneficial effects of the HRIs is a restoration of endothelial function.20 21 Multiple studies in the setting of HC have demonstrated an improvement in endothelium-dependent vasodilation after treatment,3 4 22 23 suggesting an increase in NO bioavailability. This improvement has been attributed primarily to the ability of these agents to inhibit cholesterol biosynthesis by blocking the conversion of HMGCoA to mevalonate, with a subsequent increment in LDL receptor activity, resulting in a reduction in plasma cholesterol levels. Increasing evidence is emerging that the HRIs may have benefits independent of their lipid-lowering effects. In support of this, some HC studies have shown no correlation between the extent of lipid lowering by the HRIs and improvement in endothelial function.22 In addition, a recent study in primates showed that pravastatin, in combination with a lipid-lowering diet, improved acetylcholine-mediated vasodilatation in vivo more than a lipid-lowering diet alone, despite no incremental decrease in plasma lipids.23 We have previously demonstrated that porcine experimental HC is characterized by a decrease in coronary eNOS immunoreactivity and decreased endogenous NO activity.8 In the present study, we have provided multiple lines of evidence that suggest a functional increase in NO via a direct simvastatin effect. Bradykinin, substance P, and calcium ionophore lead to vasorelaxation via different endothelial receptors, but the release of NO is involved in the action of each.24 25 Furthermore, when vessels were preincubated with L-NMMA, an NO synthase inhibitor, the enhanced vasorelaxation response to bradykinin in the simvastatin-treated group was abolished. In contrast to previous animal and human data, a recent study suggested that patients with only modestly elevated cholesterol levels did not have improvement in endothelial function after treatment with a statin.26 It may be that significantly elevated cholesterol levels need to be present at baseline to benefit from either the lipid-lowering or direct effect of the statins.
The abnormal endothelial function seen with HC also occurs in the microcirculation.27 28 Impaired vasodilation of the microcirculation may lead to myocardial perfusion abnormalities and ischemia, even without obstructive coronary disease.29 In addition, an improvement in myocardial ischemia has been demonstrated after treatment with HRIs,30 31 out of proportion with alterations in epicardial artery narrowing. However, these improvements have thus far been demonstrated only in the presence of significant plasma cholesterol lowering. We have shown a significant improvement in microvascular endothelial function after treatment with simvastatin, again despite no changes in plasma cholesterol levels. The enhanced response to bradykinin was more prominent in the microcirculation than the epicardial vessels, although this enhanced response was blocked by preincubation with L-NMMA, again suggesting a role for NO. This may have important implications for myocardial perfusion, because treatment with HRIs, even in the absence of cholesterol lowering, may lead to a decrease in the potential for myocardial ischemia.
The underlying mechanisms leading to an increase in NO bioavailability by simvastatin may be multifactorial. In vitro data have strongly suggested that the HRIs have a direct endothelial effect, preventing downregulation of eNOS protein and mRNA by oxidized LDL.5 32 The present study extends these observations and demonstrates in vivo upregulation of eNOS protein levels after simvastatin treatment in experimental HC, independent of lipid lowering. The HRIs, in addition to playing a pivotal role in alteration in cholesterol synthesis, are known to have an impact on other metabolites of the mevalonate pathway. In vitro studies have suggested a role for these metabolites in the regulation of eNOS expression,33 although the in vivo mechanism remains to be elucidated. Clearly, the upregulation of eNOS demonstrated in the present study could have significant impact on endothelial NO production.
Previous studies have demonstrated that removal of the endothelial NO synthesis pathway by denuding the endothelium results in an augmented response and increased sensitivity of the smooth muscle to NO donors.34 We have previously reported an increase in production in cGMP in the vascular smooth muscle in response to the NO donor DEA in HC in the renal vasculature.35A In the present study, we have confirmed these findings in the coronary vasculature. Furthermore, this augmented production of cGMP found in the HC group was completely reversed after treatment with simvastatin. This is consistent with simvastatin treatment leading to a chronic increase in endothelial NO bioavailability, resulting in downregulation of the smooth muscle cell responsiveness to this increased production.
Although a direct increase in eNOS protein levels and
increased production of NO may contribute to the improvement in
endothelial function seen with the HRIs, other
mechanisms may be involved. As an alternative or perhaps additional
mechanism, simvastatin may attenuate the degradation of NO
by reducing oxidative stress. The overproduction of reactive
oxygen species has previously been demonstrated in experimental
HC9 with subsequent enhanced
LDL oxidation. The HRIs have been shown to attenuate oxidative stress
in the context of lipid lowering, with decreased levels of lipid
peroxides35B and attenuation
of the response of LDL to oxidation in
vitro.36 Recent in vitro
studies have demonstrated a lipid-independent decrease in superoxide
formation by macrophages after HRI treatment,
consistent with an antioxidant
effect.7 We have now reported,
for the first time in vivo, a decrease in 2 independent
parameters of oxidative stress (isoprostanes and aldehydes)
after treatment with simvastatin, despite no reduction in
total plasma or LDL cholesterol levels. One of these
parameters, 8-epiprostaglandin
F2
, is one of a novel family of
prostaglandin isomers, the
F2-isoprostanes, which have recently been
described as a reliable measure of in vivo oxidative
stress.37 The decrease in
oxidative stress was not due to an increase in the antioxidant defense
enzymes, glutathione peroxidase or catalase. However we cannot rule out
the possibility that other mechanisms may be involved, including
alterations in the enzyme, superoxide dismutase. Although the decrease
in oxidative stress after simvastatin treatment may lead to
a diminution of NO inactivation by free radicals with enhanced NO
bioavailability and improved endothelium-dependent
vasodilatation, it is also possible that normalized production
of NO by simvastatin may itself lead to inactivation of
oxygen-derived free radicals and reduced oxidative
stress.38
In summary, the present study demonstrates that simvastatin, an HRI, preserves endothelial function in large and small coronary vessels, despite no change in plasma lipid concentrations. This increase in NO activity, involving an upregulation of eNOS and a reduction in oxidative stress, may mediate altered platelet aggregation, smooth muscle proliferation, and endothelium-leukocyte interactions, potentially regulating the atherogenic process. These alterations may play a role in the reduction of mortality and major morbidity that occurs with the HRIs beyond their effect on lipid lowering.
| Acknowledgments |
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Received September 22, 2000; accepted September 27, 2000.
| References |
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