© 2001 American Heart Association, Inc.
Vascular Biology |
HMG-CoA Reductase Inhibitors Prevent Migration of Human Coronary Smooth Muscle Cells Through Suppression of Increase in Oxidative Stress
From the Department of Cardiology, Graduate School of Medicine, Osaka City University, Osaka, Japan.
Correspondence to Kenichi Yasunari, MD, Department of Cardiology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. E-mail yasunari{at}osaka.med.or.jp
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Abstract |
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Abstract—In vitro and in vivo evidence of a decrease in vascular smooth muscle cell (SMC) migration induced by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has been reported. When added to SMC cultures for 6 hours, the HMG-CoA reductase inhibitors fluvastatin, simvastatin, and pravastatin at 1 µmol/L resulted in a 48%, 50%, and 16% suppression, respectively, of human coronary SMC migration; these reductions mirrored the suppression in oxidative stress induced by 1 µmol/L lysophosphatidylcholine (lyso-PC) of 50%, 53% and 19%, respectively. The hydroxylated metabolites of fluvastatin, M2 and M3, at 1 µmol/L also suppressed the enhancement of SMC migration by 58% and 45% and the increase in oxidative stress induced by lyso-PC of 58% and 49%, respectively. Lyso-PC activated phospholipase D and protein kinase C (PKC), and this activation was also suppressed by HMG-CoA reductase inhibitors. The inhibition of phospholipase D and PKC was reversed by 100 µmol/L mevalonate, its isoprenoid derivative, farnesol, and geranylgeraniol but not by 10 µmol/L squalene. Antisense oligodeoxynucleotides at 5 µmol/L to PKC-
,
but not those to the PKC-ß isoform, suppressed the lyso-PC–mediated
increases in SMC migration and oxidative stress. These findings suggest
that HMG-CoA reductase inhibitors have direct antimigratory
effects on the vascular wall beyond their effects on plasma lipids and
that they might exert such antimigratory effects via suppression of the
phospholipase D– and PKC (possibly PKC-)-induced increase in
oxidative stress, which might in turn prevent significant
coronary artery disease.
Key Words: lipids • atherosclerosis • smooth muscle • coronary disease
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Introduction |
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Clinical and experimental studies have shown that a reduction of plasma cholesterol, particularly LDL cholesterol, reduces the risk of cardiovascular events in both primary and secondary prevention.1 2 The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins), which are cholesterol-reducing drugs, can achieve a relatively large reduction in plasma cholesterol.3 The beneficial effects of HMG-CoA reductase inhibitors are usually assumed to result from their ability to reduce cholesterol synthesis.4 However, a variety of experimental findings suggest that statins can interfere with major events involved in the formation of atherosclerotic lesions, independent of their hypocholesterolemic properties.4 5 6
Vascular smooth muscle cell (SMC) migration in the arterial wall is an important mechanism in atherogenesis and is a possible determinant of restenosis after angioplasty.7 In addition, increased oxidative stress is reported to play an important role in SMC migration,8 suggesting a relation between oxidative stress and SMC migration. Because there exists in vitro and in vivo evidence of decreased SMC proliferation after administration of fluvastatin, simvastatin, or lovastatin,6 9 it is possible that statins have the potential to decrease oxidative stress and SMC migration.
Therefore, we examined whether suppression of lysophosphatidylcholine (lyso-PC), a major component of oxidized LDL–mediated SMC migration and oxidative stress, could be assessed after administration of therapeutic concentrations (1 µmol/L) of statins. We also examined the effects of the hydroxylated metabolites of fluvastatin, 5-hydroxyfluvastatin (M2) and 6-hydroxyfluvastatin (M3), which exist in plasma,10 on the increase in SMC migration and oxidative stress induced by lyso-PC.
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Methods |
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Cell Culture
Human coronary artery SMCs were cultured in smooth muscle basal medium (SMBM, Clonetics) containing human epidermal growth factor (0.5 ng/mL), human fibroblast growth factor (2 ng/mL), 5% fetal calf serum, 50 mg/mL gentamicin sulfate, and 50 mg/mL amphotericin B, as previously described.11 Subconfluent SMCs between the fourth and eighth passages were used for experiments.
Migration Assay
Migration of SMCs was assayed by a modification of
the Boyden chamber method with the use of microchemotaxis chambers
(Neuro Probe Inc) and polycarbonate filters (Nucleopore Corp) with
pores 12 µm in diameter, as previously
reported.12 A 200-µL SMC
suspension (
3.0x104 cells) was placed in
the upper chamber, and 40 µL of SMBM containing a migration factor
(such as lyso-PC) in the presence or absence of statins, a
phospholipase D (PLD) inhibitor, or a protein kinase C
(PKC) inhibitor was placed in the lower chamber. The
chamber was incubated at 37°C under 5% CO2 in
air for 6 hours. Migration activity was expressed as the number of
cells that had migrated in 4 high-power fields (HPF;
x400).
Assay of Intracellular Oxidative Stress
Intracellular oxidative stress was measured with a
fluorescent dye, carboxydichlorofluorescin diacetate bis-AM
ester (Molecular Probe Corp), a nonpolar compound that is converted to
a nonfluorescent polar derivative by cellular esterases after
its incorporation into cells. After 5 minutes of incubation at room
temperature, the fluorescence intensity of each point was
measured by flow cytometry as previously
reported.13
HMG-CoA Reductase Assay
The cell pellet was homogenized at 4°C
in a Dounce homogenizer in 10 mmol/L potassium
phosphate buffer (pH 7.4), 1 mmol/L EDTA, and 30 mmol/L
nicotinamide. Aliquots of the
15 000g supernatant were used
for the HMG-CoA reductase assay according to the method of Brown and
Goldstein14 for cultured
cells. The enzyme reaction was linear with time up to 60 minutes and
for protein concentrations up to 0.5 mg. Protein content was measured
by the Bradfod
method.15
PLD Activity Measured by Ethanolamine
Release
SMCs in 35-mm dishes were cultured in medium
containing [3H]ethanolamine (5 µCi/mL
per dish) for 24 hours (the latter half of the 48-hour period with
0.1% fetal calf serum) to label cellular phosphatidylethanolamine.
After a 0.5- to 1-hour incubation, the reaction was terminated by
adding 0.75 mL methanol. The cells were harvested by gentle scraping.
Fractionation of ethanolamine metabolites from the aqueous phase was
performed on Dowex 50W (H+) packed columns
as previously
described.13
Cell Fractionation and Assay of PKC
SMCs were cultured in medium with 0.1% fetal calf
serum for 48 hours. Cell fractionation was performed as previously
reported.13 PKC activity was
measured by a modification of a method with the Amersham PKC assay
system (Amersham Japan
Corp).13
Fluorescence Microscopy
SMCs were visualized through a fluorescence
microscope (Olympus IX70, x400 water-immersion objective lens)
equipped with a camera (Olympus PM-C 35DX). An automatic gain-control
mode in the camera allowed suitable transmission images to be obtained
with low-level light. To elicit fluorescent images, the
preparation was illuminated with a 200-W mercury lamp. The light was
passed through a quartz collector, heat filter, and an excitation
filter to epi-illuminate the preparation. Fluorescence emission
from the sample was passed through a band-pass filter (515 nm) and into
the camera.
Antisense
Oligonucleotides
Phosphothioate-modified
oligodeoxynucleotides for the PKC- and PKC-ß isoforms
were designed as reported by Li et
al16 and purified by
high-performance liquid chromatography by Japan
Bio Service Co: antisense PKC-, 5'-CGC CGT GGA GTC GTT GCC CG-3';
sense PKC-, 5'-CGG GCA ACG ACT CCA CGG CG-3'; antisense PKC-ß,
5'-AGC GCA CGG TGC TCT CCT CG-3'; and sense PKC-ß, 5'-CGA GGA GAG CAC
CGT GCG CT-3'. These oligodeoxynucleotides were added at a
concentration of 5 µmol/L to serum-free SMBM 24 hours before the
start of cell stimulation with platelet-derived growth factor-BB
(PDGF-BB), with transfection by use of a cationic compound.
Lipofectin reagent (Gibco BRL) and oligonucleotides
were effectively taken up by SMCs.
Immunoblotting
SMCs grown on a 6-well plate were stimulated with
agonists at 37°C in serum-free SMBM for specified durations. After
brief sonication (5 seconds), the samples were boiled for 5 minutes at
95°C and centrifuged (14 000g, 5 minutes) at 4°C, and the
supernatant (25 µL) was subjected to SDS–polyacrylamide gel
electrophoresis. Proteins in the gel were transferred to a
polyvinylidene difluoride membrane by electroblotting. The
membrane was treated with rabbit polyclonal antibodies that detect the
PKC-, PKC-ßI, or PKC-ßII isoform. After incubation with
secondary anti-rabbit antibodies, immunoreactive proteins were detected
by the CDP-Star chemiluminescence system (New England Biolabs
Inc).17
Statistical Methods
Statistical analysis was performed by ANOVA
and Scheffe’s modified t
test.18 Values of
P<0.05 were considered
significant.
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Results |
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Effects of HMG-CoA Reductase Inhibitors on SMC Migration
Fluvastatin dose-dependently suppressed the 1 µmol/L lyso-PC–mediated increase in SMC migration (control, 19.1±1.8 cells/4 HPF; lyso-PC, 42.7±1.8 cells/4 HPF; lyso-PC and 0.1 µmol/L fluvastatin, 35.5±1.8 cells/4 HPF; lyso-PC and 0.5 µmol/L fluvastatin, 26.4±1.4 cells/4 HPF; lyso-PC plus 1 µmol/L fluvastatin, 18.4±1.8 cells/4 HPF, n=8). The values in all lyso-PC/fluvastatin groups were significantly different from those in the lyso-PC–only group. Fluvastatin, simvastatin, and pravastatin, each at 1 µmol/L, suppressed the lyso-PC–induced migration of SMCs by 48%, 50%, and 16%, respectively (Table 1
), but did not affect the basal level of
migration at 1 µmol/L (control, 20.2±2.4 cells/4 HPF;
fluvastatin, 18.4±2.2 cells/4 HPF;
simvastatin, 18.0±2.6 cells/4 HPF; and
pravastatin, 19.4±2.4 cells/4 HPF; n=8). The
M2 and M3 hydroxylated
metabolites of fluvastatin, each at 1 µmol/L, also
suppressed the lyso-PC–induced migration of SMCs, by 58% and 45%,
respectively
(Table 1). Fluvastatin at 1 µmol/L also
suppressed SMC migration mediated by 5 ng/mL PDGF-BB or 10 ng/mL basic
fibroblast growth factor (bFGF) (control, 15.0±2.2 cells/4 HPF;
PDGF-BB, 40.2±4.4 cells/4 HPF; bFGF, 38.6±4.2 cells/4 HPF; PDGF-BB
and fluvastatin, 22.4±3.4 cells/4 HPF; and bFGF and
fluvastatin, 27.6±4.4 cells/4 HPF, n=8). Values for the
growth factor with fluvastatin groups were significantly
different from the values for the respective growth factor alone:
P<0.05 compared with either
PDGF-BB alone or with bFGF alone.
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Effects of HMG-CoA Reductase
Inhibitors on SMC Oxidative Stress
Fluvastatin dose-dependently suppressed the
1 µmol/L lyso-PC–mediated increase in oxidative stress (control,
59.9±10.0; lyso-PC, 133.2±6.6; lyso-PC and 0.5 µmol/L
fluvastatin, 83.3±6.6; lyso-PC and 1 µmol/L
fluvastatin, 65.0±6.0; all values in arbitrary units,
n=8). The values in all lyso-PC/fluvastatin groups were
significantly different from those of lyso-PC alone.
Fluvastatin, simvastatin, and
pravastatin, each at 1 µmol/L, suppressed the
lyso-PC–induced increase in SMC oxidative stress, by 50%, 53%, and
19%, respectively
(Table 1
), although these compounds did not affect the basal
level of SMC oxidative stress. The M2 and
M3 hydroxylated metabolites of
fluvastatin, each at 1 µmol/L, also suppressed the
lyso-PC–induced increase in SMC oxidative stress, by 58% and 49%,
respectively
(Table 1). Representative flow cytometric
findings and results of fluorescence microscopy for SMC
oxidative stress are shown in
Figure 1.
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[(fluorescence of each channel)x(cell number of the channel)]/total cell number.
Effects of HMG-CoA Reductase
Inhibitors on the HMG-CoA Reductase Activity of
Lyso-PC–Stimulated SMCs
Fluvastatin, simvastatin, and
pravastatin, each at 1 µmol/L, suppressed the
lyso-PC–induced HMG-CoA reductase activities of SMCs by 39%, 52% and
19%, respectively. However, the hydroxylated metabolites of
fluvastatin M2 and
M3, each also at 1 µmol/L, did not suppress
the lyso-PC–induced HMG-CoA reductase activities of SMCs
(Table 1).
Involvement of PLD, PKC, and Tyrosine
Kinase in the Lyso-PC–Mediated Increase in Oxidative
Stress
To assess the involvement of PLD and PKC in the
lyso-PC–mediated increase in migration and oxidative stress, PLD
activity and membrane-bound PKC activity were measured, because it has
been reported that lyso-PC stimulates vascular superoxide
production via PKC19
and that oxidative stress in human SMCs is mediated by
PLD.20 Lyso-PC increased
both PLD and PKC activities, which had been suppressed by the HMG-CoA
reductase inhibitors fluvastatin, including its
hydroxylated metabolites M2 and
M3, simvastatin, and
pravastatin
(Table 2).
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To examine the mechanisms by which lyso-PC increases
oxidative stress and migration, the effects of the specific PLD, PKC,
and tyrosine kinase inhibitors suramin, calphostin C, and
genistein on the lyso-PC–mediated increases in oxidative stress and
migration were tested. Suramin at 100 µmol/L, calphostin C at 1
µmol/L, and genistein at 100 µmol/L, each of which completely
suppressed the lyso-PC–induced increase in PLD and PKC activities
(Table 2), also suppressed the 1 µmol/L lyso-PC–induced
increases in SMC migration (control, 16.0±0.8 cells/4 HPF; lyso-PC,
32.2±1.2, cells/4 HPF; lyso-PC plus suramin, 17.0±1.0 cells/4 HPF;
lyso-PC plus calphostin C, 15.4±1.4 cells/4 HPF; and lyso-PC plus
genistein, 18.0±1.8 cells/4 HPF) and oxidative stress
(Figure 2A). The lyso-PC value was significantly different
from control, and the lyso-PC with inhibitor values were
significantly different from those of lyso-PC
alone.
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Effects of Antioxidants on Lyso-PC–Induced
Increases in SMC Oxidative Stress and Migration
The effects of the antioxidant
-tocopherol on the lyso-PC–induced increase in SMC
oxidative stress and migration were also studied.
-Tocopherol also suppressed the lyso-PC–induced
increase in SMC migration (control, 16.0±0.8 cells/4 HPF; lyso-PC,
32.2±1.2 cells/4 HPF; and lyso-PC plus -tocopherol,
18.0±1.4 cells/4 HPF) and oxidative stress
(Figure 2A). Again, the lyso-PC–only values were
significantly different from control, and the lyso-PC plus
tocopherol values were significantly different from those
of lyso-PC alone.
Effects of Mevalonate and Its Isoprenoid
Derivative on Inhibition of Coronary SMC Oxidative Stress and
Migration by Fluvastatin
Mevalonate at 100 µmol/L, its isoprenoid derivative
farnesol pyrophosphate (Sigma Aldrich) at 10 µmol/L, and
geranylgeraniol pyrophosphate (Sigma Aldrich) at 10 µmol/L, but
not squalene at 10 µmol/L, restored the 1 µmol/L
fluvastatin–mediated suppression of SMC migration
(control, 16.8±1.4 cells/4 HPF; lyso-PC, 33.4±1.4 cells/4 HPF;
lyso-PC plus fluvastatin, 18.4±1.8 cells/4 HPF; lyso-PC
plus fluvastatin plus mevalonate, 31.8±2.0 cells/4 HPF;
lyso-PC plus fluvastatin plus farnesol, 30.8±1.8 cells/4
HPF; lyso-PC plus fluvastatin plus geranylgeraniol,
30.8±2.0 cells/4 HPF; and lyso-PC plus fluvastatin plus
squalene, 18.8±1.4 cells/4 HPF) and oxidative stress
(Figure 2B) to levels induced by 1 µmol/L lyso-PC. Control
values were significantly different from those of lyso-PC only, and the
lyso-PC plus fluvastatin values were significantly
different from those of lyso-PC plus fluvastatin plus
mevalonate, farnesol, or geranylgeraniol. To investigate the roles
played by mevalonate, geranylgeraniol, farnesol, and squalene in SMC
oxidative stress and migration, PLD activity and membrane-bound PKC
activities were measured. Mevalonate, farnesol, and geranylgeraniol but
not squalene each restored the PLD and PKC activities that had been
suppressed by the HMG-CoA reductase inhibitor
fluvastatin at 1 µmol/L
(Table 2).
Inhibition of PKC- or PKC-ß Isoform
Activation by Antisense Oligonucleotide
To determine whether activation of the PKC- or
PKC-ß isoform is associated with the lyso-PC–induced increase in SMC
migration and oxidative stress, the effects of antisense
oligonucleotides to PKC- or PKC-ß isoform, which
significantly blocked the expression of PKC-, PKC- ßI, or
PKC-ßII
(Figure
I), were examined. Antisense
oligonucleotide to PKC- but not of those to the
PKC-ß isoform at 5 µmol/L significantly suppressed the
lyso-PC–induced changes
(Table 3). Sense oligonucleotides to neither
the PKC- isoform nor the PKC-ß isoform had any effects on these
lyso-PC–induced changes at 5 µmol/L
(Table 3).
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Discussion |
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The ability of HMG-CoA reductase inhibitors to suppress oxidative stress and migration of SMCs (Table 1
) at reported therapeutic concentrations (1
µmol/L)10 may be of
clinical interest, given that interference with these processes has
been considered a possible basis for antiatherosclerotic
therapy.7 Greater effects on
suppression of SMC oxidative stress and migration
(Table 1) were observed for lipophilic compounds
(simvastatin and
fluvastatin),21
consistent with previous
observations.6 8
Although the specific mechanisms by which lyso-PC acts as a
chemotactic factor for coronary SMCs remain to be determined,
the present findings suggest that lyso-PC may affect PLD activity,
leading to the activation of membrane-bound PKC
(Table 2), which in turn results in increased oxidative
stress
(Figure 2A). Lyso-PC has in fact been reported to
activate PLD in coronary endothelial
cells22 and PKC in
SMCs.23 Evidence of
activation of PKC has been observed in SMCs in atherosclerotic lesion
at autopsy.24 In addition,
PKC activation due to lyso-PC is reported to increase oxidative stress
in SMCs.20 We performed an
antisense study and found that the PKC- but not the PKC-ß isoform
may play a role in the lyso-PC–mediated increase in oxidative stress
and SMC migration. Notably, PKC- is reported to regulate human
monocyte O2 · -
production.16 We
have shown in the present study that PLD is activated by
the PKC- isoform
(Table 3), a result consistent with previous
findings.25 It has been
reported that activated PLD increases oxidative stress in
SMCs.20 Thus, it is also
possible that activation of PLD via PKC- activation increases
oxidative stress in SMCs. Increased oxidative stress is reported to
play an important role in SMC
migration.8 We have also
confirmed that tyrosine kinase may be involved in PLD activation by
lyso-PC in SMCs
(Table 2), which was already shown for oxidized LDL in
SMCs,26 and that tyrosine
kinase may be involved in migration and oxidative stress
(Figure 2A), a concept that is consistent with
findings in
neutrophils.27
We found in the present study that the HMG-CoA reductase
inhibitors fluvastatin,
simvastatin, and pravastatin directly
suppressed an increase in oxidative stress, suggesting that the
mechanism common to these drugs may underlie their effects on
regulating oxidative stress in SMCs. The addition of mevalonate to
HMG-CoA reductase inhibitor–treated SMCs restored not only
their ability to activate PLD and PKC in SMCs
(Table 1) but also their ability to suppress SMC migration
and oxidative stress
(Figure 2B). This effect of HMG-CoA reductase
inhibitors appears to be related to their inhibition of
prenylation of heterotrimeric and low-molecular-weight guanosine
triphosphate–binding proteins that are involved in signal
transduction.28 Although the
rather small effect of pravastatin on HMG-CoA reductase
activity (20%) does not necessarily explain the 20% reduction in
isoprenylation, this may be a mechanism by which HMG-CoA reductase
inhibitors at least partially inhibit PLD activity, because
mevalonate, farnesol, and geranylgeraniol but not squalene restored the
PLD activity that had been suppressed by the HMG-CoA reductase
inhibitor fluvastatin
(Table 2). In fact, it has been reported that the ß
subunits of heterometric guanosine triphosphate–binding protein and
the low-molecular-weight guanosine triphosphate–binding protein Rho
mediate PLD activation in
SMCs29 and that HMG-CoA
reductase inhibitors induce isoprenylation in
SMCs.30
In the present study, we also demonstrated for the first
time that the hydroxylated metabolites of fluvastatin,
M2 and M3, directly
decreased the increase in SMC oxidative stress induced by lyso-PC
through PLD and PKC suppression
(Table 2). However, superoxide anion–scavenging properties
of fluvastatin and its metabolites have been
reported.31 Because
PLD and PKC inhibitors completely blocked the increase in
oxidative stress and SMC migration induced by lyso-PC, the predominant
mechanism of the antioxidative and antimigratory effects of statins may
be through PLD and PKC suppression rather than by direct scavenging
properties. However, fluvastatin and its metabolites, which
have less HMG-CoA reductase inhibitory action than does
simvastatin, have antioxidation and antimigration effects
equivalent to those of simvastatin
(Table 1). This suggests that 2 different mechanisms
(suppression of mevalonate pathway–mediated PLD activation and direct
scavenging caused by 2 metabolically linked compounds) may
coincidentally result in qualitatively and quantitatively similar
effects: suppression of SMC oxidative stress and migration due to the
action of fluvastatin.
In conclusion, our findings show that HMG-CoA reductase inhibitors may, at least in experimental models, affect the early events of atherogenesis by suppressing increased SMC oxidative stress and migration, possibly through local suppression of PLD and PKC activities.
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Acknowledgments |
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The authors would like to thank Keiko Hirata and Sayuri Takagi for secretarial assistance.
Received September 18, 2000; accepted March 20, 2001.
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