© 1998 American Heart Association, Inc.
Original Contributions |
3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase and Isoprenylation Inhibitors Induce Apoptosis of Vascular Smooth Muscle Cells in Culture
From the Instituto de Investigación Médica (C.G., L.M.B.-C., M.O., A.O., J.J.P., J.E.), Fundación Jiménez Díaz, Universidad Autónoma de Madrid, Madrid, Spain; Centro de Investigación en Sanidad Animal (C.A.), INIA, Valdeolmos, Madrid, Spain; and Parke Davis Spain (C.D., G.H.), Barcelona, Spain. The current address for Dr Guijarro is the Department of Internal Medicine, Fundación Hospital Alcorcón, Alcorcón, Madrid, Spain.
Correspondence to Jesús Egido, MD, PhD, Research Laboratories, Fundación Jiménez Díaz, Avda Reyes Católicos 2, 28040 Madrid, Spain. E-mail jegido{at}uni.fjd.es
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Abstract—Recent evidence suggests that apoptosis may be involved in the control of vascular smooth muscle cell (VSMC) number in atherosclerotic lesions. 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been reported to induce apoptosis in a variety of tumor cell lines. To evaluate whether these agents also induce apoptosis of VSMCs, cultured rat VSMCs were treated with increasing doses of atorvastatin in the presence of FBS as a survival factor. The presence of apoptosis was evaluated by morphological criteria, annexin V binding, and DNA fragmentation and quantified as the proportion of hypodiploid cells by flow cytometry. Atorvastatin induced apoptosis in a dose-dependent manner, an effect also seen with simvastatin and lovastatin, but not with the hydrophilic drug pravastatin. The proapoptotic effect of statins was seen only when the inhibition of acetate incorporation into sterols was >95% and was fully reversed by mevalonate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate but not by isopentenyl adenosine, ubiquinone, or squalene, suggesting a role for prenylated proteins in the regulation of VSMC apoptosis. To further assess the role of protein prenylation, VSMCs were exposed to the prenyl transferase inhibitors perillic acid and manumycin A. Both agents induced VSMC apoptosis as evaluated by the above-mentioned criteria. Finally, VSMC treatment with lipophilic statins was associated with decreased prenylation of p21-Rho B, further supporting the role of protein prenylation inhibition in statin-induced VSMC apoptosis. The present data suggest that interference with protein prenylation by HMG-CoA reductase inhibitors or other agents may provide new strategies for the prevention of neointimal thickening.
Key Words: smooth muscle cell • apoptosis • mevalonate • protein isoprenylation • Rho • atherosclerosis
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Introduction |
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Apoptosis has recently been reported to be present in human atherosclerosis and in experimental models of vascular damage and virtually absent in normal vessels, suggesting a potential role for apoptosis in the pathophysiology of vascular injury.1 2 3 4 5 6 Human vascular smooth muscle cells (VSMCs) from atheromatous lesions are more likely to undergo apoptosis than are VSMCs derived from normal arteries.7 In addition, apoptosis is more frequent in proliferative lesions, particularly restenosis lesions.3 5 The temporal sequence of proliferation and apoptosis in experimental models is consistent with a role for apoptosis in the control of neointimal cellularity.1 Accordingly, VSMC apoptosis has been proposed to play a significant role in the control of neointimal thickening.5 6 8 In spite of its potential importance, little is known regarding the mechanisms controlling VSMC apoptosis in vivo.
3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase
inhibitors (statins, Figure 1
) have been shown to be efficacious in
reducing cardiovascular morbidity and mortality in
primary and secondary prevention clinical
trials.9 10 11 Although the salutary effects of
these agents may be explained by their beneficial actions on the lipid
profile, increasing evidence suggests that statins may also exhibit
effects unrelated to lipid reduction (Figure 1).12 13 14 In experimental models, HMG-CoA
reductase inhibitors reduced neointimal
thickening regardless of the effect of
cholesterol.15 16 17 18
Neointimal VSMC number is the result of the migration of
VSMCs from the media and their subsequent proliferation and eventual
death, including programmed cell death.19 20 Much
of the attention on the effects of statins on VSMC biology has been
devoted to migration and proliferation.16 21 22 23 24 25
Indeed, both phenomena can be attenuated by HMG-CoA reductase
inhibition and have been proposed as potential mechanisms of action in
the prevention of vascular damage by statins. HMG-CoA reductase
inhibitors induce programmed cell death in a variety of
cell lines.26 27 28 29 30 In spite of its potential
relevance, no attempts to evaluate the effect of HMG-CoA reductase
inhibitors on VSMC apoptosis have been reported. In
the present study, we show that lipophilic HMG-CoA reductase
inhibitors induce apoptosis of VSMCs even in the
presence of survival factors. This effect is paralleled by an
attenuation of the prenylation of p21-Rho B and is reversed by the
addition of mevalonate, farnesyl pyrophosphate (F-PP), and/or
geranylgeranyl pyrophosphate (GG-PP), suggesting a role for
isoprenylated proteins such as Rho B in the apoptosis of VSMCs
induced by statins. Our data suggest that interference with protein
prenylation by HMG-CoA reductase inhibitors or other agents
may provide new strategies for the prevention of neointimal
thickening.
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Reagents and Chemicals
DMEM, penicillin, streptomycin, and trypsin-EDTA were from Bio Whitaker. FBS was from GIBCO. Atorvastatin (sodium salt, dissolved in Tris borate, pH 8) was obtained from Parke Davis. Lovastatin and simvastatin (from Merck Sharp and Dohme Spain) were converted to the active compounds as described by Kita et al.31 Pravastatin was obtained from Bristol Myers-Squibb Spain and dissolved in NaCl (155 mmol/L). Perillic acid and manumycin A (from Sigma Chemical Co) were dissolved in ethanol and DMSO, respectively. FITC annexin V was kindly provided by Dr J. Turnay (School of Chemistry, Universidad Complutense, Madrid, Spain). [3H]Acetic acid (sodium salt; specific activity, 2.52 Ci/mmol) was from DuPont NEN. Rabbit anti–Rho B polyclonal antibody (Sc-180) was from Santa Cruz Biotechnology. The remaining reagents were obtained from Sigma unless specified otherwise.
VSMC Culture
Rat thoracic aorta smooth muscle cells were isolated essentially
as described by Owens et al.32 Briefly, male
Sprague-Dawley rats weighing 200 to 250 g were killed by an
overdose of pentobarbital, and the thoracic aorta was aseptically
excised and placed in PBS. Adhering fat and connective tissue were
removed by blunt dissection. The aorta was opened longitudinally and
preincubated in DMEM containing collagenase (type II, 290
U/mL), penicillin (100 U/mL), and streptomycin (100 µg/mL) for 15 to
20 minutes at 37°C in 95% air/5% CO2. After
dissection, aortas were placed in fresh enzyme solution, minced into
1-mm pieces, and incubated for an additional 1.5 to 2 hours. Pieces
were rinsed twice with PBS to remove the cells, and PBS was then
centrifuged. The cells were resuspended in DMEM with
antibiotics and 10% FBS, counted, and seeded at a concentration of
104 cells/cm2 in plastic
culture flasks (Costar). Cells were characterized as smooth muscle
cells by their typical hill-and-valley morphology by phase-contrast
microscopy and by positive immunostaining for
-smooth muscle actin (clone 1A4, Sigma). Media were replaced every 2
to 3 days. At confluence, cells were harvested for passaging with
trypsin-EDTA. Cells between passages 3 and 12 were used for all the
experiments.
Cell DNA Staining
Adherent cells were washed once with PBS and incubated with PBS
containing 9.4 µmol/L bisbenzimide (Hoechst 33342) for 20
minutes in the dark and examined under fluorescence microscopy.
Floating cells were collected by centrifugation, and a
cell smear was stained with bisbenzimide as indicated above.
Flow Cytometry Assessment of Cell Death
Cellular DNA content was assessed by flow cytometry. For this
purpose, cells were cultured in 12-well plates and treated as
appropriate. Cells attached to the plate were collected with trypsin
and 2.2 mmol/L EDTA in PBS and mixed with detached cells
present in the supernatant. Cells were spun and resuspended in a
solution containing 75 µmol/L propidium iodide, 10 mg/L RNase A,
and 0.05% Nonidet P-40 in PBS; then they were incubated at 4°C for
30 minutes in the dark and analyzed by flow cytometry (Coulter
EPICS XL-MCL flow cytometer, Hialeah, FL) using LYSIS II
software.33 34 The percentage of cells with
decreased DNA staining (A0), composed of
apoptotic cells resulting from either fragmentation or
decreased chromatin, of a minimum of 5000 to 10 000 cells per
experimental condition was counted. Cell debris were excluded from
analysis by selective gating based on anterior and right angle
scatter. As assessed by flow cytometry, none of the solvents of the
different compounds, up to the highest dose used in our experimental
conditions, induced any significant degree of apoptosis.
DNA Electrophoresis
For the evaluation of DNA fragmentation, 10 million cells were
incubated under the different experimental conditions for 48 hours and
assayed essentially as described.35 At the end of
this period, floating and adherent cells were collected, rinsed twice
with cold PBS, and lysed by incubation in 20 mmol/L EDTA, 50
mmol/L Tris-HCl (pH 8), and 0.5% (wt/vol) sodium
lauroylsarcosine for 30 minutes at 4°C. Cell debris and
high-molecular-weight DNA were precipitated by
centrifugation at 12 000g for 30 minutes at
4°C. Supernatants containing fragmented DNA were then treated with
proteinase K (0.2 g/L) at 50°C for 1 hour and 2 g/L RNase A at 50°C
for another hour and run in a 1.5% agarose gel containing ethidium
bromide. Gels were examined and photographed under UV light.
Flow Cytometry Assessment of Annexin V Binding
Annexin V binding was studied essentially as described by Vermes
et al,36 with minor modifications. For that
purpose, cells were cultured in 12-well plates and treated as
appropriate. Cells attached to the plate were carefully collected with
2.2 mmol/L EDTA in PBS and mixed with detached cells present
in the supernatant. Cells were spun, rinsed with PBS, and resuspended
in a solution containing 1.5 mg/L FITC annexin V, 10 mmol/L HEPES,
150 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L
MgCl2, and 1.8 mmol/L
CaCl2 for 15 minutes at 37°C. Cells were washed
twice with the same buffer (without FITC annexin V) and
analyzed by flow cytometry (Coulter EPICS XL-MCL flow
cytometer). Green fluorescence intensity of at least 2000 to
5000 events is displayed on a logarithmic scale against cell number.
Cell debris were excluded from analysis.
[3H]Acetate Incorporation Into Sterols
Approximately 5 million subconfluent exponentially growing
smooth muscle cells were incubated with different concentrations of
HMG-CoA reductase inhibitors in DMEM containing 10% FBS
for 3 hours. At the end of this time, 5 µCi of
[3H]sodium acetate was added, and cells were
incubated for an additional period of 21 hours. Afterward, cells were
collected and rinsed twice with PBS, and lipids were extracted by the
method of Bligh and Dyer.37 The chloroform
fractions were dried under N2, resuspended in 2
mL of 1 mol/L KOH in 95% ethanol, and saponified at 90°C for 50
minutes. Nonsaponifiable lipids were extracted twice with 1.2 mL of
hexane and dried under N2. Four hundred
micrograms of cholesterol and 2 mL of isopropanol were
added to each sample. Sterols were then precipitated by the addition of
1 mL of digitonin reagent (1% digitonin in 50% ethanol) and
incubation at 4°C for 30 minutes.38 Samples
were spun, and the precipitate was washed twice with cold acetone and
resuspended in 2 mL of methanol. Precipitates were extensively
resuspended with 2 additional washes with methanol, pooled, and dried
before counting in scintillation vials.
Immunoblotting
Cells from different experimental conditions were collected,
rinsed twice with cold PBS, and pelleted. Cells were then briefly
sonicated in 1 mL of PBS containing 2 mmol/L EDTA, 2 mmol/L
phenylmethylsulfonyl fluoride, and 1 µmol/L pepstatin
A. Cell membranes were collected by
centrifugation at 50 000g for 30 minutes
and resuspended in 100 mmol/L Tris-HCl, 300 mmol/L NaCl, 1%
Triton X-100, and 0.1% SDS containing 2 mmol/L EDTA, 2
mmol/L phenylmethylsulfonyl fluoride, and 1 µmol/L
pepstatin A. Protein in membrane or soluble extracts was calculated by
the BCA method (Pierce). Equal amounts of protein (50 to 100 µg) were
loaded into 12% acrylamide gels and electrophoresed as
described.39 The resolved proteins were
transferred onto PVDF membranes (Immobilon, Millipore). The nonspecific
sites of the membrane were blocked by incubation at room temperature 1
hour in 7.5% nonfat dry milk powder in PBS containing 0.1% Tween 20
(PBST). The membrane was incubated overnight at 4°C with rabbit
anti–Rho B polyclonal antibody (Sc-180, Santa Cruz Biotechnology) in
PBST containing 5% nonfat dry milk. The membrane was washed with PBST
and incubated 1 hour at room temperature with horseradish
peroxidase–conjugated anti-rabbit secondary antibody (Santa Cruz
Biotechnology) in PBST containing 5% nonfat dry milk. The membrane was
then washed with PBST and incubated 30 minutes in PBST containing
400 mmol/L NaCl, followed by detection with enhanced
chemiluminescence (ECL kit, Amersham). Films were scanned on a
densitometer and quantified using ImageQant software
(Molecular Dynamics).
Data Presentation
Representative data from 2 to 5 independent
experiments are presented. For quantitative data, the mean±SD
of triplicate or quadruplicate samples from 1 of 3 to 5 independent
experiments is presented. For the comparison of group means,
1-way ANOVA and Student-Newman-Keuls tests were used as appropriate. A
value P<0.05 was considered statistically significant.
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Results |
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Atorvastatin and Lipophilic HMG-CoA Reductase Inhibitors Induce Apoptosis of VSMCs
To evaluate whether HMG-CoA reductase inhibitors induce apoptosis of VSMCs, exponentially growing subconfluent rat VSMCs were exposed to increasing concentrations of the HMG-CoA reductase inhibitor atorvastatin for 24 hours in the presence of 10% FBS as a survival factor. Atorvastatin treatment was associated with cell rounding and detachment from the plate substrate and surrounding cells, as assessed by inverted phase microscopy, in a dose-dependent manner (Figure 2
).
Staining with DNA-binding dyes revealed that treatment of VSMCs with
atorvastatin was associated with the appearance of chromatin
condensation and fragmentation characteristic of apoptosis
(Figure 2). These changes were particularly prominent in detached cells
floating in the culture media. To further confirm that atorvastatin
induced apoptosis of VSMCs, the cell DNA content was assessed
by flow cytometry. Atorvastatin treatment was associated with the
appearance of a cell population with a reduced DNA content
(hypodiploid), usually referred to as sub G1 or
A0, characteristic of apoptosis (Figures 3 to 5).
Most dramatic reductions of DNA content were evident in floating cells
(not shown), mirroring the effects on chromatin fragmentation. The
reduction in cellular DNA content was paralleled by the appearance
of DNA fragmentation into multiples of 180 to 200 kb, constituting a
typical laddering pattern characteristic of apoptosis (Figure 6). Finally, atorvastatin treatment was
also associated with increased binding of annexin V, a recently
described early indicator of apoptosis (Figure 7).36 Again, most
of the increased annexin V binding was attributable to floating cells.
Mevalonate, in a dose-dependent manner, completely reversed the effects
of atorvastatin in cell morphology, DNA content, and fragmentation, and
annexin V binding (Figures 2 to 4, 6, and 7), suggesting that the
effects of atorvastatin were indeed related to the inhibition of the
mevalonate pathway. To ascertain whether the effects of atorvastatin
were shared by other HMG-CoA reductase inhibitors, VSMCs
were incubated with increasing concentrations of 2 lipophilic
(simvastatin and lovastatin) and 1 hydrophilic
(pravastatin) HMG-CoA reductase inhibitor. Both
simvastatin and lovastatin produced changes
similar to those observed with atorvastatin with an even higher
potency, whereas pravastatin (up to 100 µmol/L)
failed to induce any of the above-mentioned effects (Figures 4 and 6).
In every case, the addition of mevalonate completely reversed the
statin-induced effects (Figures 4 and 6).
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We further assessed whether the above-described effects were indeed
related to the degree of inhibition of the mevalonate pathway. First,
the antiapoptotic effect of mevalonate was specific for
statins, since mevalonate failed to prevent VSMC apoptosis
induced by other agents, such as pyrrolidinedithiocarbamate or
manumycin A (not shown). Second, the proapoptotic effect of
statins was very closely related to the degree of inhibition of the
mevalonate pathway, as assessed by the inhibition of the incorporation
of [3H]acetate into sterols. Indeed,
apoptosis was present only when the inhibition of sterol
synthesis was >95% (Figure 4
, bottom). All the above data strongly
suggest that the depletion of some mevalonate metabolite(s) is involved
in the statin-induced apoptosis of VSMCs.
Mevalonate Metabolites and Apoptosis of VSMCs
To evaluate which among mevalonate metabolites may be important in
atorvastatin-induced apoptosis, the reversal of atorvastatin
effects by several mevalonate derivatives (Figure 1) was assessed next.
Neither cholesterol (present in the serum-supplemented
media) nor its precursor, squalene (1 to 25 µmol/L), prevented
atorvastatin-induced apoptosis (not shown), suggesting that
sterols do not play a prominent role in our experimental conditions.
Similarly, neither isopentenyl adenosine (1 to 25
µmol/L) nor ubiquinone (coenzyme Q10, 1 to 25 µmol/L) reversed
the atorvastatin-induced apoptosis of VSMCs (not shown). In
contrast, both F-PP and GG-PP reversed, in a dose-dependent manner, the
atorvastatin effects in cell morphology, DNA content and fragmentation,
and annexin V binding (Figures 5 to 7).
Isoprenylation Inhibition and Apoptosis of VSMCs
Since both F-PP and GG-PP are used for the posttranslational
modification of several cell proteins, we next evaluated the effects of
2 inhibitors of protein prenylation: perillic acid and
manumycin A. In atorvastatin-treated mevalonate-repleted cells, the
weak inhibitor of prenylation perillic acid induced, in a
dose dependent manner, the appearance of the morphological changes,
annexin V binding, DNA content reduction, and laddering characteristic
of apoptosis in a significant, albeit small, proportion of
VSMCs (Figures 2, 7, and 8). In agreement
with its higher inhibitory potency, manumycin A induced, in
a dose-dependent manner, apoptosis in a higher proportion of
VSMCs (Figures 2, 7, and 8). As expected, the addition of exogenous
mevalonate (100 µmol/L) did not modify the manumycin-induced
effects (not shown). Another farnesyl transferase
inhibitor, -hydroxyfarnesyl phosphonic acid, also
induced in a dose-dependent manner the appearance of VSMC
apoptosis (not shown).
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HMG-CoA Reductase Inhibition and Protein Prenylation
The above-presented data suggest that inhibition of
protein prenylation may be important in VSMC apoptosis.
Therefore, we evaluated whether HMG-CoA reductase treatment was in fact
associated with changes in the level of prenylation of the protein
p21-Rho B, a protein that has been found to play an important role in
apoptosis. Under normal conditions p21-Rho is anchored into the
cell membrane by its covalent binding to either farnesyl or
geranylgeranyl. When protein prenylation is inhibited, Rho B remains in
the cytoplasm. The evaluation of the degree of presence of Rho B in the
membrane or cytoplasmic compartments of the cells is therefore an
indirect assessment of its degree of prenylation. As shown in Figure 9, treatment of VSMCs with lipophilic
statins, but not pravastatin, was associated with an
attenuation of the presence of Rho B in the membrane and its appearance
in the cytoplasmic fraction. Interestingly, the degree of inhibition of
Rho B prenylation (as assessed by its compartmentalization) by
different statins closely parallels the proapoptotic effect of
statins. Again, treatment with mevalonate, F-PP, or GG-PP was
associated with the disappearance of Rho from the cytoplasm and its
restoration into the membrane fraction.
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The present data show that HMG-CoA reductase inhibition by atorvastatin induces changes of cultured rat VSMCs characteristic of apoptosis: cell rounding, detachment from the culture substrata and adjacent cells, chromatin condensation and fragmentation, increased annexin V binding, reduction of cellular DNA content, and internucleosomal fragmentation.36 40 The effects are specific to the inhibition of the mevalonate pathway, since the addition of exogenous mevalonate completely reversed the atorvastatin-induced effects. Conversely, the antiapoptotic effect of mevalonate is restricted to statins: it is ineffective in reversing apoptosis induced by other agents, such as pyrrolidinedithiocarbamate or manumycin. These proapoptotic effects are not restricted to atorvastatin, as 2 other agents of the same family, namely, simvastatin and lovastatin, produced similar qualitative effects. In contrast, the hydrophilic drug pravastatin failed to induce any significant changes when used in equimolar amounts. Not surprisingly, the proapoptotic effects of statins were tightly correlated with the degree of inhibition of the mevalonate pathway (Figure 4
). Our results are quite similar to those reported regarding
the effects of different statins on VSMC
proliferation.25 It is conceivable that in both
cases, the hydrophilic properties of pravastatin and the
lack of a specific transporter in nonhepatic cells prevented an
adequate access of the drug to the cytoplasm.41
In this regard, pravastatin, compared with atorvastatin,
lovastatin, and simvastatin, was also less
efficacious in reducing vascular injury in 2 experimental models of
atherosclerosis.17 18 25 Whether
the potential benefits of a direct action of lipophilic statins on the
vascular wall may be overshadowed by an increased incidence of
extrahepatic side effects is at present unknown. HMG-CoA reductase inhibition results in the reduction of the cellular content of a variety of sterol and nonsterol mevalonate metabolites.42 43 Many of these metabolites play prominent roles in cell biology that could potentially be involved in the mechanisms of apoptosis. Our data suggest that a profound inhibition of the mevalonate pathway is required for the induction of apoptosis. Cholesterol was present in the media as a part of the FBS, at concentrations considered to be sufficient for basic cellular functions.44 Therefore, cholesterol depletion, in agreement with previous reports in other cell lines,26 27 28 29 30 does not seem to account for the induction of apoptosis in our experimental conditions. In addition, squalene, the first mevalonate metabolite committed to sterol synthesis, failed to reverse the atorvastatin-induced apoptosis of VSMCs, suggesting that nonsterol metabolites, rather than steroids, may play a major role. Similarly, isopentenyl adenosine, the precursor of isopentenyl tRNA, failed to reverse the atorvastatin effects and, consequently, does not seem to play a prominent role in our experimental conditions. In contrast, both F-PP and GG-PP, at concentrations in the micromolar range, completely abolished atorvastatin-induced effects. GG-PP is the precursor of ubiquinone, an important natural antioxidant. Since redox processes appear to be involved in the pathophysiology of VSMC apoptosis,45 46 the inhibition of ubiquinone synthesis may be of relevance. However, the addition of ubiquinone did not reverse atorvastatin-induced VSMC apoptosis, suggesting that GG-PP is acting through a different mechanism. Indeed, GG-PP and F-PP are used for the posttranslational modification of several important cell proteins.42 43 47 48 The attachment of an isoprenoid residue to these proteins is necessary for their anchorage to cell membranes and full functionality. Prenylated proteins and, more specifically, small GTP-binding proteins are key elements in signal transduction from membrane receptors involved in proliferation and survival of VSMCs, such as growth factors, endothelin, angiotensin II, and thrombin.49
Increasing attention has been directed in recent years to the development of prenyl transferase inhibitors in an attempt to inhibit tumor cell proliferation.48 50 51 52 Surprisingly, there are no reports dealing with the potential effects of prenyl transferase inhibition on VSMC apoptosis. To assess whether protein prenylation may play a role in the control of VSMC apoptosis, VSMCs were exposed to 2 unrelated inhibitors of protein prenyl transferases: perillic acid and manumycin A.53 54 Perillic acid and other limonene-related compounds are weak inhibitors of the prenylation of low-molecular-weight proteins and, for this reason, are commonly used in vitro in the presence of inhibitors of mevalonate synthesis and exogenous mevalonate repletion.53 Under these conditions, perillic acid induced apoptosis in a dose-dependent manner in a modest proportion of cells in our experimental conditions. The effects of perillic acid alone were somewhat weaker (not shown). In contrast, the farnesyl transferase inhibitor manumycin A induced changes in cell morphology, annexin V binding, and DNA content characteristic of apoptosis in a higher proportion of cells. Perillic acid inhibits the farnesylation and geranylgeranylation of low-molecular-weight proteins, whereas manumycin A is a selective inhibitor of protein farnesylation. Although prenylated proteins are modified specifically by either farnesyl or geranylgeranyl in normal conditions,47 48 little is known regarding potential modifications by a different isoprenoid when the naturally occurring reaction is interfered pharmacologically. Protein prenylation is catalyzed by 3 different enzymes that recognize both the isoprenoid and the acceptor protein: farnesyl transferase and geranylgeranyl transferases I and II.48 However, the specificity of these enzymes for their substrates is not absolute. Indeed, K-ras is normally farnesylated but can be alternatively modified by geranylgeranyl in the absence of farnesyl.55 Similarly, the low-molecular-weight protein Rho B can be either geranylgeranylated or farnesylated by geranylgeranyl transferase I.48 56 Thus, it appears that proteins usually modified by GG-PP may be prenylated by F-PP and vice versa given the appropriate circumstances. These phenomena may underlie the capacity of either F-PP or GG-PP to reverse atorvastatin effects. Nevertheless, the fact that manumycin A induces apoptosis at concentrations of >50% inhibitory concentration (IC50) for farnesyl transferase but well under the IC50 for geranylgeranyl transferase I suggests that farnesylated proteins may be of particular importance.54 More specifically, the inhibition of the prenylation of Rho may be critical. Indeed, the inhibition of Rho prenylation has very recently been described to produce apoptosis in fibroblasts,57 and the membrane restoration of Rho can overcome this effect.58 In addition, mutant constitutively active Rho prevents T-cell apoptosis,59 whereas Rho inactivation is associated with increased T-cell apoptosis.60 Therefore, we examined the effect of statins on the degree of Rho B prenylation in our experimental conditions. The effects of different statins on Rho prenylation were remarkably similar to their effects on VSMC apoptosis. In addition, the reversal of the inhibition of Rho prenylation by mevalonate or its derivatives was also associated with the abrogation of the proapoptotic effect of statins. On the whole, our results strongly suggest that the interference of the prenylation of proteins, such as Rho, by HMG-CoA reductase inhibition plays a prominent role in statin-induced VSMC apoptosis. Although Rho is an excellent marker of the effect of statins on protein prenylation and is a likely candidate to be involved in statin-induced apoptosis, our data cannot exclude that some other prenylated proteins may play a similar or even greater role.
It is difficult to ascertain how the present data may be of relevance to the in vivo situation. However, it should be noted that the concentrations of statins used in these and other experimental settings to induce apoptosis are similar to those used to inhibit cell proliferation.16 21 22 23 24 25 26 27 28 29 30 This is hardly surprising, as the mechanisms regulating cell proliferation are inextricably connected to the regulation of programmed cell death.49 61 62 In this regard, the induction of apoptosis of VSMCs might play a concurrent role with the inhibition of cell proliferation in the prevention of neointimal thickening, as proposed in experimental models.16 17 In addition, HMG-CoA reductase inhibitors were able to induce apoptosis of VSMCs even in the presence of the survival factor, ie, FBS. The effect of statins might be enhanced in circumstances in which a certain degree of apoptosis is already taking place, such as in atheromatous plaque formation. Even if statins may favor smooth muscle cell apoptosis in vivo, the potential clinical importance of this phenomenon is controversial. On the one hand, increased apoptosis may contribute to an attenuation of the neointimal thickening seen in early atherosclerosis.20 On the other hand, enhanced apoptosis may contribute to increased plaque instability and hence favor the appearance of vascular events.19 Given all these considerations, the potential role of statin-induced VSMC apoptosis in the pathophysiology of atherosclerosis deserves further investigation.
In summary, HMG-CoA reductase inhibitors induce apoptosis of VSMCs in culture, probably through a reduction of isoprenoid concentration and the subsequent protein prenylation. A better understanding of the mechanisms involved in VSMC apoptosis and its pharmacological modulation may help to provide new strategies to modify the pathophysiology of atherosclerosis.
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Acknowledgments |
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This study was supported, in part, by grants from Fondo de Investigación Sanitaria (96/2021), Ministerio de Educación (SAF 97-055, PM 97/0085), Fundación Conchita Rábago de Jiménez Díaz, Fundación Iñigo Alvarez de Toledo, and Parke Davis Spain. Dr Alonso was supported by projects PB96-0552 and SC97-066. We are grateful to Dr J. Turnay for kindly providing FITC annexin V, to Dr M.A. Lasunción for his advice on sterol synthesis studies, and to Dr J. González-Cabrero for carefully reading the manuscript.
Received May 7, 1998; accepted June 11, 1998.
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S. Wassmann, U. Laufs, K. Muller, C. Konkol, K. Ahlbory, A. T. Baumer, W. Linz, M. Bohm, and G. Nickenig Cellular Antioxidant Effects of Atorvastatin In Vitro and In Vivo Arterioscler Thromb Vasc Biol, February 1, 2002; 22(2): 300 - 305. [Abstract] [Full Text] [PDF]
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T. Ichiki, K. Takeda, T. Tokunou, N. Iino, K. Egashira, H. Shimokawa, K. Hirano, H. Kanaide, and A. Takeshita Downregulation of Angiotensin II Type 1 Receptor by Hydrophobic 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors in Vascular Smooth Muscle Cells Arterioscler Thromb Vasc Biol, December 1, 2001; 21(12): 1896 - 1901. [Abstract] [Full Text] [PDF]
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M. Takemoto and J. K. Liao Pleiotropic Effects of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Inhibitors Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1712 - 1719. [Abstract] [Full Text] [PDF]
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T. Watanabe, M. Yoshizumi, M. Akishita, M. Eto, K. Toba, M. Hashimoto, K. Nagano, Y.-Q. Liang, Y. Ohike, K. Iijima, et al. Induction of Nuclear Orphan Receptor NGFI-B Gene and Apoptosis in Rat Vascular Smooth Muscle Cells Treated With Pyrrolidinedithiocarbamate Arterioscler Thromb Vasc Biol, November 1, 2001; 21(11): 1738 - 1744. [Abstract] [Full Text] [PDF]
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 Z. A. Massy and C. Guijarro Statins: effects beyond cholesterol lowering Nephrol. Dial. Transplant., September 1, 2001; 16(9): 1738 - 1741. [Full Text] [PDF]
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B. R. Kwak and F. Mach Statins Inhibit Leukocyte Recruitment: New Evidence for Their Anti-Inflammatory Properties Arterioscler Thromb Vasc Biol, August 1, 2001; 21(8): 1256 - 1258. [Full Text] [PDF]
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R. Patel, S. F. Nagueh, N. Tsybouleva, M. Abdellatif, S. Lutucuta, H. A. Kopelen, M. A. Quinones, W. A. Zoghbi, M. L. Entman, R. Roberts, et al. Simvastatin Induces Regression of Cardiac Hypertrophy and Fibrosis and Improves Cardiac Function in a Transgenic Rabbit Model of Human Hypertrophic Cardiomyopathy Circulation, July 17, 2001; 104(3): 317 - 324. [Abstract] [Full Text] [PDF]
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 B. Wong, W. C. Lumma, A. M. Smith, J. T. Sisko, S. D. Wright, and T.-Q. Cai Statins suppress THP-1 cell migration and secretion of matrix metalloproteinase 9 by inhibiting geranylgeranylation J. Leukoc. Biol., June 1, 2001; 69(6): 959 - 962. [Abstract] [Full Text] [PDF]
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K. Yasunari, K. Maeda, M. Minami, and J. Yoshikawa HMG-CoA Reductase Inhibitors Prevent Migration of Human Coronary Smooth Muscle Cells Through Suppression of Increase in Oxidative Stress Arterioscler Thromb Vasc Biol, June 1, 2001; 21(6): 937 - 942. [Abstract] [Full Text] [PDF]
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M. Aikawa and P. Libby Vascular inflammation and activation: new targets for lipid lowering Eur. Heart J. Suppl., May 1, 2001; 3(suppl_B): B3 - B11. [Abstract] [PDF]
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G. P. van Nieuw Amerongen and V. W.M. van Hinsbergh Cytoskeletal Effects of Rho-Like Small Guanine Nucleotide-Binding Proteins in the Vascular System Arterioscler Thromb Vasc Biol, March 1, 2001; 21(3): 300 - 311. [Abstract] [Full Text] [PDF]
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S. Wassmann, U. Laufs, A. T. Bäumer, K. Müller, C. Konkol, H. Sauer, M. Böhm, and G. Nickenig Inhibition of Geranylgeranylation Reduces Angiotensin II-Mediated Free Radical Production in Vascular Smooth Muscle Cells: Involvement of Angiotensin AT1 Receptor Expression and Rac1 GTPase Mol. Pharmacol., March 1, 2001; 59(3): 646 - 654. [Abstract] [Full Text]
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Y. Fukumoto, P. Libby, E. Rabkin, C. C. Hill, M. Enomoto, Y. Hirouchi, M. Shiomi, and M. Aikawa Statins Alter Smooth Muscle Cell Accumulation and Collagen Content in Established Atheroma of Watanabe Heritable Hyperlipidemic Rabbits Circulation, February 20, 2001; 103(7): 993 - 999. [Abstract] [Full Text] [PDF]
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M. Aikawa, E. Rabkin, S. Sugiyama, S. J. Voglic, Y. Fukumoto, Y. Furukawa, M. Shiomi, F. J. Schoen, and P. Libby An HMG-CoA Reductase Inhibitor, Cerivastatin, Suppresses Growth of Macrophages Expressing Matrix Metalloproteinases and Tissue Factor In Vivo and In Vitro Circulation, January 16, 2001; 103(2): 276 - 283. [Abstract] [Full Text] [PDF]
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O. Feron, C. Dessy, J.-P. Desager, and J.-L. Balligand Hydroxy-Methylglutaryl-Coenzyme A Reductase Inhibition Promotes Endothelial Nitric Oxide Synthase Activation Through a Decrease in Caveolin Abundance Circulation, January 2, 2001; 103(1): 113 - 118. [Abstract] [Full Text] [PDF]
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W. Palinsk New Evidence for Beneficial Effects of Statins Unrelated to Lipid Lowering Arterioscler Thromb Vasc Biol, January 1, 2001; 21(1): 3 - 5. [Full Text] [PDF]
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G. P. v. N. Amerongen, M. A. Vermeer, P. Negre-Aminou, J. Lankelma, J. J. Emeis, and V. W. M. van Hinsbergh Simvastatin Improves Disturbed Endothelial Barrier Function Circulation, December 5, 2000; 102(23): 2803 - 2809. [Abstract] [Full Text] [PDF]
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H. Chen, U. Ikeda, M. Shimpo, M. Ikeda, S. Minota, and K. Shimada Fluvastatin Upregulates Inducible Nitric Oxide Synthase Expression in Cytokine-Stimulated Vascular Smooth Muscle Cells Hypertension, December 1, 2000; 36(6): 923 - 928. [Abstract] [Full Text] [PDF]
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Z. Yang, T. Kozai, B. van de Loo, H. Viswambharan, M. Lachat, M. I. Turina, T. Malinski, and T. F. Luscher HMG-CoA reductase inhibition improves endothelial cell function and inhibits smooth muscle cell proliferation in human saphenous veins J. Am. Coll. Cardiol., November 1, 2000; 36(5): 1691 - 1697. [Abstract] [Full Text] [PDF]
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D. E. Marra, T. Simoncini, and J. K. Liao Inhibition of Vascular Smooth Muscle Cell Proliferation by Sodium Salicylate Mediated by Upregulation of p21Waf1 and p27Kip1 Circulation, October 24, 2000; 102(17): 2124 - 2130. [Abstract] [Full Text] [PDF]
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 D. C. Hess, A. M. Demchuk, L. M. Brass, and F. M. Yatsu HMG-CoA reductase inhibitors (statins): A promising approach to stroke prevention Neurology, February 22, 2000; 54(4): 790 - 796. [Abstract] [Full Text] [PDF]
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T. Bourcier and P. Libby HMG CoA Reductase Inhibitors Reduce Plasminogen Activator Inhibitor-1 Expression by Human Vascular Smooth Muscle and Endothelial Cells Arterioscler Thromb Vasc Biol, February 1, 2000; 20(2): 556 - 562. [Abstract] [Full Text] [PDF]
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C. Indolfi, A. Cioppa, E. Stabile, E. Di Lorenzo, G. Esposito, A. Pisani, A. Leccia, L. Cavuto, A. M. Stingone, A. Chieffo, et al. Effects of hydroxymethylglutaryl coenzyme A reductase inhibitor simvastatin on smooth muscle cell proliferation in vitro and neointimal formation in vivo after vascular injury J. Am. Coll. Cardiol., January 1, 2000; 35(1): 214 - 221. [Abstract] [Full Text] [PDF]
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A. H. Wagner, T. Kohler, U. Ruckschloss, I. Just, and M. Hecker Improvement of Nitric Oxide-Dependent Vasodilatation by HMG-CoA Reductase Inhibitors Through Attenuation of Endothelial Superoxide Anion Formation Arterioscler Thromb Vasc Biol, January 1, 2000; 20(1): 61 - 69. [Abstract] [Full Text] [PDF]
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A. Faggiotto and R. Paoletti Statins and Blockers of the Renin-Angiotensin System : Vascular Protection Beyond Their Primary Mode of Action Hypertension, October 1, 1999; 34(4): 987 - 996. [Abstract] [Full Text] [PDF]
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R. H. WEISS, A. RAMIREZ, and A. JOO Short-Term Pravastatin Mediates Growth Inhibition and Apoptosis, Independently of Ras, via the Signaling Proteins p27Kip1 and PI3 Kinase J. Am. Soc. Nephrol., September 1, 1999; 10(9): 1880 - 1890. [Abstract] [Full Text]
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