© 2001 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Simvastatin Has Anti-Inflammatory and Antiatherosclerotic Activities Independent of Plasma Cholesterol Lowering
From the Merck Research Laboratories, Rahway, NJ.
Correspondence to Carl P. Sparrow, Merck Research Laboratories, Building 80 W, 126 E. Lincoln Ave, Rahway, NJ 07065. E-mail Carl_Sparrow{at}Merck.com
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Abstract |
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Abstract—Inhibitors of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, such as simvastatin, lower circulating cholesterol levels and prevent myocardial infarction. Several studies have shown an unexpected effect of HMG-CoA reductase inhibitors on inflammation. Here, we confirm that simvastatin is anti-inflammatory by using a classic model of inflammation: carrageenan-induced foot pad edema. Simvastatin administered orally to mice 1 hour before carrageenan injection significantly reduced the extent of edema. Simvastatin was comparable to indomethacin in this model. To determine whether the anti-inflammatory activity of simvastatin might affect atherogenesis, simvastatin was tested in mice deficient in apoE. Mice were dosed daily for 6 weeks with simvastatin (100 mg/kg body wt). Simvastatin did not alter plasma lipids. Atherosclerosis was quantified through the measurement of aortic cholesterol content. Aortas from control mice (n=20) contained 56±4 nmol total cholesterol/mg wet wt tissue, 38±2 nmol free cholesterol/mg, and 17±2 nmol cholesteryl ester/mg. Simvastatin (n=22) significantly (P<0.02) decreased these 3 parameters by 23%, 19%, and 34%, respectively. Histology of the atherosclerotic lesions showed that simvastatin did not dramatically alter lesion morphology. These data support the hypothesis that simvastatin has antiatherosclerotic activity beyond its plasma cholesterol–lowering activity.
Key Words: atherosclerosis • apoE • mice • HMG-CoA reductase • inflammation
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Introduction |
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Elevated plasma cholesterol level is a major risk factor for atherosclerosis and myocardial infarction. The statin class of drugs inhibits the enzyme 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, the first committed step of sterol synthesis, and lowers plasma cholesterol levels. In large clinical trials, the use of statins reduces coronary events (see review in Bucher et al1 ), and 1 drug in this class, simvastatin, has been shown to reduce total mortality rates in patients with coronary heart disease.2
The effectiveness and rapidity with which statins decrease coronary events have led to the speculation that statins may favorably influence vascular biology via mechanisms other than lowered plasma cholesterol. Inhibitors of HMG-CoA reductase might directly alter cellular events other than cholesterol synthesis, because the product of the enzyme, mevalonate, is an important precursor for many isoprenoids. The isoprenoids farnesyl-pyrophosphate and geranylgeranyl-pyrophosphate play important roles in signal transduction via their attachment to critical signaling proteins, such as Ras and Rho.3
In vitro studies have documented cellular effects of statins that may be beneficial in atherosclerosis; these include the inhibition of leukocyte adhesion4 5 and decreased production of cytokines.6 Atherosclerosis is clearly an inflammatory disease,7 and the in vitro observations may be regarded as anti-inflammatory. However, it is not clear that these outcomes would occur in vivo at the circulating concentrations of statins achieved after conventional oral doses. Moreover, it is difficult to determine whether statins have direct anti-inflammatory activity on human atheroma in the face of strong cholesterol lowering.
Here, we use murine models to test the potential anti-inflammatory and antiatherosclerotic effects of simvastatin. The critical feature of these models is that simvastatin does not affect plasma lipid levels, and therefore the results may be interpreted without this confounding variable. A well-characterized foot pad swelling model demonstrated a dose-dependent anti-inflammatory action of simvastatin, with efficacy observed at doses comparable to those of indomethacin, a well-known anti-inflammatory drug. In additional studies, we address the potential anti-inflammatory activity of simvastatin on atherosclerosis using the apoE knockout mouse model. Simvastatin substantially reduced aortic cholesterol accumulation in this model, suggesting an important and direct antiatheroma effect of simvastatin.
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Methods |
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Animals
Normal C57BL/6NTac female mice were obtained from Taconic. Mice homozygous for disruption of the apoE gene (apoE-/-)8 9 were bred at the Merck Research Laboratories after being originally obtained from Jackson Laboratories. All mice were barrier housed, specific pathogen free, and maintained in static microisolator cages. Autoclaved food and water were provided ad libitum. The Institutional Animal Care and Use Committee of Merck Research Laboratories approved the animal use for experimentation, and all animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals (revised 1996, National Academy Press, Washington, DC).
Carrageenan Footpad Edema Assay of
Anti-Inflammatory Activity
Normal C57BL/6NTac female mice between 8 and 12 weeks
of age were used in all tests. To induce footpad swelling, mice
received a single subplantar injection of 0.05 mL of a sterile 1%
solution of carrageenan in water. Four hours later, footpad volume was
measured with a mercury plethysmograph and compared with the
preinjection volume of the same paw. Swelling (in microliters) was then
calculated, and in drug-treated animals, percent inhibition was derived
through comparison with the vehicle (methylcellulose) control group.
Both simvastatin and indomethacin were
administered orally in aqueous methylcellulose.
Atherosclerosis Studies in
apoE-/- Mice
Male apoE-/- mice were
weaned at 4 weeks of age onto a high-fat, Western-type diet that
contained 21.22% (g/100 g) fat, 17.01% protein, 48.48% carbohydrate,
and 0.15% cholesterol (TD88137; Harlan Teklad). Three
separate studies were performed. In the first study, animals were dosed
daily via oral gavage with 10 or 100 mg/kg simvastatin in
0.5% methyl-cellulose or administered methyl-cellulose alone (vehicle
control) starting at 16 weeks of age. After 6 weeks of dosing, mice
were killed, and tissues were processed as described later. The second
study was identical to the first, except that the dosing began at 20
weeks of age. The results from these 2 studies were pooled (see
Statistical Analysis). The final study included 3 groups of
animals: 1 group was analyzed at 20 weeks of age to determine
the baseline extent of atherosclerosis. The other 2
groups were analyzed at 26 weeks of age after 6 weeks of daily
dosing with simvastatin or vehicle
control.
Analysis of Plasma Lipids
At the time the mice were killed, they were weighed.
Blood was collected from the vena cava into syringes that contained
EDTA as an anticoagulant. Plasma was prepared via
centrifugation at
850g for 15 minutes at 4°C
and stored at -20°C for later evaluation of plasma
cholesterol and triglyceride levels. Plasma
cholesterol and triglyceride measurements were
made with standard enzymatic kits (Sigma Chemical Co). For a subset of
animals, plasma was subjected to lipoprotein analysis via FPLC
size exclusion chromatography with the BioLogic
Chromatography System (Bio-Rad Life Science). Some of
the samples subjected to FPLC lipoprotein analysis were pools
of plasma from multiple animals. In all cases, 200 µL of plasma was
chromatographed onto a Superose 6 HR 10/30 column (Amersham
Pharmacia Biotech) equilibrated and run in PBS containing 1 mmol/L
EDTA, pH 7.4. The column was run at a flow rate of 0.2 mL/min, and
0.27-mL fractions were collected. Of each fraction, 100 µL was
assayed for cholesterol (Cholesterol CII Kit;
Wako Diagnostics).
Aortic Cholesterol
Measurements
After collection of the blood sample, the vasculature
was gently perfused through the left ventricle with cold PBS and 5
mmol/L EDTA. For collection from the aorta for biochemical
analysis, all branches and any adipose tissue connected to the
aorta were removed, and each aorta was carefully excised from the
aortic root to the right renal artery. The aortas were stored briefly
on ice in PBS and then blotted dry, weighed, minced, and extracted with
chloroform/methanol (2:1) according to the method of Folch et
al.10 The lipid extracts
were dried down, resuspended quantitatively in chloroform/methanol
(2:1), and stored at -20°C until the time of assay. Total and free
cholesterol levels in the aortic extracts were determined
with an enzymatic fluorometric assay based on a modification of
previously described
methods.11 12
Briefly, the solvent was evaporated from aliquots containing 1 to 16
nmol of cholesterol, and the lipid residue was
resolubilized in 100 µL of reagent grade ethanol. Aliquots of
cholesterol (Aldrich) and cholesteryl oleate
(Aldrich) standard solutions prepared in chloroform/methanol (1:1)
were treated similarly. To determine free cholesterol,
samples and standards were incubated for 1 hour at 37°C in a total
volume of 1.01 mL of 0.1 mol/L potassium phosphate buffer, pH 7.4,
containing 0.03% Triton X-100 and 0.9 mmol/L sodium cholate.
Cholesterol oxidase (0.18 U; Boehringer Mannheim),
peroxidase (2 U; Boehringer Mannheim), and
p-hydroxyphenylacetic acid (0.5
mg/mL; Aldrich) were added for an additional 1-hour incubation at
37°C. The fluorescent product was measured in a Spex
FluoroMax (SPEX Industries, Inc) (excitation 325 nm, emission 415 nm)
with acrylic UVT semimicrocuvettes (Evergreen Scientific). For total
cholesterol determinations, cholesterol
esterase (10 U; Calbiochem) was included in the first incubation step,
and cholesteryl oleate was used as a standard. The cholesteryl ester in
each sample was calculated by subtracting the value of free
cholesterol from that for total cholesterol.
Samples for each aorta were run in duplicate at 2 different
concentrations. All values are expressed as nmol/mg wet tissue
wt.
Histology
The aortas from 4 or 5 mice in each treatment group
were harvested for histological analysis by
removing the heart with 
1 mm of proximal aorta attached, and
the portion distal to the tips of the auricles was excised and
discarded. The top half of the heart containing the aortic root was
stored briefly on ice in PBS, 0.02% NaN3, and
then frozen in OCT (Optimal Cutting Temperature) embedding medium
(Fisher Scientific) over liquid nitrogen-isopentane. The fresh frozen
hearts were used to examine the morphology of lesions in the aortic
root area. Sequential 20-µm sections were cut until the aortic valve
leaflets appeared. From this point on, serial 6-µm sections were
collected on 10-well masked slides (Erie Scientific). Sections were
stained with hematoxylin-phyloxine-saffron stain (Polyscientific) for
morphology. Additional sections were stained with oil red O
(Polyscientific) for lipids. For immunohistochemistry, sections were
fixed in Nakane fixative, washed in PBS, and blocked with 1.5% normal
goat serum, followed by avidin-biotin block (Vector). For detection of
CD18, sections were reacted with monoclonal anti-CD18 antibody
(Endogen). Primary antibody was followed by incubation with
biotinylated goat anti-hamster IgG antibody in the presence of 200
µg/mL normal mouse IgG. Antibody reactivity was detected using
HRP-conjugated biotin-streptavidin complexes and developed with
diaminobenzidine tetrahydrochloride as substrate.
Statistical Analyses
ANOVA was used for the assessment of effects of
simvastatin treatment on atherosclerosis. A
2-way ANOVA model with 2 main factors, treatment and study, and
treatmentxstudy interaction was applied to the data from the first 2
studies. The results indicated that there was no significant
interaction between study and treatment
(0.10<P<0.75). Therefore,
data from the first 2 studies were combined to test the pooled
treatment effects. The final number of animals analyzed were 20
control animals, 20 animals dosed with 10 mg/kg
simvastatin, and 22 animals dosed with 100 mg/kg
simvastatin. To better meet the normality assumption
required by the model, natural logarithm transformation was applied to
4 parameters: aortic total cholesterol, aortic
free cholesterol, aortic cholesteryl ester, and body
weight. Analyses of the plasma lipid parameters
were based on the original
scale.
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Results |
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Simvastatin Exhibits Anti-Inflammatory Activity In Vivo
Simvastatin was tested in a murine model of acute inflammation. Subplantar injection of carrageenan is followed by swelling of the footpad that can be reproducibly measured after 4 hours, and inhibition of footpad swelling provides a well-characterized gauge of anti-inflammatory activity.13 Oral treatment with the cyclooxygenase inhibitor indomethacin 1 hour before challenge blocked swelling in a dose-dependent manner (Figure 1
). Oral administration of simvastatin 1
hour before carrageenan challenge produced a comparable blockade of
swelling
(Figure 1). Importantly, strong anti-inflammatory activity
was observed with as little as 3 mg/kg simvastatin, and
maximal suppression by simvastatin was comparable to that
of indomethacin. Neither simvastatin nor
indomethacin was effective when administered 24 hours
before carrageenan injection (data not shown). These observations
strongly suggest that simvastatin has acute
anti-inflammatory activity. These effects cannot depend on alterations
in plasma lipid levels, because even in sensitive species, plasma
cholesterol levels decline only after several days of
therapy.
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Simvastatin Does Not Alter Plasma
Lipids in apoE-/- Mice
We sought evidence that simvastatin affects
not only acute inflammation but also the chronic inflammation that
occurs in the arteries in atherosclerosis. The achieve
this, we needed an animal model of atherosclerosis in
which simvastatin does not alter lipid levels. Statins are
reported to be ineffective in lowering cholesterol levels
in normal mice.14 A
potential explanation for this is the very strong compensatory increase
in HMG-CoA reductase that occurs in this
species.15 To determine
whether this resistance to plasma cholesterol lowering also
occurs in hyperlipidemic mice, we tested
simvastatin in apoE-/-
animals. The mice were fed a high-fat diet on weaning and were dosed
with 10 mg/kg simvastatin (n=20), 100 mg/kg
simvastatin (n=22), or vehicle control (n=20) for the last
6 weeks of the study (see Methods for details of the study).
Simvastatin did not significantly alter plasma
cholesterol and triglyceride levels in
apoE-/- mice when dosed at either 10 or
100 mg/kg (0.18<P<0.71)
(Figure 2). To assess whether simvastatin caused
subtle changes to the lipoprotein profile in the
apoE-/- mice, plasma lipoproteins were
analyzed with FPLC. Neither the overall profile nor HDL levels
were altered by simvastatin treatment
(Figure 3). Simvastatin did decrease body weight
in a dose-dependent manner in this study; the mean weights were 35±0.8
g for the control mice, 32±0.7 for the low-dose group
(P=0.01 versus control), and
31±0.7 for the high-dose group
(P=0.0002 versus
control).
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Simvastatin Decreases Aortic
Cholesterol Accumulation in
apoE-/- Mice
The extent of atherosclerosis in the
apoE-/- mice was quantified by measurement
of the aortic content of cholesterol. This
parameter has been used previously to quantify
atherosclerosis in
mice.16 In the rabbit model,
many studies have found good correlations between aortic
cholesterol content and atherosclerosis
measured according to other criteria, including intima/media
ratio17 18 and
lesion area as percent of total aortic surface
area.18 19 20 21 22 23 24 25
Furthermore, there are reports in both
rabbits24 25 and
apoE-/-
mice26 that drug
interventions show larger effects in the thoracic aorta than in the
arch, presumably because the area available for lesion development in
the arch "saturates" quickly and therefore there is a smaller
window available to detect changes. Aortic cholesterol
content is less likely to saturate, as evidenced by the
report27 of essentially
linear accumulation of aortic cholesterol over 12 months in
both cholesterol-fed and Watanabe heritable
hyperlipidemic rabbits.
The data in
Figure 4 show that simvastatin decreased aortic
cholesterol accumulation. Aortas from the control mice
contained 56±4 nmol/mg (mean±SEM) total cholesterol,
38±2 nmol/mg free cholesterol, and 17±2 nmol/mg
cholesteryl ester. Simvastatin at 100 mg/kg decreased total
aortic cholesterol by 23%, free cholesterol by
19%, and cholesteryl ester by 34%
(P<0.015 for all 3 effects)
(Figure 4). The decrease in aortic cholesterol
was dose responsive, as the aortic cholesterol in the group
of mice administered 10 mg/kg simvastatin was intermediate
between the control group and the 100 mg/kg group. None of the values
for the low-dose group were significantly different from those of
either of the other 2 groups
(0.11<P<0.28).
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Significantly different from control group (P=0.015).
Aortic cholesterol content is only 1 possible
measure of atherosclerosis. Aortic weight has been
proposed as a valid surrogate for
atherosclerosis.28
In the study shown in
Figure 4
, simvastatin decreased aortic weight in
a dose-dependent manner; the values were 6.7±0.5 mg in the control
group, 6.4±0.3 mg in the low-dose simvastatin group, and
6.2±0.3 mg in the high-dose simvastatin group. Although
aortic weights trended down in a dose-dependent fashion, neither the 10
mg/kg nor the 100 mg/kg data points alone attained statistical
significance
(0.21<P<0.37).
Simvastatin significantly decreased aortic
cholesterol accumulation without significantly decreasing
plasma cholesterol levels. Simvastatin did,
however, tend to lower cholesterol levels, and it seemed
possible that this tendency may have influenced the progression of
disease. To test whether plasma cholesterol levels
correlated with aortic cholesterol accumulation, we
calculated Pearson correlation coefficients between these 2
parameters for each treatment group and found no
significant correlation
(P>0.66). The correlation
coefficients were also calculated by combining all 3 treatment groups,
with residuals (ie, responses minus the group mean) used to remove the
differences due to treatment effects. Again, no correlation was found
(P>0.71). The lack of
correlation is shown graphically in
Figure 5, which compares plasma cholesterol and
aortic cholesteryl ester for each animal in the control and high-dose
groups. There clearly is no correlation between these 2
parameters, even though the largest effect seen with
simvastatin was for cholesteryl ester.
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Simvastatin Dramatically Decreased
Accumulation of Aortic Cholesterol During the 6-Week
Dosing Period
Atherosclerotic lesions in
apoE-/- mice increase in size throughout
the life span of the
animals.8 9 16
The data in
Figure 4 show that simvastatin decreased aortic
cholesterol accumulation even though the mice were dosed
for only 25% of their lifetime. To evaluate the suppression of
atherosclerotic lesion formation during the 6 weeks of
simvastatin dosing, we measured aortic
cholesterol accumulation in 3 groups of
apoE-/- mice: 20-week-old animals that
were never dosed (baseline group), animals administered 50 mg/kg
simvastatin for 6 weeks starting at 20 weeks of age
(progression with simvastatin group), and animals dosed in
parallel with vehicle only (progression control).
Simvastatin did not alter plasma lipids or body weights in
this study (0.35<P<0.87). The
aortic total cholesterol, free cholesterol, and
cholesteryl ester values in the vehicle control group were all
approximately twice those found in the baseline group
(P<0.0002)
(Figure 6). Simvastatin dramatically attenuated
the increase in lesion size during the 6-week dosing period (66%
decrease for total cholesterol,
P=0.005; 59% for free
cholesterol,
P=0.02; and 77% for
cholesteryl ester, P=0.0008)).
Simvastatin treatment essentially halted the deposition of
cholesteryl ester in the aorta, because this value was not different
from the baseline group
(P=0.20).
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The changes in aortic cholesterol content in the study were mirrored by changes in aortic weight. Aortic weight in the baseline group was 6.3±0.5 mg, and this parameter increased during the 6 weeks of progression to 8.3±0.8 mg (P=0.04 versus baseline). The aortic weight in the simvastatin-treated group was 7.1±0.3 mg (P=0.16 versus progression control group). These data show that during the 6 weeks of dosing, simvastatin treatment slowed the increase in aortic weight by 60%. The magnitude of this effect is very similar to the 59% to 77% decrease in aortic cholesterol accumulation caused by simvastatin.
Histology of Aortic Root Lesions From
Simvastatin-Treated and Control Animals Reveals Similar
Morphology
The data that show simvastatin prevented an
increase in aortic cholesterol content implied that
atherosclerotic lesion size was diminished. To determine whether
simvastatin altered the morphology of the lesions, the
aortic root areas of apoE-/- mice treated
with 100 mg/kg simvastatin were examined
histologically and compared with those from untreated
animals
(Figure 7). The overall morphology of lesions was very
similar with or without simvastatin treatment, with fibrous
areas, areas rich in foam cells, and necrotic areas present in both
(Figures 7A and 7B). Oil red O staining showed a similar
localization of lipid in lesions from untreated or
simvastatin-treated animals
(Figures 7C and 7D). Macrophage-derived foam cells
were identified through immunohistochemical staining of sections for
the macrophage marker CD18
(Figures 7E and 7F). Macrophages were present both
beneath overlying fibrous caps and at the surface of complex lesions
from both simvastatin-treated and control animals. The
effect of simvastatin treatment on lesion morphology in the
aortic root was therefore less apparent than the effect on the overall
deposition of cholesteryl esters within the aorta. This is perhaps due
to the lesions reaching a near-maximal size in the aortic root during
the course of the study, because the time for analysis was
chosen to obtain optimal biochemical measurements of aortic
cholesterol. Many studies have shown that lesions develop
faster in the aortic arch, and lesion development proceeds more slowly
in the more distal portions of the aorta, in both
rabbits24 25 29
and apoE-/-
mice.26 30 31
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Here, we show that simvastatin has anti-inflammatory activity in a well-characterized acute animal model: carrageenan-induced foot pad edema. The potency and effectiveness of simvastatin in this model were comparable to that of indomethacin, a classic anti-inflammatory agent. Both simvastatin and indomethacin were effective acutely (ie, when administered 1 hour before carrageenan injection), and neither compound was effective when administered 24 hours before injection. This anti-inflammatory effect could not have been caused by plasma lipid lowering, because simvastatin did not change lipid levels in these mice. Importantly, the strong effect of statins in causing changes in footpad swelling in mice occurred within 4 hours, well before any lipid changes could possibly occur. These observations suggest that although plasma lipids may affect inflammation, they cannot account for all of the anti-inflammatory effects observed for statins. These data strongly support the hypothesis that simvastatin has anti-inflammatory activity beyond its cholesterol-lowering activity.
The footpad swelling affected by simvastatin represents an acute inflammatory response characterized by the influx of polymorphonuclear leukocytes. Other workers have also observed an effect of simvastatin on acute inflammatory responses. Lefer et al32 demonstrated that a single administration of simvastatin (25 µg/rat) blocked the influx of polymorphonuclear leukocytes into heart muscle after ischemia and reperfusion. Furthermore, Endres et al33 showed that the administration of simvastatin to mice induces aortic endothelial NO synthase (eNOS) activity and renders animals resistant to cerebral ischemia/reperfusion injury. Additional observations in the literature suggest that simvastatin affects not only acute inflammation and the movement of cells of the innate immune system but also chronic inflammation and the action of cells of the adaptive immune system. Stanislaus et al6 showed that the long-term administration of 2 mg/kg lovastatin blocked neuroinflammation after challenge of the rats with myelin basic protein, a response that requires T lymphocytes. Moreover, additional work has shown that statins block the rejection of islet transplants in animals.34 Importantly, clinical studies have shown that both simvastatin and pravastatin decrease the incidence of cardiac allograft vasculopathy in cardiac transplant patients.35 36 These observations suggest a broad range of anti-inflammatory activities of statins.
Atherosclerosis is clearly an inflammatory
process,7 and interruption of
the function of inflammatory mediators can decrease atherosclerotic
lesion size in mice. This has been shown for
interferon-
,37 as well as
for monocyte chemoattractant
protein-138 and its
receptor, CCR2.39 In humans,
serum levels of C-reactive protein predict vascular
disease,40 implying that
low-level inflammation accelerates atherosclerosis. The
anti-inflammatory properties of statins may thus contribute to the
observed effects on coronary heart disease. However,
determination of the magnitude of the potential contribution is made
difficult by the strong effects of statins on LDL
cholesterol, an undisputed contributor to atherogenesis. To
overcome this difficulty, we tested simvastatin in a model
of atherosclerosis that is resistant to
statin-mediated plasma cholesterol lowering. Using the
apoE-/- mouse model, we show that
simvastatin decreased aortic cholesterol
accumulation in mice without lowering cholesterol levels or
altering lipoprotein profile. These data strongly support the
hypothesis that simvastatin has antiatherosclerotic
activity beyond its cholesterol-lowering
activity.
In our studies of apoE-/- mice, the extent of atherosclerosis did not correlate with plasma cholesterol levels. This lack of correlation has been previously observed in this model. Dansky et al41 showed that plasma cholesterol did not correlate with atherosclerotic lesion area in the F2 progeny of C57/apoE-/- x FVB/apoE-/- mice. Despite the failure of small changes in plasma cholesterol levels to correlate with atherosclerosis in apoE-/- mice, interventions that dramatically lower cholesterol levels can decrease atherosclerosis in apoE-/- mice.42 43 Therefore, plasma cholesterol does contribute to atherosclerosis in this model.
What is the mechanism by which statins achieve blockade of inflammation leading to decreased atherosclerotic lesion size? The most frequently proposed model is that statins interrupt proinflammatory signaling by blocking the geranyl-geranylation of proteins such as the GTPase Rho-A.44 Members of the Rho family and related proteins have well-documented roles in signaling a variety of cellular functions, including the cytoskeletal rearrangements required for cell migration. Interruption of geranyl-geranylation is plausible, because statins block the synthesis of mevalonate, the precursor of farnesol, geranyl-geraniol, and cholesterol. Simvastatin-induced changes in protein prenylation might have their greatest influence on vascular biology by altering the production of NO. Statins have been shown to increase both the expression and the activity of eNOS in vitro45 and in vivo.45 46 47 This increase may be related to changes in prenylation, because the in vitro effects could be overcome by the addition of mevalonate or geranylgeranyl pyrophosphate to the media.45 NO from endothelial cells is thought to be anti-inflammatory. For example, Fox-Robichaud et al48 observed that inflammatory cells move into tissues on inhibition of eNOS, suggesting an anti-inflammatory tone produced by eNOS action. The NO produced from eNOS is probably also antiatherosclerotic, as evidenced by the favorable effects of arginine on atherosclerosis.49 This hypothesis is also consistent with the observation that fluvastatin decreased atherosclerosis and increased eNOS mRNA in rabbit aorta without altering plasma lipids.47
Our results with simvastatin support the hypothesis that simvastatin has anti-inflammatory activity that is relevant to the prevention of atherosclerosis by this drug. Although the mechanism is not yet established, further research may lead to new understanding of the actions of statins and new therapeutic interventions for atherosclerosis.
Received September 5, 2000; accepted September 27, 2000.
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