Eur J Clin Pharmacol (2003) 58: 719–731
DOI 10.1007/s00228-002-0556-0
R EV IE W A RT I C L E
Ulrich Laufs
Beyond lipid-lowering: effects of statins on endothelial nitric oxide
Received: 2 September 2002 / Accepted: 15 December 2002 / Published online: 18 February 2003
Ó Springer-Verlag 2003
Abstract Endothelial dysfunction is now recognised as
an important process in the pathogenesis of atheroscle-
rosis. Nitric oxide (NO) release by the endothelium
regulates blood flow, inflammation and platelet aggre-
gation, and consequently its disruption during endo-
thelial dysfunction can decrease plaque stability and
encourage the formation of atherosclerotic lesions and
thrombi. Inhibitors of 3-hydroxy-3-methylglutaryl
coenzyme A reductase (statins) are often utilised in the
prevention of coronary heart disease due to their efficacy
at lowering lipid levels. However, statins may also pre-
vent atherosclerotic disease by non-lipid or pleiotropic
effects, for example, improving endothelial function by
promoting the production of NO. There are various
mechanisms whereby statins may alter NO release, such
as inhibiting the production of mevalonate and impor-
tant isoprenoid intermediates, thereby preventing the
isoprenylation of the small GTPase Rho, which nega-
tively regulates the expression of endothelial nitric oxide
synthase (eNOS). Furthermore, statins may also in-
crease eNOS activity via post-translational activation of
the phosphatidylinositol 3-kinase/protein kinase Akt
(PI3 K/Akt) pathway and/or through an interaction
with the molecular chaperone heat-shock protein 90
(HSP90). Data suggest that statins may vary in their
efficacy for enhancing the release of NO, and the
mechanisms dictating these differences are not yet clear.
By increasing NO production, statins may interfere with
atherosclerotic lesion development, stabilise plaque, in-
hibit platelet aggregation, improve blood flow and pro-
tect against ischaemia. Therefore, the ability of statins to
improve endothelial function through the release of NO
may partially account for their beneficial effects at re-
ducing the incidence of cardiovascular events.
U. Laufs
Medizinische Klinik und Poliklinik der Universität des Saarlandes,
Innere Medizin III, 66421, Homburg/Saar, Germany
E-mail: ulrich@laufs.com
Tel.: +49-6841-1623000
Fax: +49-6841-1623437
Keywords Atherosclerosis Æ Endothelial dysfunction Æ
Nitric oxide
Introduction
Atherosclerosis is the underlying cause of many car-
diovascular diseases, such as stroke, myocardial infarc-
tion and peripheral artery disease. There is increasing
support for the premise that atherosclerosis is the result
of a chronic inflammation perpetuated by a dysfunc-
tional endothelium [1]. Atherosclerotic lesions develop
as a result of monocyte migration and subsequent foam-
cell formation, combined with vascular smooth-muscle
cell proliferation. As the endothelium regulates vascular
tone, endothelial permeability, platelet aggregation, and
leucocyte activation and extravasation, it has been
implicated in the development of atherosclerosis [2].
Indeed, endothelial dysfunction can promote athero-
sclerosis by affecting production of vasoactive media-
tors, such as nitric oxide (NO) and prostacyclin (PGI2),
which regulate these processes. The most important
and well-known endothelium-derived mediator is NO.
Disrupted production and release of endothelium-
derived NO is a major consequence of endothelial dys-
function [3].
The aim of this review was to examine the role of NO
in the development of atherosclerosis and how statins
may improve endothelial function.
Endothelial dysfunction and the role
of NO in atherosclerosis
Endothelial dysfunction occurs early in the development
of atherosclerosis as a result of exposure to cardiovas-
cular risk factors, such as age, smoking, diabetes, hy-
pertension or dyslipidaemia [1, 2]. A dysfunctional
endothelium predisposes blood vessels to processes
favouring the development of atherosclerosis (Fig. 1).
720
Fig. 1 Scheme through which cardiovascular risk factors may
promote atherosclerosis via endothelial dysfunction. LDL low-
density lipoprotein
For example, macrophage accumulation, a key process
in atherosclerotic lesion development, may occur when a
dysfunctional endothelium: (1) disrupts anti-inflamma-
tory processes, leading to increased monocyte activa-
tion, adhesion and migration; (2) increases endothelial
permeability thereby increasing access of monocytes and
lipoproteins to the vessel wall; and/or (3) reduces
availability of vasodilators, thereby reducing blood flow
and aiding monocyte adhesion and migration.
Following monocyte migration there are several steps
in the development of an atherosclerotic lesion (Fig. 1).
Once within the vessel wall, macrophages can take up
and store oxidised low-density lipoprotein (oxLDL),
forming a foam cell and creating the characteristic ‘‘fatty
streak’’ of an early atherosclerotic lesion [118]. Release
of growth factors from these inflammatory cells can
cause proliferation of vascular smooth muscle cells
within the intima, further increasing lesion size and
decreasing lumen diameter. Finally, release of matrix
metalloproteases from leucocytes can break down col-
lagen and cause atherosclerotic plaque to destabilise and
rupture, leading to thrombus formation and triggering
an acute cardiovascular event [4].
One consequence of endothelial dysfunction is
reduced production of the vasoactive mediator NO,
which is created during the conversion of L-arginine to
L-citrulline by endothelial nitric oxide synthase (eNOS).
Production and release of NO by endothelial cells occurs
at both a basal level and in response to stimuli by
vasoactive mediators such as endothelin-1, bradykinin
and acetylcholine. In addition, flow of blood over en-
dothelial cells (shear stress) can also stimulate release of
NO [5]. Indeed, it is notable that atherosclerotic lesions
tend to develop at vascular bifurcations, where turbu-
lence disturbs the flow of blood over the endothelium.
Several anti-atherosclerotic processes are mediated by
NO (Fig. 2). NO can inhibit vascular smooth muscle cell
proliferation [6], induce vasodilation [7] and increase
blood flow [8], promote angiogenesis, increase endothe-
lial cell survival [9], reduce platelet aggregation [10],
inhibit inflammation [11] and regulate leucocyte–endo-
thelium interactions [12, 13]. For example, NO can
reduce adhesion of monocytes to human endothelial
cells (Fig. 2) by counteracting the induction of vascular
cell adhesion molecule-1 (VCAM-1) by cytokines [14]
and by downregulating the chemokine monocyte
chemoattractant protein-1 (MCP-1) at a transcriptional
level [15]. Considering the pivotal role played by NO in
regulating atherosclerotic processes, it is of little surprise
that an imbalance in production has been shown to
promote the development of coronary artery disease in
individuals with multiple risk factors [16]. Mice lacking
the gene for eNOS show increased blood pressure,
impaired vascular response to injury and suffer from
larger cerebral infarcts [17, 18, 19]. Furthermore, it has
been reported that chronic inhibition of eNOS induces
early vascular inflammation and development of ath-
erosclerosis in rats [20].
Improving endothelial dysfunction and NO release
may represent a new treatment paradigm for the pre-
vention of coronary heart disease (CHD). The 3-hy-
droxy-3-methylglutaryl coenzyme A (HMG-CoA)
reductase inhibitors (statins), a class of lipid-modifying
agents used in the treatment and prevention of athero-
sclerosis, have been shown to have beneficial effects on
these processes.
Use of statins for the treatment of atherosclerosis
There is a strong association between elevated plasma
cholesterol levels and atherosclerosis [21, 22]. Conse-
quently, physicians recognise the importance of lowering
total cholesterol and LDL cholesterol levels to prevent
CHD. The cholesterol-modifying agents of choice for
prevention of CHD are the statins [23, 24].
HMG-CoA reductase catalyses the rate-limiting step
of cholesterol biosynthesis, a four-electron reductive
deacylation of HMG-CoA to CoA and mevalonate.
Statins are HMG-CoA reductase inhibitors with inhi-
bition constant values in the nanomolar range. All sta-
tins share a HMG-like moiety and inhibit the reductase
by the same mechanism. Recently, the structure of the
catalytic portion of human HMG-CoA reductase com-
plexed with different statins was determined [25]. The
 721

Fig. 2 Diagram indicating the

influence of oxidised low-
density lipoprotein (LDL) on
nitric oxide (NO) production
and its effect on various
atherosclerotic processes.
Dotted lines indicate an
inhibitory effect. eNOS
endothelial nitric oxide
synthase, L-ArgL-arginine,
L-Cit L-citrulline, MCP-1
monocyte chemoattractant
protein-1, ICAM-1 intercellular
adhesion molecule-1, VCAM-1
vascular cell adhesion molecule-
1, oxLDL oxidised low-density
lipoprotein

bulky, hydrophobic compounds of statins occupy the
HMG-binding pocket and block access of the substrate
HMG. The tight binding of statins is due to the large
number of van der Waals interactions between inhibitors
and HMG-CoA reductase. The structurally diverse,
rigid, hydrophobic groups of the different statins are
accommodated in a shallow non-polar groove that is
present only when COOH-terminal residues of HMG-
CoA reductase are disordered. There are subtle differ-
ences in the modes of binding between the various
statins, with the synthetic compounds atorvastatin and
rosuvastatin having the greatest number of bonding
interactions with HMG-CoA reductase [25]. These
differences account for the diverse inhibition constant
values. The currently available statins (atorvastatin,
fluvastatin, lovastatin, pravastatin and simvastatin) can
reduce LDL cholesterol levels by 24–60% (Table 1) [26].
New synthetic statins, such as rosuvastatin, are more
effective at reducing LDL cholesterol (Table 1) [27, 116,
117, 119], possibly due to their improved binding to
HMG-CoA reductase. The effectiveness of statins at
reducing cholesterol levels and cardiovascular events has
been reported in large outcome-based clinical trials [28,
29, 30, 31, 32, 33].
More recently, additional benefits of statins have
been identified that may be independent of their

Table 1 Comparison of lipophilicity and the selectivity of choles- not available, IC50 inhibition of cholesterol synthesis by 3-hydroxy
terol synthesis for various statins in hepatocytes and endothelial 3-methylglutaryl coenzyme A reductase, HUVEC human umbilical
cells. LDL-C low-density lipoprotein cholesterol, NA information vein endothelial cells, NO nitric oxide

Statin Maximum Reduction Lipophilicityc Plasma Purified IC50 (nM) HUVECe NO releasef
,c
dose in LDL-C at half-life enzyme *
maximum dose
(mg) (%) Log D at (h) Rat hepatocytese (% of A23187-
pH 7.4 stimulated release)**

Atorvastatin 80 55a 1.11 14d 8.2 0.8 5.5 27

Cerivastatin
0.4 34a 1.69 23d 10 2.5 3.1 NA
Fluvastatin 80 34a 1.27 1.2d 27.6 4.8 0.6 NA
Lovastatin 80 41a NA 2d NA NA NA 23
Pravastatin 40 34a 0.84 12d 44.1 5.0 1900 28
Rosuvastatin 40 63b 0.33 20c 5.4 0.3 41 NA
Simvastatin 80 48a 1.6 12d 11.2 5.2 1.0 56

a
Cerivastatin is no longer available Maron et al. 2000 [26]
b
*Human HMG-CoA reductase catalytic domain Olsson et al. 2001 [27]
c
**NO release is the peak NO concentration after stimulation of McTaggart et al. 2001 [28] and Hanefield 2001 [29]
d
bovine endothelial cells with of 1 lmol/l of statin compared as a Knopp 1999 [30]
e
percentage decrease from the peak concentration after stimulation Buckett et al. 2000 [31]
f
with calcium ionophore, A23187 (1 lmol/l) Dobrucki et al. 2001 [32]
722
cholesterol-lowering effects. Pleiotropic effects, i.e. ac-
tions not dependent on cholesterol lowering, have been
observed in several studies. For example, in the West of
Scotland Coronary Prevention Study (WOSCOPS), the
Scandinavian Simvastatin Survival Study (4S) and the
recent Heart Protection Study (HPS), it was noted that
clinical benefit associated with statin therapy was inde-
pendent of LDL cholesterol levels [33, 34, 35]. Fur-
thermore, although LDL cholesterol is not considered a
major risk factor for stroke [36], statin treatment can
reduce the risk of cerebrovascular events [33, 37, 38].
However, the clinical importance of non-cholesterol or
‘‘pleiotropic’’ effects of statins in humans in relation to
cardiovascular mortality is difficult to determine in a
prospective clinical trial because statins effectively re-
duce cholesterol levels even in individuals with low
baseline cholesterol levels. In cynomolgus monkeys,
cholesterol-independent effects of statins were demon-
strated using a ‘‘clamped-cholesterol’’ design, where
dietary cholesterol was regulated to maintain equal
changes in lipoprotein levels in control and pravastatin-
treated animals [39]. In addition, a similar study has
shown a cholesterol-independent reduction in the
development of atherosclerosis with rosuvastatin in
APOE*-Leiden transgenic mice [40]. One explanation
for the pleiotropic effects of statins is their ability to
influence endothelial function, and NO production in
particular.
Effects of statins on NO synthesis
Increased release of NO from bovine endothelial cells in
culture has been demonstrated with lovastatin, ator-
vastatin, pravastatin, simvastatin and rosuvastatin [32,
41]. Enhanced NO release by statins appears to be as-
sociated with dose-dependent increases in eNOS in hu-
man endothelial cells in culture [42, 43]. This effect of
statins on eNOS expression has been observed in several
species, including human cells in culture [42, 43, 44] and
in mice [45], rats [46], rabbits [47], dogs [48] and primates
in vivo [39]. The NO synthase inhibitor, L-nitro arginine
methyl ester (L-NAME), inhibited the increased gener-
ation of NO with rosuvastatin [49], pravastatin and
simvastatin [13, 50] in rats and rat tissues in vitro, con-
firming an effect on synthesis by eNOS. Both basal and
stimulated NO release is increased with statin treatment.
For example, basal and stimulated release of NO was
increased by 45% and 107%, respectively, from cultured
bovine aortic endothelial cells treated with atorvastatin
[51]. These studies show that statins increase the bio-
availability of NO.
The mechanisms whereby statins improve NO pro-
duction are being elucidated. It has been suggested that
this beneficial effect of statins may be a result of reduced
formation or availability of mevalonate within endothe-
lial cells [52]. Indeed, statin-induced increases in eNOS
expression are reversed by addition of mevalonate to
human endothelial cells in culture [42, 53]. Mevalonate is
an important precursor in the biosynthesis of cholesterol
(Fig. 3) and its production is inhibited by statins.
However, mevalonate is a substrate for compounds other
than cholesterol [54]. This may indicate a potential
mechanism for the cholesterol-independent effects
observed with statins. It is now apparent that the
mechanisms whereby statins increase NO production can
be both cholesterol dependent and cholesterol indepen-
dent (Table 2, Fig. 3).
Cholesterol-dependent regulation of NO by statins
Regulation of NO production and endothelial function
may be dependent on LDL cholesterol concentration
and oxidation status [120]. LDL cholesterol apheresis
has been shown to rapidly improve endothelial-depen-
dent vasodilation [55] and coronary blood flow [56] in
hypercholesterolaemic patients. While this could argu-
ably be a result of decreased blood viscosity and a cor-
responding improvement in shear stress-mediated NO
release, in vitro experiments indicate other possible
mechanisms. In cultured human endothelial cells, eNOS
mRNA and protein was downregulated by oxidised
LDL cholesterol [57] as well as by atherogenic concen-
trations of human native LDL [58]. Therefore, by re-
ducing LDL cholesterol levels, statins may prevent this
downregulation of eNOS. Indeed, such an effect has
been reported with simvastatin treatment in human en-
dothelial cells in culture [53].
Oxidation status of LDL also appears to be an im-
portant factor in the regulation of eNOS. It has been
reported that expression of eNOS mRNA in human and
bovine endothelial cells was decreased by oxLDL in a
concentration-dependent manner [57, 59]. Simvastatin,
atorvastatin and lovastatin treatment attenuated this
reduction in eNOS expression in the presence of oxLDL
[59, 60]. Therefore, the reduction of LDL and hence
oxLDL, may be a mechanism for statins to regulate
eNOS in vivo. However, in cell-culture experiments,
statins do not decrease the concentration of the exter-
nally added cholesterol, indicating that additional path-
ways are also likely to contribute to eNOS upregulation.
Oxidised LDL appears to regulate eNOS through its
action on caveolin-1, a primary coat protein of cellular
invaginations called caveolae that are necessary for the
assembly and trafficking of cholesterol [61]. Caveolin-1
has been shown to bind to eNOS in caveolae and act as a
negative regulator of the enzyme [61, 62]. Exposure of
bovine endothelial cells to hypercholesterolaemic serum
has been shown to upregulate caveolin-1 abundance and
promotes association of caveolin-1 and eNOS into in-
hibitory complexes, thereby decreasing NO production
[63]. From these data, it is possible to reason that statins
may improve eNOS activity by reducing cholesterol and,
consequently, abundance and expression of caveolin-1.
Indeed, statins have been shown to reduce caveolin-1
abundance in cultured bovine endothelial cells [51] and
mice in vivo [64] and thereby decrease its inhibitory

Fig. 3 Mevalonate-dependent
inhibition of endothelial nitric
oxide synthase (eNOS) may
occur through cholesterol-
dependent and/or independent
mechanisms. Acetyl-CoA acetyl
coenzyme A, HMG-CoA
3-hydroxy-3-methylglutaryl
coenzyme A, NO nitric oxide,
oxLDL oxidised low density
lipoprotein, PP pyrophosphate
action on both basal and agonist-stimulated eNOS ac-
tivity [51]. Feron and colleagues have reported that basal
NO release was potentiated by statin treatment more
when cholesterol levels were low, whereas stimulated
release was greater when cholesterol levels were high
[51]. This varying ability of statin treatment to influence
caveolin-1 and eNOS with cholesterol level may be
suggestive of a combination of cholesterol-dependent
and independent effects. Thus, statins may regulate NO
production through lowering cholesterol and decreasing
abundance of caveolin-1.
Receptors for LDL also mediate cholesterol-depen-
dent regulation of eNOS. For example, the oxLDL
class-B scavenger receptor, CD36, has been shown to
mediate the effects of oxLDL on caveolae composition
and eNOS activation in cultured human microvascular
endothelial cells [65]. Antibodies for CD36 prevented
oxLDL-induced redistribution of eNOS and increased
acetylcholine-induced eNOS activation. Upregulation of
the lectin-like oxidised LDL receptor 1 (LOX-1) by ox-
LDL was associated with a reduction in eNOS expres-
sion in human coronary artery endothelial cells in
culture [66]. Thus, statins could improve eNOS expres-
sion by reducing LDL. Treatment of human coronary
artery endothelial cells in culture with simvastatin or
atorvastatin reduced the oxLDL-induced LOX-1 upre-
gulation and increased eNOS expression. However, as
statins are unable to influence extracellular cholesterol in
these cell culture experiments, cholesterol-independent
effects may also have an important role to play in in-
creasing eNOS expression.
723
Cholesterol-independent regulation of NO by statins
In addition to their effects on NO through cholesterol
synthesis, statins can upregulate eNOS expression in-
dependent of plasma cholesterol levels (Fig. 3) [67]. In-
hibition of mevalonate synthesis by statins prevents the
synthesis of important isoprenoid intermediates such as
geranylgeranylpyrophosphate (GGPP). Rho GTPases,
members of the Ras superfamily of GTP-binding pro-
teins, consist of at least 14 proteins that can be separated
into Rho, Rac and Cdc42 [68]. Rho proteins cycle be-
tween an active GTP-bound state and an inactive GDP-
bound state (Fig. 4). Translocation of inactive Rho in
the cytosol to the membrane where it is active is de-
pendent on geranylgeranylation [69]. The active form of
Rho can induce changes in the actin cytoskeleton and
influence intracellular transport, gene transcription and
mRNA stability [70]. Rho can negatively regulate eNOS
expression [69, 71] and it is the inhibition of Rho
isoprenylation by statins that was shown to be respon-
sible for the upregulation of eNOS mRNA expression
within human endothelial cells in culture [72]. Indeed,
the effects of statins on Rho isoprenylation and eNOS
expression can be reversed by GGPP. The inhibition of
Rho isoprenylation by statins has been shown to in-
crease eNOS mRNA stability in cultured human endo-
thelial cells [44, 72, 71]. While statins prevent the
activation of Rho by geranylgeranylation, it has been
noted that a negative feedback signal from the actin
cytoskeleton results in increased Rho gene transcription
724

Table 2 Mechanism of action of statins on nitric oxide pathways. LDL low-density lipoprotein, LOX-1 lectin-like oxidised LDLreceptor-1, oxLDL oxidised LDL,NO nitric oxide, and accumulation. Therefore, if statin treatment is

Feron et al. 1999 [63]; Everson & Smart 2001 [61]; Sessa 2001 [62]
ceased, the increase in active Rho may induce a rebound

Liao et al. 1995 [57], Vidal et al. 1998 [58], Hernandez-Perera

Laufs & Liao 1998 [72], 2000 [69]; Takemoto et al. 2002 [71]
inhibition of NO [73].

Oxidative stress, NO and statins

Dimmeler et al. 1999 [91]; Fulton et al. 1999 [92]

Feron et al. 2001 [51]; Brouet et al. 2001 [95]

Endres et al. 1998 [42]; Scalia et al. 2001 [52]
Oxygen radicals impair endothelial function and accel-

Wagner et al. 2000 [84]; Wassman 2002 [85]

Feron et al. 2001 [51]; Pelat et al. 2002 [64]
erate the progression of atherosclerotic lesions by
et al. 1998 [59]; Mehta et al. 2001 [66]

promoting lipid oxidation, the expression of proinflam-

matory genes and by oxidative inactivation of endothe-
Laufs & Liao 1998 [72], 2000 [69]

lial NO [74, 75, 76]. Superoxide radicals can react with

Garcia-Cardena et al. 1998 [94]

NO to form peroxynitrite (ONOO)). As ONOO) does
not activate guanylyl cyclase as effectively as NO, the
bioavailability of NO is limited [77]. ONOO) has also
Kureishi et al. 2000 [90]
Hattori et al. 2002 [83]
Wang et al. 1998 [74]

been shown to transform LDL cholesterol into its pro-

atherogenic oxidised form [75, 78]. In addition to the
inactivation of NO by superoxide, there is evidence to
suggest that oxLDL cholesterol can also inhibit the ac-
Reference

tivity of NO in cultured bovine aortic endothelial cells

eNOS endothelial nitric oxide synthase, NADPH nicotinamide adenine dinucleotide phosphate, HSP heat shock protein

[79]. There are several sources of superoxide radicals

within vascular cells, including NADH/NADPH oxid-
ases, xanthine oxidase and NOS [80]. When the avail-
LDL cholesterol increases the abundance ofcaveolin-1 (an inhibitor of eNOS)

ability of its co-factor tetrahydrobiopterin is limited,

production may determine their effectiveness at improving NO production

eNOS can uncouple and generate superoxide anions [81,

The ability of statins to influence circulating or endothelial mevalonate

82, 121, 122]. Interestingly, statins have been shown to

potentiate the synthesis of tetrahydrobiopterin in rat
Statins potentiate the synthesis of tetrahydrobiopterin (a cofactor

Mevalonate, a precurser of cholesterol and isoprenoid synthesis,

aortic smooth muscle cells in culture [83], which may

Rho negatively regulates eNOS mRNA expression and stability

Statins decrease production of superoxide by NADPH oxidase

Statins promote the phosphorylation and activation of eNOS

shift the balance away from NOS-generated superoxide

for eNOS that reduces production of superoxide by eNOS)
Oxidative stress and oxLDL can inhibit the activityof NO

production to the generation of NO. Statins such as

Protein kinase Akt phosphorylates and activates eNOS

atorvastatin appear to have further indirect anti-oxidant

Statins promote the recruitment of HSP90 to eNOS

effects by preventing isoprenylation of the GTP-binding

OxLDL/native LDL downregulates eNOS mRNA

can limit the effect of statins on NO production

protein p21 Rac, which reduces NADPH oxidase-

Statins may decrease the abundance of caveolin

HSP90 binds to, and rapidly activates, eNOS

dependent superoxide formation by rat endothelial cells

[84] and smooth muscle cells [85] in culture and in vivo
[46, 85]. Fluvastatin treatment in hypercholesterolaemic
rabbits has been shown to reduce superoxide production
Statins inhibit Rho isoprenylation

and improve NO availability in arteries [86]. Thus,

statins may prolong the availability of NO through an
anti-oxidant capacity. The relevance of this anti-oxidant
effect of statins in humans has not yet been clarified
since anti-oxidant vitamin supplements have no effect on
coronary events [87]. Another recent study indicates that
anti-oxidants may limit the beneficial effect of statins on
coronary events [88]. In contrast, a prior study with anti-
oxidants has shown that flow-mediated dilation, a
Details

measure of endothelial function, can be improved when

used in combination with a statin in hypercholestero-
laemic men [89]. Therefore, whether anti-oxidants are
Regulation of NO independent

beneficial and an anti-oxidant activity is a clinically

relevant effect of statins has yet to be demonstrated
satisfactorily.
Additional mechanisms

Mevalonate hypothesis
Cholesterol-dependent

of serum cholesterol

and NO regulation
regulation of NO

Oxidative stress

Additional mechanisms of NO regulation

Mechanism

There are further mechanisms whereby NO release

may be improved by statin treatment. For example,
statins may increase eNOS activity via post-translational

Fig. 4 Regulation of small G-
proteins: Rho isoprenylation a
key event in cellular events. PP
pyrophosphate, GG
geranylgeranyl, GEF guanine
nucleotide exchange factors,
GTP guanine triphosphate,
GAP GTPase activating
proteins, HMG-Co A
3-hydroxy-3-methylglutaryl
coenzyme A
activation of the phosphatidylinositol 3-kinase/protein
kinase Akt (PI3 K/Akt) pathway, as has been reported
in cultured human endothelial cells [90]. Phosphoryla-
tion of Akt is an important event in several cellular ac-
tivities. Indeed, production of NO by the endothelium
can be regulated by phosphorylation and activation of
eNOS by Akt [91, 92]. Simvastatin has been shown to
promote phosphorylation and activation of eNOS by
Akt, thereby increasing angiogenesis in normocholeste-
rolaemic rabbits [90]. Interestingly, the activation of Akt
by statins in total mononuclear cells from human blood
is also thought to increase the release of endothelial
progenitor cells [93], which may play a role in angio-
genesis and the revascularisation of ischaemic tissue and
contribute to the benefits of statin therapy in the sec-
ondary prevention of coronary events.
Statins may also increase eNOS activity through an
interaction with the molecular chaperone, heat shock
protein 90 (HSP90). Studies have shown that binding of
HSP90 to eNOS is associated with rapid activation and
release of NO [94]. Statins promote the recruitment of
HSP90 to the eNOS protein in cultured bovine endo-
thelial cells [51], an effect that may involve the loss of
plasma membrane cholesterol and/or caveolin-1 [62].
Atorvastatin has been shown to stimulate NO-depen-
dent angiogenesis independent of eNOS expression. In
cultured human endothelial cells, statin-induced phos-
phorylation of eNOS was directly dependent on the
ability of HSP90 to recruit Akt to the eNOS complex,
and this potentiated NO-mediated angiogenesis [95].
Taken together, the different cholesterol-independent
pathways leading to upregulation of endothelial NO
bioavailability may explain the observation that one of
the earliest clinical effects of statin treatment is a rapid
improvement of NO-dependent vasodilatation [96, 97,
98, 99]. Indeed, increased endothelial NO release has
been reported as early as 3 days after initiation of statin
treatment in patients with elevated LDL cholesterol
levels [97]. Conversely, retrospective analysis suggests
that withdrawal of statin treatment in a vulnerable
725
clinical situation, such as an acute coronary syndrome,
may increase the rate of myocardial infarction [100].
Differences among statins
Statins differ in their ability to increase NO production
in vitro [32, 101]. However, comparative studies of the
pleiotropic effects of statins are few. One study of cul-
tured bovine aortic endothelial cells has shown that
simvastatin induced the release of more NO than pra-
vastatin, atorvastatin and lovastatin (Table 1) [32]. In
contrast, another study has shown pravastatin to be
more effective than simvastatin at releasing NO from
cultured bovine endothelial cells and producing NO-
mediated vasorelaxation of endothelium-intact rat aortic
rings [50].
The reasons why statins differ in their ability to re-
lease NO are unclear. Various factors could influence
their effectiveness at increasing NO production. For
example, it has been shown that regulation of NO syn-
thesis by endothelial cells can, at least in part, be cho-
lesterol dependent. Therefore, the ability of a statin
to lower LDL cholesterol levels (Table 1) may be rele-
vant in determining the degree that NO production may
be improved, particularly in hypercholesterolaemic
patients.
The mevalonate hypothesis
Mevalonate appears to play a major role in the regula-
tion of NO production. Statin-induced increases in
eNOS within cultured human endothelial cells can be
reversed by addition of mevalonate [42, 53]. Therefore,
increasing mevalonate concentrations within endothelial
cells could limit the NO-mediated benefits of statins and
may explain why there are differences in pleiotropic ef-
ficacy between statins. If circulating mevalonate does
limit the activity of eNOS in endothelial cells, what
726
would be the source and how would the different statins
regulate it? Endothelial cell mevalonate concentration
may be dependent on extracellular sources or intracel-
lular production (Fig. 5). It seems most likely that the
liver, being involved in the synthesis of cholesterol, may
be a major source of mevalonate. Hence, it may be hy-
pothesised that circulating mevalonate levels are most
likely influenced by those statins that easily access, and
are potent at inhibiting, hepatocyte HMG-CoA reduc-
tase. Unfortunately, comparative data for the effect of
statins on circulating mevalonate is scarce. Similarly,
there is insufficient data currently available on the role of
circulating mevalonate at limiting the extra-hepatic ef-
fects of statins.
Another source of mevalonate that would influence
the pleiotropic activity of statins is the endothelial cell
itself. Intracellular mevalonate production is likely to be
dependent on a statin’s ability to enter endothelial cells
and its potency for inhibiting endothelial HMG-CoA
reductase. It has been suggested that the varying lipo-
philicity of statins affects their uptake into target organs
and, therefore, their ability to elicit pleiotropic effects
[69, 102]. The relative lipophilicity of some commonly
available statins and their potency on hepatocyte and
endothelial cell HMG-CoA reductase are listed for
comparison in Table 1. Lipophilic statins, such as lo-
vastatin and cerivastatin, are considered more likely to
enter endothelial cells by passive diffusion than hydro-
philic statins, such as pravastatin and rosuvastatin,
which are targeted to the liver. However, in bovine en-
dothelial cells in culture, where external sources of
cholesterol and mevalonate are absent, pravastatin was
at least as effective as more lipophilic statins such as
atorvastatin, lovastatin [32] and simvastatin [50] at
stimulating the release of NO from cultured bovine
endothelial cells. Moreover, it would also seem that
Fig. 5 Possible mechanisms whereby both lipophilic and hydro-
philic statins may improve endothelial release of nitric oxide (NO).
EC endothelial cell, Acetyl-Co A acetyl coenzyme A, HMG-Co A
3-hydroxy-3-methylglutaryl coenzyme A, eNOS endothelial nitric
oxide synthase
pravastatin is the least potent statin at inhibiting HMG-
CoA reductase from human endothelial cells in culture
(Table 1) [31]. Similarly, despite being less lipophilic and
less potent at inhibiting endothelial HMG-CoA reduc-
tase than atorvastatin and simvastatin (Table 1) [28, 31],
rosuvastatin is at least as effective and potent as these
agents at increasing eNOS activity in mouse aorta [41].
Thus, the lipophilicity and potency within endothelial
cells does not entirely predict the ability of statins to
improve NO production/release, and so other unidenti-
fied factors may play a role. Indeed, vascular MCP-1
expression can also be downregulated by statins in hy-
percholesterolaemic pigs, regardless of their lipophilicity
[103]. It may be that there are specific mechanisms for
hydrophilic statins to enter endothelial cells. Such a
mechanism is present in the liver, where the hepatic or-
ganic anion transporter (OATP-C) helps hydrophilic
statins enter hepatocytes [104], and a similar process in
endothelial cells would explain why the lipophilicity of a
statin does not always predict its pleiotropic efficacy.
More data on how different statins enter endothelial
cells are needed to address this issue.
It is likely that there is a complex interplay of
mechanisms determining the effectiveness of statins for
increasing NO release. Further comparative and mech-
anistic studies are required if this pleiotropic effect is to
be understood fully and predictions made on the utility
of different statins for increasing NO in humans.
Benefits of statins by improving endothelial function
Animal experiments
By increasing NO production, statins can interfere with
atherosclerotic lesion development, thereby improving
vascular health and reducing the risk of atherosclerotic
disease. The beneficial effect of increasing NO release
with statins has been shown in several animal models of
cardiovascular disease. For example, a study with flu-
vastatin has demonstrated regression of atherosclerosis
in rabbits, an effect associated with upregulation of
eNOS mRNA and restoration of endothelial function in
cholesterol-fed rabbits [47]. In addition, simvastatin
provided protection against stroke by increasing cere-
bral blood flow in wild-type mice, although this benefit
was not observed in eNOS ()/)) mice [42], indicating
that NO mediates this effect of statin treatment. Simi-
larly, treatment with the synthetic statins, atorvastatin
or rosuvastatin significantly reduced cerebral infarct
volume after an hour of cerebral artery occlusion in
normocholesterolaemic mice [41, 105]. This protective
effect was mediated in part by upregulation of eNOS
and a subsequent decrease in platelet activation. Other
experiments show protection from ischaemic damage
with statin treatment. Following an episode of hindlimb
ischaemia in mice, collateral vessel growth and enhanced
blood flow was promoted by cerivastatin, and this was
associated with an increase in eNOS activity [106].

Simvastatin has been reported to limit contractile dys-
function in isolated rat hearts subjected to global myo-
cardial ischaemia and reperfusion [13]. This protective
effect was attributed to a NO-mediated attenuation of
neutrophil infiltration into the myocardium [13]. Simi-
larly, a reduction in myocardial injury following ischa-
emia/reperfusion in rats in vivo has been observed with
rosuvastatin treatment [107]. These latter effects are
likely a result of decreased leucocyte–endothelium in-
teractions. For example, rosuvastatin reduced expres-
sion of P-selectin and, consequently, leucocyte adhesion
and migration in rats [49]. This effect with rosuvastatin
was not observed in eNOS-deficient mice [49, 52], indi-
cating that the anti-inflammatory benefits with statin
treatment are a result of an improvement in endothelial
function and NO release.
Human studies
Statins have been shown to increase NO production in
humans. For example, fluvastatin increased the bio-
availability of NO in a randomised, double-blind, pla-
cebo-controlled trial of 29 hypercholesterolaemic
patients [108]. Similarly, simvastatin has also been
shown to improve endothelial function, augment basal
and acetylcholine-stimulated vasodilation, over 4 weeks
in a double-blind, placebo-controlled, crossover trial
[98]. Moreover, the NO enhancing effects of statins oc-
cur at clinically relevant doses [109].
However, it is difficult to determine whether this
ability of statin treatment to increase NO production is
clinically important. In a clinical setting, it would not
be easy to differentiate between benefits derived from
improved NO release and the well-reported advantages
of lipid lowering, particularly as cholesterol may
modulate the production of NO. Indeed, changes in
forearm blood flow were reported to be inversely re-
lated to alterations in LDL cholesterol levels in humans
[108]. Furthermore, both the inhibition of cholesterol
synthesis and the cholesterol-independent regulation of
NO by statins are dependent on the production of a
common precurser, mevalonate (Fig. 3). Consequently,
it is unsurprising that the two effects are often inversely
related. However, despite these difficulties, a short-term
study with pravastatin has demonstrated a NO-medi-
ated improvement in forearm blood flow and an in-
crease in urinary nitrite/nitrate production, indicative
of an increase in NO production, which occurred in-
dependently of changes in cholesterol levels in normo-
cholesterolaemic patients with other risk factors for
CHD [110]. In another study, short-term (2 weeks)
lipid-lowering therapy with cerivastatin improved en-
dothelial function and NO bioavailability in patients
with hypercholesterolaemia [97]. This improvement in
endothelium-dependent vasodilation was no longer
observed when the NO synthase inhibitor N(G)-
monomethyl-L-arginine was co-infused. Similarly, in
normocholesterolaemic men with normal vascular
727
function, treatment with high-dose atorvastatin (80 mg)
increased forearm blood flow within 24 h, whereas se-
rum cholesterol and high-sensitivity C reactive protein
(hs-CRP) were not decreased until 2 days of treatment.
Cessation of statin treatment after 30 days resulted in a
rebound of forearm blood flow within 24 h of with-
drawal, whereas cholesterol and hs-CRP slowly and
steadily returned to baseline [111]. Therefore, the po-
tential importance of cholesterol-independent effects of
statins in humans may also relate to the time course of
their effects, for example, after withdrawal of treatment
[101].
Theoretically, it may be possible to observe a rela-
tionship between the cholesterol-independent effect of
statins on NO and cardiovascular outcomes by com-
paring statin-treated patients with individuals whose
cholesterol was lowered to the same degree with a non-
statin, lipid-lowering agent that does not influence NO.
In cynomolgus monkeys, who develop atherosclerosis
very similar to humans, cholesterol-independent effects
of statins, including upregulation of endothelial NO,
were demonstrated using a ‘‘clamped-cholesterol’’ de-
sign, where dietary cholesterol was adjusted to equalise
cholesterol levels between groups [39, 112]. Inhibitors of
NO production are unlikely to be useful to differentiate
between NO-mediated and lipid-dependent effects of
statins in long-term studies since NO plays such an in-
tegral role in normal vascular function. Unfortunately,
many potential studies are not currently possible be-
cause of a lack of suitable pharmacological tools. NO
does however appear to have some clinical relevance in
atherosclerotic disease. For example, there is a reduced
contribution of NO to vasodilation in patients with
hypercholesterolaemia [113] or risk factors for athero-
sclerosis [114]. In addition, restoring vascular NO for-
mation can improve symptoms of intermittent
claudication in patients with peripheral arterial disease
[115]. A prospective trial to test a potentially negative
effect of withdrawal of statin treatment mediated by
reduced NO production is ethically impossible. Thus,
there is compelling circumstantial evidence that im-
proved NO production by statins may be clinically rel-
evant, but it may be some time before such an effect is
proven to contribute to the reduction in cardiovascular
events.
Conclusions
Statins improve endothelial dysfunction by enhancing
the production of NO, which may partially account for
their beneficial effects in reducing the incidence of car-
diovascular events. The mechanisms of increased NO
production by statins may be cholesterol dependent and/
or cholesterol independent.
Statins vary in their efficacy for increasing NO pro-
duction. The exact mechanisms dictating these differences
have yet to be fully elucidated, but may be related to their
ability to reduce LDL cholesterol, to lower circulating
728
and/or endothelial cell mevalonate or by their lipophilic-
ity and ability to access endothelial cells.
By improving endothelial function and NO produc-
tion, statin therapy may limit the development of ath-
erosclerosis and improve cardiovascular clinical
outcomes for patients. Furthermore, in patients with
atherosclerosis, increasing NO levels with statins may
decrease the likelihood of an acute event by stabilising
plaques, increasing blood flow and reducing platelet
aggregation.
Acknowledgements Thanks to G. Allcock for editorial assistance in
the preparation of this manuscript. U.L. received financial support
from and has been a speaker for AstraZeneca, Bayer, MSD,
Novartis, Pfizer and Sankyo. U.L.’s research was supported by the
Deutsche Forschungsgemeinschaft and the Universität des Saar-
landes.
References
1. Libby P, Ridker PM, Maseri A (2002) Inflammation and
atherosclerosis. Circulation 105:1135–1143
2. Kinlay S, Ganz P (2000) Relation between endothelial dys-
function and the acute coronary syndrome: implications for
therapy. Am J Cardiol 86[Suppl]:10J–13J
3. Liao JK (1998) Endothelium and acute coronary syndromes.
Clin Chem 44:1799–1808
4. Libby P (2001) Current concepts of the pathogenesis of the
acute coronary syndromes. Circulation 104:365–372
5. Joannides R, Haefeli WE, Linder L, Richard V, Bakkali EH,
Thuillez C, Luscher TF (1995) Nitric oxide is responsible for
flow-dependent dilatation of human peripheral conduit ar-
teries in vivo. Circulation 91:1314–1319
6. Garg UC, Hassid A (1989) Nitric oxide-generating vasodila-
tors and 8-bromo-cyclic guanosine monophosphate inhibit
mitogenesis and proliferation of cultured rat vascular smooth
muscle cells. J Clin Invest 83:1774–1777
7. Fleming I, Busse R (1999) NO: the primary EDRF. J Mol
Cell Cardiol 31:514
8. Vallance P, Collier J, Moncada S (1989) Nitric oxide syn-
thesised from L-arginine mediates endothelium dependent
dilatation in human veins in vivo. Cardiovasc Res 23:1053–
1057
9. Dimmeler S, Zeiher AM (1999) Nitric oxide – an endothelial
cell survival factor. Cell Death Differ 6:964–968
10. Radomski MW, Palmer RM, Moncada S (1987) Endogenous
nitric oxide inhibits human platelet adhesion to vascular
endothelium. Lancet 2:1057–1058
11. Peng HB, Libby P, Liao JK (1995) Induction and stabilization
of I kappa B alpha by nitric oxide mediates inhibition of NF-
kappa B. J Biol Chem 270:14214–14219
12. Kubes P, Suzuki M, Granger DN (1991) Nitric oxide: an
endogenous modulator of leukocyte adhesion. Proc Natl Acad
Sci USA 88:4651–4655
13. Lefer AM, Campbell B, Shin YK, Scalia R, Hayward R, Lefer
DJ (1999) Simvastatin preserves the ischemic-reperfused
myocardium in normocholesterolemic rat hearts. Circulation
100:178–184
14. De Caterina R, Libby P, Peng HB, Thannickal VJ, Rajav-
ashisth TB, Gimbrone MA Jr, Shin WS, Liao JK (1995) Nitric
oxide decreases cytokine-induced endothelial activation.
Nitric oxide selectively reduces endothelial expression of
adhesion molecules and proinflammatory cytokines. J Clin
Invest 96:60–68
15. Tsao PS, Wang B, Buitrago R, Shyy JY, Cooke JP (1997)
Nitric oxide regulates monocyte chemotactic protein-1.
Circulation 96:934–940
16. Ijem J, Granlie C (2000) More than cholesterol: the com-
plexity of coronary artery disease. S D J Med 53:489–491
17. Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz
MA, Bevan JA, Fishman MC (1995) Hypertension in mice
lacking the gene for endothelial nitric oxide synthase. Nature
377:239–242
18. Huang Z, Huang PL, Ma J, Meng W, Ayata C, Fishman MC,
Moskowitz MA (1996) Enlarged infarcts in endothelial nitric
oxide synthase knockout mice are attenuated by nitro-L-arg-
inine. J Cereb Blood Flow Metab 16:981–987
19. Huang PL (1998) Disruption of the endothelial nitric oxide
synthase gene: effect on vascular response to injury. Am J
Cardiol 82:57S–59S
20. Ni W, Egashira K, Kataoka C, Kitamoto S, Koyanagi M,
Inoue S, Takeshita A (2001) Antiinflammatory and antiarte-
riosclerotic actions of HMG-CoA reductase inhibitors in a rat
model of chronic inhibition of nitric oxide synthesis. Circ Res
89:415–421
21. LaRosa JC, Hunninghake D, Bush D, Criqui MH, Getz GS,
Gotto AM Jr, Grundy SM, Rakita L, Robertson RM, Weis-
feldt ML, Cleeman JI (1990) The cholesterol facts. A summary
of the evidence relating dietary fats, serum cholesterol, and
coronary heart disease. A joint statement by the American
Heart Association and the National Heart, Lung and Blood
Institute. The Task Force on Cholesterol Issues, American
Heart Association. Circulation 81:1721–1733
22. Coresh J, Kwiterovich PO Jr (1996) Small, dense low-density
lipoprotein particles and coronary heart disease risk: a clear
association with uncertain implications. JAMA 276:914–915
23. Wood D, De Backer G, Faergeman O, Graham I, Mancia G,
Pyörälä K (1998) Prevention of coronary heart disease in
clinical practice: recommendations of the second joint task
force of European and other societies on coronary prevention.
Eur Heart J 19:1434–1503
24. Gotto AM Jr, Kuller LH (2002) Eligibility for lipid-lowering
drug therapy in primary prevention: how do the Adult
Treatment Panel II and Adult Treatment Panel III guidelines
compare? Circulation 105:136–139
25. Istvan ES, Deisenhofer J (2001) Structural mechanism for
statin inhibition of HMG-CoA reductase. Science 292:1160–
1164
26. Maron DJ, Fazio S, Linton MF (2000) Current perspectives
on statins. Circulation 101:207–213
27. Olsson AG, Pears J, McKellar J, Mizan J, Raza A (2001)
Effect of rosuvastatin on low-density lipoprotein cholesterol in
patients with hypercholesterolemia. Am J Cardiol 88:504–508
28. Scandinavian Simvastatin Survival Study Group (1994)
Randomised trial of cholesterol lowering in 4444 patients with
coronary heart disease: the Scandinavian Simvastatin Survival
Study (4S). Lancet 344:1383–1389
29. Shepherd J, Cobbe SM, Ford I, Isles CG, Lorimer AR,
MacFarlane PW, McKillop JH, Packard CJ, for the West of
Scotland Coronary Prevention Study Group (1995) Preven-
tion of coronary heart disease with pravastatin in men with
hypercholesterolemia. N Engl J Med 333:1301–1307
30. Sacks FM, Pfeffer MA, Moye LA, Rouleau JL, Rutherford
JD, Cole TG, Brown L, Warnica JW, Arnold JM, Wun C-C,
Davis BR, Braunwald E (1996) The effect of pravastatin on
coronary events after myocardial infarction in patients with
average cholesterol levels. Cholesterol and Recurrent Events
Trial investigators. N Engl J Med 335:1001–1009
31. Downs JR, Clearfield M, Weis S, Whitney E, Shapiro DR,
Beere PA, Langendorfer A, Stein EA, Kruyer W, Gotto AM
Jr (1998) Primary prevention of acute coronary events with
lovastatin in men and women with average cholesterol levels:
results of AFCAPS/TexCAPS. Air Force/Texas Coronary
Atherosclerosis Prevention Study. JAMA 279:1615–1622
32. The Long-term Intervention with Pravastatin in Ischemic
Disease (LIPID) Study Group (1998) Prevention of cardio-
vascular events and death with pravastatin in patients with
coronary heart disease and a broad range of initial cholesterol
levels. N Engl J Med 339:1349–1357

33. Heart Protection Study Collaborative Group (2002) MRC/
BHF heart protection study of cholesterol lowering with
simvastatin in 20,536 high-risk individuals: a randomised
placebo-controlled trial. Lancet 360:722
34. West of Scotland Coronary Prevention Study Group (1998)
Influence of pravastatin and plasma lipids on clinical events in
the West of Scotland coronary prevention study (WOSCOPS).
Circulation 97:1440–1445
35. Scandinavian Simvastatin Survival Study Group (1995) Base-
line serum cholesterol and treatment effect in the Scandinavian
simvastatin survival study (4S). Lancet 345:1274–1275
36. Prospective Studies Collaboration (1995) Cholesterol, dia-
stolic blood pressure, and stroke: 13,000 strokes in 450,000
people in 45 prospective cohorts. Lancet 346:1647–1653
37. Pedersen TR, Kjekshus J, Pyörälä K, Olsson AG, Cook TJ,
Musliner TA, Tobert JA, Haghfelt T (1998) Effect of sim-
vastatin on ischemic signs and symptoms in the Scandinavian
simvastatin survival study (4S). Am J Cardiol 81:333–335
38. Liao JK (2002) Statins and ischemic stroke. Atherosclerosis
Suppl 3:21–25
39. Williams JK, Sukhova GK, Herrington DM, Libby P (1998)
Pravastatin has cholesterol-lowering independent effects on
the artery wall of atherosclerotic monkeys. J Am Coll Cardiol
31:684–691
40. Havekes LM, van Duyvenvoorde W, Maas MCE, van der
Boom H, van den Hoogen CM, Emeis JJ, Princen HMG
(2002) Rosuvastatin reduces atherosclerosis independently of
its cholesterol-lowering effect in APOE*3 Leiden transgenic
mice. Atherosclerosis 3:187
41. Laufs U, Gertz K, Dirnagl U, Bohm M, Nickenig G, Endres
M (2002) Rosuvastatin, a new HMG-CoA reductase inhibitor,
upregulates endothelial nitric oxide synthase and protects
from ischemic stroke in mice. Brain Res 942:23–30
42. Endres M, Laufs U, Huang Z, Nakamura T, Huang P,
Moskowitz MA, Liao JK (1998) Stroke protection by 3-hy-
droxy-3-methylglutaryl (HMG)-CoA reductase inhibitors
mediated by endothelial nitric oxide synthase. Proc Natl Acad
Sci U S A 95:8880–8885
43. Mueck AO, Seeger H, Wallwiener D (2001) Further evidence
for direct vascular actions of statins: effect on endothelial
nitric oxide synthase and adhesion molecules. Exp Clin
Endocrinol Diabetes 109:181–183
44. Laufs U, Fata VL, Liao JK (1997) Inhibition of 3-hydroxy-
3-methylglutaryl (HMG)-CoA reductase blocks hypoxia-
mediated down-regulation of endothelial nitric oxide synthase.
J Biol Chem 272:31725–31729
45. Yamada M, Huang Z, Dalkara T, Endres M, Laufs U,
Waeber C, Huang PL, Liao JK, Moskowitz MA (2000)
Endothelial nitric oxide synthase-dependent cerebral blood
flow augmentation by L-arginine after chronic statin treat-
ment. J Cereb Blood Flow Metab 20:709–717
46. Wassmann S, Laufs U, Baumer AT, Muller K, Ahlbory K,
Linz W, Itter G, Rosen R, Bohm M, Nickenig G (2001)
HMG-CoA reductase inhibitors improve endothelial dys-
function in normocholesterolemic hypertension via reduced
production of reactive oxygen species. Hypertension 37:1450–
1457
47. Kano H, Hayashi T, Sumi D, Esaki T, Asai Y, Thakur NK,
Jayachandran M, Iguchi A (1999) A HMG-CoA reductase
inhibitor improved regression of atherosclerosis in the rabbit
aorta without affecting serum lipid levels: possible relevance of
up-regulation of endothelial NO synthase mRNA. Biochem
Biophys Res Commun 259:414–419
48. Mital S, Zhang X, Zhao G, Bernstein RD, Smith CJ, Fulton
DL, Sessa WC, Liao JK, Hintze TH (2000) Simvastatin
upregulates coronary vascular endothelial nitric oxide pro-
duction in conscious dogs. Am J Physiol Heart Circ Physiol
279:H2649–H2657
49. Stalker TJ, Lefer AM, Scalia R (2001) A new HMG-CoA
reductase inhibitor, rosuvastatin, exerts anti-inflammatory
effects on the microvascular endothelium: the role of meva-
lonic acid. Br J Pharmacol 133:406–412
729
50. Kaesemeyer WH, Caldwell RB, Huang J, Caldwell RW (1999)
Pravastatin sodium activates endothelial nitric oxide synthase
independent of its cholesterol-lowering actions. J Am Coll
Cardiol 33:234–241
51. Feron O, Dessy C, Desager JP, Balligand JL (2001) Hydroxy-
methylglutaryl-coenzyme A reductase inhibition promotes
endothelial nitric oxide synthase activation through a decrease
in caveolin abundance. Circulation 103:113–118
52. Scalia R, Stalker TJ, Lefer AM (2001) Mechanisms of the
anti-inflammatory action of rosuvastatin, a new HMG-CoA
reductase inhibitor. Atherosclerosis Suppl 2:37
53. Martinez-Gonzalez J, Raposo B, Rodriguez C, Badimon L
(2001) 3-hydroxy-3-methylglutaryl coenzyme a reductase
inhibition prevents endothelial NO synthase downregulation
by atherogenic levels of native LDLs: balance between tran-
scriptional and posttranscriptional regulation. Arterioscler
Thromb Vasc Biol 21:804–809
54. Grunler J, Ericsson J, Dallner G (1994) Branch-point
reactions in the biosynthesis of cholesterol, dolichol, ubiq-
uinone and prenylated proteins. Biochim Biophys Acta
1212:259–277
55. Tamai O, Matsuoka H, Itabe H, Wada Y, Kohno K, Imaiz-
umi T (1997) Single LDL apheresis improves endothelium-
dependent vasodilation in hypercholesterolemic humans.
Circulation 95:76–82
56. Mellwig KP, Baller D, Gleichmann U, Moll D, Betker S,
Weise R, Notohamiprodjo G (1998) Improvement of coro-
nary vasodilatation capacity through single LDL apheresis.
Atherosclerosis 139:173–178
57. Liao JK, Shin WS, Lee WY, Clark SL (1995) Oxidized low-
density lipoprotein decreases the expression of endothelial
nitric oxide synthase. J Biol Chem 270:319–324
58. Vidal F, Colome C, Martinez-Gonzalez J, Badimon L (1998)
Atherogenic concentrations of native low-density lipoproteins
down-regulate nitric-oxide-synthase mRNA and protein levels
in endothelial cells. Eur J Biochem 252:378–384
59. Hernandez-Perera O, Perez-Sala D, Navarro-Antolin J,
Sanchez-Pascuala R, Hernandez G, Diaz C, Lamas S (1998)
Effects of the 3-hydroxy-3-methylglutaryl-CoA reductase in-
hibitors, atorvastatin and simvastatin, on the expression of
endothelin-1 and endothelial nitric oxide synthase in vascular
endothelial cells. J Clin Invest 101:2711–2719
60. Laufs U, La Fata V, Plutzky J, Liao JK (1998) Upregulation
of endothelial nitric oxide synthase by HMG CoA reductase
inhibitors. Circulation 97:1129–1135
61. Everson WV, Smart EJ (2001) Influence of caveolin, choles-
terol, and lipoproteins on nitric oxide synthase: implications
for vascular disease. Trends Cardiovasc Med 11:246–250
62. Sessa WC (2001) Can modulation of endothelial nitric oxide
synthase explain the vasculoprotective actions of statins?
Trends Mol Med 7:189–191
63. Feron O, Dessy C, Moniotte S, Desager JP, Balligand JL
(1999) Hypercholesterolemia decreases nitric oxide production
by promoting the interaction of caveolin and endothelial nitric
oxide synthase. J Clin Invest 103:897–905
64. Pelat M, Dessy C, Feron O, Balligand J-L (2002) HMG-CoA
reductase inhibition decreases caveolin-1 independently of
plasma lipid lowering in heart and blood vessels of apoE ()/))
mice. Int J Clin Pract Suppl 124:4
65. Uittenbogaard A, Shaul PW, Yuhanna IS, Blair A, Smart EJ
(2000) High density lipoprotein prevents oxidized low density
lipoprotein-induced inhibition of endothelial nitric-oxide
synthase localization and activation in caveolae. J Biol Chem
275:11278–11283
66. Mehta JL, Li DY, Chen HJ, Joseph J, Romeo F (2001)
Inhibition of LOX-1 by statins may relate to upregulation of
eNOS. Biochem Biophys Res Commun 289:857–861
67. Chen H, Ikeda U, Shimpo M, Shimada K (2000) Direct effects
of statins on cells primarily involved in atherosclerosis.
Hypertens Res 23:187–192
68. Hall A (1998) Rho GTPases and the actin cytoskeleton.
Science 279:509–514
730
69. Laufs U, Liao JK (2000) Direct vascular effects of HMG-CoA
reductase inhibitors. Trends Cardiovasc Med 10:143–148
70. Laufs U, Liao JK (2000) Targeting Rho in cardiovascular
disease. Circ Res 87:526–528
71. Takemoto M, Sun J, Hiroki J, Shimokawa H, Liao JK (2002)
Rho-kinase mediates hypoxia-induced downregulation of
endothelial nitric oxide synthase. Circulation 106:57–62
72. Laufs U, Liao JK (1998) Post-transcriptional regulation of
endothelial nitric oxide synthase mRNA stability by Rho
GTPase. J Biol Chem 273:24266–24271
73. Laufs U, Endres M, Custodis F, Gertz K, Nickenig G, Liao
JK, Böhm M (2000) Suppression of endothelial nitric oxide
production after withdrawal of statin treatment is mediated by
negative feedback regulation of Rho GTPase gene transcrip-
tion. Circulation 102:3104–3110
74. Wang HD, Pagano PJ, Du YT, Cayatte AJ, Quinn MT,
Brecher P, Cohen RA (1998) Superoxide anion from the ad-
ventitia of the rat thoracic aorta inactivates nitric oxide. Circ
Res 82:810–818
75. Darley-Usmar VM, Hogg N, O’Leary VJ, Wilson MT,
Moncada S (1992) The simultaneous generation of superoxide
and nitric oxide can initiate lipid peroxidation in human low
density lipoprotein. Free Radic Res Commun 17:920
76. Nickenig G, Harrison DG (2002) The AT(1)-type angiotensin
receptor in oxidative stress and atherogenesis. Part I: oxidative
stress and atherogenesis. Circulation 105:393–396
77. Gryglewski RJ, Palmer RM, Moncada S (1986). Superoxide
anion is involved in the breakdown of endothelium-derived
vascular relaxing factor. Nature 320:454–456
78. Hogg N, Darley-Usmar VM, Graham A, Moncada S (1993)
Peroxynitrite and atherosclerosis. Biochem Soc Trans 21:358–
362
79. Chin JH, Azhar S, Hoffman BB (1992) Inactivation of endo-
thelial-derived relaxing factor by oxidized lipoproteins. J Clin
Invest 89:10–18
80. Kojda G, Harrison D (1999) Interactions between NO and
reactive oxygen species: pathophysiological importance in
atherosclerosis, hypertension, diabetes and heart failure.
Cardiovasc Res 43:562–571
81. Xia Y, Dawson VL, Dawson TM, Snyder SH, Zweier JL
(1996) Nitric oxide synthase generates superoxide and nitric
oxide in arginine-depleted cells leading to peroxynitrite-me-
diated cellular injury. Proc Natl Acad Sci U S A 93:6770–6774
82. Stuehr D, Pou S, Rosen GM (2001) Oxygen reduction by
nitric oxide synthases. J Biol Chem 276:14533–14536
83. Hattori Y, Nakanishi N, Kasai K (2002) Statin enhances
cytokine-mediated induction of nitric oxide synthesis in
vascular smooth muscle cells. Cardiovasc Res 54:649–658
84. Wagner AH, Kohler T, Ruckschloss U, Just I, Hecker M
(2000) Improvement of nitric oxide-dependent vasodilatation
by HMG-CoA reductase inhibitors through attenuation of
endothelial superoxide anion formation. Arterioscler Thromb
Vasc Biol 20:61–69
85. Wassman S, Laufs U, Muller K, Konkol C, Ahlbory K,
Baumer AT, Linz W, Bohm M, Nickenig G (2002) Cellular
antioxidant effects of atorvastatin in vitro and in vivo. Arte-
rioscler Thromb Vasc Biol 22:300–305
86. Sumi D, Hayashi T, Thankur NK, Jayachandran M, Asai Y,
Kano H, Matsui H, Iguchi A (2001) A HMG-CoA reductase
inhibitor possesses a potent anti-atherosclerotic effect other
than serum lipid lowering effects the relevance of endothelial
nitric oxide synthase and superoxide anion scavenging action.
Atherosclerosis 155:347–357
87. Heart Protection Study Collaborative Group (2002) MRC/
BHF heart protection study of antioxidant vitamin supple-
mentation in 20,536 high-risk individuals: a randomised
placebo-controlled trial. Lancet 360:23–33
88. Brown BG, Zhao X-Q, Chait A, Fisher LD, Cheung MC,
Morse JS, Dowdy AA, Marino EK, Bolson EL, Alaupovic P,
Frohlich J, Albers JJ (2001) Simvastatin and niacin, antioxi-
dant vitamins, or the combination for the prevention of
coronary disease. N Engl J Med 345:1583–1592
89. Neunteufl T, Kostner K, Katzenschlager R, Zehetgruber M,
Maurer G, Weidinger F (1998) Additional benefit of vitamin E
supplementation to simvastatin therapy on vasoreactivity of
the brachial artery of hypercholesterolemic men. J Am Coll
Cardiol 32:711–716
90. Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ,
Sessa WC, Walsh K (2000) The HMG-CoA reductase inhib-
itor simvastatin activates the protein kinase Akt and promotes
angiogenesis in normocholesterolemic animals. Nat Med
6:1004–1010
91. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R,
Zeiher AM (1999) Activation of nitric oxide synthase in en-
dothelial cells by Akt-dependent phosphorylation. Nature
399:601–605
92. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y,
Walsh K, Franke TF, Papapetropoulos A, Sessa WC (1999)
Regulation of endothelium-derived nitric oxide production by
the protein kinase Akt. Nature 399:597–601
93. Dimmeler S, Aicher A, Vasa M, Mildner-Rihm C, Alder K,
Tiemann M, Rutten H, Fichtlscherer S, Martin H, Zeiher AM
(2001) HMG-CoA reductase inhibitors (statins) increase en-
dothelial progenitor cells via the PI 3-kinase/Akt pathway. J
Clin Invest 108:391–397
94. Garcia-Cardena G, Fan R, Shah V, Sorrentino R, Cirino G,
Papapetropoulos A, Sessa WC (1998) Dynamic activation of
endothelial nitric oxide synthase by Hsp90. Nature 392:821–824
95. Brouet A, Sonveaux P, Dessy C, Moniotte S, Balligand JL,
Feron O (2001) Hsp90 and caveolin are key targets for the
proangiogenic nitric oxide-mediated effects of statins. Circ Res
89:866–873
96. Anderson TJ, Meredith IT, Yeung AC, Frei B, Selwyn AP,
Ganz P (1995) The effect of cholesterol-lowering and antiox-
idant therapy on endothelium-dependent coronary vasomo-
tion. N Engl J Med 332:488–493
97. John S, Delles C, Jacobi J, Schlaich MP, Schneider M, Sch-
mitz G, Schmieder RE (2001) Rapid improvement of nitric
oxide bioavailability after lipid-lowering therapy with ceri-
vastatin within two weeks. J Am Coll Cardiol 37:1351–1358
98. O’Driscoll G, Green D, Taylor RR (1997) Simvastatin, an
HMG-coenzyme A reductase inhibitor, improves endothelial
function within 1 month. Circulation 95:1126–1131
99. Tsunekawa T, Hayashi T, Kano H, Sumi D, Matsui-Hirai H,
Thakur NK, Egashira K, Iguchi A (2001) Cerivastatin, a hy-
droxymethylglutaryl coenzyme a reductase inhibitor, im-
proves endothelial function in elderly diabetic patients within
3 days. Circulation 104:376–379
100. Heeschen C, Hamm CW, Laufs U, Snapinn S, Böhm M,
White HD. Platelet and Receptor Inhibition in Ischaemic
Syndrome Management (PRISM) Investigators (2002) With-
drawal of statins increases event rates in patients with acute
coronary syndromes. Circulation 105:1446–1452
101. Aengevaeren WR (1999) Beyond lipids the role of the endo-
thelium in coronary artery disease. Atherosclerosis 147[Suppl
1]:S11–S16
102. Fukumoto Y, Libby P, Rabkin E, Hill CC, Enomoto M,
Hirouchi Y, Shiomi M, Aikawa M (2001) Statins alter smooth
muscle cell accumulation and collagen content in established
atheroma of Watanabe heritable hyperlipidemic rabbits. Cir-
culation 103:993–999
103. Martinez-Gonzalez J, Alfon J, Berrozpe M, Badimon L (2001)
HMG-CoA reductase inhibitors reduce vascular monocyte
chemotactic protein-1 expression in early lesions from hyper-
cholesterolemic swine independently of their effect on plasma
cholesterol levels. Atherosclerosis 159:27–33
104. Corsini A, Bellosta S, Baetta R, Fumagalli R, Paoletti R, Bernini
F (1999) New insights into the pharmacodynamic and phar-
macokinetic properties of statins. Pharmacol Ther 84:413–428
105. Laufs U, Gertz K, Huang P, Nickenig G, Bohm M, Dirnagl
U, Endres M (2000) Atorvastatin upregulates type III nitric
oxide synthase in thrombocytes, decreases platelet activation,
and protects from cerebral ischemia in normocholesterolemic
mice. Stroke 31:2442–2449

106. Sata M, Nishimatsu H, Suzuki E, Sugiura S, Yoshizumi M,
Ouchi Y, Hirata Y, Nagai R (2001) Endothelial nitric oxide
synthase is essential for the HMG-CoA reductase inhibitor
cerivastatin to promote collateral growth in response to isch-
emia. FASEB J 15:2530–2532
107. Li W, Asagami T, McTaggart F, Tsao PS (2002) Rosuvastatin
diminishes myocardial injury after ischemia/reperfusion. Int
J Clin Pract Suppl 124:6
108. John S, Schlaich M, Langenfeld M, Weihprecht H, Schmitz G,
Weidinger G, Schmieder RE (1998) Increased bioavailability
of nitric oxide after lipid-lowering therapy in hypercholeste-
rolemic patients: a randomized, placebo-controlled, double-
blind study. Circulation 98:211–216
109. Lefer AM, Scalia R, Lefer DJ (2001) Vascular effects of HMG
CoA-reductase inhibitors (statins) unrelated to cholesterol
lowering: new concepts for cardiovascular disease. Cardiovasc
Res 49:281–287
110. Masumoto A, Hirooka Y, Hironaga K, Eshima K, Setoguchi
S, Egashira K, Takeshita A (2001) Effect of pravastatin on
endothelial function in patients with coronary artery disease
(cholesterol-independent effect of pravastatin). Am J Cardiol
88:1291–1294
111. Laufs U, Wassmann S, Hilgers S, Ribaudo N, Bohm M,
Nickenig G (2001) Rapid effects on vascular function after
initiation and withdrawal of atorvastatin in healthy, normo-
cholesterolemic men. Am J Cardiol 88:1306–1307
112. Sukhova GK, Williams JK, Libby P (2002) Statins reduce
inflammation in atheroma of nonhuman primates independent
of effects on serum cholesterol. Arterioscler Thromb Vasc Biol
22:1452–1458
113. Preik M, Kelm M, Schoebel F, Schottenfeld Y, Leschke M,
Strauer BE (1996) Selective impairment of nitric oxide
dependent vasodilation in young adults with hypercholeste-
rolaemia. J Cardiovasc Risk 3:465–471
731
114. Quyyumi AA, Dakak N, Andrews NP, Gilligan DM, Panza
JA, Cannon RO III (1995) Contribution of nitric oxide to
metabolic coronary vasodilation in the human heart. Circu-
lation 92:320–326
115. Boger RH, Bode-Boger SM, Thiele W, Creutzig A, Alexander
K, Frolich JC (1998) Restoring vascular nitric oxide forma-
tion by L-arginine improves the symptoms of intermittent
claudication in patients with peripheral arterial occlusive dis-
ease. J Am Coll Cardiol 32:1336–1344
116. McTaggart F, Buckett L, Davidson R, Holdgate G, McCor-
mick A, Schneck R, Smith G, Warwick M (2001) Preclinical
and clinical pharmacology of rosuvastatin, a new 3-hydroxy-
3-methylglutaryl coenzyme A reductase inhibitor. Am J Car-
diol 87 [Suppl]:28B–32B
117. Hanefield M (2001) Clinical rationale for rosuvastatin, a
potent new HMG-CoA reductase inhibitor. Int J Clin Pract
55:399–405
118. Knopp RH (1999) Drug treatment of lipid disorders. N Engl
J Med 341:498–511
119. Buckett L, Ballard P, Davidson R, Dunkley C, Martin L,
Stafford J, McTaggart F (2000) Selectivity of ZD4522 for
inhibition of cholesterol synthesis in hepatic versus non-he-
patic cells. Atherosclerosis 151:41
120. Dobrucki LW, Kalinowski L, Dobrucki IT, Malinski T (2001)
Statin-stimulated nitric oxide release from endothelium. Med
Sci Monit 7:622–627
121. Griendling KK, Harrison DG (2001) Out, damned dot:
studies of the NADPH oxidase in atherosclerosis. J Clin
Invest 108:1423–1424
122. Heitzer T, Schlinzig T, Krohn K, Meinertz T, Münzel T
(2001) Endothelial dysfunction, oxidative stress, and risk of
cardiovascular events in patients with coronary artery disease.
Circulation 104:2673–2678
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