RESEARCH PAPERbph_1416 598..

606
Rosuvastatin improves
endothelial function in
db/db mice: role of
angiotensin II type 1
receptors and oxidative
stress
XY Tian
1
, WT Wong
1
, A Xu
4
, ZY Chen
2
, Y Lu
1
, LM Liu
1
, VW Lee
3
,
CW Lau
1
, X Yao
1
and Y Huang
1
1
Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, School of Biomedical
Sciences, Hong Kong, China,
2
Department of Biochemistry,
3
School of Pharmacy, Chinese
University of Hong Kong, Hong Kong, China, and
4
Departments of Medicine and Pharmacology
& Pharmacy, University of Hong Kong, Hong Kong, China
Correspondence
Yu Huang, School of Biomedical
Sciences, Chinese University of
Hong Kong, Shatin, NT, Hong
Kong, China. E-mail:
yu-huang@cuhk.edu.hk
----------------------------------------------------------------
Keywords
statin; diabetes; endothelial
function; oxidative stress;
vasodilatation
----------------------------------------------------------------
Received
19 August 2010
Revised
19 February 2011
Accepted
29 March 2011
BACKGROUND AND PURPOSE
HMG-CoA reductase inhibitors, statins, with lipid-reducing properties combat against atherosclerosis and diabetes. The
favourable modulation of endothelial function may play a signicant role in this effect. The present study aimed to investigate
the cellular mechanisms responsible for the therapeutic benets of rosuvastatin in ameliorating diabetes-associated endothelial
dysfunction.
EXPERIMENTAL APPROACH
Twelve-week-old db/db diabetic mice were treated with rosuvastatin at 20 mgkg
-1
day
-1
p.o.for 6 weeks. Isometric force was
measured in isolated aortae and renal arteries. Protein expressions including angiotensin II type 1 receptor (AT
1
R), NOX4,
p22
phox
, p67
phox
, Rac-1, nitrotyrosine, phospho-ERK1/2 and phospho-p38 were determined by Western blotting, while reactive
oxygen species (ROS) accumulation in the vascular wall was evaluated by dihydroethidium uorescence and lucigenin assay.
KEY RESULTS
Rosuvastatin treatment of db/db mice reversed the impaired ACh-induced endothelium-dependent dilatations in both renal
arteries and aortae and prevented the exaggerated contractions to angiotensin II and phenylephrine in db/db mouse renal
arteries and aortae. Rosuvastatin reduced the elevated expressions of AT
1
R, p22
phox
and p67
phox
, NOX4, Rac1, nitrotyrosine and
phosphorylation of ERK1/2 and p38 MAPK and inhibited ROS production in aortae from db/db mice.
CONCLUSIONS AND IMPLICATIONS
The vasoprotective effects of rosuvastatin are attributed to an increase in NO bioavailability, which is probably achieved by its
inhibition of ROS production from the AT
1
R-NAD(P)H oxidase cascade.
Abbreviations
Ang II, angiotensin II; AT
1
R, angiotensin II type 1 receptor; DHE, dihydroethidium; eNOS, endothelial NOS; HMG-CoA
reductase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; L-NAME, N
G
-nitro-L-arginine methyl ester; RAAS,
reninangiotensinaldosterone system; ROS, reactive oxygen species; SNP, sodium nitroprusside
BJP
British Journal of
Pharmacology
DOI:10.1111/j.1476-5381.2011.01416.x
www.brjpharmacol.org
598 British Journal of Pharmacology (2011) 164 598606 2011 The Authors
British Journal of Pharmacology 2011 The British Pharmacological Society
Introduction
Endothelial dysfunction, a hallmark in diabetes, initiates vas-
cular pathogenesis leading to diabetic vasculopathy (De
Vriese et al., 2000), and it is closely associated with reduced
bioavailability of endothelium-derived NO, which can result
from the reduced NO-generating capacity or exaggerated
reactive oxygen species (ROS) formation (Wong et al., 2010a).
Endothelial dysfunction usually occurs early in diabetes, and
therapeutic strategies targeting endothelial dysfunction
might help to combat the cardiovascular complications
(Thomas et al., 2008).
Arteries from diabetic db/db mice show impaired endot-
helial NO-mediated dilatation, which is accompanied by
the up-regulation of local vascular reninangiotensin
aldosterone system (RAAS) and related over-production of
ROS (Wong et al., 2010b). It is well documented that angio-
tensin II (Ang II)-induced activation of NAD(P)H oxidase, a
multi-subunit enzyme complex, is an important source for
the production of ROS in the vasculature (Gao et al., 2009).
Hyperglycaemia impairs NO production and enhances ROS
accumulation in blood vessels (Wang et al., 2009), and ROS
are implicated in the initiation and progression of diabetic
vascular complications usually associated with endothelial
dysfunction (Cohen et al., 2007; Thomas et al., 2008). Given
the importance of oxidative stress in hyperglycaemia-related
vascular events and lack of overall benet of antioxidants in
the treatment of cardiovascular complications (Jialal et al.,
2003), specic therapies targeting ROS-generating enzymes
and their upstream regulators may become more effective in
reversing endothelial dysfunction and thus reducing the
related vascular morbidity and mortality in diabetes.
3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA)
reductase inhibitors, such as rosuvastatin, improve endothe-
lial function in streptozotocin-induced diabetic mice (Nangle
et al., 2003) or reduce oxidative stress in glomeruli of trans-
genic TG(mRen2)27 (Ren2) rats (Whaley-Connell et al., 2008)
through a cholesterol-lowering independent mechanism.
Statins reduce angiotensin type 1 receptor (AT
1
R) expression
in hypercholesterolaemic individuals (Nickenig et al., 1999).
Although statins are found to improve endothelial function
in diabetic animals (Nangle et al., 2003; Schafer et al., 2007)
and in patients with diabetes (Brunetti et al., 2007; Tomizawa
et al., 2009), it is still unclear how this vascular benet is
achieved. With the use of type 2 diabetic db/db mouse model,
we hypothesized that rosuvastatin treatment ameliorates
endothelial dysfunction and increases NO bioavailability,
which is associated with the inhibition of AT
1
R and NAD(P)H
oxidases.
Methods
Animal and drug treatment
Type 2 diabetic mice (C57BL/KSJ background) lacking the
gene encoding for leptin receptor (db/db) and heterozygote
(db/m
+
) were provided by the Chinese University of Hong
Kong (CUHK) Laboratory Animal Service Center. All animal
care and experimental procedures were approved by the
CUHK Animal Experimentation Ethics Committee. Mice were
kept in a temperature-controlled holding room (2224C)
with a 12 h light/dark cycle and fed with a standard diet and
water ad libitum. At the age of 12 weeks, adult male db/db
mice were treated for 6 weeks with rosuvastatin at
20 mgkg
-1
(body weight)day
-1
or vehicle by oral gavage. The
rosuvastatin dosage and treatment duration were comparable
with those used by other research groups on rats and mice.
Oral glucose tolerance test (OGTT)
After 8 h of food deprivation, mice were loaded with glucose
solution (1.2 gkg
-1
; body weight) by oral gavage. Blood was
drawn from the mouse tail, and blood glucose was measured
at times 0, 15, 30, 60 and 120 min with a commercial glu-
cometer (Ascensia ELITE, Bayer, Mishawaka, IN).
Plasma lipid prole and insulin level
Plasma levels of total cholesterol, triglyceride, high-density
lipoprotein (HDL) and non-HDL were determined using
enzymatic methods (Stanbio, Boerne, TX). Plasma insulin
concentration was determined by enzyme immunoassay
(Mercodia, Sweden).
Functional studies in myograph
After mice were killed by CO
2
inhalation, the thoracic aortae
and renal arteries were rapidly dissected out and placed in
oxygenated ice-cold KrebsHenseleit solution (mmolL
-1
: 119
NaCl, 4.7 KCl, 2.5 CaCl
2
, 1 MgCl
2
, 25 NaHCO
3
, 1.2 KH
2
PO
4
and 11 D-glucose). Changes in isometric tension of arteries
were measured in a multi-myograph system (Danish Myo
Technology, Aarhus, Denmark) as described previously
(Wong et al., 2010b) and changes in isometric tension were
recorded. The rings were stretched to an optimal baseline
tension of 3 mN for aortae and 1 mN for renal arteries.
Experimental protocols
Each ring was equilibrated for 1 h before the experiment
started. The ring was rst contracted by 60 mmolL
-1
KCl and
rinsed in Krebs solution for several times before phenyleph-
rine (1 mmolL
-1
) was used to evoke a stable contraction
and then relaxed by accumulative concentrations of ACh
(10 nmolL
-1
to 10 mmolL
-1
). Endothelium-independent
relaxations to sodium nitroprusside (SNP) (1 nmolL
-1
to
10 mmolL
-1
) were recorded in endothelium-denuded rings.
To illustrate the ability of the increased NO bioavailability
to inhibit vasoconstriction, phenylephrine was used to
trigger concentration-dependent contractions for compari-
son in aortae and renal arteries from db/m
+
, db/db and
rosuvastatin-treated db/db mice. To conrm the role of
basal NO production in the reduced vasoconstriction, the
same experiments were performed in some arterial rings
after 30 min exposure to N
G
-nitro-L-arginine (L-NAME,
100 mmolL
-1
).
To determine the contribution of the reduced expression
and function of AT
1
R in rosuvastatin-treated db/db mice, con-
traction to Ang II (100 nmolL
-1
) was compared in arteries
from the three treatment groups of mice in the presence of
100 mmolL
-1
L-NAME.
Western blotting
Protein samples (20 mg each lane) prepared from aorta
homogenates were electrophoresed through a 10%
BJP
Statins and endothelial function in db/db mice
British Journal of Pharmacology (2011) 164 598606 599
SDS-poly-acrylamide gel and transferred onto an
immobilon-P polyvinylidene diuoride membrane (Millipore
Corp., Bedford, MA). Nonspecic binding sites were blocked
with 5% non-fat milk or 1% BSA in Tris-buffered saline con-
taining 0.05% Tween-20. The blots were incubated overnight
at 4C with the primary antibodies: monoclonal anti-AT
1
R
(1:1000; Abcam, Cambridge, UK); monoclonal anti-
nitrotyrosine (1:1000; Abcam); anti-p22
phox
and anti-p67
phox
(1:1000; Santa Cruz, CA); polyclonal anti-NOX4 and anti-
Rac1 (Abcam); monoclonal anti-phosphor-p38 MAPK (Thr
180
/
Tyr
182
), polyclonal anti-p38 MAPK, monoclonal anti-
phospho-p44/42 MAPK (ERK1/2) (Thr
202
/Tyr
204
), monoclonal
anti-p44/42 MAPK, polyclonal anti-phospho-PKC (bII Ser
660
),
anti-phospho-PKCa/b
II
(Thr
638/641
), anti-PKCa, anti-phospho-
MYPT1 (Thr
853
) (Cell Signaling, Beverly, MA); polyclonal
anti-MYPT1 (Covance, Princeton, NJ); followed by horse-
radish peroxidase-conjugated secondary antibody (1:4000;
DakoCytomation, Carpinteria, CA). Monoclonal anti-b-actin
(1:5000; Ambion, Cambridge, UK) was used as a housekeep-
ing protein. Densitometry was performed using a documen-
tation programme (Flurochem, Alpha Innotech Corp., San
Leandro, CA).
Measurement of intracellular
oxidant formation
The amount of intracellular oxidant formation was deter-
mined using dihydroethidium (DHE) (Molecular Probes, OR)
(Wong et al., 2010b). Aortic rings from db/m
+
, db/db and
rosuvastatin-treated db/db mice were dissected out and frozen
in OCT compound. Frozen sections of the aortic ring were cut
at 10 mm thickness using a cryostat microtome (Leica
CM1100, Leica Instruments, Germany) and incubated for
10 min at 37C in Krebs solution containing 5 mmolL
-1
DHE.
Fluorescence intensity was measured by confocal microscope
(FV1000, Olympus, Tokyo, Japan) at excitation/emission of
488/605 nm to visualize the signal. The images were analysed
by the Fluoview software (Olympus, Tokyo, Japan).
In addition, the amount of superoxide production was
also measured by lucigenin chemiluminescence (Wassmann
et al., 2001; Ejiri et al., 2003). Mouse aortae were cut into
3 mm segments and incubated with 10 mmolL
-1
lucigenin in
Krebs solution bubbled with 95% O
2
and 5% CO
2
for 30 min
at 37C. Samples were transferred into vials containing 1 mL
Krebs solution with 10 mmolL
-1
lucigenin, and chemilunines-
cence was repeatedly measured using a Wallace Victor lumi-
nometer (PerkinElmer, Boston, MA) over 10 min, at 1 min
interval. The count per minute was determined by the
average of all readings and normalized to dry weight of the
aortic segment.
Chemicals
Rosuvastatin was provided by AstraZeneca (Cheshire UK).
ACh, L-NAME, phenylephrine, angiotensin II, SNP and
lucigenin were purchased from Sigma-Aldrich Chemical (St
Louis, MO). All the drugs used were dissolved in double
distilled water.
Data analysis
Results are means SEMfromdifferent mice. Concentration
relaxation curves were analysed by non-linear regression
curve tting using GraphPad Prism software (version 4.0, San
Diego, CA) to estimate E
max
as the maximal response and pD
2
as the negative logarithm of the drug concentration that
produced 50% of E
max
. Contractions were presented as active
tension [force (mN) recorded / (2 length (mm) of ring)].
Statistical signicance was determined by Students two-tailed
t-test or one-way ANOVA followed by the Bonferroni post hoc
test when more than two treatments were compared. P < 0.05
indicates statistically signicant difference.
Results
Statin improves the endothelium-dependent
relaxation (EDR) in renal arteries and aortae
of db/db mice
EDRs in response to accumulative concentration of ACh in
phenylephrine-precontracted segments of renal arteries were
signicantly reduced in db/db mice compared with db/m
+
mice, as shown in representative traces (Figure 1A) and sum-
marized data (Figure 1B). Rosuvastatin treatment in db/db
mice improved EDRs in renal arteries (Figure 1A and B). The
increased initial active tension induced by phenylephrine in
arteries from db/db mice was reduced by rosuvastatin treat-
ment (Supporting Figure S1). EDRs to SNP were similar in
renal arteries from all groups (Figure 1C). In aortae, reduced
EDRs in db/db mice were also improved after rosuvastatin
treatment (Figure 1D and E). Again, SNP-induced relaxations
were similar in aortic rings from all groups (Figure 1F).
Statin reduces phenylephrine-induced
contraction through an increase in
NO bioavailability
Phenylephrine-induced contractions in renal arteries
(Figure 2A) and aortae (Figure 2B, Supporting Figure S1) of
db/db mice were signicantly larger than those from db/m
+
mice. The enhanced contraction was reduced by chronic
rosuvastatin treatment (Figure 2A and B). After pre-
incubation with L-NAME, contractions to phenylephrine
were similar in renal arteries (Figure 2C) and aortae
(Figure 2D) in the three groups.
Statin suppresses Ang II-induced contraction
in arteries of db/db mice
Ang II (100 nmolL
-1
)-induced contractions were markedly
augmented in db/db mouse renal arteries (Figure 3A) and
aortae (Figure 3B) compared to those from db/m
+
mice, but
were reduced in arteries from rosuvastatin-treated db/db
mice. In contrast, concentration-dependent contractions to
increasing concentrations of KCl were comparable in renal
arteries and aortae from the three groups (Figure 3C and D).
Statin reduces the up-regulated expressions of
AT
1
R, NAD(P)H oxidase subunits and
oxidative stress markers in aortae of
db/db mice
The protein expressions of angiotensin type 1 receptor
(AT
1
R), NAD(P)H oxidase subunits p22
phox
and p67
phox
increased in aortae from db/db mice than db/m
+
mice;
BJP
XY Tian et al.
600 British Journal of Pharmacology (2011) 164 598606
rosuvastatin treatment normalized the increased expression
of AT
1
R, p22
phox
and p67
phox
, NOX4 and Rac1 (Figure 4AC,
EF) but not NOX2 (Supporting Figure S2). In addition, rosu-
vastatin treatment in db/db mice also reduced the increased
levels of nitrotyrosine (Figure 4D). The intracellular oxidant
formation, measured by DHE uorescence, and superoxide
production, measured by lucigenin assay, were higher in the
sections of aortic rings from db/db mice than in those from
db/m
+
mice; this increase was inhibited by rosuvastatin treat-
ment (Figure 5).
Statin reduces the pro-inammatory
signalling pathways in aortae of db/db mice
Western blotting showed increased phosphorylations of
ERK1/2 (Figure 6A) and p38 MAPK (Figure 6B) in db/db mouse
aortae, which were prevented by rosuvastatin treatment
(Figure 6A and B).
Basic metabolic parameters are not modied
by statin treatment
Rosuvastatin treatment did not change body weights of db/db
mice, which were signicantly higher than age-matched
db/m
+
control littermates (Table 1). The levels of fasting blood
glucose and plasma insulin were higher in db/db mice than
those from db/m
+
mice (Table 1). Rosuvastatin treatment did
not alter these parameters nor affect the glucose tolerance in
db/db mice (Supporting Figure S3, Table 1). In addition, the
elevated levels of total cholesterol, triglyceride, HDL and
non-HDL in db/db mice were not modied by rosuvastatin
treatment (Table 1).
Discussions and conclusions
The present study demonstrates that a 6-week treatment with
rosuvastatin, a HMG-CoA reductase inhibitor, restores the
impaired EDRs in renal arteries and aortae in diabetic db/db
mouse model without affecting the plasma lipids or glucose
tolerance. This effect is associated with an increase in NO
bioavailability. We showed for the rst time that statin treat-
Figure 1
Statin improves EDRs in renal arteries and aortae of db/db
mice. Representative traces showing the impaired ACh-induced
endothelium-dependent relaxations of renal arteries (A) and aortae
(D) from db/db mice were restored by chronic treatment with rosu-
vastatin. Summarized graphs show that rosuvastatin treatment
improved EDRs in renal arteries (B) and aortae (E) of db/db mice.
Comparable endothelium-independent relaxations to SNP were
shown in renal arteries (C) and aortae (F) from the three groups of
mice. Results are means SEM of six mice. *P < 0.05 between db/m
+
and db/db; #P < 0.05 between db/db and rosuvastatin-treated db/db
mice.
Figure 2
Statin reduces phenylephrine-induced vasoconstriction in arteries of
db/db mice through an increase in NO bioavailability. The aug-
mented contractions to phenylephrine (Phe) in renal arteries (A) and
aortae (B) in db/db mice were prevented by chronic treatment with
rosuvastatin. This inhibitory effect was absent in renal arteries (C) and
aortae (D) treated with 100 mmolL
-1
L-NAME. Results are means
SEM of six mice. *P < 0.05 between db/m
+
and db/db; #P < 0.05
between db/db and rosuvastatin-treated db/db mice.
BJP
Statins and endothelial function in db/db mice
British Journal of Pharmacology (2011) 164 598606 601
ment reduces oxidative stress in arteries of db/db mice, which
is associated with the inhibition of the RAAS cascade involv-
ing AT
1
R and NAD(P)H oxidases. Furthermore, the statin
treatment diminished the nitrotyrosine formation and pro-
inammatory signalling molecules including ERK1/2 and p38
MAPK. Taken together, the present novel ndings reveal that
rosuvastatin inhibits AT
1
R-dependent oxidative stress and
restores NO bioavailability in arteries of db/db mouse beyond
its effect on plasma lipid levels.
Over the past few years, statins have been increasingly
identied to possess vasoprotective effects in diabetes, which
are attributed to actions independent of their effects on
lipids. Clinical studies have demonstrated that statins
improve ow-mediated vasodilatation in patients with type 2
diabetes (Brunetti et al., 2007; Tomizawa et al., 2009) and
hyperlipidaemia (ter Avest et al., 2005). In animal studies,
consistent results were also reported showing the vasoprotec-
tive effects of statins. Nangle et al. (2003) rst demonstrated
that rosuvastatin treatment improved endothelium-
dependent NO-mediated relaxations in aortae of
streptozotocin-induced diabetic mice. Recently, rosuvastatin
has been shown to normalize endothelial function of aortae
in streptozotocin-induced diabetic rats (Schafer et al., 2007;
Tarhzaoui et al., 2008). Treatment with simvastatin preserved
endothelial function in large and small coronary vessels of
high cholesterol-fed pigs (Wilson et al., 2001). However,
limited data are available regarding the effects of statins on
vascular function in a type 2 diabetic db/db mouse model. The
present study thus shows for the rst time that rosuvastatin,
administered for 6 weeks, improved vascular function in
db/db mouse model without modifying the lipid proles,
which included the triglycerides and cholesterols levels. This
unique property might be useful for the characterization of
the pleiotropic effects of statins in the alleviation of diabetic
vasculopathy.
It is estimated that around 70% of patients with diabetes
will die of vascular events (Wing et al., 2001). One of the
major causes of cardiovascular mortality in this population is
due to diabetic nephropathy (de Zeeuw et al., 2004). Endot-
helial dysfunction usually occurs early in diabetes, and thera-
peutic strategies targeting endothelial function might help to
combat the cardiovascular complications (Thomas et al.,
2008). Statins are known to reduce the cardiovascular mor-
bidity and mortality in diabetic patients (Goldberg et al.,
Figure 3
Statin suppresses Ang II-induced vasoconstriction in arteries of db/db
mice. Elevated contractions to Ang II (100 nmolL
-1
) in renal arteries
(A) and aortae (B) in db/db mice were inhibited by chronic treatment
with rosuvastatin, while contractions to raised extracellular KCl were
identical in renal arteries (C) and aortae (D) from the three groups of
mice. Contractions were presented as active tension [force (mN)
recorded / (2 length (mm) of ring)]. Results are means SEM of six
mice. *P < 0.05 between db/m
+
and db/db; #P < 0.05 between db/db
and rosuvastatin-treated db/db mice.
Figure 4
Statin reduces the up-regulated expressions of AT
1
R, NAD(P)H
oxidase subunits and nitrotyrosine in aortae of db/db mice. The
elevated protein expression of AT
1
R (A), p22
phox
(B), p67
phox
(C),
nitrotyrosine (Ntyr, D), NOX4 (E), Rac1 (F) in aortae of db/db mice
was reduced after chronic treatment with rosuvastatin. Results are
means SEM of four to ve mice. *P < 0.05 between db/m
+
and
db/db; #P < 0.05 between db/db and rosuvastatin-treated db/db
mice.
BJP
XY Tian et al.
602 British Journal of Pharmacology (2011) 164 598606
1998; Haffner et al., 1999; Keech et al., 2003). In the present
study, we assessed the vascular function using a myograph to
examine whether treatment with rosuvastatin could reverse
vascular dysfunction in aortae and renal arteries of db/db
mice. Impaired EDRs in aortae in db/db mice were reported
previously (Zhong et al., 2007; Moien-Afshari et al., 2008;
Wong et al., 2010b); in contrast, the renovascular function in
diabetes is less well dened. The present study demonstrated
that ACh-induced EDRs are reduced in renal arteries and
aortae of db/db mice compared with the non-diabetic db/m
+
mice. Treatment with rosuvastatin for 6 weeks restored the
impaired EDRs in db/db mice. On the other hand, the EDRs to
SNP were similar in renal arteries and aortae from db/m
+
,
db/db and rosuvastatin-treated db/db mice, suggesting that the
sensitivity of vascular smooth muscle to the relaxing effect of
NO is not modulated under hyperglycaemic conditions or by
rosuvastatin. Previous reports demonstrated that pitavastatin
ameliorated diabetic nephropathy in db/db mouse model
(Fujii et al., 2007), and rosuvastatin exerts renoprotective
effects in transgenic TG(mRen2)27 rats (Whaley-Connell
et al., 2008). However, no study has attempted to elucidate
whether statins could protect the renovascular function in
Table 1
Basic parameters of db/m
+
, db/db and rosuvastatin-treated db/db mice
db/m
+
db/db db/db + Rosuvastatin
n 6 8 8
Parameter
Body weight (g) 29.8 0.7 57.3 3.6* 59.6 4.4*
Plasma levels of:
Fasting glucose (mmolL
-1
) 3.3 0.3 16.2 1.2* 16.1 1.8*
Insulin (ngmL
-1
) 1.5 0.3 22.5 1.9* 22.9 3.9*
Total cholesterol (mgmL
-1
) 0.78 0.02 1.31 0.10* 1.24 0.14*
Triglyceride (mgmL
-1
) 0.95 0.04 2.58 0.25* 2.56 0.40*
HDL (mgmL
-1
) 0.46 0.02 0.65 0.03* 0.59 0.04*
non-HDL (mgmL
-1
) 0.30 0.04 0.71 0.05* 0.60 0.09*
Results are means SEM. *P < 0.01 versus db/m
+
group.
Figure 5
Statin suppresses the increased ROS production in aortic walls of
db/db mice. (A) Representative images showing ROS accumulation in
the vascular wall of db/db mouse aortae from the three groups.
Chronic treatment with rosuvastatin reduced ROS generation indi-
cated by DHE uorescence (B) and lucigenin assay (C) in db/db mice.
Results are means SEM of four to six mice. *P < 0.05 between
db/m
+
and db/db; #P < 0.05 between db/db and rosuvastatin-treated
db/db mice.
Figure 6
Statin reduces the pro-inammatory signalling pathways in aortae of
db/db mice. Increased levels of phosphorylation of ERK1/2 (A) and
p38MAPK (B) in db/db mouse aortae were normalized by the chronic
treatment with rosuvastatin without affecting the total protein
expressions of ERK1/2 and p38MAPK. Results are means SEM of
four to six mice. *P < 0.05 between db/m
+
and db/db; #P < 0.05
between db/db and rosuvastatin-treated db/db mice.
BJP
Statins and endothelial function in db/db mice
British Journal of Pharmacology (2011) 164 598606 603
diabetes. The present results reveal that the impaired ACh-
induced EDRs and augmented phenylephrine-induced vaso-
constrictions in renal arteries and aortae of db/db mice were
prevented by rosuvastatin treatment. One possible explana-
tion for this effect is that it is due to an increase in NO
bioavailability after statin treatment, as the effects of statin
on EDRs and phenylephrine-induced vasoconstrictions were
abolished in the presence of L-NAME. Since the endothelial
NOS (eNOS) levels in aortae were comparable in the three
groups of mice (Supporting Figure S4), the increase in NO
bioavailability might be attributed to the reduction in ROS
production by rosuvastatin treatment. The present ndings
thus provide novel evidence to support the renovascular pro-
tective effects of statins in diabetic nephropathy.
The mechanisms underlying the pleiotropic effects of
statins on vascular function remain largely unknown. Direct
vasorelaxation effects of statins including rosuvastatin have
been reported in rat aortic rings (Lopez et al., 2008), although
the concentrations needed to induce relaxation were far
above therapeutically achievable levels in humans. The pleio-
tropic effects of statins on vascular and renal function have
been associated with the up-regulation of eNOS (Wilson et al.,
2001) and superoxide dismutase (Desjardins et al., 2008),
inhibition of the production of NAD(P)H oxidase-dependent
superoxide anions (Li et al., 2005; Erdos et al., 2006; Fujii
et al., 2007; Whaley-Connell et al., 2008) or the reduction of
pro-inammatory genes including VCAM-1, MCP-1 and
MMP-9. Furthermore, rosuvastatin normalizes platelet aggre-
gation and ROS production in a hamster model of pre-
diabetic insulin resistance induced by fructose feeding
(Miersch et al., 2007) and prevents apoptosis of human
umbilical vein endothelial cells induced by high glucose by
reducing NAD(P)H oxidase-derived oxidative stress (Piconi
et al., 2008). We have previously demonstrated a critical role
for AT
1
R-mediated ROS in the impairments of EDRs in renal
arteries from diabetic patients and aortae from db/db mice.
The impaired vasodilator responses could be reversed by the
AT
1
R blocker, NAD(P)H oxidase inhibitors and ROS scaven-
gers (Wong et al., 2010b). It is postulated that elevated ROS
levels derived from AT
1
R-mediated NAD(P)H oxidases lower
the bioavailability of NO by either directly scavenging NO or
by reducing the biosynthesis of NO catalysed by eNOS.
Uncoupling of eNOS could be another source of ROS accu-
mulation in the vascular wall of db/db mice, as we have
recently shown that both L-NAME and a NAD(P)H oxidase
inhibitor inhibit the ROS levels in these arteries, indicating
that ROS derived from NADPH oxidase is probably required
for stimulation of eNOS uncoupling to further increase intra-
cellular ROS formation in vascular cells.
The effects of statins on the RAAS cascade are not known.
The present study shows for the rst time that rosuvastatin
restores vascular function in db/db mice, which was associ-
ated with favourable modulation of the RAAS cascade based
on the following observations. First, the augmented contrac-
tion to Ang II in renal arteries and aortae of db/db mice were
normalized by rosuvastatin treatment without affecting
the KCl-induced vasoconstrictions. An augmented Ang
II-induced contraction indicates an increased expression or
activity of AT
1
R. Indeed, the up-regulation of AT
1
R in aortae
from db/db mice was normalized by rosuvastatin treatment.
Second, rosuvastatin treatment reduced the expressions of
NAD(P)H oxidase subunits p22
phox
(membranous subunit),
p67
phox
(cytosolic subunit) and Rac1 in aortae of db/db mouse,
which were associated with a reduction of nitrotyrosine for-
mation (a marker of peroxynitrite formation) and oxidant
formation. In addition, rosuvastatin treatment also reduced
the Ang II level in the db/db mouse aorta as shown by immu-
nostaining (Supporting Figure S5). Third, MAPKs including
p44/42 and p38 can be activated by Ang II and have been
implicated as downstream signalling molecules in response to
many pro-inammatory factors (Xu et al., 2005). The present
results also reveal that the activations of p38 and ERK1/2
MAPK in aortae of db/db mice were normalized by rosuvasta-
tin treatment. This helps to explain the anti-atherosclerotic
effects of statins in the vasculature. Although statin was
reported to stimulate MAPK/ERK (Lee et al., 2004; Merla et al.,
2007; Cho et al., 2011), the MAPK/ERK activation is mediated
via an adenosine-dependentmechanism, while the induction
of MAPK/ERK and downstream haemoxygenase-1 up-
regulation by statin occurs mainly in vascular smooth muscle
cells. Taken together, we provide novel evidence that rosuv-
astatin restores vascular function in db/db mice, which is
related to the inhibition of the RAAS-oxidative stress axis.
Statins are known to inhibit the activity of RhoA/Rho-
kinase (Ohnaka et al., 2001; Turner et al., 2005; Ruperez et al.,
2007; Rashid et al., 2009). The present results do not suggest
a signicant role for RhoA/Rho-kinase in the inhibitory effect
of rosuvastatin that results in improved endothelial function
based on the following observations. In aortas from treated
db/db mice, rosuvastatin does not affect (a) contraction to
U46619, which is known to stimulate RhoA/Rho-kinase and
(b) the phosphorylation of MYPT1 and PKC, the downstream
targets regulated by RhoA/Rho-kinase (Supporting Figure S5).
We have yet to determine whether a longer treatment with
this statin or treatment at higher dosages could inhibit RhoA/
Rho-kinase activity in vascular smooth muscle cells if the
plasma statin concentration can reach a level that is compa-
rable with those used in in vitro assays.
In summary, the present study demonstrates that rosuv-
astatin, an HMG-CoA reductase inhibitor preserves endothe-
lial function in renal arteries and aortae of db/db mice, despite
not changing plasma lipid concentrations. The vasoprotec-
tive effects of rosuvastatin are attributed to an increase in NO
bioavailability, which is at least in part achieved by its inhi-
bition of ROS production from the AT
1
R-NAD(P)H oxidase
cascade. Other contributing mechanisms require further
investigation.
Acknowledgements
This study was supported by Research Grants (4653/08 M,
466110) and Collaborative Research Fund (HKU2/07C and
HKU4/CRF10) from the Research Council of Hong Kong,
CUHK Focused Investment Scheme and CUHK Li Ka Shing
Institute of Health Sciences.
Conicts of interest
The authors declare that there is no duality of interest asso-
ciated with this manuscript.
BJP
XY Tian et al.
604 British Journal of Pharmacology (2011) 164 598606
References
ter Avest E, Abbink EJ, Holewijn S, de Graaf J, Tack CJ,
Stalenhoef AF (2005). Effects of rosuvastatin on endothelial
function in patients with familial combined hyperlipidaemia (FCH).
Curr Med Res Opin 21: 14691476.
Brunetti, ND, Maulucci, G, Casavecchia, GP, Distaso, C,
De Gennaro, L, Pellegrino, PL et al. (2007). Improvement in
endothelium dysfunction in diabetics treated with statins: a
randomized comparison of atorvastatin 20 mg versus rosuvastatin
10 mg. J Interv Cardiol 20: 481487.
Cho KJ, Hill MM, Chigurupati S, Du G, Parton RG, Hancock JF
(2011). Therapeutic levels of the HMG-CoA reductase inhibitor
lovastatin activate Ras signaling via phospholipase D2. Mol Cell
Biol 31: 11101120.
Cohen G, Riahi Y, Alpert E, Gruzman A, Sasson S (2007). The roles
of hyperglycaemia and oxidative stress in the rise and collapse of
the natural protective mechanism against vascular endothelial cell
dysfunction in diabetes. Arch Physiol Biochem 113: 259267.
De Vriese AS, Verbeuren TJ, Van de Voorde J, Lameire NH,
Vanhoutte PM (2000). Endothelial dysfunction in diabetes. Br J
Pharmacol 130: 963974.
Desjardins F, Sekkali B, Verreth W, Pelat M, De Keyzer D, Mertens A
et al. (2008). Rosuvastatin increases vascular endothelial
PPARgamma expression and corrects blood pressure variability in
obese dyslipidaemic mice. Eur Heart J 29: 128137.
Ejiri J, Inoue N, Tsukube T, Munezane T, Hino Y, Kobayashi S et al.
(2003). Oxidative stress in the pathogenesis of thoracic aortic
aneurysm: protective role of statin and angiotensin II type 1
receptor blocker. Cardiovasc Res 59: 988996.
Erdos B, Snipes JA, Tulbert CD, Katakam P, Miller AW, Busija DW
(2006). Rosuvastatin improves cerebrovascular function in Zucker
obese rats by inhibiting NAD(P)H oxidase-dependent superoxide
production. Am J Physiol Heart Circ Physiol 290: H1264H1270.
Fujii M, Inoguchi T, Maeda Y, Sasaki S, Sawada F et al. (2007).
Pitavastatin ameliorates albuminuria and renal mesangial
expansion by downregulating NOX4 in db/db mice. Kidney Int 72:
473480.
Gao L, Mann GE (2009). Vascular NAD(P)H oxidase activation in
diabetes: a double-edged sword in redox signalling. Cardiovasc Res
82: 920.
Goldberg RB, Mellies MJ, Sacks FM, Moye LA, Howard BV,
Howard WJ et al. (1998). Cardiovascular events and their reduction
with pravastatin in diabetic and glucose-intolerant myocardial
infarction survivors with average cholesterol levels: subgroup
analyses in the cholesterol and recurrent events (CARE) trial. The
Care Investigators. Circulation 98: 25132519.
Haffner SM, Alexander CM, Cook TJ, Boccuzzi SJ, Musliner TA,
Pedersen TR et al. (1999). Reduced coronary events in
simvastatin-treated patients with coronary heart disease and
diabetes or impaired fasting glucose levels: subgroup analyses in the
Scandinavian Simvastatin Survival Study. Arch Intern Med 159:
26612667.
Jialal I, Devaraj S (2003). Role of C-reactive protein in the
assessment of cardiovascular risk. Am J Cardiol 91: 200202.
Keech A, Colquhoun D, Best J, Kirby A, Simes RJ, Hunt D et al.
(2003). Secondary prevention of cardiovascular events with
long-term pravastatin in patients with diabetes or impaired fasting
glucose: results from the LIPID trial. Diabetes Care 26: 27132721.
Lee TS, Chang CC, Zhu Y, Shyy JY (2004). Simvastatin induces
heme oxygenase-1: a novel mechanism of vessel protection.
Circulation 110: 12961302.
Li W, Asagami T, Matsushita H, Lee KH, Tsao PS (2005).
Rosuvastatin attenuates monocyte-endothelial cell interactions and
vascular free radical production in hypercholesterolemic mice. J
Pharmacol Exp Ther 313: 557562.
Lopez J, Mendoza R, Cleva Villanueva G, Martinez G, Castillo EF,
Castillo C (2008). Participation of K+ channels in the endothelium-
dependent and endothelium-independent components of the
relaxant effect of rosuvastatin in rat aortic rings. J Cardiovasc
Pharmacol Ther 13: 207213.
Merla R, Ye Y, Lin Y, Manickavasagam S, Huang MH, Perez-Polo RJ
et al. (2007). The central role of adenosine in statin-induced
ERK1/2, Akt, and eNOS phosphorylation. Am J Physiol Heart Circ
Physiol 293: H1918H1928.
Miersch S, Sliskovic I, Raturi A, Mutus B (2007). Antioxidant and
antiplatelet effects of rosuvastatin in a hamster model of
prediabetes. Free Radic Biol Med 42: 270279.
Moien-Afshari F, Ghosh S, Khazaei M, Kieffer TJ, Brownsey RW,
Laher I (2008). Exercise restores endothelial function independently
of weight loss or hyperglycaemic status in db/db mice. Diabetologia
51: 13271337.
Nangle MR, Cotter MA, Cameron NE (2003). Effects of rosuvastatin
on nitric oxide-dependent function in aorta and corpus
cavernosum of diabetic mice: relationship to cholesterol
biosynthesis pathway inhibition and lipid lowering. Diabetes 52:
23962402.
Nickenig G, Baumer AT, Temur Y, Kebben D, Jockenhovel F,
Bohm M (1999). Statin-sensitive dysregulated AT1 receptor function
and density in hypercholesterolemic men. Circulation 100:
21312134.
Ohnaka K, Shimoda S, Nawata H, Shimokawa H, Kaibuchi K,
Iwamoto Y et al. (2001). Pitavastatin enhanced BMP-2 and
osteocalcin expression by inhibition of Rho-associated kinase in
human osteoblasts. Biochem Biophys Res Commun 287: 337342.
Piconi L, Corgnali M, Da Ros R, Assaloni R, Piliego T, Ceriello A
(2008). The protective effect of rosuvastatin in human umbilical
endothelial cells exposed to constant or intermittent high glucose. J
Diabetes Complications 22: 3845.
Rashid M, Tawara S, Fukumoto Y, Seto M, Yano K, Shimokawa H
(2009). Importance of Rac1 signaling pathway inhibition in the
pleiotropic effects of HMG-CoA reductase inhibitors. Circ J 73:
361370.
Ruperez M, Rodrigues-Diez R, Blanco-Colio LM, Sanchez-Lopez E,
Rodriguez-Vita J, Esteban V et al. (2007). HMG-CoA reductase
inhibitors decrease angiotensin II-induced vascular brosis: role of
RhoA/ROCK and MAPK pathways. Hypertension 50: 377383.
Schafer A, Fraccarollo D, Vogt C, Flierl U, Hemberger M, Tas P et al.
(2007). Improved endothelial function and reduced platelet
activation by chronic HMG-CoA-reductase inhibition with
rosuvastatin in rats with streptozotocin-induced diabetes mellitus.
Biochem Pharmacol 73: 13671375.
Tarhzaoui K, Valensi P, Boulakia FC, Lestrade R, Albertini JP,
Behar A (2008). Effect of rosuvastatin on capillary ltration of
albumin and blood pressure in rats with streptozotocin-induced
diabetes. Diabetes Res Clin Pract 80: 335343.
Thomas SR, Witting PK, Drummond GR (2008). Redox control of
endothelial function and dysfunction: molecular mechanisms and
therapeutic opportunities. Antioxid Redox Signal 10: 17131765.
BJP
Statins and endothelial function in db/db mice
British Journal of Pharmacology (2011) 164 598606 605
Tomizawa A, Hattori Y, Suzuki K, Okayasu T, Kase H, Satoh H et al.
(2009). Effects of statins on vascular endothelial function in
hypercholesterolemic patients with type 2 diabetes mellitus:
Fluvastatin vs. rosuvastatin. Int J Cardiol 144: 108109.
Turner NA, ORegan DJ, Ball SG, Porter KE (2005). Simvastatin
inhibits MMP-9 secretion from human saphenous vein smooth
muscle cells by inhibiting the RhoA/ROCK pathway and reducing
MMP-9 mRNA levels. FASEB J 19: 804806.
Wang Y, Huang Y, Lam KS, Li Y, Wong WT, Ye H et al. (2009).
Berberine prevents hyperglycemia-induced endothelial injury and
enhances vasodilatation via adenosine monophosphate-activated
protein kinase and endothelial nitric oxide synthase. Cardiovasc
Res 82: 484492.
Wassmann S, Laufs U, Baumer AT, Muller K, Ahlbory K, Linz W
et al. (2001). HMG-CoA reductase inhibitors improve endothelial
dysfunction in normocholesterolemic hypertension via reduced
production of reactive oxygen species. Hypertension 37:
14501457.
Whaley-Connell A, Habibi J, Nistala R, Cooper SA, Karuparthi PR,
Hayden MR et al. (2008). Attenuation of NADPH oxidase activation
and glomerular ltration barrier remodeling with statin treatment.
Hypertension 51: 474480.
Wilson SH, Simari RD, Best PJ, Peterson TE, Lerman LO, Aviram M
et al. (2001). Simvastatin preserves coronary endothelial function in
hypercholesterolemia in the absence of lipid lowering. Arterioscler
Thromb Vasc Biol 21: 122128.
Wing RR, Goldstein MG, Acton KJ, Birch LL, Jakicic JM, Sallis JF
et al. (2001). Behavioral science research in diabetes: lifestyle
changes related to obesity, eating behavior, and physical activity.
Diabetes Care 24: 117123.
Wong WT, Wong SL, Tian XY, Huang Y (2010a). Endothelial
dysfunction: the common consequence in diabetes and
hypertension. J Cardiovasc Pharmacol 55: 300307.
Wong WT, Tian XY, Xu A, Ng CF, Lee HK, Chen ZY et al. (2010b).
Angiotensin II type 1 receptor-dependent oxidative stress mediates
endothelial dysfunction in type 2 diabetic mice. Antioxid Redox
Signal 13: 757768.
Xu ZG, Lanting L, Vaziri ND, Li Z, Sepassi L, Rodriguez-Iturbe B
et al. (2005). Upregulation of angiotensin II type 1 receptor,
inammatory mediators, and enzymes of arachidonate metabolism
in obese Zucker rat kidney: reversal by angiotensin II type 1
receptor blockade. Circulation 111: 19621969.
de Zeeuw D, Remuzzi G, Parving HH, Keane WF, Zhang Z et al.
(2004). Albuminuria, a therapeutic target for cardiovascular
protection in type 2 diabetic patients with nephropathy.
Circulation 110: 921927.
Zhong JC, Yu XY, Huang Y, Yung LM, Lau CW, Lin SG (2007).
Apelin modulates aortic vascular tone via endothelial nitric oxide
synthase phosphorylation pathway in diabetic mice. Cardiovasc Res
74: 388395.
Supporting information
Additional Supporting Information may be found in the
online version of this article:
Figure S1 Contractions to phenylephrine (Phe, 1 mM) before
cumulative concentration of acetylcholine were calculated
and normalized to active tension. Phe-induced contractions
from db/db mice were larger than those from db/m
+
, which
were reduced by rosuvastatin (RSV) treatment, in both aorta
and renal arteries. Data are means SEM. P < 0.05 versus
db/m
+
(n = 8). P < 0.05 versus db/db (n = 8). db/db
+
RSV (n = 6).
Figure S2 Chronic rosuvastatin (RSV) treatment did not
affect the elevated expression of NOX2 in the aortas from
treated db/db mice. Data are means SEM. *P < 0.05 versus
db/m
+
(n = 5). P < 0.05 versus db/db. db/db
+
RSV (n = 6).
Figure S3 (a) The lack of effect of chronic treatment with
rosuvastatin (RSV) on oral glucose tolerance test (OGTT) in
db/db mice. (b) Area under curves for OGTT. Data are means
SEM of 6 mice. *P < 0.05 versus db/m
+
mice.
Figure S4 The total eNOS of the aortae were comparable
between the three groups of mice. Data are means SEM of
four mice.
Figure S5 Immunohistochemical staining of angiotensin II
in the wall of mouse aortas. Chronic rosuvastatin (RSV) treat-
ment reduced the elevated angiotensin II level in db/db
mouse aortas as compared with those from db/m
+
mice. Data
are means SEM of four to ve mice. *P < 0.05 versus db/m
+
;
#P < 0.05 versus db/db.
Figure S6 Chronic rosuvastatin (RSV) treatment did not
modify (a) contractions induced by U46619, thromboxane
A2 analogue in db/db mouse aortas, and RSV did not affect the
level of phosphorylation MYPY1 (Thr853) (b), PKCa/b
(Thr638/641) and PKC (Ser660) (c and d) in db/db mouse
aortas. Data are means SEM of ve to six mice. The U46619-
induced contraction was abolished by 1 mM S18886, the TP
receptor antagonist (data not shown).
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials supplied
by the authors. Any queries (other than missing material)
should be directed to the corresponding author for the article.
BJP
XY Tian et al.
606 British Journal of Pharmacology (2011) 164 598606