Clinical Science (2015) 128, 411423 (Printed in Great Britain) doi: 10.

1042/CS20140456

Vascular injury in diabetic db/db mice is

ameliorated by atorvastatin: role of
Rac1/2-sensitive Nox-dependent pathways

Clinical Science

www.clinsci.org

Thiago Bruder-Nascimento , Glaucia E. Callera, Augusto C. Montezano, Ying He, Tayze T. Antunes,
Aurelie Nguyen Dinh Cat, Rita C. Tostes and Rhian M. Touyz

Department of Pharmacology, Ribeirao Preto Medical School, University of Sao Paulo, Sao Paulo, Brazil
Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ottawa, Canada
Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, U.K.

Abstract
Oxidative stress [increased bioavailability of reactive oxygen species (ROS)] plays a role in the endothelial
dysfunction and vascular inflammation, which underlie vascular damage in diabetes. Statins are
cholesterol-lowering drugs that are vasoprotective in diabetes through unknown mechanisms. We tested the
hypothesis that atorvastatin decreases NADPH oxidase (Nox)-derived ROS generation and associated vascular injury
in diabetes. Leprdb /Leprdb (db/db) mice, a model of Type 2 diabetes and control Leprdb /Lepr+ (db/+) mice were
administered atorvastatin (10 mg/kg per day, 2 weeks). Atorvastatin improved glucose tolerance in db/db mice.
Systemic and vascular oxidative stress in db/db mice, characterized by increased plasma TBARS (thiobarbituric
acid-reactive substances) levels and exaggerated vascular Nox-derived ROS generation respectively, were inhibited
by atorvastatin. Cytosol-to-membrane translocation of the Nox regulatory subunit p47phox and the small GTPase
Rac1/2 was increased in vessels from db/db mice compared with db/+ mice, an effect blunted by atorvastatin.
The increase in vascular Nox1/2/4 expression and increased phosphorylation of redox-sensitive mitogen-activated
protein kinases (MAPKs) was abrogated by atorvastatin in db/db mice. Pro-inflammatory signalling (decreased IB-
and increased NF-B p50 expression, increased NF-B p65 phosphorylation) and associated vascular inflammation
[vascular cell adhesion molecule-1 (VCAM-1) expression and vascular monocyte adhesion], which were increased in
aortas of db/db mice, were blunted by atorvastatin. Impaired acetylcholine (Ach)- and insulin (INS)-induced
vasorelaxation in db/db mice was normalized by atorvastatin. Our results demonstrate that, in diabetic mice,
atorvastatin decreases vascular oxidative stress and inflammation and ameliorates vascular injury through
processes involving decreased activation of Rac1/2 and Nox. These findings elucidate redox-sensitive and
Rac1/2-dependent mechanisms whereby statins protect against vascular injury in diabetes.
Key words: inflammation, NADPH oxidase (Nox), oxidative stress, statin, Type 2 diabetes, vascular function

INTRODUCTION
Statins, 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase
inhibitors, inhibit cholesterol biosynthesis and are widely used in
the treatment of hypercholesterolaemia [1]. Statins also exhibit
actions beyond cholesterol-lowering, the so-called pleiotropic effects, including vasoprotection [2,3]. Recent evidence indicates
that statins influence redox-sensitive processes through putative
antioxidant properties and by inhibiting generation of reactive

oxygen species (ROS) [4,5]. Statins also promote an increase

in nitric oxide (NO) production by stimulating endothelial nitric
oxide synthase (NOS) activity [6]. As such, statins may be protective in conditions associated with vascular oxidative stress,
including hypertension, atherosclerosis and diabetes, possibly by
improving endothelial dysfunction and by decreasing vascular
inflammation and remodelling [7,8]. Statins have been shown to
ameliorate endothelial dysfunction in experimental and clinical
diabetes, through unknown mechanisms [911].

Abbreviations: ACh, acetylcholine; Duox, dual oxidase; ERK, extracellular-signal-regulated protein kinase; HMG-CoA, 3-hydroxy-3-methylglutaryl CoA; INS, insulin; IB-, inhibitor of
B-; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MDA, malondialdehyde; NE, noradrenaline (norepinephrine); NF-B, nuclear factor B; NO, nitric oxide;
NOS, nitric oxide synthase; Nox, NADPH oxidase; OGTT, oral glucose tolerance test; ROS, reactive oxygen species; SBP, systolic blood pressure; TBARS, thiobarbituric acid-reactive
substances; VCAM-1, vascular cell adhesion molecule-1.
Correspondence: Professor Rhian M. Touyz (email Rhian.Touyz@glasgow.ac.uk).


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Previous studies demonstrated that statins decrease activation

of NADPH oxidase (Nox)1, Nox2 and Nox4 (ROS-generating
oxidases) in the cardiovascular system and evidence indicates
an inhibitory effect of rosuvastatin on vascular Nox4 in diabetes
[1214]. The pleiotropic effects of statins are related, in part,
to reduced formation of isoprenoids, lipid moieties responsible
for post-translational modification of specific signalling proteins.
Isoprenoids promote hydrophobic modifications of small GTPases, such as Rac1/2, which plays a critical role in the activation of
Noxs [15]. Rac1/2 also regulates NOS [16]. Inhibition of hydrophobic modification of Rac1/2 by statins has a significant effect
on Nox activation and subsequent ROS generation [15,17,18].
Noxs are the main source of ROS in the vasculature and participate not only in the normal cell function, but also trigger the
development of injury in pathological conditions [19,20].
The Nox family is composed of catalytic subunits [Nox1Nox5,
dual oxidase (Duox)1 and Duox2] and the docking subunit
p22phox , all present in the cell membrane. The regulatory subunits Nox organizer 1 (Noxo1), Nox activator 1 (Noxa1), p67phox ,
p47phox and p40phox , are located in the cytosol. Activation of
Rac1/2 regulates the translocation and assembly of the Nox subunits in the plasma membrane and is a key event in Nox activation
[21,22].
Oxidative stress, due to exaggerated Nox-induced ROS formation and decreased NO bioavailability, is a hallmark of diabetic vasculopathy [2325]. We previously demonstrated an important role for Nox1 in accelerated atherosclerosis in diabetes
[26]. Considering that the beneficial effects of statins are mediated, in part, by reducing ROS bioavailability, the aim of the
present study was to determine whether atorvastatin, a lipid soluble cholesterol-lowering drug of the statin family, decreases
ROS-associated vascular dysfunction and inflammation in db/db
mice, through Nox-dependent processes. We thus tested the hypothesis that atorvastatin reduces Nox activity and redox signalling
in db/db mice. Since statins target small GTPases through posttranslational modifications, we also questioned whether changes
in Nox activity by atorvastatin are associated with altered activity
of vascular Rac1/2 in diabetes.

MATERIALS AND METHODS

Animals
The study was approved by the Animal Ethics Committee of
the Ottawa Hospital Research Institute, University of Ottawa.
Experiments were conducted in accordance with the guidelines
from the National Institutes of Health Guide for the Care and
Use of Laboratory Animals and with Institutional guidelines.
Male Leprdb /Lepr+ (db/+) and Leprdb /Leprdb (db/db) mice
[B6.BKS(D)-Leprdb/J] were purchased from Jackson Laboratories at the age of 6 weeks. Mice were treated with atorvastatin
(10 mg/kg per day atorvastatin; Millipore) for 2 weeks. This dose
of atorvastatin was selected as it is a relatively low dose that has
been well described for rodents in the literature [27,28]. Atorvastatin was incorporated in the chow (2018 Teklad Global 18 %
Protein Rodent Diet, Harlan Laboratories) according to the pre-

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vious food intake assessment (db/+: 0.063 g of atorvastatin/kg

of chow; db/db: 0.056 g of atorvastatin/kg of chow). Food intake
during the treatment was evaluated daily. Systolic blood pressure
(SBP) was measured weekly by tail-cuff plethysmography. At the
end of the treatment, mice were killed by isoflurane inhalation
and subsequently decapitated.

Plasma biochemistry
Blood was collected immediately prior to killing by cardiac puncture. Plasma glucose, triacylglycerols (triglycerides) and cholesterol were determined by auto-analyser (Beckman Coulter
AU5800).

Oral glucose tolerance test

The oral glucose tolerance test (OGTT) was performed to evaluate glucose tolerance in db/db and db/+ mice treated with control
or atorvastatin diet. Mice were deprived of food for 6 h. Blood
was sampled from the lateral saphenous vein immediately before
(baseline, t0 ) and after (t15 , t30 , t60 , t90 , t120 min) administration of
2 g of glucose/kg by oral gavage. Glucose levels were determined
using a glucose analyser (Accu-Check, Roche Diagnostics).

Vascular function
Mesenteric vascular beds were isolated from db/db and db/+ mice
treated with control or atorvastatin diet. Second-order branches
of superior mesenteric artery were dissected and mounted on
a wire myograph (DMT, Danish Myo Technology). Vessel segments (2 mm in length) were mounted on 25 m wires in a vessel
bath chamber for isometric tension recording and equilibrated
for 30 min in KrebsHenseleit-modified physiological salt solution (120 mmol/l NaCl, 25 mmol/l NaHCO3 , 4.7 mmol/l KCl,
1.18 mmol/l KH2 PO4 , 1.18 mmol/l MgSO4 , 2.5 mmol/l CaCl2 ,
0.026 mmol/l EDTA and 5.5 mmol/l glucose), at 37 C, continuously bubbled with 95 % O2 and 5 % CO2 , pH 7.4. At the beginning of each experiment, arteries were contracted with 10 mol/l
noradrenaline (norepinephrine; NE) or 90 mmol/l KCl to test
for functional integrity. In some experiments, the vascular endothelium was removed by gently rubbing the lumen side of
the ring segments. The integrity of the endothelium or its removal was assessed by the presence or absence of relaxation in
response to 1 mol/l acetylcholine (ACh) of NE pre-contracted
arteries respectively. Endothelium-dependent relaxation was assessed by the concentrationresponse curves to ACh (0.1 nmol/l
to 10 mol/l) and insulin (INS; 0.110 000 ng/dl) in vessels precontracted with NE at a concentration to achieve approximately
70 % of maximal response. Contractile responses mediated by NE
(0.1 nmol/l to 10 mol/l) were evaluated in endothelium-intact
and endothelium-denuded arteries.

Vascular structural and mechanical studies

Second-order branches of superior mesenteric artery (23 mm in
length) were slipped on to two glass microcannulas, one of which
was positioned until vessel walls were parallel, in a pressure
myograph (Living Systems). Vessel segments were equilibrated
in KrebsHenseleit-modified physiological salt solution, at 37 C,
continuously bubbled with 95 % O2 and 5 % CO2, pH 7.4, under
constant intraluminal pressure (45 mmHg). Vascular structure

Atorvastatin decreases oxidative stress in diabetic mice

and mechanical studies were assessed under Ca2+ -free conditions

to eliminate the effects of myogenic tone. Vessels were perfused
for 30 min with Ca2+ -free Krebs solution containing 10 mmol/l
EGTA. Measurements of media thickness and lumen diameter
were taken at stepwise increments of luminal pressure (3120
mmHg). Vascular, structural and mechanical parameters were
calculated as we previously described [29].

nuclear factor B (NF-B) p65 (Ser536 ) (Cell Signaling); antiNox1 (Sigma); anti-Nox2, anti-Nox4, anti-vascular cell adhesion
molecule-1 (VCAM-1), anti-NF-B p50, anti-inhibitor of B-
(IB-; Santa Cruz Biotechnology). Antibody to -actin (Sigma)
was used as internal housekeeping control. After incubation with
secondary antibodies, signals were revealed with chemiluminescence, visualized by autoradiography and quantified densitometrically.

Measurement of plasma lipid peroxidation products

Plasma lipid peroxidation was determined by quantifying thiobarbituric acid-reactive substances (TBARS) [30]. Plasma samples
were mixed with 2 % butylated hydroxytoluene and quintanilla
reagent (26 mmol/l thiobarbituric acid and 15 % trichloroacetic
acid). The mixture was boiled for 15 min. Subsequently, the mixture was cooled (4 C) and centrifuged at 3000 g for 10 min.
TBARS formed in each of the samples was assessed by measuring absorbance of the supernatant at 535 nm with an absorbance
microplate reader (BioTek ELx808). In parallel, MDA (malondialdehyde) standards were diluted in a range of 06 nmol/ml.
TBARS were calculated by plotting the obtained absorbance
against an MDA concentration standard curve.

Lucigenin-enhanced chemiluminescence
Vascular ROS generation was measured by a luminescence assay
with lucigenin as the electron acceptor and NADPH as the substrate. Aortic segments from db/db and db/+ mice treated with
control or atorvastatin diet were homogenized in assay buffer
(50 mmol/l KH2 PO4 , 1 mmol/l EGTA and 150 mmol/l sucrose,
pH 7.4) with a glass-to-glass homogenizer. The assay was performed with 100 l of sample, 1.25 l of lucigenin (5 mol/l),
25 l of NADPH (0.1 mmol/l) and assay buffer to a total volume
of 250 l. Luminescence was measured for 30 cycles of 18 s each
by a luminometer (Lumistar Galaxy, BMG Labtechnologies).
Basal readings were obtained prior to the addition of NADPH to
the assay. The reaction was started by the addition of the substrate.
Basal and buffer blank values were subtracted from the NADPHderived luminescence. Superoxide production was expressed as
relative luminescence unit (RLU)/g of protein.

Western blotting
Total protein was extracted from aortas. Frozen tissues were homogenized in 50 mmol/l Tris/HCl (pH 7.4) lysis buffer (containing 1 % Nonidet P-40, 0.5 % sodium deoxycholate, 150 mmol/l
NaCl, 1 mmol/l EDTA, 0.1 % SDS, 2 mmol/l Na3 VO4 , 1 mmol/l
PMSF, 1 g/ml pepstatin A, 1 g/ml leupeptin and 1 g/ml
aprotinin). Total protein extracts were cleared by centrifugation
at 12 000 g for 10 min and pellet was discarded. Proteins from
homogenates of vascular tissues (20 g) were separated by electrophoresis on a polyacrylamide gel (10 %) and transferred on
to a nitrocellulose membrane. Non-specific binding sites were
blocked with 5 % skim milk or 1 % BSA in Tris-buffered saline solution with Tween for 1 h at 24 C. Membranes were then
incubated with specific antibodies overnight at 4 C. Antibodies were as follows: anti-p38 mitogen-activated protein kinase
(MAPK) (Thr180 /Tyr182 ), anti-extracellular-signal-regulated protein kinases 1 and 2 (ERK1/2) MAPK (Thr202 /Tyr204 ), antic-Jun N-terminal kinase (JNK) MAPK (Thr183 /Tyr185 ), anti-

Cytosol and membrane fractionation

Mesenteric arterial beds were homogenized in 50 mM Tris/HCl,
pH 7.4, lysis buffer containing 5 mmol/l EGTA and 2 mmol/l
EDTA, 0.1 mmol/l PMSF, 0.2 mmol/l Na3 VO4 , 1 mol/l pepstatin A, 1 mol/l leupeptin and 1 mol/l aprotinin. Homogenates were centrifuged at 100 000 g for 1 h at 4 C. The supernatant
(cytosolic fraction) was collected. The pellet, containing the particulate fraction, was re-suspended in lysis buffer containing 1 %
Triton X-100 and centrifuged at 10 000 g for 10 min at 4 C. The
resultant supernatant was collected (membrane-enriched fraction). Protein analysis was performed by Western blotting as
described above using anti-p47phox (1:500 dilution, Santa Cruz)
and anti-Rac1/2 (1:1000 dilution, Cell Signaling) antibodies. Antibody to -actin (Sigma) was used as internal housekeeping
control. Results are expressed as membrane to cytosol ratio of
protein content in the cell fractions as an index of translocation
and activation.

Vascular inflammatory response: macrophage

adhesion
The adhesion assay was performed in a 24-well plate coated with
4 % agarose. Aortic rings (5 mm) were cleaned and opened longitudinally. The vascular segments were positioned endotheliumside up in the solid agarose surface (one segment/well), fixed with
sharp pointed pins and kept in F12 medium at 37 C. Murinederived J774A.1 monocyte/macrophage cell line was obtained
from the American Type Culture Collection. J774A.1 adherent
cells were cultured in Dulbeccos modified Eagles medium and
10 % heat-inactivated FBS. For cell fluorescent labelling, macrophages (105 cells/ml) were suspended in 1 % BSA supplemented
PBS containing 1 mol/l calcein-AM (Molecular Probes) and incubated for 20 min at 37 C. Labelled macrophages were washed
twice with PBS and suspended in Hanks buffered salt solution.
Fluorescence-labelled cells (105 cells/well) were then added to
the wells containing the vascular segments and were allowed
to adhere for 30 min at 37 C in 5 % CO2 . Non-adherent cells
were removed by gently washing with pre-warmed Hanks buffered salt solution. The number of adherent cells was counted using fluorescence microscopy. Four fields were evaluated
per segment. Imaging was acquired with an Axiovert Imaging
System.

Data analysis
Results are presented as means +
S.E.M. Comparisons were performed by one-way ANOVA followed by the Bonferroni test or
the NewmanKeuls test, when appropriate. P < 0.05 was considered statistically significant.


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Table 1

Plasma biochemistry
Biochemistry analysis of plasma from db/+ and db/db treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks).

Results are means +

S.E.M. of 12 mice in each experimental group. P < 0.05 compared with db/+ control diet; P < 0.05
db/db atorvastatin diet compared with db/db control diet.
Group
Parameter

db/+

db/db

db/+ +atorvastatin

db/db +atorvastatin

Cholesterol (mmol/l)

2.37 +
0.6
0.93 +
0.35
11.79 +
2.1

3.16 +
0.4
+
2.14 0.9

32.05 +
13.9

2.25 +
0.3
1.07 +
0.1
11.89 +
3.0

2.53 +
0.2
+
1.28 0.5

27.58 +
7.1

Triacylglycerols (mmol/l)
Glucose (mmol/l)

Figure 1

Atorvastatin improves glucose tolerance in db/db mice

OGTT was performed in db/+ and db/db mice treated with control or atorvastatin (10 mg/kg per day) diet (2 weeks). After
6 h fasting, baseline blood glucose was measured. Mice received 2 mg/kg glucose by gavage and blood samples were
collected at 15, 30, 60, 90 and 120 min after the challenge. Results are means +
S.E.M. of 12 mice in each experimental
group. Line graphs, time course of blood glucose after the challenge. Bar graph, area under the curve (AUC) in the plot of

blood glucose concentration against time. P < 0.05 compared with db/+ control diet, P < 0.05 db/db atorvastatin diet
compared with db/db control diet.

RESULTS
Metabolic parameters in db/+ and db/db mice
treated with control or atorvastatin diet
Treatment with atorvastatin did not affect body weight or food
intake in db/+ and db/db mice, compared with control counterparts (Supplementary Figure S1). Table 1 shows that atorvastatin decreased plasma cholesterol and triacylglycerol levels in
db/db mice compared with control-diet-treated db/db mice. Nonfasting db/db mice treated with atorvastatin displayed slightly
reduced, but non-significant, plasma glucose levels as compared
with db/db mice on control diet. Glucose tolerance was determined by OGTT (Figure 1). No differences were observed in the
fasting blood glucose levels at baseline between db/db mice receiving atorvastatin or control diet. Overall, db/db mice receiving
control diet displayed impaired glucose tolerance as compared
with db/+ mice. Atorvastatin-treated db/db mice showed a decrease in blood glucose compared with control-diet-treated db/db
mice at 60, 90 and 120 min after the glucose challenge. The statin

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treatment had no effect on fasting blood glucose levels at baseline

or glucose tolerance in db/+ mice.

Systolic blood pressure in db/+ and db/db mice

treated with control or atorvastatin diet
Diabetic db/db mice and db/+ control mice had similar SBP
levels (Supplementary Figure S2). Atorvastatin did not affect the
SBP in either db/+ or db/db mice.

Status of oxidative stress in arteries from db/+

and db/db mice
The potential antioxidant effect of atorvastatin was evaluated
in db/db and db/+ mice. Figure 2(A) demonstrates that plasma
TBARS levels were significantly higher in db/db mice compared with db/+ and this increase was inhibited by atorvastatin
treatment. NADPH-dependent superoxide anion generation was
measured in aortic homogenates from db/+ and db/db mice. Figure 2(B) shows that lucigenin-derived luminescence was significantly higher in arteries from db/db mice compared with db/+.

Atorvastatin decreases oxidative stress in diabetic mice

Figure 2

Atorvastatin reduced vascular oxidative stress in db/db mice

Plasma, aorta and mesenteric arteries were obtained from db/+ and db/db mice treated with atorvastatin (10 mg/kg per
day) or control diet (2 weeks). ROS status was assessed by plasma TBARS levels (A) and lucigenin-enhanced chemiluminescence (B). Translocation of p47phox (C) and Rac1/2 (D) was assessed by the protein expression in membrane and
+ S.E.M. of 68 mice in each
cytosolic fractions isolated from mesenteric arterial bed homogenates. Results are means
experimental group. P < 0.05 compared with db/+ control diet, P < 0.05 db/db atorvastatin diet compared with db/db
control diet.

Atorvastatin reduced the increase in superoxide anion generation in db/db mice. Translocation of the Nox cytosolic subunit
p47phox and the small GTPase Rac1/2 from the cytosol to the cell
membrane was evaluated as an index of the oxidase activation.
Expression of p47phox (Figure 2C) and Rac1/2 (Figure 2D) in
membrane-enriched fractions was increased in mesenteric arteries from db/db mice. This effect was abrogated by atorvastatin.

Expression of Nox isoforms in arteries from db/+

and db/db mice
Since enhanced oxidative stress was observed in the vasculature
of db/db mice, we evaluated the protein expression of the Nox
isoforms in aortas from of db/+ and db/db mice. Figure 3 demonstrates that the protein expression of Nox1, 2 and 4 was upregulated in aortas from db/db mice compared with the db/+
group. Atorvastatin reduced expression of the catalytic subunit
of the oxidase isoforms in the vasculature of diabetic mice.

Effects of atorvastatin treatment on MAPK

phosphorylation in arteries from db/+ and db/db
mice
The effect of atorvastatin on the phosphorylation levels of
MAPKs, the downstream signalling targets of ROS, is demonstrated in Figure 4. In the aorta, p38 MAPK phosphorylation
(Figure 4A) was increased in db/db mice compared with db/+
mice. Similar results were observed for ERK1/2 MAPK (Figure 4B) and JNK MAPK (Figure 4C). Atorvastatin inhibited the
increase in MAPK phosphorylation observed in the db/db mice.

Pro-inflammatory responses in arteries from db/+

and db/db mice treated with atorvastatin
The redox-sensitive NF-B family of transcription factors regulates multiple cellular processes including inflammation. Increased phosphorylation levels of the NF-B p65 subunit were
observed in aortas from db/db mice (Figure 5A). Aortas from


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Figure 3

Effects of atorvastatin on Nox protein expression in the vasculature of db/m and db/db mice
Protein levels of Nox1 (A), Nox2 (B) and Nox4 (C) were evaluated in mesenteric arteries isolated from db/+ and db/db
mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). Side panels, representative immunoblots
of Nox1, Nox2, Nox4 and -actin. Results are presented as means +
S.E.M. of seven mice in each experimental group.

P < 0.05 compared with db/+ control diet, P < 0.05 db/db atorvastatin diet compared with db/db control diet.

db/db mice exhibited increased protein expression of the NF-B

subunit p50 (Figure 5B) with reduced expression of the regulatory protein IB- compared with db/+ mice (Figure 5C). These
effects were normalized by atorvastatin.
One of the initial steps in vascular inflammation is expression of adhesion molecules such as VCAM-1. VCAM-1 content was increased in the vasculature of db/db mice versus
controls (Figure 6A). Figure 6(B) shows adherent green fluorescent macrophages in aortic segments. Increased numbers of
adherent macrophages were observed in aortic segments of db/db
mice compared with db/+ mice. Atorvastatin reduced vascular
inflammatory responses as evidenced by decreased VCAM-1 expression and macrophage adhesion in db/db mice.

Effect of atorvastatin on vascular function in

arteries from db/+ and db/db mice
Figure 7 and Tables 2 and 3 show the effects of atorvastatin on
vascular function of db/+ and db/db mice. Endothelium-intact
mesenteric arteries from db/db mice were more sensitive to NE
compared with those from db/+ mice, as evidenced by the leftward shift in the concentrationresponse curve to the agonist.
Atorvastatin abolished the increased sensitivity to NE in arteries
from db/db mice (Figure 7A). No differences in the response to
NE were observed in endothelium-denuded arteries from db/+
and db/db mice (Figure 7B). Relaxation in response to ACh was
significantly reduced in arteries from db/db mice, effects that

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were restored by atorvastatin (Figure 7C). Maximum relaxation

in response to INS was decreased in mesenteric arteries from
db/db mice compared with those from db/+ mice. Atorvastatin
improved the endothelium-dependent relaxation in response to
INS in arteries from db/db mice (Figure 7D). Atorvastatin did
not affect the endothelium-dependent relaxant responses in arteries from db/+ mice.

Vascular morphology and mechanics in arteries

from db/+ and db/db mice
Mesenteric arteries from db/+ and db/db mice presented similar wall thickness, lumen diameter and cross-sectional area in
response to stepwise increments of intraluminal pressure (Supplementary Figures S3AS3C). No differences were observed in
the stressstrain relationship curve between arteries from db/+
and db/db mice (Supplementary Figure S3D). Atorvastatin did
not affect morphological parameters and mechanical properties
in all experimental groups.

DISCUSSION
Major findings in the present study demonstrate that INS resistance, endothelial dysfunction and vascular inflammation in
diabetic db/db mice are ameliorated by atorvastatin through

Atorvastatin decreases oxidative stress in diabetic mice

Figure 4

Increased MAPK phosphorylation is inhibited by atorvastatin in the vasculature of db/db mice

Phosphorylation levels of p38 MAPK (A), ERK1/2 MAPK (B) and JNK MAPK (C) were evaluated in mesenteric arteries
isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). Side panels,
representative immunoblots of p38 MAPK (Thr180 /Tyr182 ), ERK1/2 MAPK (Thr202 /Tyr204 ), JNK MAPK (Thr183 /Tyr185 ) and
+ S.E.M. of seven mice in each experimental group. P < 0.05 compared with
-actin. Results are presented as means
db/+ control diet, P < 0.05 db/db atorvastatin diet compared with db/db control diet.

processes associated with reduced vascular oxidative stress and

decreased pro-inflammatory signalling. These phenomena were
associated with decreased activation of Rac1/2, down-regulation
of Nox isoforms and decreased generation of ROS. Our data
provide some novel insights involving Rac1/2-regulated Noxs
whereby statins, such as atorvastatin, protect against vasculopathy in diabetes. Statins as adjuvant therapy in the management
of diabetes may have beneficial metabolic and vascular effects
beyond lipid lowering.
The major morbidities associated with diabetes are cardiovascular disease and nephropathy due in large part to vascular injury
[31]. In our study, db/db mice exhibited significant endothelial
dysfunction and vascular inflammation as evidenced by reduced
ACh- and INS-induced endothelium-dependent vasorelaxation
and increased monocyte adhesion with augmented VCAM-1 expression. Vascular dysfunction has been described in patients
with diabetes [32,33] and in experimental models of Type 1 diabetes (OVE26 mice and streptozotocin-induced diabetes) [34,35]
and Type 2 diabetes (db/db, fat-fed) [36,37]. Our findings support others that have shown improved endothelium-dependent
NO-mediated vasorelaxation by another statin, rosuvastatin, in
experimental diabetes and are in line with clinical studies demonstrating that statins improve flow-mediated vasodilation in pa-

tients with Type 2 diabetes [12,38,39]. Although these vascular

effects have been attributed to mechanisms independent of lipidlowering effects [12], the metabolic and lipid profiles of db/db
mice in our study were improved by atorvastatin. Accordingly, we
cannot exclude the possibility that some vasoprotection occurs
through lipid lowering.
Exact mechanisms whereby statins affect directly on vascular function still remain elusive. However, growing evidence indicates that these drugs influence signalling pathways involved
in the generation of ROS and NO [40]. Statins inhibit HMGCoA reductase [1], which normally catalyses the biosynthesis
of mevalonate, the main intermediate fatty acid in cholesterol
biosynthesis. Mevalonate is also the primary precursor of lipid
isoprenoid intermediates, such as farnesyl pyrophosphate (FPP)
and geranylgeranyl pyrophosphate (GGPP), which are essential
for the prenylation and activation of small GTPases [15,41], such
as Rac1/2. Hence, statins have the capacity to inhibit Rac1/2 activation, which could affect Rac-dependent signalling, including
Nox-ROS pathways [42]. db/db mice exhibited increased activation of Rac1/2 as evidenced by increased cytosol-to-membrane
translocation, with associated increased p47phox translocation and
activation of Noxs. Increased Rac1/2 activation has also been
demonstrated in streptozotocin-induced diabetic rats [43] and we


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Figure 5

Atorvastatin affects NF-B proteins phosphorylation and expression in the vasculature of db/db mice
Phosphorylation of NF-B p65 (A) and the expression of NF-B p50 (B) and IB- (C) were evaluated in mesenteric
arteries isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks).
Side panels, representative immunoblots of NF-B p65 (Ser536 ), NF-B p50, IB- and -actin. Results are presented as
+ S.E.M. of seven mice in each experimental group. P < 0.05 compared with db/+ control diet, P < 0.05 db/db
means
atorvastatin diet compared with db/db control diet.

Figure 6

Atorvastatin reduces VCAM-1 expression and macrophages adhesion in aortas from db/db mice
Aortas were isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks).
VCAM-1 expression in aortic homogenates (A) and the number of adherent fluorescent macrophages to aortic segments (B)

were evaluated. Results are presented as means +

S.E.M. of seven mice in each experimental group. P < 0.05 compared
with db/+ control diet P < 0.05 db/db atorvastatin diet compared with db/db control diet.


C The Authors Journal compilation 
C 2015 Biochemical Society

Atorvastatin decreases oxidative stress in diabetic mice

Figure 7

Atorvastatin decreases the sensitivity to NE and improves endothelium dependent relaxation in small mesenteric arteries from db/db mice
Resistance mesenteric arteries were isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) or
control diet (2 weeks) and mounted in a wire myograph. Concentrationresponse curves to NE (0.1 nmol/l to 10 mol/l)
were performed in endothelium-intact (A) or endothelium-denuded (B) arteries. Endothelium-intact arteries were precontracted with NE (106 mol/l) and concentrationresponse curves to (C) ACh (1 nmol/l to 10 mol/l) and (D) INS

(0.110 000 ng/dl) were performed. Results are mean +

S.E.M. of 58 mice in each experimental group. P < 0.05
compared with db/+ control diet, P < 0.05 db/db atorvastatin diet compared with db/db control diet.

showed that vascular and renal Nox-mediated ROS generation

is amplified in streptozotocin-induced and db/db diabetic mice
[26,44]. Of the Nox isoforms, we previously demonstrated an
important role for Nox1 and Nox4 in cardiovascular and renal
injury in diabetic mice [26,42]. Other studies have implied a role
for Nox2 [45,46]. In the present study, we found increased expression of all three Nox isoforms in db/db vessels and as such
cannot identify which Nox is primarily responsible for the increased ROS generation in our model. However, since Nox1 and
Nox2 have an obligatory need for p47phox , which was also activated in db/db mice, these Noxs may be particularly important,
similar to what was previously reported.
In atorvastatin-treated db/db mice, Nox activation was
attenuated as evidenced by reduced NADPH-induced generation
of ROS, down-regulation of Nox isoforms, reduced p47phox
translocation and decreased levels of TBARS. These effects
may relate to Rac1/2 inhibition since atorvastatin blocked
translocation of Rac from the cytosol to the membrane. Rac1/2
normally regulates Noxs by promoting translocation of the

p47phox /p67phox /p40phox cytosolic complex to the Nox/p22phox

complex in the cell membrane and is important in the initial steps
of the electron transfer reaction for superoxide anion generation.
Hence, statins probably blunt Nox activity, at least in part,
through inhibitory effects on Rac1/2 and possibly by modulating
assembly of oxidase subunits. Statins have been shown to
regulate Rac activity through various mechanisms including
stimulation of small GTP-binding protein GDP dissociation
stimulator (SmgGDS), which negatively regulates Rac1/2, and
by inhibiting post-translational modifications [47]. Our findings,
in the present paper, expand those observations and provide a
putative molecular mechanism whereby statins interfere with
Rac-regulated Nox-derived ROS in diabetes, which probably
reduces oxidative stress, normalizes endothelial function and
prevents vascular inflammation in db/db mice. These processes
probably involve reduced pro-inflammatory signalling through
MAPKs and NF-B [4850], pathways that were downregulated by treatment in diabetic mice. Such effects may relate
to blunted Rac1/2 and Nox activation. It is also possible


C The Authors Journal compilation 
C 2015 Biochemical Society

419

T. Bruder-Nascimento and others

Table 2

Vascular reactivity to NE, ACh and INS: maximum effect

Effects of atorvastatin treatment (10 mg/kg per day for 2 weeks) on vascular responses [maximum effect (Emax ) of second
order mesenteric arteries)] to NE, ACh and INS in db/+ and db/db mice. Concentrationeffect curves were performed in
endothelium-intact (+E) and endothelium-denuded (E) mesenteric rings. Results are means +
S.E.M. of seven mice in each
experimental group. Emax values for NE are presented as a percentage of KCl (60 mM) responses, whereas ACh and INS
are presented in relation of pre-contraction of 0.1 M NE. P < 0.05 compared with db/+ control diet, P < 0.05 db/db
atorvastatin diet compared with db/db control diet.
Group (Emax )
Agonist

db/+

db/db

db/+ +atorvastatin

db/db +atorvastatin

NE (+E)

101.4 +
13.3
91.1 +
10.8
85.2 +
5.6
68.1 +
5.4

110.0 +
10.9
98.7 +
11.8

48.0 +
5.7

42.2 +
5.2

119.3 +
12.53
91.5 +
9.0
87.9 +
5.8
77.2 +
8.2

97.3 +
14.80
89.8 +
16.42

79.6 +
11.3

65.9 +
7.8

NE (-E)
ACh
INS

Table 3

Vascular reactivity to NE, ACh and INS: pD2

Effects of atorvastatin treatment (10 mg/kg per day for 2 weeks) on vascular responses [pD2 (negative logarithm of the
EC50 of second-order mesenteric arteries)] to NE, ACh and INS in db/+ and db/db mice. Concentrationeffect curves were
performed in endothelium-intact (+E) and endothelium-denuded (E) mesenteric rings. Results are means +
S.E.M. of seven
mice in each experimental group. P < 0.05 compared with db/+ control diet, P < 0.05 db/db atorvastatin diet compared
with db/db control diet.
Group (pD2 )
Agonist

db/+

db/db

db/+ +atorvastatin

db/db +atorvastatin

NE (+E)

7.53 +
0.3
7.25 +
0.3
7.36 +
0.2
6.72 +
0.2

8.55 +
0.3
7.66 +
0.3

8.66 +
0.9

7.32 +
0.5

6.78 +
0.5
7.53 +
0.2
7.73 +
0.2
6.61 +
0.1

7.41 +
0.1
7.69 +
0.2

7.23 +
0.2
7.17 +
0.2

NE (E)
ACh
INS

that
atorvastatin
improved
vasorelaxation
through
NOS/NO-dependent pathways, since statins have been shown to
stimulate activity of NOS with increased NO generation [51,52].
Statin treatment had a positive effect on the metabolic profile and INS sensitivity in db/db mice, which could also have
an effect on improved vascular status in these mice, as previously suggested [53]. In our experiments, atorvastatin decreased
vascular sensitivity to NE and improved endothelium-dependent
vasorelaxation in db/db mice. These phenomena were associated
with reduced vascular oxidative damage and inflammation and
may have been due, at least in part, to changes in INS sensitivity
in vascular cells.
In support of our results in db/db mice others have shown in
experimental rat models of diabetes [53,54] and in patients with
diabetes [55] that atorvastatin improves INS sensitivity. However, data from some clinical studies reported that statins actually
worsen diabetes and that they promote new onset diabetes and
INS resistance. Reasons for these conflicting data are unclear, but
it should be stressed that studies reporting pro-diabetic actions of
statins were primarily retrospective analyses of large clinical trials that demonstrated associations between statin treatment and
development of diabetes [5659]. In such associative studies,
causality cannot be established and in none of those investigations was a direct negative effect of statins on INS sensitivity actually demonstrated. Further in depth investigations are required
to determine whether statins directly influence INS metabolism
in diabetes.
In conclusion, we elucidate some molecular mechanisms
whereby atorvastatin protects against vascular damage in dia-

420


C The Authors Journal compilation 
C 2015 Biochemical Society

betes. We also demonstrate that statin treatment improves INS

sensitivity, which in its own right could positively influence vascular status. Taken together, our data suggest that statins may
have important vasoprotective effects in diabetes beyond lipid
lowering.

CLINICAL PERSPECTIVES

r
r

Statins, cholesterol-lowering drugs, improve vascular function

in diabetes, but exact mechanisms are elusive.
We demonstrated that, in a mouse model of Type 2 diabetes
(db/db), systemic and vascular oxidative stress, endothelial
dysfunction and vascular inflammation were normalized by
atorvastatin. These processes were associated with downregulation of Rac1/2, decreased Nox-derived ROS generation
and reduced pro-inflammatory signalling.
Our data identify some molecular mechanisms involving
Rac1/2-regulated Noxs whereby statins may protect against
vascular dysfunction in diabetes. Clinically, statins may have
vasoprotective therapeutic potential beyond lipid-lowering in
diabetes.

AUTHOR CONTRIBUTION

The study was conceived by Rhian Touyz and Rita Tostes, and developed by Thiago Bruder-Nascimento and Glaucia Callera. Thiago
Bruder-Nascimento conducted the studies with help from Glaucia
Callera and Ying He. The paper was written by Thiago BruderNascimento, Glaucia Callera, Rita Tostes and Rhian Touyz, with

Atorvastatin decreases oxidative stress in diabetic mice

contributions from Augusto Montezano, Tayze Antunes and Aurelie

Nguyen Dinh Cat.

13

FUNDING

This work was supported by the S

ao Paulo Research Foundation
(FAPESP) [grant numbers 2010/52214-6 (to R.C.T); 2011/017856; 2011/22035-5 (to T.B.-N.)]; the Agence Universitaire de la Francophonie [grant number 58145FT103]; the Juvenile Diabetes Research Foundation [grant number 4-2010-528]; the Canadian Institutes of Health Research [grant number 44018]; and the British
Heart Foundation [grant number RG/13/7/30099].

14

15

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Received 29 July 2014/30 September 2014; accepted 31 October 2014

Published as Immediate Publication 31 October 2014, doi: 10.1042/CS20140456


C The Authors Journal compilation 
C 2015 Biochemical Society

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