Clinical Science (2015) 128, 411–423 (Printed in Great Britain) doi: 10.1042/CS20140456

Vascular injury in diabetic db/db mice isameliorated by atorvastatin: role ofRac1/2-sensitive Nox-dependent pathwaysThiago 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.

AbstractOxidative stress [increased bioavailability of reactive oxygen species (ROS)] plays a role in the endothelialdysfunction and vascular inflammation, which underlie vascular damage in diabetes. Statins arecholesterol-lowering drugs that are vasoprotective in diabetes through unknown mechanisms. We tested thehypothesis that atorvastatin decreases NADPH oxidase (Nox)-derived ROS generation and associated vascular injuryin diabetes. Leprdb/Leprdb (db/db) mice, a model of Type 2 diabetes and control Leprdb/Lepr+ (db/+) mice wereadministered 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 (thiobarbituricacid-reactive substances) levels and exaggerated vascular Nox-derived ROS generation respectively, were inhibitedby atorvastatin. Cytosol-to-membrane translocation of the Nox regulatory subunit p47phox and the small GTPaseRac1/2 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-activatedprotein kinases (MAPKs) was abrogated by atorvastatin in db/db mice. Pro-inflammatory signalling (decreased IκB-αand increased NF-κB p50 expression, increased NF-κB p65 phosphorylation) and associated vascular inflammation[vascular cell adhesion molecule-1 (VCAM-1) expression and vascular monocyte adhesion], which were increased inaortas of db/db mice, were blunted by atorvastatin. Impaired acetylcholine (Ach)- and insulin (INS)-inducedvasorelaxation 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 throughprocesses involving decreased activation of Rac1/2 and Nox. These findings elucidate redox-sensitive andRac1/2-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) reductaseinhibitors, inhibit cholesterol biosynthesis and are widely used inthe treatment of hypercholesterolaemia [1]. Statins also exhibitactions beyond cholesterol-lowering, the so-called pleiotropic ef-fects, including vasoprotection [2,3]. Recent evidence indicatesthat statins influence redox-sensitive processes through putativeantioxidant properties and by inhibiting generation of reactive

Abbreviations: ACh, acetylcholine; Duox, dual oxidase; ERK, extracellular-signal-regulated protein kinase; HMG-CoA, 3-hydroxy-3-methylglutaryl CoA; INS, insulin; IκB-α, inhibitor ofκB-α; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MDA, malondialdehyde; NE, noradrenaline (norepinephrine); NF-κB, nuclear factor κB; 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-reactivesubstances; VCAM-1, vascular cell adhesion molecule-1.

Correspondence: Professor Rhian M. Touyz (email Rhian.Touyz@glasgow.ac.uk).

oxygen species (ROS) [4,5]. Statins also promote an increasein nitric oxide (NO) production by stimulating endothelial nitricoxide synthase (NOS) activity [6]. As such, statins may be pro-tective in conditions associated with vascular oxidative stress,including hypertension, atherosclerosis and diabetes, possibly byimproving endothelial dysfunction and by decreasing vascularinflammation and remodelling [7,8]. Statins have been shown toameliorate endothelial dysfunction in experimental and clinicaldiabetes, through unknown mechanisms [9–11].

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Previous studies demonstrated that statins decrease activationof NADPH oxidase (Nox)1, Nox2 and Nox4 (ROS-generatingoxidases) in the cardiovascular system and evidence indicatesan inhibitory effect of rosuvastatin on vascular Nox4 in diabetes[12–14]. The pleiotropic effects of statins are related, in part,to reduced formation of isoprenoids, lipid moieties responsiblefor post-translational modification of specific signalling proteins.Isoprenoids promote hydrophobic modifications of small GTP-ases, such as Rac1/2, which plays a critical role in the activation ofNoxs [15]. Rac1/2 also regulates NOS [16]. Inhibition of hydro-phobic modification of Rac1/2 by statins has a significant effecton Nox activation and subsequent ROS generation [15,17,18].

Noxs are the main source of ROS in the vasculature and par-ticipate not only in the normal cell function, but also trigger thedevelopment of injury in pathological conditions [19,20].The Nox family is composed of catalytic subunits [Nox1–Nox5,dual oxidase (Duox)1 and Duox2] and the docking subunitp22phox, all present in the cell membrane. The regulatory sub-units Nox organizer 1 (Noxo1), Nox activator 1 (Noxa1), p67phox,p47phox and p40phox, are located in the cytosol. Activation ofRac1/2 regulates the translocation and assembly of the Nox sub-units in the plasma membrane and is a key event in Nox activation[21,22].

Oxidative stress, due to exaggerated Nox-induced ROS form-ation and decreased NO bioavailability, is a hallmark of dia-betic vasculopathy [23–25]. We previously demonstrated an im-portant role for Nox1 in accelerated atherosclerosis in diabetes[26]. Considering that the beneficial effects of statins are me-diated, in part, by reducing ROS bioavailability, the aim of thepresent study was to determine whether atorvastatin, a lipid sol-uble cholesterol-lowering drug of the statin family, decreasesROS-associated vascular dysfunction and inflammation in db/dbmice, through Nox-dependent processes. We thus tested the hypo-thesis that atorvastatin reduces Nox activity and redox signallingin db/db mice. Since statins target small GTPases through post-translational modifications, we also questioned whether changesin Nox activity by atorvastatin are associated with altered activityof vascular Rac1/2 in diabetes.

MATERIALS AND METHODS

AnimalsThe study was approved by the Animal Ethics Committee ofthe Ottawa Hospital Research Institute, University of Ottawa.Experiments were conducted in accordance with the guidelinesfrom the National Institutes of Health Guide for the Care andUse 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 Laborat-ories at the age of 6 weeks. Mice were treated with atorvastatin(10 mg/kg per day atorvastatin; Millipore) for 2 weeks. This doseof atorvastatin was selected as it is a relatively low dose that hasbeen well described for rodents in the literature [27,28]. Ator-vastatin was incorporated in the chow (2018 Teklad Global 18 %Protein Rodent Diet, Harlan Laboratories) according to the pre-

vious food intake assessment (db/+: 0.063 g of atorvastatin/kgof chow; db/db: 0.056 g of atorvastatin/kg of chow). Food intakeduring the treatment was evaluated daily. Systolic blood pressure(SBP) was measured weekly by tail-cuff plethysmography. At theend of the treatment, mice were killed by isoflurane inhalationand subsequently decapitated.

Plasma biochemistryBlood was collected immediately prior to killing by cardiac punc-ture. Plasma glucose, triacylglycerols (triglycerides) and cho-lesterol were determined by auto-analyser (Beckman CoulterAU5800).

Oral glucose tolerance testThe oral glucose tolerance test (OGTT) was performed to evalu-ate glucose tolerance in db/db and db/+ mice treated with controlor atorvastatin diet. Mice were deprived of food for 6 h. Bloodwas sampled from the lateral saphenous vein immediately before(baseline, t0) and after (t15, t30, t60, t90, t120 min) administration of2 g of glucose/kg by oral gavage. Glucose levels were determinedusing a glucose analyser (Accu-Check, Roche Diagnostics).

Vascular functionMesenteric vascular beds were isolated from db/db and db/+ micetreated with control or atorvastatin diet. Second-order branchesof superior mesenteric artery were dissected and mounted ona wire myograph (DMT, Danish Myo Technology). Vessel seg-ments (2 mm in length) were mounted on 25 μm wires in a vesselbath chamber for isometric tension recording and equilibratedfor 30 min in Krebs–Henseleit-modified physiological salt solu-tion (120 mmol/l NaCl, 25 mmol/l NaHCO3, 4.7 mmol/l KCl,1.18 mmol/l KH2PO4, 1.18 mmol/l MgSO4, 2.5 mmol/l CaCl2,0.026 mmol/l EDTA and 5.5 mmol/l glucose), at 37 ◦C, continu-ously bubbled with 95 % O2 and 5 % CO2, pH 7.4. At the begin-ning of each experiment, arteries were contracted with 10 μmol/lnoradrenaline (norepinephrine; NE) or 90 mmol/l KCl to testfor functional integrity. In some experiments, the vascular en-dothelium was removed by gently rubbing the lumen side ofthe ring segments. The integrity of the endothelium or its re-moval was assessed by the presence or absence of relaxation inresponse to 1 μmol/l acetylcholine (ACh) of NE pre-contractedarteries respectively. Endothelium-dependent relaxation was as-sessed by the concentration–response curves to ACh (0.1 nmol/lto 10 μmol/l) and insulin (INS; 0.1–10 000 ng/dl) in vessels pre-contracted with NE at a concentration to achieve approximately70 % of maximal response. Contractile responses mediated by NE(0.1 nmol/l to 10 μmol/l) were evaluated in endothelium-intactand endothelium-denuded arteries.

Vascular structural and mechanical studiesSecond-order branches of superior mesenteric artery (2–3 mm inlength) were slipped on to two glass microcannulas, one of whichwas positioned until vessel walls were parallel, in a pressuremyograph (Living Systems). Vessel segments were equilibratedin Krebs–Henseleit-modified physiological salt solution, at 37 ◦C,continuously bubbled with 95 % O2 and 5 % CO2, pH 7.4, underconstant intraluminal pressure (45 mmHg). Vascular structure

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and mechanical studies were assessed under Ca2+-free conditionsto eliminate the effects of myogenic tone. Vessels were perfusedfor 30 min with Ca2+-free Krebs solution containing 10 mmol/lEGTA. Measurements of media thickness and lumen diameterwere taken at stepwise increments of luminal pressure (3–120mmHg). Vascular, structural and mechanical parameters werecalculated as we previously described [29].

Measurement of plasma lipid peroxidation productsPlasma lipid peroxidation was determined by quantifying thiobar-bituric acid-reactive substances (TBARS) [30]. Plasma sampleswere mixed with 2 % butylated hydroxytoluene and quintanillareagent (26 mmol/l thiobarbituric acid and 15 % trichloroaceticacid). The mixture was boiled for 15 min. Subsequently, the mix-ture was cooled (4 ◦C) and centrifuged at 3000 g for 10 min.TBARS formed in each of the samples was assessed by measur-ing absorbance of the supernatant at 535 nm with an absorbancemicroplate reader (BioTek ELx808). In parallel, MDA (malondi-aldehyde) standards were diluted in a range of 0–6 nmol/ml.TBARS were calculated by plotting the obtained absorbanceagainst an MDA concentration standard curve.

Lucigenin-enhanced chemiluminescenceVascular ROS generation was measured by a luminescence assaywith lucigenin as the electron acceptor and NADPH as the sub-strate. Aortic segments from db/db and db/+ mice treated withcontrol or atorvastatin diet were homogenized in assay buffer(50 mmol/l KH2PO4, 1 mmol/l EGTA and 150 mmol/l sucrose,pH 7.4) with a glass-to-glass homogenizer. The assay was per-formed with 100 μl of sample, 1.25 μl of lucigenin (5 μmol/l),25 μl of NADPH (0.1 mmol/l) and assay buffer to a total volumeof 250 μl. Luminescence was measured for 30 cycles of 18 s eachby a luminometer (Lumistar Galaxy, BMG Labtechnologies).Basal readings were obtained prior to the addition of NADPH tothe assay. The reaction was started by the addition of the substrate.Basal and buffer blank values were subtracted from the NADPH-derived luminescence. Superoxide production was expressed asrelative luminescence unit (RLU)/μg of protein.

Western blottingTotal protein was extracted from aortas. Frozen tissues were ho-mogenized in 50 mmol/l Tris/HCl (pH 7.4) lysis buffer (contain-ing 1 % Nonidet P-40, 0.5 % sodium deoxycholate, 150 mmol/lNaCl, 1 mmol/l EDTA, 0.1 % SDS, 2 mmol/l Na3VO4, 1 mmol/lPMSF, 1 μg/ml pepstatin A, 1 μg/ml leupeptin and 1 μg/mlaprotinin). Total protein extracts were cleared by centrifugationat 12 000 g for 10 min and pellet was discarded. Proteins fromhomogenates of vascular tissues (20 μg) were separated by elec-trophoresis on a polyacrylamide gel (10 %) and transferred onto a nitrocellulose membrane. Non-specific binding sites wereblocked with 5 % skim milk or 1 % BSA in Tris-buffered sa-line solution with Tween for 1 h at 24 ◦C. Membranes were thenincubated with specific antibodies overnight at 4 ◦C. Antibod-ies were as follows: anti-p38 mitogen-activated protein kinase(MAPK) (Thr180/Tyr182), anti-extracellular-signal-regulated pro-tein kinases 1 and 2 (ERK1/2) MAPK (Thr202/Tyr204), anti-c-Jun N-terminal kinase (JNK) MAPK (Thr183/Tyr185), anti-

nuclear factor κB (NF-κB) p65 (Ser536) (Cell Signaling); anti-Nox1 (Sigma); anti-Nox2, anti-Nox4, anti-vascular cell adhesionmolecule-1 (VCAM-1), anti-NF-κB p50, anti-inhibitor of κB-α(IκB-α; Santa Cruz Biotechnology). Antibody to β-actin (Sigma)was used as internal housekeeping control. After incubation withsecondary antibodies, signals were revealed with chemilumines-cence, visualized by autoradiography and quantified densitomet-rically.

Cytosol and membrane fractionationMesenteric arterial beds were homogenized in 50 mM Tris/HCl,pH 7.4, lysis buffer containing 5 mmol/l EGTA and 2 mmol/lEDTA, 0.1 mmol/l PMSF, 0.2 mmol/l Na3VO4, 1 μmol/l pep-statin A, 1 μmol/l leupeptin and 1 μmol/l aprotinin. Homogen-ates were centrifuged at 100 000 g for 1 h at 4 ◦C. The supernatant(cytosolic fraction) was collected. The pellet, containing the par-ticulate fraction, was re-suspended in lysis buffer containing 1 %Triton X-100 and centrifuged at 10 000 g for 10 min at 4 ◦C. Theresultant supernatant was collected (membrane-enriched frac-tion). Protein analysis was performed by Western blotting asdescribed above using anti-p47phox (1:500 dilution, Santa Cruz)and anti-Rac1/2 (1:1000 dilution, Cell Signaling) antibodies. An-tibody to β-actin (Sigma) was used as internal housekeepingcontrol. Results are expressed as membrane to cytosol ratio ofprotein content in the cell fractions as an index of translocationand activation.

Vascular inflammatory response: macrophageadhesionThe adhesion assay was performed in a 24-well plate coated with4 % agarose. Aortic rings (5 mm) were cleaned and opened lon-gitudinally. The vascular segments were positioned endothelium-side up in the solid agarose surface (one segment/well), fixed withsharp pointed pins and kept in F12 medium at 37 ◦C. Murine-derived J774A.1 monocyte/macrophage cell line was obtainedfrom the American Type Culture Collection. J774A.1 adherentcells were cultured in Dulbecco’s modified Eagle’s medium and10 % heat-inactivated FBS. For cell fluorescent labelling, macro-phages (105 cells/ml) were suspended in 1 % BSA supplementedPBS containing 1 μmol/l calcein-AM (Molecular Probes) and in-cubated for 20 min at 37 ◦C. Labelled macrophages were washedtwice with PBS and suspended in Hanks’ buffered salt solution.Fluorescence-labelled cells (105 cells/well) were then added tothe wells containing the vascular segments and were allowedto adhere for 30 min at 37 ◦C in 5 % CO2. Non-adherent cellswere removed by gently washing with pre-warmed Hanks’ buf-fered salt solution. The number of adherent cells was coun-ted using fluorescence microscopy. Four fields were evaluatedper segment. Imaging was acquired with an Axiovert ImagingSystem.

Data analysisResults are presented as means +− S.E.M. Comparisons were per-formed by one-way ANOVA followed by the Bonferroni test orthe Newman–Keuls test, when appropriate. P < 0.05 was con-sidered statistically significant.

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Table 1 Plasma biochemistryBiochemistry 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 < 0.05db/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 3.16 +− 0.4∗ 2.25 +− 0.3 2.53 +− 0.2∗∗

Triacylglycerols (mmol/l) 0.93 +− 0.35 2.14 +− 0.9∗ 1.07 +− 0.1 1.28 +− 0.5∗∗

Glucose (mmol/l) 11.79 +− 2.1 32.05 +− 13.9∗ 11.89 +− 3.0 27.58 +− 7.1∗∗

Figure 1 Atorvastatin improves glucose tolerance in db/db miceOGTT was performed in db/+ and db/db mice treated with control or atorvastatin (10 mg/kg per day) diet (2 weeks). After6 h fasting, baseline blood glucose was measured. Mice received 2 mg/kg glucose by gavage and blood samples werecollected at 15, 30, 60, 90 and 120 min after the challenge. Results are means +− S.E.M. of 12 mice in each experimentalgroup. Line graphs, time course of blood glucose after the challenge. Bar graph, area under the curve (AUC) in the plot ofblood glucose concentration against time. ∗P < 0.05 compared with db/+ control diet, ∗∗P < 0.05 db/db atorvastatin dietcompared with db/db control diet.

RESULTS

Metabolic parameters in db/+ and db/db micetreated with control or atorvastatin dietTreatment with atorvastatin did not affect body weight or foodintake in db/+ and db/db mice, compared with control counter-parts (Supplementary Figure S1). Table 1 shows that atorvast-atin decreased plasma cholesterol and triacylglycerol levels indb/db mice compared with control-diet-treated db/db mice. Non-fasting db/db mice treated with atorvastatin displayed slightlyreduced, but non-significant, plasma glucose levels as comparedwith db/db mice on control diet. Glucose tolerance was determ-ined by OGTT (Figure 1). No differences were observed in thefasting blood glucose levels at baseline between db/db mice re-ceiving atorvastatin or control diet. Overall, db/db mice receivingcontrol diet displayed impaired glucose tolerance as comparedwith db/+ mice. Atorvastatin-treated db/db mice showed a de-crease in blood glucose compared with control-diet-treated db/dbmice at 60, 90 and 120 min after the glucose challenge. The statin

treatment had no effect on fasting blood glucose levels at baselineor glucose tolerance in db/+ mice.

Systolic blood pressure in db/+ and db/db micetreated with control or atorvastatin dietDiabetic db/db mice and db/+ control mice had similar SBPlevels (Supplementary Figure S2). Atorvastatin did not affect theSBP in either db/+ or db/db mice.

Status of oxidative stress in arteries from db/+and db/db miceThe potential antioxidant effect of atorvastatin was evaluatedin db/db and db/+ mice. Figure 2(A) demonstrates that plasmaTBARS levels were significantly higher in db/db mice com-pared with db/+ and this increase was inhibited by atorvastatintreatment. NADPH-dependent superoxide anion generation wasmeasured in aortic homogenates from db/+ and db/db mice. Fig-ure 2(B) shows that lucigenin-derived luminescence was signi-ficantly higher in arteries from db/db mice compared with db/+.

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Figure 2 Atorvastatin reduced vascular oxidative stress in db/db micePlasma, aorta and mesenteric arteries were obtained from db/+ and db/db mice treated with atorvastatin (10 mg/kg perday) or control diet (2 weeks). ROS status was assessed by plasma TBARS levels (A) and lucigenin-enhanced chemilu-minescence (B). Translocation of p47phox (C) and Rac1/2 (D) was assessed by the protein expression in membrane andcytosolic fractions isolated from mesenteric arterial bed homogenates. Results are means +− S.E.M. of 6–8 mice in eachexperimental group. ∗P < 0.05 compared with db/+ control diet, ∗∗P < 0.05 db/db atorvastatin diet compared with db/dbcontrol diet.

Atorvastatin reduced the increase in superoxide anion genera-tion in db/db mice. Translocation of the Nox cytosolic subunitp47phox and the small GTPase Rac1/2 from the cytosol to the cellmembrane was evaluated as an index of the oxidase activation.Expression of p47phox (Figure 2C) and Rac1/2 (Figure 2D) inmembrane-enriched fractions was increased in mesenteric arter-ies from db/db mice. This effect was abrogated by atorvastatin.

Expression of Nox isoforms in arteries from db/+and db/db miceSince enhanced oxidative stress was observed in the vasculatureof db/db mice, we evaluated the protein expression of the Noxisoforms in aortas from of db/+ and db/db mice. Figure 3 demon-strates that the protein expression of Nox1, 2 and 4 was up-regulated in aortas from db/db mice compared with the db/+group. Atorvastatin reduced expression of the catalytic subunitof the oxidase isoforms in the vasculature of diabetic mice.

Effects of atorvastatin treatment on MAPKphosphorylation in arteries from db/+ and db/dbmiceThe effect of atorvastatin on the phosphorylation levels ofMAPKs, the downstream signalling targets of ROS, is demon-strated 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 (Fig-ure 4B) and JNK MAPK (Figure 4C). Atorvastatin inhibited theincrease in MAPK phosphorylation observed in the db/db mice.

Pro-inflammatory responses in arteries from db/+and db/db mice treated with atorvastatinThe redox-sensitive NF-κB family of transcription factors reg-ulates multiple cellular processes including inflammation. In-creased phosphorylation levels of the NF-κB p65 subunit wereobserved 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 miceProtein levels of Nox1 (A), Nox2 (B) and Nox4 (C) were evaluated in mesenteric arteries isolated from db/+ and db/dbmice treated with atorvastatin (10 mg/kg per day) or control diet (2 weeks). Side panels, representative immunoblotsof Nox1, Nox2, Nox4 and β -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-κBsubunit p50 (Figure 5B) with reduced expression of the regulat-ory protein IκB-α compared with db/+ mice (Figure 5C). Theseeffects were normalized by atorvastatin.

One of the initial steps in vascular inflammation is expres-sion of adhesion molecules such as VCAM-1. VCAM-1 con-tent was increased in the vasculature of db/db mice versuscontrols (Figure 6A). Figure 6(B) shows adherent green fluor-escent macrophages in aortic segments. Increased numbers ofadherent macrophages were observed in aortic segments of db/dbmice compared with db/+ mice. Atorvastatin reduced vascularinflammatory responses as evidenced by decreased VCAM-1 ex-pression and macrophage adhesion in db/db mice.

Effect of atorvastatin on vascular function inarteries from db/+ and db/db miceFigure 7 and Tables 2 and 3 show the effects of atorvastatin onvascular function of db/+ and db/db mice. Endothelium-intactmesenteric arteries from db/db mice were more sensitive to NEcompared with those from db/+ mice, as evidenced by the left-ward shift in the concentration–response curve to the agonist.Atorvastatin abolished the increased sensitivity to NE in arteriesfrom db/db mice (Figure 7A). No differences in the response toNE were observed in endothelium-denuded arteries from db/+and db/db mice (Figure 7B). Relaxation in response to ACh wassignificantly reduced in arteries from db/db mice, effects that

were restored by atorvastatin (Figure 7C). Maximum relaxationin response to INS was decreased in mesenteric arteries fromdb/db mice compared with those from db/+ mice. Atorvastatinimproved the endothelium-dependent relaxation in response toINS in arteries from db/db mice (Figure 7D). Atorvastatin didnot affect the endothelium-dependent relaxant responses in arter-ies from db/+ mice.

Vascular morphology and mechanics in arteriesfrom db/+ and db/db miceMesenteric arteries from db/+ and db/db mice presented sim-ilar wall thickness, lumen diameter and cross-sectional area inresponse to stepwise increments of intraluminal pressure (Sup-plementary Figures S3A–S3C). No differences were observed inthe stress–strain relationship curve between arteries from db/+and db/db mice (Supplementary Figure S3D). Atorvastatin didnot affect morphological parameters and mechanical propertiesin all experimental groups.

DISCUSSION

Major findings in the present study demonstrate that INS res-istance, endothelial dysfunction and vascular inflammation indiabetic db/db mice are ameliorated by atorvastatin through

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Figure 4 Increased MAPK phosphorylation is inhibited by atorvastatin in the vasculature of db/db micePhosphorylation levels of p38 MAPK (A), ERK1/2 MAPK (B) and JNK MAPK (C) were evaluated in mesenteric arteriesisolated 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) andβ -actin. Results are presented as means +− S.E.M. of seven mice in each experimental group. ∗P < 0.05 compared withdb/+ control diet, ∗∗P < 0.05 db/db atorvastatin diet compared with db/db control diet.

processes associated with reduced vascular oxidative stress anddecreased pro-inflammatory signalling. These phenomena wereassociated with decreased activation of Rac1/2, down-regulationof Nox isoforms and decreased generation of ROS. Our dataprovide some novel insights involving Rac1/2-regulated Noxswhereby statins, such as atorvastatin, protect against vasculo-pathy in diabetes. Statins as adjuvant therapy in the managementof diabetes may have beneficial metabolic and vascular effectsbeyond lipid lowering.

The major morbidities associated with diabetes are cardiovas-cular disease and nephropathy due in large part to vascular injury[31]. In our study, db/db mice exhibited significant endothelialdysfunction and vascular inflammation as evidenced by reducedACh- and INS-induced endothelium-dependent vasorelaxationand increased monocyte adhesion with augmented VCAM-1 ex-pression. Vascular dysfunction has been described in patientswith diabetes [32,33] and in experimental models of Type 1 dia-betes (OVE26 mice and streptozotocin-induced diabetes) [34,35]and Type 2 diabetes (db/db, fat-fed) [36,37]. Our findings sup-port others that have shown improved endothelium-dependentNO-mediated vasorelaxation by another statin, rosuvastatin, inexperimental diabetes and are in line with clinical studies demon-strating that statins improve flow-mediated vasodilation in pa-

tients with Type 2 diabetes [12,38,39]. Although these vasculareffects have been attributed to mechanisms independent of lipid-lowering effects [12], the metabolic and lipid profiles of db/dbmice in our study were improved by atorvastatin. Accordingly, wecannot exclude the possibility that some vasoprotection occursthrough lipid lowering.

Exact mechanisms whereby statins affect directly on vascu-lar function still remain elusive. However, growing evidence in-dicates that these drugs influence signalling pathways involvedin the generation of ROS and NO [40]. Statins inhibit HMG-CoA reductase [1], which normally catalyses the biosynthesisof mevalonate, the main intermediate fatty acid in cholesterolbiosynthesis. Mevalonate is also the primary precursor of lipidisoprenoid intermediates, such as farnesyl pyrophosphate (FPP)and geranylgeranyl pyrophosphate (GGPP), which are essentialfor the prenylation and activation of small GTPases [15,41], suchas Rac1/2. Hence, statins have the capacity to inhibit Rac1/2 ac-tivation, which could affect Rac-dependent signalling, includingNox-ROS pathways [42]. db/db mice exhibited increased activ-ation of Rac1/2 as evidenced by increased cytosol-to-membranetranslocation, with associated increased p47phox translocation andactivation of Noxs. Increased Rac1/2 activation has also beendemonstrated in streptozotocin-induced diabetic rats [43] and we

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Figure 5 Atorvastatin affects NF-κB proteins phosphorylation and expression in the vasculature of db/db micePhosphorylation of NF-κB p65 (A) and the expression of NF-κB p50 (B) and IκB-α (C) were evaluated in mesentericarteries 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-κB p65 (Ser536), NF-κB p50, IκB-α and β -actin. Results are presented asmeans +− S.E.M. of seven mice in each experimental group. ∗P < 0.05 compared with db/+ control diet, ∗∗P < 0.05 db/dbatorvastatin diet compared with db/db control diet.

Figure 6 Atorvastatin reduces VCAM-1 expression and macrophages adhesion in aortas from db/db miceAortas 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 comparedwith db/+ control diet ∗∗P < 0.05 db/db atorvastatin diet compared with db/db control diet.

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Figure 7 Atorvastatin decreases the sensitivity to NE and improves endothelium dependent relaxation in small mesen-teric arteries from db/db miceResistance mesenteric arteries were isolated from db/+ and db/db mice treated with atorvastatin (10 mg/kg per day) orcontrol diet (2 weeks) and mounted in a wire myograph. Concentration–response curves to NE (0.1 nmol/l to 10 μmol/l)were performed in endothelium-intact (A) or endothelium-denuded (B) arteries. Endothelium-intact arteries were pre-contracted with NE (10−6 mol/l) and concentration–response curves to (C) ACh (1 nmol/l to 10 μmol/l) and (D) INS(0.1–10 000 ng/dl) were performed. Results are mean +− S.E.M. of 5–8 mice in each experimental group. ∗P < 0.05compared 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 generationis amplified in streptozotocin-induced and db/db diabetic mice[26,44]. Of the Nox isoforms, we previously demonstrated animportant role for Nox1 and Nox4 in cardiovascular and renalinjury in diabetic mice [26,42]. Other studies have implied a rolefor Nox2 [45,46]. In the present study, we found increased ex-pression of all three Nox isoforms in db/db vessels and as suchcannot identify which Nox is primarily responsible for the in-creased ROS generation in our model. However, since Nox1 andNox2 have an obligatory need for p47phox, which was also activ-ated in db/db mice, these Noxs may be particularly important,similar to what was previously reported.

In atorvastatin-treated db/db mice, Nox activation wasattenuated as evidenced by reduced NADPH-induced generationof ROS, down-regulation of Nox isoforms, reduced p47phox

translocation and decreased levels of TBARS. These effectsmay relate to Rac1/2 inhibition since atorvastatin blockedtranslocation of Rac from the cytosol to the membrane. Rac1/2normally 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 stepsof 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 modulatingassembly of oxidase subunits. Statins have been shown toregulate Rac activity through various mechanisms includingstimulation of small GTP-binding protein GDP dissociationstimulator (SmgGDS), which negatively regulates Rac1/2, andby inhibiting post-translational modifications [47]. Our findings,in the present paper, expand those observations and provide aputative molecular mechanism whereby statins interfere withRac-regulated Nox-derived ROS in diabetes, which probablyreduces oxidative stress, normalizes endothelial function andprevents vascular inflammation in db/db mice. These processesprobably involve reduced pro-inflammatory signalling throughMAPKs and NF-κB [48–50], pathways that were down-regulated by treatment in diabetic mice. Such effects may relateto blunted Rac1/2 and Nox activation. It is also possible

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Table 2 Vascular reactivity to NE, ACh and INS: maximum effectEffects of atorvastatin treatment (10 mg/kg per day for 2 weeks) on vascular responses [maximum effect (Emax) of secondorder mesenteric arteries)] to NE, ACh and INS in db/+ and db/db mice. Concentration–effect curves were performed inendothelium-intact (+E) and endothelium-denuded (−E) mesenteric rings. Results are means +− S.E.M. of seven mice in eachexperimental group. Emax values for NE are presented as a percentage of KCl (60 mM) responses, whereas ACh and INSare presented in relation of pre-contraction of 0.1 μM NE. ∗P < 0.05 compared with db/+ control diet, ∗∗P < 0.05 db/dbatorvastatin diet compared with db/db control diet.

Group (Emax)

Agonist db/+ db/db db/+ +atorvastatin db/db +atorvastatin

NE (+E) 101.4 +− 13.3 110.0 +− 10.9 119.3 +− 12.53 97.3 +− 14.80

NE (-E) 91.1 +− 10.8 98.7 +− 11.8 91.5 +− 9.0 89.8 +− 16.42

ACh 85.2 +− 5.6 48.0 +− 5.7∗ 87.9 +− 5.8 79.6 +− 11.3∗∗

INS 68.1 +− 5.4 42.2 +− 5.2∗ 77.2 +− 8.2 65.9 +− 7.8∗∗

Table 3 Vascular reactivity to NE, ACh and INS: pD2Effects of atorvastatin treatment (10 mg/kg per day for 2 weeks) on vascular responses [pD2 (negative logarithm of theEC50 of second-order mesenteric arteries)] to NE, ACh and INS in db/+ and db/db mice. Concentration–effect curves wereperformed in endothelium-intact (+E) and endothelium-denuded (−E) mesenteric rings. Results are means +− S.E.M. of sevenmice in each experimental group. ∗P < 0.05 compared with db/+ control diet, ∗∗P < 0.05 db/db atorvastatin diet comparedwith db/db control diet.

Group (pD2)

Agonist db/+ db/db db/+ +atorvastatin db/db +atorvastatin

NE (+E) 7.53 +− 0.3 8.55 +− 0.3∗ 6.78 +− 0.5 7.41 +− 0.1∗∗

NE (−E) 7.25 +− 0.3 7.66 +− 0.3 7.53 +− 0.2 7.69 +− 0.2

ACh 7.36 +− 0.2 8.66 +− 0.9∗ 7.73 +− 0.2 7.23 +− 0.2∗∗

INS 6.72 +− 0.2 7.32 +− 0.5∗ 6.61 +− 0.1 7.17 +− 0.2

that atorvastatin improved vasorelaxation throughNOS/NO-dependent pathways, since statins have been shown tostimulate activity of NOS with increased NO generation [51,52].

Statin treatment had a positive effect on the metabolic pro-file and INS sensitivity in db/db mice, which could also havean effect on improved vascular status in these mice, as previ-ously suggested [53]. In our experiments, atorvastatin decreasedvascular sensitivity to NE and improved endothelium-dependentvasorelaxation in db/db mice. These phenomena were associatedwith reduced vascular oxidative damage and inflammation andmay have been due, at least in part, to changes in INS sensitivityin vascular cells.

In support of our results in db/db mice others have shown inexperimental rat models of diabetes [53,54] and in patients withdiabetes [55] that atorvastatin improves INS sensitivity. How-ever, data from some clinical studies reported that statins actuallyworsen diabetes and that they promote new onset diabetes andINS resistance. Reasons for these conflicting data are unclear, butit should be stressed that studies reporting pro-diabetic actions ofstatins were primarily retrospective analyses of large clinical tri-als that demonstrated associations between statin treatment anddevelopment of diabetes [56–59]. In such associative studies,causality cannot be established and in none of those investiga-tions was a direct negative effect of statins on INS sensitivity ac-tually demonstrated. Further in depth investigations are requiredto determine whether statins directly influence INS metabolismin diabetes.

In conclusion, we elucidate some molecular mechanismswhereby atorvastatin protects against vascular damage in dia-

betes. We also demonstrate that statin treatment improves INSsensitivity, which in its own right could positively influence vas-cular status. Taken together, our data suggest that statins mayhave important vasoprotective effects in diabetes beyond lipidlowering.

CLINICAL PERSPECTIVES

� Statins, cholesterol-lowering drugs, improve vascular functionin diabetes, but exact mechanisms are elusive.

� We demonstrated that, in a mouse model of Type 2 diabetes(db/db), systemic and vascular oxidative stress, endothelialdysfunction and vascular inflammation were normalized byatorvastatin. These processes were associated with down-regulation of Rac1/2, decreased Nox-derived ROS generationand reduced pro-inflammatory signalling.

� Our data identify some molecular mechanisms involvingRac1/2-regulated Noxs whereby statins may protect againstvascular dysfunction in diabetes. Clinically, statins may havevasoprotective therapeutic potential beyond lipid-lowering indiabetes.

AUTHOR CONTRIBUTION

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

420 C© The Authors Journal compilation C© 2015 Biochemical Society

Atorvastatin decreases oxidative stress in diabetic mice

contributions from Augusto Montezano, Tayze Antunes and AurelieNguyen Dinh Cat.

FUNDING

This work was supported by the Sao Paulo Research Foundation(FAPESP) [grant numbers 2010/52214-6 (to R.C.T); 2011/01785-6; 2011/22035-5 (to T.B.-N.)]; the Agence Universitaire de la Fran-cophonie [grant number 58145FT103]; the Juvenile Diabetes Re-search Foundation [grant number 4-2010-528]; the Canadian Insti-tutes of Health Research [grant number 44018]; and the BritishHeart Foundation [grant number RG/13/7/30099].

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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 423