RESEARCH PAPERbph_1416 598..606

Rosuvastatin improvesendothelial function indb/db mice: role ofangiotensin II type 1receptors and oxidativestressXY Tian1, WT Wong1, A Xu4, ZY Chen2, Y Lu1, LM Liu1, VW Lee3,CW Lau1, X Yao1 and Y Huang1

1Institute of Vascular Medicine, Li Ka Shing Institute of Health Sciences, School of Biomedical

Sciences, Hong Kong, China, 2Department of Biochemistry, 3School of Pharmacy, Chinese

University of Hong Kong, Hong Kong, China, and 4Departments of Medicine and Pharmacology

& Pharmacy, University of Hong Kong, Hong Kong, China

CorrespondenceYu Huang, School of BiomedicalSciences, Chinese University ofHong Kong, Shatin, NT, HongKong, China. E-mail:yu-huang@cuhk.edu.hk----------------------------------------------------------------

Keywordsstatin; diabetes; endothelialfunction; oxidative stress;vasodilatation----------------------------------------------------------------

Received19 August 2010Revised19 February 2011Accepted29 March 2011

BACKGROUND AND PURPOSEHMG-CoA reductase inhibitors, statins, with lipid-reducing properties combat against atherosclerosis and diabetes. Thefavourable modulation of endothelial function may play a significant role in this effect. The present study aimed to investigatethe cellular mechanisms responsible for the therapeutic benefits of rosuvastatin in ameliorating diabetes-associated endothelialdysfunction.

EXPERIMENTAL APPROACHTwelve-week-old db/db diabetic mice were treated with rosuvastatin at 20 mg·kg-1·day-1 p.o.for 6 weeks. Isometric force wasmeasured in isolated aortae and renal arteries. Protein expressions including angiotensin II type 1 receptor (AT1R), NOX4,p22phox, p67phox, Rac-1, nitrotyrosine, phospho-ERK1/2 and phospho-p38 were determined by Western blotting, while reactiveoxygen species (ROS) accumulation in the vascular wall was evaluated by dihydroethidium fluorescence and lucigenin assay.

KEY RESULTSRosuvastatin treatment of db/db mice reversed the impaired ACh-induced endothelium-dependent dilatations in both renalarteries and aortae and prevented the exaggerated contractions to angiotensin II and phenylephrine in db/db mouse renalarteries and aortae. Rosuvastatin reduced the elevated expressions of AT1R, p22phox and p67phox, NOX4, Rac1, nitrotyrosine andphosphorylation of ERK1/2 and p38 MAPK and inhibited ROS production in aortae from db/db mice.

CONCLUSIONS AND IMPLICATIONSThe vasoprotective effects of rosuvastatin are attributed to an increase in NO bioavailability, which is probably achieved by itsinhibition of ROS production from the AT1R-NAD(P)H oxidase cascade.

AbbreviationsAng II, angiotensin II; AT1R, angiotensin II type 1 receptor; DHE, dihydroethidium; eNOS, endothelial NOS; HMG-CoAreductase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase; L-NAME, NG-nitro-L-arginine methyl ester; RAAS,renin–angiotensin–aldosterone system; ROS, reactive oxygen species; SNP, sodium nitroprusside

BJP British Journal ofPharmacology

DOI:10.1111/j.1476-5381.2011.01416.xwww.brjpharmacol.org

598 British Journal of Pharmacology (2011) 164 598–606 © 2011 The AuthorsBritish Journal of Pharmacology © 2011 The British Pharmacological Society

IntroductionEndothelial dysfunction, a hallmark in diabetes, initiates vas-cular pathogenesis leading to diabetic vasculopathy (DeVriese et al., 2000), and it is closely associated with reducedbioavailability of endothelium-derived NO, which can resultfrom the reduced NO-generating capacity or exaggeratedreactive oxygen species (ROS) formation (Wong et al., 2010a).Endothelial dysfunction usually occurs early in diabetes, andtherapeutic strategies targeting endothelial dysfunctionmight 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 bythe up-regulation of local vascular renin–angiotensin–aldosterone system (RAAS) and related over-production ofROS (Wong et al., 2010b). It is well documented that angio-tensin II (Ang II)-induced activation of NAD(P)H oxidase, amulti-subunit enzyme complex, is an important source forthe production of ROS in the vasculature (Gao et al., 2009).Hyperglycaemia impairs NO production and enhances ROSaccumulation in blood vessels (Wang et al., 2009), and ROSare implicated in the initiation and progression of diabeticvascular complications usually associated with endothelialdysfunction (Cohen et al., 2007; Thomas et al., 2008). Giventhe importance of oxidative stress in hyperglycaemia-relatedvascular events and lack of overall benefit of antioxidants inthe treatment of cardiovascular complications (Jialal et al.,2003), specific therapies targeting ROS-generating enzymesand their upstream regulators may become more effective inreversing endothelial dysfunction and thus reducing therelated 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 (Nangleet 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 (AT1R) expressionin hypercholesterolaemic individuals (Nickenig et al., 1999).Although statins are found to improve endothelial functionin diabetic animals (Nangle et al., 2003; Schafer et al., 2007)and in patients with diabetes (Brunetti et al., 2007; Tomizawaet al., 2009), it is still unclear how this vascular benefit isachieved. With the use of type 2 diabetic db/db mouse model,we hypothesized that rosuvastatin treatment amelioratesendothelial dysfunction and increases NO bioavailability,which is associated with the inhibition of AT1R and NAD(P)Hoxidases.

Methods

Animal and drug treatmentType 2 diabetic mice (C57BL/KSJ background) lacking thegene encoding for leptin receptor (db/db) and heterozygote(db/m+) were provided by the Chinese University of HongKong (CUHK) Laboratory Animal Service Center. All animalcare and experimental procedures were approved by theCUHK Animal Experimentation Ethics Committee. Mice were

kept in a temperature-controlled holding room (22–24°C)with a 12 h light/dark cycle and fed with a standard diet andwater ad libitum. At the age of 12 weeks, adult male db/dbmice were treated for 6 weeks with rosuvastatin at20 mg·kg-1(body weight)·day-1 or vehicle by oral gavage. Therosuvastatin dosage and treatment duration were comparablewith 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 glucosesolution (1.2 g·kg-1; body weight) by oral gavage. Blood wasdrawn from the mouse tail, and blood glucose was measuredat times 0, 15, 30, 60 and 120 min with a commercial glu-cometer (Ascensia ELITE®, Bayer, Mishawaka, IN).

Plasma lipid profile and insulin levelPlasma levels of total cholesterol, triglyceride, high-densitylipoprotein (HDL) and non-HDL were determined usingenzymatic methods (Stanbio, Boerne, TX). Plasma insulinconcentration was determined by enzyme immunoassay(Mercodia, Sweden).

Functional studies in myographAfter mice were killed by CO2 inhalation, the thoracic aortaeand renal arteries were rapidly dissected out and placed inoxygenated ice-cold Krebs–Henseleit solution (mmol·L-1: 119NaCl, 4.7 KCl, 2.5 CaCl2, 1 MgCl2, 25 NaHCO3, 1.2 KH2PO4

and 11 D-glucose). Changes in isometric tension of arterieswere measured in a multi-myograph system (Danish MyoTechnology, Aarhus, Denmark) as described previously(Wong et al., 2010b) and changes in isometric tension wererecorded. The rings were stretched to an optimal baselinetension of 3 mN for aortae and 1 mN for renal arteries.

Experimental protocolsEach ring was equilibrated for 1 h before the experimentstarted. The ring was first contracted by 60 mmol·L-1 KCl andrinsed in Krebs solution for several times before phenyleph-rine (1 mmol·L-1) was used to evoke a stable contractionand then relaxed by accumulative concentrations of ACh(10 nmol·L-1 to 10 mmol·L-1). Endothelium-independentrelaxations to sodium nitroprusside (SNP) (1 nmol·L-1 to10 mmol·L-1) were recorded in endothelium-denuded rings.

To illustrate the ability of the increased NO bioavailabilityto inhibit vasoconstriction, phenylephrine was used totrigger concentration-dependent contractions for compari-son in aortae and renal arteries from db/m+, db/db androsuvastatin-treated db/db mice. To confirm the role ofbasal NO production in the reduced vasoconstriction, thesame experiments were performed in some arterial ringsafter 30 min exposure to NG-nitro-L-arginine (L-NAME,100 mmol·L-1).

To determine the contribution of the reduced expressionand function of AT1R in rosuvastatin-treated db/db mice, con-traction to Ang II (100 nmol·L-1) was compared in arteriesfrom the three treatment groups of mice in the presence of100 mmol·L-1 L-NAME.

Western blottingProtein samples (20 mg each lane) prepared from aortahomogenates were electrophoresed through a 10%

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SDS-poly-acrylamide gel and transferred onto animmobilon-P polyvinylidene difluoride membrane (MilliporeCorp., Bedford, MA). Nonspecific binding sites were blockedwith 5% non-fat milk or 1% BSA in Tris-buffered saline con-taining 0.05% Tween-20. The blots were incubated overnightat 4°C with the primary antibodies: monoclonal anti-AT1R(1:1000; Abcam, Cambridge, UK); monoclonal anti-nitrotyrosine (1:1000; Abcam); anti-p22phox and anti-p67phox

(1:1000; Santa Cruz, CA); polyclonal anti-NOX4 and anti-Rac1 (Abcam); monoclonal anti-phosphor-p38 MAPK (Thr180/Tyr182), polyclonal anti-p38 MAPK, monoclonal anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), monoclonalanti-p44/42 MAPK, polyclonal anti-phospho-PKC (bII Ser660),anti-phospho-PKCa/bII (Thr638/641), anti-PKCa, anti-phospho-MYPT1 (Thr853) (Cell Signaling, Beverly, MA); polyclonalanti-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., SanLeandro, CA).

Measurement of intracellularoxidant formationThe 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 androsuvastatin-treated db/db mice were dissected out and frozenin OCT compound. Frozen sections of the aortic ring were cutat 10 mm thickness using a cryostat microtome (LeicaCM1100, Leica Instruments, Germany) and incubated for10 min at 37°C in Krebs solution containing 5 mmol·L-1 DHE.Fluorescence intensity was measured by confocal microscope(FV1000, Olympus, Tokyo, Japan) at excitation/emission of488/605 nm to visualize the signal. The images were analysedby the Fluoview software (Olympus, Tokyo, Japan).

In addition, the amount of superoxide production wasalso measured by lucigenin chemiluminescence (Wassmannet al., 2001; Ejiri et al., 2003). Mouse aortae were cut into3 mm segments and incubated with 10 mmol·L-1 lucigenin inKrebs solution bubbled with 95% O2 and 5% CO2 for 30 minat 37°C. Samples were transferred into vials containing 1 mLKrebs solution with 10 mmol·L-1 lucigenin, and chemilunines-cence was repeatedly measured using a Wallace Victor lumi-nometer (PerkinElmer, Boston, MA) over 10 min, at 1 mininterval. The count per minute was determined by theaverage of all readings and normalized to dry weight of theaortic segment.

ChemicalsRosuvastatin was provided by AstraZeneca (Cheshire UK).ACh, L-NAME, phenylephrine, angiotensin II, SNP andlucigenin were purchased from Sigma-Aldrich Chemical (StLouis, MO). All the drugs used were dissolved in doubledistilled water.

Data analysisResults are means � SEM from different mice. Concentration–relaxation curves were analysed by non-linear regression

curve fitting using GraphPad Prism software (version 4.0, SanDiego, CA) to estimate Emax as the maximal response and pD2

as the negative logarithm of the drug concentration thatproduced 50% of Emax. Contractions were presented as activetension [force (mN) recorded / (2 ¥ length (mm) of ring)].Statistical significance was determined by Student’s two-tailedt-test or one-way ANOVA followed by the Bonferroni post hoctest when more than two treatments were compared. P < 0.05indicates statistically significant difference.

Results

Statin improves the endothelium-dependentrelaxation (EDR) in renal arteries and aortaeof db/db miceEDRs in response to accumulative concentration of ACh inphenylephrine-precontracted segments of renal arteries weresignificantly 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/dbmice improved EDRs in renal arteries (Figure 1A and B). Theincreased initial active tension induced by phenylephrine inarteries from db/db mice was reduced by rosuvastatin treat-ment (Supporting Figure S1). EDRs to SNP were similar inrenal arteries from all groups (Figure 1C). In aortae, reducedEDRs in db/db mice were also improved after rosuvastatintreatment (Figure 1D and E). Again, SNP-induced relaxationswere similar in aortic rings from all groups (Figure 1F).

Statin reduces phenylephrine-inducedcontraction through an increase inNO bioavailabilityPhenylephrine-induced contractions in renal arteries(Figure 2A) and aortae (Figure 2B, Supporting Figure S1) ofdb/db mice were significantly larger than those from db/m+

mice. The enhanced contraction was reduced by chronicrosuvastatin treatment (Figure 2A and B). After pre-incubation with L-NAME, contractions to phenylephrinewere similar in renal arteries (Figure 2C) and aortae(Figure 2D) in the three groups.

Statin suppresses Ang II-induced contractionin arteries of db/db miceAng II (100 nmol·L-1)-induced contractions were markedlyaugmented in db/db mouse renal arteries (Figure 3A) andaortae (Figure 3B) compared to those from db/m+ mice, butwere reduced in arteries from rosuvastatin-treated db/dbmice. In contrast, concentration-dependent contractions toincreasing concentrations of KCl were comparable in renalarteries and aortae from the three groups (Figure 3C and D).

Statin reduces the up-regulated expressions ofAT1R, NAD(P)H oxidase subunits andoxidative stress markers in aortae ofdb/db miceThe protein expressions of angiotensin type 1 receptor(AT1R), NAD(P)H oxidase subunits p22phox and p67phox

increased in aortae from db/db mice than db/m+ mice;

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rosuvastatin treatment normalized the increased expressionof AT1R, p22phox and p67phox, NOX4 and Rac1 (Figure 4A–C,E–F) but not NOX2 (Supporting Figure S2). In addition, rosu-vastatin treatment in db/db mice also reduced the increasedlevels of nitrotyrosine (Figure 4D). The intracellular oxidantformation, measured by DHE fluorescence, and superoxideproduction, measured by lucigenin assay, were higher in thesections of aortic rings from db/db mice than in those fromdb/m+ mice; this increase was inhibited by rosuvastatin treat-ment (Figure 5).

Statin reduces the pro-inflammatorysignalling pathways in aortae of db/db miceWestern blotting showed increased phosphorylations ofERK1/2 (Figure 6A) and p38 MAPK (Figure 6B) in db/db mouseaortae, which were prevented by rosuvastatin treatment(Figure 6A and B).

Basic metabolic parameters are not modifiedby statin treatmentRosuvastatin treatment did not change body weights of db/dbmice, which were significantly higher than age-matcheddb/m+ control littermates (Table 1). The levels of fasting bloodglucose and plasma insulin were higher in db/db mice thanthose from db/m+ mice (Table 1). Rosuvastatin treatment didnot alter these parameters nor affect the glucose tolerance indb/db mice (Supporting Figure S3, Table 1). In addition, theelevated levels of total cholesterol, triglyceride, HDL andnon-HDL in db/db mice were not modified by rosuvastatintreatment (Table 1).

Discussions and conclusions

The present study demonstrates that a 6-week treatment withrosuvastatin, a HMG-CoA reductase inhibitor, restores theimpaired EDRs in renal arteries and aortae in diabetic db/dbmouse model without affecting the plasma lipids or glucosetolerance. This effect is associated with an increase in NObioavailability. We showed for the first time that statin treat-

Figure 1Statin improves EDRs in renal arteries and aortae of db/dbmice. Representative traces showing the impaired ACh-inducedendothelium-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 treatmentimproved EDRs in renal arteries (B) and aortae (E) of db/db mice.Comparable endothelium-independent relaxations to SNP wereshown in renal arteries (C) and aortae (F) from the three groups ofmice. 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/dbmice.

Figure 2Statin reduces phenylephrine-induced vasoconstriction in arteries ofdb/db mice through an increase in NO bioavailability. The aug-mented contractions to phenylephrine (Phe) in renal arteries (A) andaortae (B) in db/db mice were prevented by chronic treatment withrosuvastatin. This inhibitory effect was absent in renal arteries (C) andaortae (D) treated with 100 mmol·L-1 L-NAME. Results are means �

SEM of six mice. *P < 0.05 between db/m+ and db/db; #P < 0.05between db/db and rosuvastatin-treated db/db mice.

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ment reduces oxidative stress in arteries of db/db mice, whichis associated with the inhibition of the RAAS cascade involv-ing AT1R and NAD(P)H oxidases. Furthermore, the statintreatment diminished the nitrotyrosine formation and pro-inflammatory signalling molecules including ERK1/2 and p38MAPK. Taken together, the present novel findings reveal thatrosuvastatin inhibits AT1R-dependent oxidative stress andrestores NO bioavailability in arteries of db/db mouse beyondits effect on plasma lipid levels.

Over the past few years, statins have been increasinglyidentified to possess vasoprotective effects in diabetes, whichare attributed to actions independent of their effects onlipids. Clinical studies have demonstrated that statinsimprove flow-mediated vasodilatation in patients with type 2diabetes (Brunetti et al., 2007; Tomizawa et al., 2009) andhyperlipidaemia (ter Avest et al., 2005). In animal studies,consistent results were also reported showing the vasoprotec-tive effects of statins. Nangle et al. (2003) first demonstratedthat rosuvastatin treatment improved endothelium-dependent NO-mediated relaxations in aortae ofstreptozotocin-induced diabetic mice. Recently, rosuvastatinhas been shown to normalize endothelial function of aortaein streptozotocin-induced diabetic rats (Schafer et al., 2007;Tarhzaoui et al., 2008). Treatment with simvastatin preservedendothelial function in large and small coronary vessels ofhigh cholesterol-fed pigs (Wilson et al., 2001). However,limited data are available regarding the effects of statins onvascular function in a type 2 diabetic db/db mouse model. Thepresent study thus shows for the first time that rosuvastatin,administered for 6 weeks, improved vascular function in

db/db mouse model without modifying the lipid profiles,which included the triglycerides and cholesterols levels. Thisunique property might be useful for the characterization ofthe pleiotropic effects of statins in the alleviation of diabeticvasculopathy.

It is estimated that around 70% of patients with diabeteswill die of vascular events (Wing et al., 2001). One of themajor causes of cardiovascular mortality in this population isdue 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 tocombat 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 3Statin suppresses Ang II-induced vasoconstriction in arteries of db/dbmice. Elevated contractions to Ang II (100 nmol·L-1) in renal arteries(A) and aortae (B) in db/db mice were inhibited by chronic treatmentwith rosuvastatin, while contractions to raised extracellular KCl wereidentical in renal arteries (C) and aortae (D) from the three groups ofmice. Contractions were presented as active tension [force (mN)recorded / (2 ¥ length (mm) of ring)]. Results are means � SEM of sixmice. *P < 0.05 between db/m+ and db/db; #P < 0.05 between db/dband rosuvastatin-treated db/db mice.

Figure 4Statin reduces the up-regulated expressions of AT1R, NAD(P)Hoxidase subunits and nitrotyrosine in aortae of db/db mice. Theelevated protein expression of AT1R (A), p22phox (B), p67phox (C),nitrotyrosine (Ntyr, D), NOX4 (E), Rac1 (F) in aortae of db/db micewas reduced after chronic treatment with rosuvastatin. Results aremeans � SEM of four to five mice. *P < 0.05 between db/m+ anddb/db; #P < 0.05 between db/db and rosuvastatin-treated db/dbmice.

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1998; Haffner et al., 1999; Keech et al., 2003). In the presentstudy, we assessed the vascular function using a myograph toexamine whether treatment with rosuvastatin could reversevascular dysfunction in aortae and renal arteries of db/dbmice. 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 indiabetes is less well defined. The present study demonstratedthat ACh-induced EDRs are reduced in renal arteries andaortae of db/db mice compared with the non-diabetic db/m+

mice. Treatment with rosuvastatin for 6 weeks restored theimpaired EDRs in db/db mice. On the other hand, the EDRs toSNP were similar in renal arteries and aortae from db/m+,db/db and rosuvastatin-treated db/db mice, suggesting that thesensitivity of vascular smooth muscle to the relaxing effect ofNO is not modulated under hyperglycaemic conditions or byrosuvastatin. Previous reports demonstrated that pitavastatinameliorated diabetic nephropathy in db/db mouse model(Fujii et al., 2007), and rosuvastatin exerts renoprotectiveeffects in transgenic TG(mRen2)27 rats (Whaley-Connellet al., 2008). However, no study has attempted to elucidatewhether statins could protect the renovascular function in

Table 1Basic 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 (mmol·L-1) 3.3 � 0.3 16.2 � 1.2* 16.1 � 1.8*

Insulin (ng·mL-1) 1.5 � 0.3 22.5 � 1.9* 22.9 � 3.9*

Total cholesterol (mg·mL-1) 0.78 � 0.02 1.31 � 0.10* 1.24 � 0.14*

Triglyceride (mg·mL-1) 0.95 � 0.04 2.58 � 0.25* 2.56 � 0.40*

HDL (mg·mL-1) 0.46 � 0.02 0.65 � 0.03* 0.59 � 0.04*

non-HDL (mg·mL-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 5Statin suppresses the increased ROS production in aortic walls ofdb/db mice. (A) Representative images showing ROS accumulation inthe vascular wall of db/db mouse aortae from the three groups.Chronic treatment with rosuvastatin reduced ROS generation indi-cated by DHE fluorescence (B) and lucigenin assay (C) in db/db mice.Results are means � SEM of four to six mice. *P < 0.05 betweendb/m+ and db/db; #P < 0.05 between db/db and rosuvastatin-treateddb/db mice.

Figure 6Statin reduces the pro-inflammatory signalling pathways in aortae ofdb/db mice. Increased levels of phosphorylation of ERK1/2 (A) andp38MAPK (B) in db/db mouse aortae were normalized by the chronictreatment with rosuvastatin without affecting the total proteinexpressions of ERK1/2 and p38MAPK. Results are means � SEM offour to six mice. *P < 0.05 between db/m+ and db/db; #P < 0.05between db/db and rosuvastatin-treated db/db mice.

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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 wereprevented by rosuvastatin treatment. One possible explana-tion for this effect is that it is due to an increase in NObioavailability after statin treatment, as the effects of statinon EDRs and phenylephrine-induced vasoconstrictions wereabolished in the presence of L-NAME. Since the endothelialNOS (eNOS) levels in aortae were comparable in the threegroups of mice (Supporting Figure S4), the increase in NObioavailability might be attributed to the reduction in ROSproduction by rosuvastatin treatment. The present findingsthus provide novel evidence to support the renovascular pro-tective effects of statins in diabetic nephropathy.

The mechanisms underlying the pleiotropic effects ofstatins on vascular function remain largely unknown. Directvasorelaxation effects of statins including rosuvastatin havebeen reported in rat aortic rings (Lopez et al., 2008), althoughthe concentrations needed to induce relaxation were farabove therapeutically achievable levels in humans. The pleio-tropic effects of statins on vascular and renal function havebeen 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-dependentsuperoxide anions (Li et al., 2005; Erdos et al., 2006; Fujiiet al., 2007; Whaley-Connell et al., 2008) or the reduction ofpro-inflammatory genes including VCAM-1, MCP-1 andMMP-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 humanumbilical vein endothelial cells induced by high glucose byreducing NAD(P)H oxidase-derived oxidative stress (Piconiet al., 2008). We have previously demonstrated a critical rolefor AT1R-mediated ROS in the impairments of EDRs in renalarteries from diabetic patients and aortae from db/db mice.The impaired vasodilator responses could be reversed by theAT1R blocker, NAD(P)H oxidase inhibitors and ROS scaven-gers (Wong et al., 2010b). It is postulated that elevated ROSlevels derived from AT1R-mediated NAD(P)H oxidases lowerthe bioavailability of NO by either directly scavenging NO orby 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 haverecently shown that both L-NAME and a NAD(P)H oxidaseinhibitor inhibit the ROS levels in these arteries, indicatingthat ROS derived from NADPH oxidase is probably requiredfor 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 first time that rosuvastatinrestores vascular function in db/db mice, which was associ-ated with favourable modulation of the RAAS cascade basedon the following observations. First, the augmented contrac-tion to Ang II in renal arteries and aortae of db/db mice werenormalized by rosuvastatin treatment without affectingthe KCl-induced vasoconstrictions. An augmented AngII-induced contraction indicates an increased expression oractivity of AT1R. Indeed, the up-regulation of AT1R in aortaefrom db/db mice was normalized by rosuvastatin treatment.Second, rosuvastatin treatment reduced the expressions of

NAD(P)H oxidase subunits p22phox (membranous subunit),p67phox (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 oxidantformation. In addition, rosuvastatin treatment also reducedthe Ang II level in the db/db mouse aorta as shown by immu-nostaining (Supporting Figure S5). Third, MAPKs includingp44/42 and p38 can be activated by Ang II and have beenimplicated as downstream signalling molecules in response tomany pro-inflammatory factors (Xu et al., 2005). The presentresults also reveal that the activations of p38 and ERK1/2MAPK in aortae of db/db mice were normalized by rosuvasta-tin treatment. This helps to explain the anti-atheroscleroticeffects of statins in the vasculature. Although statin wasreported to stimulate MAPK/ERK (Lee et al., 2004; Merla et al.,2007; Cho et al., 2011), the MAPK/ERK activation is mediatedvia an adenosine-dependentmechanism, while the inductionof MAPK/ERK and downstream haemoxygenase-1 up-regulation by statin occurs mainly in vascular smooth musclecells. Taken together, we provide novel evidence that rosuv-astatin restores vascular function in db/db mice, which isrelated 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 suggesta significant role for RhoA/Rho-kinase in the inhibitory effectof rosuvastatin that results in improved endothelial functionbased on the following observations. In aortas from treateddb/db mice, rosuvastatin does not affect (a) contraction toU46619, which is known to stimulate RhoA/Rho-kinase and(b) the phosphorylation of MYPT1 and PKC, the downstreamtargets regulated by RhoA/Rho-kinase (Supporting Figure S5).We have yet to determine whether a longer treatment withthis statin or treatment at higher dosages could inhibit RhoA/Rho-kinase activity in vascular smooth muscle cells if theplasma 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, despitenot changing plasma lipid concentrations. The vasoprotec-tive effects of rosuvastatin are attributed to an increase in NObioavailability, which is at least in part achieved by its inhi-bition of ROS production from the AT1R-NAD(P)H oxidasecascade. Other contributing mechanisms require furtherinvestigation.

Acknowledgements

This study was supported by Research Grants (4653/08 M,466110) and Collaborative Research Fund (HKU2/07C andHKU4/CRF10) from the Research Council of Hong Kong,CUHK Focused Investment Scheme and CUHK Li Ka ShingInstitute of Health Sciences.

Conflicts of interest

The authors declare that there is no duality of interest asso-ciated with this manuscript.

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Supporting information

Additional Supporting Information may be found in theonline version of this article:

Figure S1 Contractions to phenylephrine (Phe, 1 mM) beforecumulative concentration of acetylcholine were calculatedand normalized to active tension. Phe-induced contractionsfrom db/db mice were larger than those from db/m+, whichwere reduced by rosuvastatin (RSV) treatment, in both aortaand renal arteries. Data are means � SEM. P < 0.05 versusdb/m+ (n = 8). P < 0.05 versus db/db (n = 8). db/db+ RSV (n = 6).Figure S2 Chronic rosuvastatin (RSV) treatment did notaffect the elevated expression of NOX2 in the aortas fromtreated db/db mice. Data are means � SEM. *P < 0.05 versusdb/m+ (n = 5). P < 0.05 versus db/db. db/db+ RSV (n = 6).Figure S3 (a) The lack of effect of chronic treatment withrosuvastatin (RSV) on oral glucose tolerance test (OGTT) indb/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 comparablebetween the three groups of mice. Data are means � SEM offour mice.Figure S5 Immunohistochemical staining of angiotensin IIin the wall of mouse aortas. Chronic rosuvastatin (RSV) treat-ment reduced the elevated angiotensin II level in db/dbmouse aortas as compared with those from db/m+ mice. Dataare means � SEM of four to five mice. *P < 0.05 versus db/m+;#P < 0.05 versus db/db.Figure S6 Chronic rosuvastatin (RSV) treatment did notmodify (a) contractions induced by U46619, thromboxaneA2 analogue in db/db mouse aortas, and RSV did not affect thelevel of phosphorylation MYPY1 (Thr853) (b), PKCa/b(Thr638/641) and PKC (Ser660) (c and d) in db/db mouseaortas. Data are means � SEM of five to six mice. The U46619-induced contraction was abolished by 1 mM S18886, the TPreceptor antagonist (data not shown).

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BJP XY Tian et al.

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