Experimental and Toxicologic Pathology 63 (2011) 473–478

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Experimental and Toxicologic Pathology

0940-29

doi:10.1

Abbre

tase; NO

hydroch

treated

choleste

adenine

BM, bod

wall ten

TS, tens

interest

acetate

spontan

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END, en

mean; An Corr

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journal homepage: www.elsevier.de/etp

Beneficial effects of rosuvastatin on aortic adverse remodeling innitric oxide-deficient rats

Rodrigo Neto-Ferreira a, Vinıcius Novaes Rocha a, Thiago da Silva Torres b,Carlos Alberto Mandarim-de-Lacerda b, Jorge Jose de Carvalho a,n

a Laboratory of Ultrastructure and Tecidual Biology, Biomedical Center, Institute of Biology, State University of Rio de Janeiro, Brazilb Laboratory of Morphometry and Cardiovascular Morphology, Biomedical Center, Institute of Biology, State University of Rio de Janeiro, Brazil

a r t i c l e i n f o

Article history:

Received 14 January 2010

Accepted 17 March 2010

Keywords:

Hypertension

L-NAME

Nitric oxide

Statin

Rats

Vascular remodeling

93/$ - see front matter & 2010 Elsevier Gmb

016/j.etp.2010.03.007

viations: HMG-CoA reductase, 3-hydroxy-3-

, nitric oxide; C, control; CR, control treated

loride of NG-nitro-metil-ester-L-arginine; LN

with rosuvastatin; BP, blood pressure; LDL-C

rol; HDL-C, high-density lipoprotein- cholest

dinucleotide phosphate; eNOS, endothelial n

y mass; TM, tunica media; IMA, intima–medi

sion; MSBP, media systolic blood pressure; I

ile stress; IMT, intima–media thickness; I-M,

; VSMC, vascular smooth muscle cells; DOCA

(DOCA)-salt hypertensive rats; mRNA, messe

eously hypertensive rats; I, intima; HEC, hete

lasmatic vesicles; SMC, smooth muscle cells;

dothelial cells; ECM, extracellular matrix; SE

NOVA, one-way analysis of variance

esponding author. Tel./fax:+55 21 2587 6468

ail addresses: rnvetferreira@hotmail.com (R. N

@hotmail.com (V. Novaes Rocha), thiago_tor

ilva Torres), mandarim@uerj.br (C.A. Mandar

@uerj.br (J.J. de Carvalho).

a b s t r a c t

This study aimed to evaluate the effect of rosuvastatin upon structural and ultrastructural aortic

remodeling in a rat model of hypertension induced by NO synthase blockade. Wistar rats were divided

into 4 groups: Control group (C); control treated with rosuvastatin 20 mg/kg/day (CR); L-NAME group

40 mg/kg/day (LN) and L-NAME treated with rosuvastatin (LNR) (same doses). Body mass and blood

pressure were measured weekly; the experiment lasted 5 weeks. L-NAME administration augmented

blood pressure (BP) in the LN group in comparison to the C group (123.3 vs. 180.5 mmHg at week 5). In

LNR rats, rosuvastatin slightly attenuated BP rise, but it had no effect on the BP of CR group. Intima and

media thickening of the thoracic aorta were observed in the LN group, and increased elastic fiber

content as well. Rosuvastatin prevented all these alterations as seen in the LNR group. Ultrastructural

changes due to L-NAME intake (intracellular vesicles and altered membrane morphology in endothelial

cells, extracellular matrix deposition, and cytoplasmatic projections from smooth muscle cells toward

the internal elastic lamina) were also prevented by rosuvastatin. All in all, rosuvastatin administration

is capable of attenuating ultrastructural aortic wall remodeling in NO-deficient rats despite small

changes in blood pressure.

& 2010 Elsevier GmbH. All rights reserved.

Introduction

Excessive adiposity and increased blood pressure, beginning atchildhood, together with accelerated adverse longitudinalchanges in risk variables of metabolic syndrome throughout

H. All rights reserved.

methyl-glutaryl-CoA reduc-

with rosuvastatin; L-NAME,

, L-NAME; LNR, L-NAME

, low-density lipoprotein-

erol; NADPH, nicotinamide

itric oxide synthase;

a area; CWT, circumferential

D, internal diameter;

intima–media; AOI, area of

-salt, deoxycorticosterone

nger ribonucleic acid; SHR,

rogeneous endothelial cells;

IEL, internal elastic lamina;

M, standard error of the

.

eto-Ferreira),

res@hotmail.com

im-de-Lacerda),

young adulthood, characterize the early natural history ofhypertension (Srinivasan et al., 2006).

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reduc-tase, referred to as statins, are some of the most widely prescribedmedications in the United States for treating dyslipidemia (Belayet al., 2007). Their benefits are derived both from reducingatherogenic lipoprotein levels (LDL-C) and from increasingantiatherogenic lipoproteins (HDL-C) (Nicholls et al., 2007).Recent clinical and experimental studies have shown that statinshave pleiotropic effects, besides modulating lipid, such asanti-inflammatory, anti-proliferative, anti-thrombotic effects,attenuation of NADPH oxidase-mediated superoxide generationand improving endothelial vasomotor function, all of which mayaffect cardiovascular outcomes in high risk patients includinghypertension (Suh et al., 2010). Thus, statin prescription togetherwith antihypertensive agents is a therapeutic strategy forreducing cardiovascular events (Girerd and Giral, 2004).

Recent studies have shown that chronic administration ofL-arginine analogues such as L-NAME to rats induces a dosedependent systemic arterial hypertension, blocking the endothe-lial nitric oxide synthase (eNOS) isoform and therefore nitricoxide (NO) biosynthesis leads to endothelial dysfunction andimpaired vasodilatation and inflammatory phenotypic changesin the coronary vascular wall (Ribeiro et al., 1992; Rossi andColombini-Netto, 2001). The major target organ for statin action

R. Neto-Ferreira et al. / Experimental and Toxicologic Pathology 63 (2011) 473–478474

is the vascular endothelium, which plays an important role onboth atherosclerosis and angiogenesis development. Cholesterol-independent vascular effects of statins seem to involve eitherdirectly restoring or improvement of endothelial function byincreasing NO production, promoting re-endothelization afterarterial injury, and inhibiting inflammatory responses within thevessel wall that are thought to contribute to atherosclerosis(Ii and Losordo, 2007).

Vascular endothelial cells have a crucial role in the pathogen-esis of the arterial wall alterations and hypertension. All in all,here we evaluate the effect of rosuvastatin on structural andultrastructural aortic remodeling in a rat model of arterialhypertension induced by NO synthase blockade. The focus of thestudy was to evaluate the pleiotropic action of rosuvastatin uponaortic endothelial and muscle layers in hypertensive normocho-lesterolemic animals.

Materials and methods

Animals and treatment

Wistar rats were housed in a temperature and humiditycontrolled facility, being exposed to a 12 h light/dark cycle.Animals received water and standard chow (Nuvilab, Parana,Brazil) ad libitum. The investigation agrees with the ‘‘Guide forCare and Use of Laboratory Animals’’ published by the US NationalInstitutes of Health (NHI Publication No. 85-23, revised 1996) andwas approved by the local committee.

Animals started the experiment with four months of age andwere divided into four groups, 6 animals each: control group (C);control treated group (CR), animals received rosuvastatin (Crestor,AstraZeneca, Brazil) 20 mg/kg/day by orogastric gavage; L-NAMEgroup (LN), animals received L-NAME (hydrochloride of NG-nitro-metil-ester-L-arginine – Sigma-Aldrich Chemical) 40 mg/kg/daydissolved in the drinking water, and L-NAME treated group (LNR),animals received L-NAME and rosuvastatin, as described forprevious groups. Animals were individually housed, and theexperiment lasted five weeks.

Blood pressure (BP) and body mass (BM) were verified weekly.BP was measured in conscious rats through the non-invasivemethod of tail-cuff plethysmography (Letica LE 5100, Panlab,Spain).

Euthanasia and technical preparation of the material

At the end of the experiment, animals were deeply anaes-thetized (intraperitonial sodium pentobarbital, 15 mg/kg) andexsanguinated. Later, a fixative solution (freshly prepared for-maldehyde 1 �27 mol/l in 0.1 M phosphate buffer; pH 7 �2) wasperfused along the vascular system with a constant pressure(90 mmHg) through a catheter placed into the left ventricle(Miniplus 3: Gilson S.A.S., Villiers le Bel, France) until bodyrigidity.

Plasma levels of triglycerides and cholesterol were analyzed bya kinetic-colorimetric method according to the manufacturer’sinstructions (Bioclin System II, Quibasa, Belo Horizonte,MG, Brazil). The thoracic aorta was excised and arterial rings(5 mm long, close to the first intercostal artery) were rapidlyisolated from adhered adipose and connective tissues. Somenon-consecutive rings were immersed in freshly prepared 4% w/vformaldehyde (0.1 M phosphate buffer pH 7.2 for 48 h) for lightmicroscopy, then sectioned according to vertical section method(Pereira et al., 2004). The aortic rings were processed according tohistological routine procedures, embedded in Paraplast plus

(Sigma-Aldrich, St. Louis, USA), and then sectioned at 5 mm thick.Sections were stained by orcinol-neo fuchsin in order to identifyelastic fibers (Fullmer and Lillie, 1956). The remaining aortic ringswere immediately fixed in 2.5% glutaraldehyde in 0.1 M cacody-late buffer (pH 7.2) and 0.25% tannic acid for 3 h. Later, fragmentswere immersed in 1% osmium tetroxide with 0.8% potassiumferricyanide in 0.1 M cacodylate buffer for 1 h, and thendehydrated in acetone and embedded in Epon (Embed-812).Semithin sections (1 mm) were stained with toluidine blueand observed under light microscope to select areas for ultrathinsections (Leica Ultracut-UCT with diamond knife), counter-stained with uranyl acetate and lead citrate, and then examinedin a Zeiss EM 906 transmission electron microscope (Carls Zeiss,Oberkochen, Germany) at 80 KV.

Morphometry and data analysis

Five non-consecutive aortic digital images were acquired (TIFFformat, 36-bit color) with an LC Evolution camera and anOlympus BX51 light microscope. Tunica media (TM), defined bythe region delimited between the internal and external elasticlaminae, was measured to determine TM thickness (four mea-sures obtained at 01, 901, 1801, and 2701). Elastic fibers densitywas estimated on five non-consecutive aortic sections per animal,and expressed as a percentage of total wall area. The total amountof elastic fibers for aortic cross-section was calculated as theproduct of intima–media area (IMA) and the percentage of theorcinol-stained area, expressed as mm2 (Mendonca et al., 2007)(Image-Pro Plus software, version 5, Media Cybernetics, SilverSpring, USA).

Mean circumferential wall tension (CWT) was calculated byLaplace’s law according to the following formula: CWT¼MSBP�(ID/2), where CWT is expressed as dyne/cm, MSBP is the meansystolic blood pressure (dynes/cm2) and ID is the internaldiameter (cm). Tensile stress (TS) was computed as TS¼CWT/IMT,where TS is expressed as dyne/cm2, CWT is the circumferentialwall tension (dyne/cm) and IMT is intima–media thickness (cm)(Carallo et al., 1999).

Immunohistochemistry

Sections (0.5 mm) of the aortic rings were incubated withendothelial nitric oxide synthase antibody (NOS3 (H-159):sc-8311; Santa Cruz), at 4 1C overnight, both at dilutions of1:100. The sections were rinsed with phosphate-buffered saline.A biotinylated antibody (K0679, Universal DakoCytomation LSAB+Peroxidase Kit; DakoCytomation, Glostrup, Denmark) was used asa secondary antibody detected by reaction with horseradishperoxidase–streptavidin–biotin complex. Positive immunoreac-tion was identified after incubation with 3,30-diaminobenzidinetetrachloride (K3466, DAB; DakoCytomation) and counterstainingwith Mayer hematoxylin.

Image analysis

Digital images from the endothelial layer in tunica intima wereobtained and studied by image analysis. A selection tool wasused to identify the endothelial layer area with positiveimmunoreactions, and this selection was segmented in a black-and-white image, where white shows the immunostained area.The endothelial layer was delimited using an irregular AOI tool,and inside this delimited area, the endothelial area occupiedby white color was quantified using the image histogram tool.It was expressed as density stained per endothelial layer (%)

R. Neto-Ferreira et al. / Experimental and Toxicologic Pathology 63 (2011) 473–478 475

(Image-Pro Plus version 7.0, Media Cybernetics, Silver Spring, MD)(Mandarim-de-Lacerda et al., 2010).

Data are shown as mean and standard error of the mean.Differences among groups were analyzed by one-way analysis ofvariance and post-hoc test of Tukey. A P-value of 0.05 was consideredstatistically significant (GraphPad Prism version 5.0, San Diego, USA).

Table 2Parameters of aortic remodeling.

Parameters C CR LN LNR

TM thickness (mm) 81.070.2 77.270.8y 109.171.1n 89.070.7y

Results

Body mass and blood pressure

There was no significant difference among groups for BM after5 weeks of the experiment: C group 440.0744.0 g, CR group 424.0741.0 g, LN group 447.0712.0 g, and LNR group, 430.0743.0 g.

Animals started the experiment without a difference in BPlevels (Fig. 1). Animals from C and CR groups remained normo-tensives throughout the experiment. At the fifth week, the BPwas significantly higher in both LN and LNR groups thanin matched control groups (plus 35% and 25%, respectively, incomparison to C and CR groups). Despite the high BP in LNR rats, itwas 9% lower than that in the LN group.

Fig. 1. Blood pressure at the beginning (baseline week shown in the white bar)

and at the end (final week shown in the black bar) of the experiment.

C – normotensive untreated; CR – normotensive treated with rosuvastatin;

LN – hypertensive untreated and LNR – hypertensive treated with rosuvastatin

groups. Values are given as mean and 7SEM. One-way ANOVA and post-hoc test

of Tukey: n Po0.001 if compared with the C group; yPo0.001 if compared with

the CR group and yPo0.001 if compared with the LN group.

Table 1Plasma levels of cholesterol, triglycerides.

Parameters C CR LN LNR

Cholesterol (mg/dl) 96.671.56 88.872.65 105.071.36 101.171.03

Triglycerides (mg/dl) 22.470.43 12.870.41n 27.070.57 18.270.91nz

Values are given as mean and 7SEM. One-way ANOVA and post-hoc test of Tukey:nPo0.05 if compared with the C group; yPo0.05 if compared with the LN group;zPo0.05 if compared with CR. Total cholesterol was not different among groups.

Triglycerides and cholesterol levels

Total cholesterol was not different among groups (Table 1).However, lower triglyceride levels were found in statin-treatedgroups in comparison to the non-treated one (less than 43% and33%, respectively, in CR and LNR groups).

Arterial wall remodeling

L-NAME intake induced media hypertrophy, evidenced bythe increased TM thickness and I–M area (plus 35% and 51%,respectively, in comparison to the C group) (Table 2). The amountof elastic fibers was also increased in this group (plus 73% thanthe C group). Rosuvastatin treatment resulted not only in athinner TM in the LNR group in comparison to the LN group (less18%) but also in a smaller I–M area as well (less 23%).

Fig. 2. The eNOS was positively expressed in the aortic wall (endothelial layer in

tunica intima). (a) C – normotensive untreated; (b) CR – normotensive treated

with rosuvastatin; (c) LN–hypertensive untreated; (d) LNR – hypertensive treated

with rosuvastatin. The intensity of expression was greater in the LNR group than in

the LN one.

I–M area (mm2) 0.3770.01 0.3770.01 0.5670.03n 0.4370.02y

Elastic fibers (%) 39.871.4 36.270.5 36.171.8 41.271.5

Elastic fibers (mm2) 0.3070.02 0.3470.02 0.5270.04n 0.4270.03

Values are given as mean and 7SEM. Differences were analyzed using one-way

ANOVA and post-hoc test of Tukey: nPo0.05 when compared with the C group;yPo0.05 when compared with the LN group.

R. Neto-Ferreira et al. / Experimental and Toxicologic Pathology 63 (2011) 473–478476

eNOS immunostaining

In the aortic wall, the immunostaining of eNOS (Fig. 2) waspositive. The eNOS was positive in the endothelial layer of allgroups. LN animals showed a reduction of eNOS expression incomparison to C animals (less 27%), but this expression was

Fig. 3. eNOS expression in endothelial layer in tunica intima. C – normotensive

untreated; CR – normotensive treated with rosuvastatin; LN – hypertensive

untreated and LNR – hypertensive treated with rosuvastatin. Same symbol over

the bars indicates significant difference between the groups. Values are given as

mean and 7SEM. One-way ANOVA and post-hoc test of Tukey: nPo0.0001 if

compared with the C group, yPo0.0001 if compared with the CR group, andyPo0.0001 if compared with the LNR group.

Fig. 4. Circumferential wall tension (CWT) and tensile stress (TS); C-normotensive untr

and LNR – hypertensive treated with rosuvastatin. Same symbol over the bars indicate

One-way ANOVA and post-hoc test of Tukey: nPo0.05 if compared with the C group a

higher in LNR animals in comparison to LN (plus 52%) and stillhigher compared to C (plus 34%) and CR (plus 29%) groups (Fig. 3).

Circumferential wall tension and tensile stress

Circumferential wall tension in NO-deficient animals wasgreater than that in matched control groups (plus 40%, Po0.001in the LN group than the C group, and plus 31%, Po0.01 in theLNR than the CR group) (Fig.4). Rosuvastatin administration tothe LNR group slightly reduced CWT (less than 13%) incomparison to the LN group. Tensile stress was greater in LNand LNR groups in comparison to the C group.

Ultrastructural morphology

Ultrastructural alterations on the aortic wall were remarkablein NO-deficient rats (LN group). This group had large cytoplasmicvesicles inside endothelial cells, and irregular nuclear andcytoplasmatic boundaries as well (Fig. 5). The tunica media wasthick and internal elastic lamina was splited with abundantextracellular matrix content. Smooth muscle cells hadcytoplasmatic projections, which go toward the internal elasticlamina at the largest underlying subendothelial space (Fig. 6).Finally, ultrastructural characteristics of the LNR (Fig. 7) groupwere similar to C and CR groups.

Discussion

The dose of 20 mg/kg/day of rosuvastatin used in the presentstudy is known to produce beneficial effects upon endothelialdysfunction and systemic and regional hemodynamic and hasbeen used previously (Susic et al., 2003). Likewise, the dose of40 mg/kg/day of L-NAME is efficient to induce hypertension andincrease the aortic wall thickness (Bernatova et al., 1999; Pereiraet al., 1998).

Present results showed, as expected, media thickening inNO-deficient rats, agreeing with the previous works (Babal

eated; CR – normotensive treated with rosuvastatin; LN – hypertensive untreated

s significant difference between the groups. Values are given as mean and 7SEM.

nd yPo0.05 if compared with the LNR group.

Fig. 5. Transmission electron microscopy. Aorta from the LN untreated group. The intima (I) is thickened; the heterogeneous endothelial cells (HEC) normally have large

citoplasmatic vesicles (V) and irregular nuclear and citoplasmatic boundaries. It was observed the accumulation of elastic tissue (arrow) between the endothelial cells and

internal elastic lamina (panel a). It was perceived evagination of smooth muscle cell (SMC) directed by endothelial cells, through the fenestrae of the internal elastic lamina

(IEL) (panel b). (scale bars¼2 mm).

Fig. 6. Transmission electron microscopy. Aorta from the control group (C). It was

seen beneath the endothelial cells (END) the internal elastic lamina (IEL) that

delimitated the intima and smooth muscle cell of media tunica (SMC) (scale

bar¼2 mm).

Fig. 7. Illustrates the endothelial cells of the LNR group (hypertensive treated with

rosuvastatin) under transmission electron microscopy, most of them with a large

number of small micropinocytosis vesicles (n), irregular nuclear and citoplasmatic

boundaries and well preserved internal elastic lamina (IEL). ECM (extracellular

matrix) accumulated in the subendothelial space (arrow) (scale bar¼2 mm).

R. Neto-Ferreira et al. / Experimental and Toxicologic Pathology 63 (2011) 473–478 477

et al., 1997; Bernatova et al., 1999; Pereira et al., 2004). It is likelya result of increased tensile forces acting on the aorticwall (CWT and TS) pursuant to arterial hypertension andthus increased wall stretching (Prado and Rossi, 2006). However,aortic wall did not alter significantly when LN rats were treatedwith rosuvastatin, suggesting that there is a BP-independentfactor protecting the aortic wall from stretching-inducedremodeling.

There is evidence that in animal models and in humans,impaired endothelial function and a decrease in NO bioavailabilitymay occur in hypertension, diabetes and hypercholesterolemia,despite normal or increased NO production by the endothelium.In a number of animal models of disease, including hypertensionand hypercholesterolemia, an increase in superoxide occursconcurrent to the decrease in NO bioavailability (Davignon andGanz, 2004).

Chronic NO synthesis blockade induces a time-dependentBP elevation that leads to significant cardiac and arterialwall damage (Pereira et al., 2004; Rossi and Colombini-Netto,2001). In NO-deficient rats treated with rosuvastatin, there wereslightly attenuated BP rise and ameliorated adverse structuraland ultrastructural remodeling of the aortic wall. The interestingfact is that this beneficial effect was achieved independent ofBP levels, since LNR rats were still hypertensive after rosuva-statin treatment. The rosuvastatin attenuates cardiovascularremodeling, especially aortic medial thickening, in DOCA-salthypertensive rats without lowering of blood pressure Statins havebeen reported to reduce blood pressure in a randomized, double-blind crossover trial in humans (pravastatin) and hypertensiverodent models (simvastatin and lovastatin), including DOCA-salthypertensive mice (lovastatin) (Loch et al., 2006). Reduced eNOSactivity could explain the impaired endothelium-dependent

R. Neto-Ferreira et al. / Experimental and Toxicologic Pathology 63 (2011) 473–478478

vasodilatation response associated with the aging process(Cernadas et al., 1998).

It is known that vascular endothelial cells have a role in thepathogenesis of the arterial wall alterations and represent a keycellular target for statin therapy (Greenwood and Mason, 2007).Statins control the growth and apoptosis of VSMC during vascularremodeling, when the inhibition of proliferation, hypertrophy andmigration of these cells occur and facilitate apoptosis. The actionof rosuvastatin is known to change the phenotype of VSMC andsubsequently its proliferation and migration in the artery (Kiyanet al., 2007), which could explain, at least in part, the presentresults.

The endothelial NO synthase is regulated by both the HMG-CoA reductase pathway and endotelin. Statins improve endothe-lial NO synthase, an endogenous vasodilator and inhibitor ofvascular smooth muscle growth (Zhou et al., 2004), and anti-apoptotic agent (Maejima et al., 2005). Furthermore, statinsinhibit the mRNA expression of pre-pro-endotelin-1 and decreaseendotelin-1 synthesis, resulting in vasoconstriction and inhibitionof the VSMC proliferation (Alvarez de Sotomayor et al., 1999).

The present findings showed lower expression of eNOS in the LNgroup. The administration of rosuvastatin (in the LNR group) hadbeneficial effects by increasing the eNOS expression and reducingthe BP. Statins restore NO availability by increasing phosphorylatedextracellular signal-regulated kinase 1/2, pAkt, peNOS, and inducibleNO synthase levels (Virdis et al., 2009) and, although the eNOSexpression does not differ between the rosuvastatin-treated andcontrol groups, the level of phosphorylated eNOS is much higher inthe rosuvastatin group (Suh et al., 2010).

We have also observed that rosuvastatin administration toNO-deficient animals diminished serum triglyceride but didnot alter total cholesterol, corroborating with the previousresults in spontaneously hypertensive rats treated with either

simvastatin or pravastatin (Bezerra and Mandarim-de-Lacerda,2005). It is interesting to remark that, besides their lipid-lowering effect, statins also possess broad immunomodu-latory and anti-inflammatory properties (Greenwood and Mason,2007).

The elucidation of the regulatory effects of endothelial cellgrowth on eNOS and NAD(P)H oxidase expression and activitiesmay have significant implications in the pathogenesis of athero-sclerosis. The proliferation and migration of VSMC contribute tothe formation of atheroma. Activation of NAD(P)H oxidaseenzyme and, therefore, generation of excess quantities of super-oxide in the VSMC increase cellular metabolism, and thus anincreased amount of free radicals may reduce the beneficialeffects of NO and accelerate disease formation (Bayraktutan,2004). The antioxidant activity and the impairment of vascularatherosclerosis development are both the mechanisms contribut-ing to the beneficial effects of statins (Sicard et al., 2008).

Conclusions

In conclusion, rosuvastatin intake by NO-deficient ratsdiminishes ultrastructural adverse aortic wall remodeling, despitesmall changes in blood pressure. Therefore, this medicationmight be useful for protecting arterial structure, thus preservingarterial function, in patients with concomitant hypertension andhypercholesterolemia.

Competing interests

The author(s) declare that they have no competing interests.

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