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

Experimental and Toxicologic Pathology 63 (2011) 473–478

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Experimental and Toxicologic Pathology journal homepage: www.elsevier.de/etp

Beneficial effects of rosuvastatin on aortic adverse remodeling in nitric oxide-deficient rats Rodrigo Neto-Ferreira a, Vinı´cius Novaes Rocha a, Thiago da Silva Torres b, Carlos Alberto Mandarim-de-Lacerda b, Jorge Jose de Carvalho a,n a b

Laboratory of Ultrastructure and Tecidual Biology, Biomedical Center, Institute of Biology, State University of Rio de Janeiro, Brazil 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 in fo

abstract

Article history: Received 14 January 2010 Accepted 17 March 2010

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.

Keywords: Hypertension L-NAME Nitric oxide Statin Rats Vascular remodeling

Introduction Excessive adiposity and increased blood pressure, beginning at childhood, together with accelerated adverse longitudinal changes in risk variables of metabolic syndrome throughout

Abbreviations: HMG-CoA reductase, 3-hydroxy-3-methyl-glutaryl-CoA reductase; NO, nitric oxide; C, control; CR, control treated with rosuvastatin; L-NAME, hydrochloride of NG-nitro-metil-ester-L-arginine; LN, L-NAME; LNR, L-NAME treated with rosuvastatin; BP, blood pressure; LDL-C, low-density lipoproteincholesterol; HDL-C, high-density lipoprotein- cholesterol; NADPH, nicotinamide adenine dinucleotide phosphate; eNOS, endothelial nitric oxide synthase; BM, body mass; TM, tunica media; IMA, intima–media a´rea; CWT, circumferential wall tension; MSBP, media systolic blood pressure; ID, internal diameter; TS, tensile stress; IMT, intima–media thickness; I-M, intima–media; AOI, area of interest; VSMC, vascular smooth muscle cells; DOCA-salt, deoxycorticosterone acetate (DOCA)-salt hypertensive rats; mRNA, messenger ribonucleic acid; SHR, spontaneously hypertensive rats; I, intima; HEC, heterogeneous endothelial cells; V, citoplasmatic vesicles; SMC, smooth muscle cells; IEL, internal elastic lamina; END, endothelial cells; ECM, extracellular matrix; SEM, standard error of the mean; ANOVA, one-way analysis of variance n Corresponding author. Tel./fax: + 55 21 2587 6468. E-mail addresses: [email protected] (R. Neto-Ferreira), [email protected] (V. Novaes Rocha), [email protected] (T. da Silva Torres), [email protected] (C.A. Mandarim-de-Lacerda), [email protected] (J.J. de Carvalho). 0940-2993/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2010.03.007

young adulthood, characterize the early natural history of hypertension (Srinivasan et al., 2006). Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, referred to as statins, are some of the most widely prescribed medications in the United States for treating dyslipidemia (Belay et al., 2007). Their benefits are derived both from reducing atherogenic lipoprotein levels (LDL-C) and from increasing antiatherogenic lipoproteins (HDL-C) (Nicholls et al., 2007). Recent clinical and experimental studies have shown that statins have pleiotropic effects, besides modulating lipid, such as anti-inflammatory, anti-proliferative, anti-thrombotic effects, attenuation of NADPH oxidase-mediated superoxide generation and improving endothelial vasomotor function, all of which may affect cardiovascular outcomes in high risk patients including hypertension (Suh et al., 2010). Thus, statin prescription together with antihypertensive agents is a therapeutic strategy for reducing cardiovascular events (Girerd and Giral, 2004). Recent studies have shown that chronic administration of L-arginine analogues such as L-NAME to rats induces a dose dependent systemic arterial hypertension, blocking the endothelial nitric oxide synthase (eNOS) isoform and therefore nitric oxide (NO) biosynthesis leads to endothelial dysfunction and impaired vasodilatation and inflammatory phenotypic changes in the coronary vascular wall (Ribeiro et al., 1992; Rossi and Colombini-Netto, 2001). The major target organ for statin action

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is the vascular endothelium, which plays an important role on both atherosclerosis and angiogenesis development. Cholesterolindependent vascular effects of statins seem to involve either directly restoring or improvement of endothelial function by increasing NO production, promoting re-endothelization after arterial injury, and inhibiting inflammatory responses within the vessel wall that are thought to contribute to atherosclerosis (Ii and Losordo, 2007). Vascular endothelial cells have a crucial role in the pathogenesis of the arterial wall alterations and hypertension. All in all, here we evaluate the effect of rosuvastatin on structural and ultrastructural aortic remodeling in a rat model of arterial hypertension induced by NO synthase blockade. The focus of the study was to evaluate the pleiotropic action of rosuvastatin upon aortic endothelial and muscle layers in hypertensive normocholesterolemic animals.

Materials and methods Animals and treatment Wistar rats were housed in a temperature and humidity controlled 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 for Care and Use of Laboratory Animals’’ published by the US National Institutes of Health (NHI Publication No. 85-23, revised 1996) and was approved by the local committee. Animals started the experiment with four months of age and were 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-NAME group (LN), animals received L-NAME (hydrochloride of NG-nitrometil-ester-L-arginine – Sigma-Aldrich Chemical) 40 mg/kg/day dissolved in the drinking water, and L-NAME treated group (LNR), animals received L-NAME and rosuvastatin, as described for previous groups. Animals were individually housed, and the experiment lasted five weeks. Blood pressure (BP) and body mass (BM) were verified weekly. BP was measured in conscious rats through the non-invasive method 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 anaesthetized (intraperitonial sodium pentobarbital, 15 mg/kg) and exsanguinated. Later, a fixative solution (freshly prepared formaldehyde 1  27 mol/l in 0.1 M phosphate buffer; pH 7  2) was perfused 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 body rigidity. Plasma levels of triglycerides and cholesterol were analyzed by a kinetic-colorimetric method according to the manufacturer’s instructions (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 rapidly isolated from adhered adipose and connective tissues. Some non-consecutive rings were immersed in freshly prepared 4% w/v formaldehyde (0.1 M phosphate buffer pH 7.2 for 48 h) for light microscopy, then sectioned according to vertical section method (Pereira et al., 2004). The aortic rings were processed according to histological 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 identify elastic fibers (Fullmer and Lillie, 1956). The remaining aortic rings were immediately fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) and 0.25% tannic acid for 3 h. Later, fragments were immersed in 1% osmium tetroxide with 0.8% potassium ferricyanide in 0.1 M cacodylate buffer for 1 h, and then dehydrated in acetone and embedded in Epon (Embed-812). Semithin sections (1 mm) were stained with toluidine blue and observed under light microscope to select areas for ultrathin sections (Leica Ultracut-UCT with diamond knife), counterstained with uranyl acetate and lead citrate, and then examined in 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 (TIFF format, 36-bit color) with an LC Evolution camera and an Olympus BX51 light microscope. Tunica media (TM), defined by the region delimited between the internal and external elastic laminae, was measured to determine TM thickness (four measures obtained at 01, 901, 1801, and 2701). Elastic fibers density was estimated on five non-consecutive aortic sections per animal, and expressed as a percentage of total wall area. The total amount of elastic fibers for aortic cross-section was calculated as the product of intima–media area (IMA) and the percentage of the orcinol-stained area, expressed as mm2 (Mendonca et al., 2007) (Image-Pro Plus software, version 5, Media Cybernetics, Silver Spring, USA). Mean circumferential wall tension (CWT) was calculated by Laplace’s law according to the following formula: CWT ¼MSBP  (ID/2), where CWT is expressed as dyne/cm, MSBP is the mean systolic blood pressure (dynes/cm2) and ID is the internal diameter (cm). Tensile stress (TS) was computed as TS¼CWT/IMT, where TS is expressed as dyne/cm2, CWT is the circumferential wall 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 with endothelial nitric oxide synthase antibody (NOS3 (H-159): sc-8311; Santa Cruz), at 4 1C overnight, both at dilutions of 1:100. The sections were rinsed with phosphate-buffered saline. A biotinylated antibody (K0679, Universal DakoCytomation LSAB+ Peroxidase Kit; DakoCytomation, Glostrup, Denmark) was used as a secondary antibody detected by reaction with horseradish peroxidase–streptavidin–biotin complex. Positive immunoreaction was identified after incubation with 3,30 -diaminobenzidine tetrachloride (K3466, DAB; DakoCytomation) and counterstaining with Mayer hematoxylin. Image analysis Digital images from the endothelial layer in tunica intima were obtained and studied by image analysis. A selection tool was used to identify the endothelial layer area with positive immunoreactions, and this selection was segmented in a blackand-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 occupied by white color was quantified using the image histogram tool. It was expressed as density stained per endothelial layer (%)

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(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 of variance and post-hoc test of Tukey. A P-value of 0.05 was considered statistically significant (GraphPad Prism version 5.0, San Diego, USA).

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Triglycerides and cholesterol levels Total cholesterol was not different among groups (Table 1). However, lower triglyceride levels were found in statin-treated groups in comparison to the non-treated one (less than 43% and 33%, respectively, in CR and LNR groups).

Arterial wall remodeling

Results Body mass and blood pressure There was no significant difference among groups for BM after 5 weeks of the experiment: C group 440.0744.0 g, CR group 424.07 41.0 g, LN group 447.0712.0 g, and LNR group, 430.0743.0 g. Animals started the experiment without a difference in BP levels (Fig. 1). Animals from C and CR groups remained normotensives throughout the experiment. At the fifth week, the BP was significantly higher in both LN and LNR groups than in matched control groups (plus 35% and 25%, respectively, in comparison to C and CR groups). Despite the high BP in LNR rats, it was 9% lower than that in the LN group.

L-NAME intake induced media hypertrophy, evidenced by the increased TM thickness and I–M area (plus 35% and 51%, respectively, in comparison to the C group) (Table 2). The amount of elastic fibers was also increased in this group (plus 73% than the C group). Rosuvastatin treatment resulted not only in a thinner TM in the LNR group in comparison to the LN group (less 18%) but also in a smaller I–M area as well (less 23%). Table 2 Parameters of aortic remodeling. Parameters

C

CR

LN

TM thickness (mm) I–M area (mm2) Elastic fibers (%) Elastic fibers (mm2)

81.0 7 0.2 0.37 7 0.01 39.8 7 1.4 0.30 7 0.02

77.2 7 0.8 0.37 7 0.01 36.2 7 0.5 0.34 7 0.02 y

LNR

109.1 7 1.1 0.56 7 0.03n 36.1 7 1.8 0.52 7 0.04n

n

89.0 7 0.7y 0.43 7 0.02y 41.2 7 1.5 0.42 7 0.03

Values are given as mean and 7 SEM. Differences were analyzed using one-way ANOVA and post-hoc test of Tukey: nP o 0.05 when compared with the C group; y Po 0.05 when compared with 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 Po 0.001 if compared with the C group; yP o 0.001 if compared with the CR group and yP o 0.001 if compared with the LN group.

Table 1 Plasma levels of cholesterol, triglycerides. Parameters

C

CR

LN

LNR

Cholesterol (mg/dl) Triglycerides (mg/dl)

96.6 7 1.56 22.4 7 0.43

88.8 7 2.65 12.8 7 0.41n

105.0 7 1.36 27.0 7 0.57

101.1 7 1.03 18.2 7 0.91nz

Values are given as mean and 7 SEM. One-way ANOVA and post-hoc test of Tukey: n Po 0.05 if compared with the C group; yP o0.05 if compared with the LN group; z Po 0.05 if compared with CR. Total cholesterol was not different among groups.

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.

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

higher in LNR animals in comparison to LN (plus 52%) and still higher 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 was greater than that in matched control groups (plus 40%, Po0.001 in the LN group than the C group, and plus 31%, Po0.01 in the LNR than the CR group) (Fig.4). Rosuvastatin administration to the LNR group slightly reduced CWT (less than 13%) in comparison to the LN group. Tensile stress was greater in LN and LNR groups in comparison to the C group. Ultrastructural morphology Ultrastructural alterations on the aortic wall were remarkable in NO-deficient rats (LN group). This group had large cytoplasmic vesicles inside endothelial cells, and irregular nuclear and cytoplasmatic boundaries as well (Fig. 5). The tunica media was thick and internal elastic lamina was splited with abundant extracellular matrix content. Smooth muscle cells had cytoplasmatic projections, which go toward the internal elastic lamina at the largest underlying subendothelial space (Fig. 6). Finally, ultrastructural characteristics of the LNR (Fig. 7) group were similar to C and CR groups.

Discussion

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: nPo 0.0001 if compared with the C group, yPo 0.0001 if compared with the CR group, and y Po 0.0001 if compared with the LNR group.

The dose of 20 mg/kg/day of rosuvastatin used in the present study is known to produce beneficial effects upon endothelial dysfunction and systemic and regional hemodynamic and has been used previously (Susic et al., 2003). Likewise, the dose of 40 mg/kg/day of L-NAME is efficient to induce hypertension and increase the aortic wall thickness (Bernatova et al., 1999; Pereira et al., 1998). Present results showed, as expected, media thickening in NO-deficient rats, agreeing with the previous works (Babal

Fig. 4. Circumferential wall tension (CWT) and tensile stress (TS); 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 7 SEM. One-way ANOVA and post-hoc test of Tukey: nP o0.05 if compared with the C group and yPo 0.05 if compared with the LNR group.

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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 bars ¼ 2 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 bar ¼2 mm).

et al., 1997; Bernatova et al., 1999; Pereira et al., 2004). It is likely a result of increased tensile forces acting on the aortic wall (CWT and TS) pursuant to arterial hypertension and thus increased wall stretching (Prado and Rossi, 2006). However, aortic wall did not alter significantly when LN rats were treated with rosuvastatin, suggesting that there is a BP-independent factor protecting the aortic wall from stretching-induced remodeling. There is evidence that in animal models and in humans, impaired endothelial function and a decrease in NO bioavailability may occur in hypertension, diabetes and hypercholesterolemia, despite normal or increased NO production by the endothelium. In a number of animal models of disease, including hypertension and hypercholesterolemia, an increase in superoxide occurs concurrent to the decrease in NO bioavailability (Davignon and Ganz, 2004).

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 bar ¼ 2 mm).

Chronic NO synthesis blockade induces a time-dependent BP elevation that leads to significant cardiac and arterial wall damage (Pereira et al., 2004; Rossi and Colombini-Netto, 2001). In NO-deficient rats treated with rosuvastatin, there were slightly attenuated BP rise and ameliorated adverse structural and ultrastructural remodeling of the aortic wall. The interesting fact is that this beneficial effect was achieved independent of BP levels, since LNR rats were still hypertensive after rosuvastatin treatment. The rosuvastatin attenuates cardiovascular remodeling, especially aortic medial thickening, in DOCA-salt hypertensive rats without lowering of blood pressure Statins have been reported to reduce blood pressure in a randomized, doubleblind crossover trial in humans (pravastatin) and hypertensive rodent models (simvastatin and lovastatin), including DOCA-salt hypertensive mice (lovastatin) (Loch et al., 2006). Reduced eNOS activity could explain the impaired endothelium-dependent

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vasodilatation response associated with the aging process (Cernadas et al., 1998). It is known that vascular endothelial cells have a role in the pathogenesis of the arterial wall alterations and represent a key cellular target for statin therapy (Greenwood and Mason, 2007). Statins control the growth and apoptosis of VSMC during vascular remodeling, when the inhibition of proliferation, hypertrophy and migration of these cells occur and facilitate apoptosis. The action of rosuvastatin is known to change the phenotype of VSMC and subsequently its proliferation and migration in the artery (Kiyan et al., 2007), which could explain, at least in part, the present results. The endothelial NO synthase is regulated by both the HMGCoA reductase pathway and endotelin. Statins improve endothelial NO synthase, an endogenous vasodilator and inhibitor of vascular smooth muscle growth (Zhou et al., 2004), and antiapoptotic agent (Maejima et al., 2005). Furthermore, statins inhibit the mRNA expression of pre-pro-endotelin-1 and decrease endotelin-1 synthesis, resulting in vasoconstriction and inhibition of the VSMC proliferation (Alvarez de Sotomayor et al., 1999). The present findings showed lower expression of eNOS in the LN group. The administration of rosuvastatin (in the LNR group) had beneficial effects by increasing the eNOS expression and reducing the BP. Statins restore NO availability by increasing phosphorylated extracellular signal-regulated kinase 1/2, pAkt, peNOS, and inducible NO synthase levels (Virdis et al., 2009) and, although the eNOS expression does not differ between the rosuvastatin-treated and control groups, the level of phosphorylated eNOS is much higher in the rosuvastatin group (Suh et al., 2010). We have also observed that rosuvastatin administration to NO-deficient animals diminished serum triglyceride but did not alter total cholesterol, corroborating with the previous results in spontaneously hypertensive rats treated with either simvastatin or pravastatin (Bezerra and Mandarim-de-Lacerda, 2005). It is interesting to remark that, besides their lipidlowering effect, statins also possess broad immunomodulatory and anti-inflammatory properties (Greenwood and Mason, 2007). The elucidation of the regulatory effects of endothelial cell growth on eNOS and NAD(P)H oxidase expression and activities may have significant implications in the pathogenesis of atherosclerosis. The proliferation and migration of VSMC contribute to the formation of atheroma. Activation of NAD(P)H oxidase enzyme and, therefore, generation of excess quantities of superoxide in the VSMC increase cellular metabolism, and thus an increased amount of free radicals may reduce the beneficial effects of NO and accelerate disease formation (Bayraktutan, 2004). The antioxidant activity and the impairment of vascular atherosclerosis development are both the mechanisms contributing to the beneficial effects of statins (Sicard et al., 2008).

Conclusions In conclusion, rosuvastatin intake by NO-deficient rats diminishes ultrastructural adverse aortic wall remodeling, despite small changes in blood pressure. Therefore, this medication might be useful for protecting arterial structure, thus preserving arterial function, in patients with concomitant hypertension and hypercholesterolemia.

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

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