Auto-inhibitory regulation of angiotensin II functionality in hamster aorta during the early phases of dyslipidemia

European Journal of Pharmacology 781 (2016) 1–9

Contents lists available at ScienceDirect

European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular pharmacology

Auto-inhibitory regulation of angiotensin II functionality in hamster aorta during the early phases of dyslipidemia Priscila Cristina Pereira a,n, Larissa Pernomian b, Hariane Côco a, Mayara Santos Gomes b, João José Franco c, Kátia Colombo Marchi a, Ulisses Vilela Hipólito d, Sergio Akira Uyemura c, Carlos Renato Tirapelli d, Ana Maria de Oliveira b a

Laboratory of Pharmacology, Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil Laboratory of Vascular Injury, Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil c Department of Clinical, Toxicological and Bromatological Analysis, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil d Laboratory of Pharmacology, School of Nursing of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo, Brazil b

art ic l e i nf o

a b s t r a c t

Article history: Received 24 November 2015 Received in revised form 31 March 2016 Accepted 7 April 2016 Available online 8 April 2016

Emerging data point the crosstalk between dyslipidemia and renin-angiotensin system (RAS). Advanced dyslipidemia is described to induce RAS activation in the vasculature. However, the interplay between early dyslipidemia and the RAS remains unexplored. Knowing that hamsters and humans have a similar lipid profile, we investigated the effects of early and advanced dyslipidemia on angiotensin II-induced contraction. Cumulative concentration-response curves for angiotensin II (1.0pmol/l to 1.0mmol/l) were obtained in the hamster thoracic aorta. We also investigated the modulatory action of NAD(P)H oxidase on angiotensin II-induced contraction using ML171 (Nox-1 inhibitor, 0.5mmol/l) and VAS2870 (Nox-4 inhibitor, 5mmol/l). Early dyslipidemia was detected in hamsters treated with a cholesterol-rich diet for 15 days. Early dyslipidemia decreased the contraction induced by angiotensin II and the concentration of Nox-4-derived hydrogen peroxide. Advanced dyslipidemia, observed in hamsters treated with cholesterol-rich diet for 30 days, restored the contractile response induced by angiotensin II by compensatory mechanism that involves Nox-4-mediated oxidative stress. The hyporresponsiveness to angiotensin II may be an auto-inhibitory regulation of the angiotensinergic function during early dyslipidemia in an attempt to reduce the effects of the upregulation of the vascular RAS during the advanced stages of atherogenesis. The recovery of vascular angiotensin II functionality during the advanced phases of dyslipidemia is the result of the upregulation of redox-pro-inflammatory pathway that might be most likely involved in atherogenesis progression rather than in the recovery of vascular function. Taken together, our findings show the early phase of dyslipidemia may be the most favorable moment for effective atheroprotective therapeutic interventions. & 2016 Elsevier B.V. All rights reserved.

Keywords: Dyslipidemia Angiotensin AT1 receptors NAD(P)H oxidase Hydrogen peroxide Golden Syrian hamsters

1. Introduction Recent findings suggest the existence of a crosstalk between dyslipidemia and the renin-angiotensin system (RAS) during atherogenesis. Activation of vascular angiotensin type 1 (AT1) receptors increases the generation of NAD(P)H oxidase-derived reactive oxygen species (ROS) during hypercholesterolemia (Warnholtz et al., 1999). Superoxide anion (O2
) is the primary ROS generated by NAD(P)H oxidase and the reaction of nitric oxide n Correspondence to: Laboratory of Vascular Injury, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Avenue of Café s/n, 14040-903 Ribeirão Preto, SP, Brazil. E-mail address: [email protected] (P.C. Pereira).

http://dx.doi.org/10.1016/j.ejphar.2016.04.008 0014-2999/& 2016 Elsevier B.V. All rights reserved.

(NO) with O2
may reduce NO bioavailability (Beckman and Koppenol, 1996; Pernomian et al., 2014a). In this sense, AT1 receptors antagonists and angiotensin-converting enzyme (ACE) inhibitors are described to reduce vascular oxidative stress, recover endothelial function and reduce the progression of the atherogenic plate (Schiffrin et al., 2000). ROS generation and RAS activation are two important events associated with hypercholesterolemia since both increase oxidative stress in different cells (Griendling et al., 1994). In this line, Azumi et al. (2002) reported that activation of NAD(P)H oxidase plays a role on ROS generation and the oxidation of low-density lipoprotein (LDL) in human coronary atheroma. This finding suggests that ROS evoke the oxidation of LDL and their uptake by macrophages, further triggering the formation of foam cells

2

P.C. Pereira et al. / European Journal of Pharmacology 781 (2016) 1–9

(Channon, 2002). Moreover, ROS act as intracellular signaling molecules during inflammation, inducing the expression of endothelial adhesion molecules (Kunsch and Medford, 1999), which play an important role in the initiation and progression of atherosclerosis (Pernomian and da Silva, 2015). Animal models of cardiovascular diseases contribute to the understanding of the pathophysiology of atherosclerosis and are essential tools to evaluate new therapeutic approaches to predict and prevent cardiovascular complications induced by atherosclerosis. However, the animal species and the diet used to induce cholesterolemia are important factors that influence the changes in lipid profile and the development of the atherogenic plate (Getz and Reardon, 2006; Dillard et al., 2010). These variations on the animal model jeopardize the comprehension of the mechanisms underlying cholesterolemia and atherosclerosis, especially when the experimental model is not similar to the atherogenic process in humans. Golden Syrian hamster (Mesocricetus auratus) has been used to study atherosclerosis due to several characteristics that make it an excellent animal model for this purpose (Pien et al., 2002; Xiangdong et al., 2011). As observed in humans, hamsters exhibit increased cholesteryl-ester transfer protein activity when fed with a cholesterol-rich diet (Stein et al., 1990; Tsutsumi et al., 2001). Hamsters also exhibit low rate of endogenous cholesterol synthesis (Dietschy et al., 1993), receptor-mediated uptake of LDL, secretion of apolipoprotein B-100 from liver and apolipoprotein B-48 from small intestine (Liu et al., 1991), and uptake of LDL mediated by the LDL receptor pathway (Nistor et al., 1987). Moreover, the morphological characteristics of aortic foam cells and lesions from hamsters fed with atherogenic diets are similar to those found in human lesions (Kahlon et al., 1996). Finally, unlike rats and mice, hamsters develop atherosclerotic lesions in the vascular wall in response to a hypercholesterolemic diet (Martinello et al., 2006). Although current evidence has shown the existence of a crosstalk between ROS and RAS activation in the atherogenic process, there are no reports describing this relation in early phases of dyslipidemia. Thus, our aim was to investigate the correlation of ROS generation and RAS activation in the initial stage of dyslipidemia in hamsters. Such approach will certainly contribute to the understanding of the mechanisms underlying human atherosclerosis.

2. Material and methods 2.1. Animals Experimental protocols were approved by the animal ethics committee from the University of São Paulo (#141/2011). Male Golden Syrian hamsters (Mesocricetus auratus) (75 days old) were provided by the vivarium of the School of Pharmaceutical Sciences of Ribeirão Preto / University of São Paulo. The animals were housed in colony cages (5-6 animals per cage) at controlled room temperature (22 °C) and a 12 h/12 h light/dark cycle. The cholesterolemic diet was prepared by adding cholesterol (1%) to the rodent chow (Nuvilabs) (Librandi et al., 2007). Animals from the control group were fed with a standard diet (i.e., the cholesterol-free Nuvilab rodent chow). Animals from the cholesterolemic group were fed with a cholesterol-rich diet (i.e., the cholesterol 1%-enriched Nuvilab rodent chow) for 5, 10, 15, 20, 25 or 30. Hamsters treated with the cholesterolemic diet for 5, 10, 15, 20, 25 and 30 days received the cholesterol-rich chow at 70, 65, 60, 55, 50 and 45 days old, respectively. Thus, hamsters from both control and cholesterolemic diet-fed groups were 75 days old at the end of the experimental stage.

2.2. Biochemical analysis Serum levels of total cholesterol, high-density lipoprotein (HDL)-cholesterol and LDL-cholesterol were evaluated for the determination of cholesterolemia (Pernomian et al., 2015). Serum lipid profile was evaluated using commercially available kits (Labtests Diagnostica, Montes Claros, MG, Brazil). 2.3. Vascular reactivity studies 2.3.1. Vessel ring preparation Hamsters were anesthetized with isoflurane and killed by decapitation. The thoracic aorta was removed and cut into rings (4 mm in length). The vascular endothelium was preserved or mechanically removed by gently rolling the lumen of the vessel on a thin wire. Aortic rings were placed in 5.0 ml organ chambers containing Krebs solution (composition in nmol/l: NaCl 119.0; KCl 4.7; CaCl2 2.5; KH2PO4 1.5; MgCl2 1.0; NaHCO3 25.0; glucose 11.1; pH 7.4), gassed with 95% O2 and 5% CO2 and maintained at 37 °C. After 60 min of stabilization at a resting tension of 2 g (which was previously determined by resting tension curves), the rings were stimulated with phenylephrine (0.3 mmol/l). Endothelial integrity was assessed qualitatively by the degree of relaxation induced by acetylcholine (0.3 mmol/l) as described previously (Pernomian et al., 2014b). 2.3.2. Experimental protocols Cumulative concentration-response curves for angiotensin II (1.0 pmol/l to 1.0 mmol/l) were obtained in endothelium-intact or endothelium-denuded aortic rings in the absence or after incubation for 30 min with ML171 (a selective Nox-1 inhibitor, 0.5 mmol/l) (Pernomian et al., 2015) or VAS2870 (a selective Nox-4 inhibitor, 5.0 mmol/l) (Moreira et al., 2015). Contraction is expressed as g/mg of dry tissue. 2.4. Hydroxyl radical (OH)-mediated lipid peroxidation measurement in the hamster aorta Thiobarbituric acid reactive substances (TBARS) content was colorimetrically determined at 540 nm using a commercially available kit (#10009055, Cayman Chemical, Ann Arbor, MI, USA). TBARS levels were determined using a standard curve for malondialdehyde bis (MDA) (range of the kit: 0-50 nmol/ml). Results are expressed as nmol/mg of protein. Protein concentration in all experiments was determined using the Lowry protein assay (BioRad Laboratories, Hercules, CA, USA). 2.5. NO measurement by confocal microscopy Aortic rings (150 mm thick) were placed vertically on a coverslip covered with poly-L-lysine (de Oliveira et al., 2012). The tissue was loaded with the selective dye for NO 4,5-diaminofluorescein-2diacetate (DAF-2DA, 50 mmol/l) for 30 min at room temperature (Simplicio et al., 2014). After entering the cell, 4,5-diaminofluorescein-2-diacetate is hydrolyzed to 4,5-diaminofluorescein (DAF-2), which is converted by nitrosation into the fluorescent derivative 4,5-diaminofluorescein triazole (DAF-2T) that emits green fluorescence upon excitation at 490–495 nm (Restini et al., 2014). After washing, the DAF-2DA-loaded rings were placed in a chamber with a volume of 400 ml. The chamber was placed on the stage of a confocal microscope, and images were taken from the bottom of the chamber through a water-immersion objective (63  ). NO generation was assessed using a confocal scanning laser microscope (Leica TCS-SP5). DAF-2DA fluorescence was excited with the 488 nm line of an argon ion laser, and the emitted fluorescence was measured at 520 nm. A time course response

P.C. Pereira et al. / European Journal of Pharmacology 781 (2016) 1–9

was evaluated using a software that captures images of the cells at intervals of 1.314 s (xyt) in Live Data Mode (Pereira et al., 2014). NO generation was measured in vascular smooth muscle cells in the absence or presence of the selective endothelial NOS synthase (eNOS) inhibitor L-NNA (1 mmol/l; 30 min). The maximum and minimum intracellular fluorescence intensities of DAF-2T were measured using the LSCM software. 2.6. Determination of O2
levels in the hamster aorta The lucigenin-derived chemiluminescence assay was used to evaluate O2
generation in aorta homogenates. The tissue was homogenized in phosphate buffer as previously described (Yogi et al., 2010). Luminescence was read in a luminometer (Orion II luminometer, Berthold Detection Systems, Pforzheim, Germany) and the results are expressed as relative light units (RLU)/mg protein. 2.7. Determination of hydrogen peroxide (H2O2) levels in the hamster aorta H2O2 levels were measured in the supernatants of aorta homogenates using the Amplex Reds assay kit (#A22188, Molecular Probes, Invitrogen, Carlsbad, CA, USA) as previously described (Côco et al., 2016). In brief, the isolated aortas were frozen in liquid nitrogen (
196 °C) and stored at
80 °C. The frozen samples were homogenized in Krebs–Henseleit bicarbonate buffer (composition in mmol/l: NaCl 118.4; KCl 4.7; CaCl2 1.9; KH2PO4 1.2; MgSO4  7 H2O 1.2; NaHCO3 25.0; glucose 11.6, pH 7.4, 37 °C) and centrifuged at 10.000  g under refrigeration (4 °C). A standard solution of H2O2 was incubated with the Ultra Red working solution (100 μmol/l) at 37 °C to obtain a standard curve on a 96-well plate. Fluorescence emission was measured at an excitation of 530 nm and emission of 590 nm on a Biotek Synergy HT plate reader. H2O2 levels are expressed as μmol/g of protein. 2.8. Determination of superoxide dismutase (SOD) activity in the hamster aorta Superoxide dismutase (SOD) activity in aorta homogenates was measured using a SOD determination kit (#19160, Sigma-Aldrich, St Louis, MO, USA). Aorta homogenates were prepared in phosphate buffer (composition in mmol/l: NaCl 68.9; Na2HPO4 4.08; KH2PO4 0.73; KCl 1.34; pH 7.4) with a glass-to-glass homogenizer. SOD activity was measured in a suspension containing 20 μl of the sample according to the manufacturer's protocol. The absorbance was measured at 450 nm on a microplate reader and SOD activity is expressed as inhibition rat%/mg protein. This assay provides a measurement of general SOD activity since it does not specify the SOD isoform. 2.9. Determination of catalase activity in the hamster aorta Catalase activity was measured by H2O2 consumption as

3

previously described (Gonzaga et al., 2014). In brief, aortas were homogenized in 200 ml of phosphate buffer (composition in mmol/ l: NaCl 68.9; Na2HPO4 4.08; KH2PO4 0.73; KCl 1.34; pH 7.4) with a glass-to-glass homogenizer. The homogenates were centrifugated at 7800  g for 10 min at 4 °C. Reaction buffer [2.5 ml of Tris ethylenediaminetetraacetic acid buffer (1 mol/l of Trizma and 5 mmol/l of ethylenediaminetetracetic acid), 47.35 ml of MilliQ water and 175.5 μl of H2O2 at 30%] was used to read the samples. Reaction buffer (980 μl) was added to quartz cuvettes containing 20 μl of the supernatant. The absorbance was read for 30 s at 240 nm and catalase activity is expressed as one catalase unit (U) per milligram of protein. 2.10. Statistical analysis Data are expressed as mean 7 standard error of the mean (SEM). The differences between the mean values were assessed using one-way or two-way ANOVA followed by Bonferroni posthoc test as indicated in tables and figures. Results were plotted in graphics using the software GraphPad prism version 5. The significance level considered in all tests was 0.05.

3. Results 3.1. Effects of the cholesterolemic diet on serum lipid profile An altered serum lipid profile was detected in hamsters fed with the cholesterol-rich chow. Serum levels of total cholesterol and LDL-cholesterol were increased in hamsters fed with the cholesterol-rich diet for 5, 10, 15, 20, 25 and 30 days. Interestingly, serum HDL-cholesterol levels were also increased in hamsters fed with the cholesterolemic diet for 15, 20, 25 and 30 days. Serum triglycerides levels were increased only in hamsters that were fed with the cholesterol-rich diet for 25 and 30 days (Table 1). 3.2. Consequences of cholesterolemia on angiotensin II-induced contraction in the hamster aorta The maximal contraction induced by angiotensin II was reduced in endothelium-intact aortic rings from hamsters treated with the cholesterolemic diet for 10, 15 and 20 days (Fig. 1A and C). Angiotensin II-induced contraction was also decreased in endothelium-denuded rings from hamsters treated with the cholesterolemic diet for 15 days (Fig. 1B and C). No difference on angiotensin II-induced contraction was detected in endothelium-intact or endothelium-denuded rings isolated from hamsters treated with the cholesterolemic diet for 30 days (Fig. 1C). The more accentuated effect of hypercholesterolemia on angiotensin II-induced contraction was observed on aortas from animals treated with the cholesterolemic diet for 15 days. Thus, the experiments designed to study the mechanisms underlying the vascular effects of the cholesterolemic diet were

Table 1 Serum lipid profile of control or cholesterolemic diet-fed hamsters. Groups

Cholesterolemic diet Control

TC HDL-C LDL-C Triglycerides a

85.8 7 4.6 46.5 7 4.1 26.2 7 4.3 84.9 7 6.5

5 days

10 days a

170.5 7 4.0 55.4 7 5.6 97.8 7 5.3a 87.0 7 10.2

15 days a

185.8 7 7.9 58.7 7 8.5 129.8 7 16.3a 118.0 7 21.4

20 days a

211.2 7 11.5 75.8 7 5.6a 135.77 12.7a 120.7 7 26.4

25 days a

239.3 7 22.8 101.4 7 2.9a 145.7 7 21.7a 169.0 7 24.3

Significantly different from control group. Po 0.05, two-way ANOVA followed by Bonferroni (n¼ 10 for each group).

30 days a

225.6 76.4 97.8 75.5a 139.2 74.5a 171.5 730.2a

293.0 7 15.0a 90.7 7 8.8a 173.0 7 8.9a 180.3 7 19.9a

4

P.C. Pereira et al. / European Journal of Pharmacology 781 (2016) 1–9

Fig. 1. Concentration-response curves (A and B) and maximum contractile effect (Emax) values (C) for angiotensin II in endothelium-intact (E þ) or endothelium-denuded (E-) aorta from hamsters fed with control or cholesterolemic diets for 5, 10, 15, 20, 25 or 30 days. Data represent mean 7 S.E.M. Significantly different from endotheliumintact (*) or endothelium-denuded (#) control hamster aorta, from endothelium-intact aorta of 10 days-lasting (§) or 15 days-lasting (Ψ) cholesterolemic diet-fed hamsters, or from endothelium-denuded aorta of 15 days-lasting cholesterolemic diet-fed hamsters (ρ). Po 0.05, two-way ANOVA followed by Bonferroni (n¼10 for Fig. 1A–C).

conducted in aortas from hamsters treated with the diet for 15 or 30 days. 3.3. Modulation of Nox-1 or Nox-4 on angiotensin II-induced contraction in aortas from cholesterolemic hamsters ML171 (a selective Nox-1 inhibitor) reduced the maximal contraction induced by angiotensin II in endothelium-intact aortas from control and 30 days-treated cholesterolemic hamsters. However, ML171 did not alter angiotensin II-induced contraction in endothelium-intact aortas from hamsters treated for 15 days with the cholesterolemic diet (Fig. 2A and C). ML171 had no effect on angiotensin II-induced contraction in endothelium-denuded aortas from control or cholesterolemic hamsters (Fig. 2B and C). VAS2870 (a selective Nox-4 inhibitor) reduced the maximal contraction induced by angiotensin II in endothelium-intact rings from control hamsters. However, VAS2870 did not alter angiotensin II-induced contraction in endothelium-intact aortas from hamsters treated for 15 or 30 days with the cholesterolemic diet (Fig. 3A and C). VAS287 reduced the contraction induced by angiotensin II in endothelium-denuded aortic rings from control and 30 days-treated cholesterolemic hamsters. However, VAS2870 did not alter angiotensin II-induced contraction in endothelium-denuded aortas from hamsters treated for 15 days with the cholesterolemic diet (Fig. 3B and D).

3.4. Effect of cholesterolemia on TBARS concentration in the hamster aorta Cholesterolemia had no effect on TBARS concentration in aortas from hamsters fed with the cholesterolemic diet for 15 days. On the other hand, treatment of the hamsters with the cholesterolemic diet for 30 days significantly increased TBARS concentration in the hamster aorta (Fig. 4). 3.5. Effect of cholesterolemia on NO levels in the hamster aorta Treatment for 15 and 30 days with the cholesterolemic diet reduced the fluorescence intensity of DAF-2T in the hamster aorta. L-NNA (an eNOS inhibitor) reduced the fluorescence intensity of DAF2T in aortas from control hamsters. However, L-NNA did not alter the fluorescence intensity of DAF-2T in aortas from hamsters treated with the cholesterolemic diet for 15 or 30 days (Fig. 5A and B). 3.6. Effect of cholesterolemia on O2
and H2O2 levels in the hamster aorta Treatment for 15 days with the cholesterolemic diet did not alter lucigenin-derived luminescence in the hamster aorta. Conversely, treatment of the hamsters for 30 days with the cholesterolemic diet increased lucigenin-derived luminescence in the aorta (Fig. 6). The concentration of H2O2 was reduced in aortas

P.C. Pereira et al. / European Journal of Pharmacology 781 (2016) 1–9

5

Fig. 2. Effect of ML171 (Nox-1 inhibitor) on angiotensin II-induced contraction in endothelium-intact (E þ) (A and C) or endothelium-denuded (E-) (B and D) aorta from hamsters fed with control or cholesterolemic diets for 15 or 30 days. Data represent mean 7S.E.M. Significantly different from endothelium-intact (*) or endotheliumdenuded (Ψ) control hamster aorta in the absence of ML171, or from endothelium-intact aorta of15 days-lasting (§) or 30 days-lasting (#) cholesterolemic diet-fed hamster in the absence of ML171. Po 0.05, two-way ANOVA followed by Bonferroni (n ¼11 for Fig. 2A–D).

from hamsters treated with the cholesterolemic diet for 15 or 30 days (Fig. 7). 3.7. Effect of cholesterolemia on the activity of SOD and catalase in the hamster aorta SOD activity was not altered in aortas from hamsters fed with the cholesterolemic diet for 15 days. On the other hand, treatment for 30 days with the cholesterolemic diet reduced SOD activity in the hamster aorta (Fig. 8). Catalase activity was reduced in aortas from hamsters fed with the cholesterolemic diet for 15 days. However, no difference on catalase activity was observed in aortas from hamsters fed with the cholesterolemic diet for 30 days (Fig. 9).

4. Discussion Treatment of hamsters for 15 days with a cholesterolemic diet triggered a moderate hypercholesterolemia, which was followed by a moderate hypertriglyceridemia at the 30th day of treatment. These findings corroborate previous studies showing that a moderate increase in LDL-cholesterol fraction is observed in hamsters fed with cholesterol (1%)-rich chow for 15–30 days (Martinello et al., 2006; Librandi et al., 2007). Moreover, pro-inflammatory cytokines gradually reduce the effectiveness of lipoprotein lipase in hydrolyzing triglycerides, whose serum levels increase during advanced hypercholesterolemia (Gregoire et al., 1998; Eder et al., 2009).

Experimental evidence has shown that dyslipidemia increases the activation of the ACE–angiotensin II–AT1 axis in vessels affected by atherosclerotic lesions. In this line, hypercholesterolemia was described to increase plasma angiotensin II levels (Daugherty et al., 2004), ACE expression (Dietschy et al., 1993), and the functional responses mediated by AT1 receptors in the vasculature (Yang et al., 1998; Schieffer et al., 2000; Pernomian et al., 2015). However, the majority of these studies were performed in animals in which the atherogenic process has already been established. For this reason, this experimental approach provides an inaccurate conclusion regarding the factor(s) responsible for the induction of the angiotensinergic dysfunction. The latter could be the result of the dyslipidemia or the vascular inflammation. In order to clarify this point, here we investigated the effects of dyslipidemia on the angiotensinergic function before the development of atherosclerotic lesions. Our findings show a decrease on angiotensin II-induced contraction during the dyslipidemic phase that predates the atherogenic process. This finding contrasts previous results showing hyperreactivity to angiotensin II during hypercholesterolemia associated with atherosclerosis (Yang et al., 1998; Schieffer et al., 2000; Pernomian et al., 2015). Importantly, the hyporresponsiveness to angiotensin II was found to be more evident in aortas from hamsters fed with the cholesterolemic diet for 15 days, further suggesting that vascular dysfunction is already apparent on early phases of dyslipidemia. At 15 days of treatment with the cholesterolemic diet the generation of vascular relaxing factors seems to be enhanced while the production of contractile factors may be

6

P.C. Pereira et al. / European Journal of Pharmacology 781 (2016) 1–9

Fig. 3. Effect of VAS2870 (Nox-4 inhibitor) on angiotensin II-induced contraction in endothelium-intact (E þ) (A and C) or endothelium-denuded (E-) (B and D) aorta from hamsters fed with control or cholesterolemic diets for 15 or 30 days. Data represent mean 7S.E.M. Significantly different from endothelium-intact (*) or endotheliumdenuded (#) control hamster aorta in the absence of VAS2870, from endothelium-intact aorta of 15 days-lasting cholesterolemic diet-fed hamster in the absence of VAS2870 (§) or from endothelium-denuded aorta of 30 days-lasting (Ψ) cholesterolemic diet-fed hamster in the absence of VAS2870. Po 0.05, two-way ANOVA followed by Bonferroni (n¼ 11 for Fig. 3A–D).

Fig. 4. Consequences of cholesterolemia on aortic content of thiobarbituric acid reactive species (TBARS) in hamsters fed with control or cholesterolemic diets for 15 or 30 days. Data represent mean 7 S.E.M. *Significantly different from control hamster aorta. P o0.05, one-way ANOVA followed by Bonferroni (n¼ 7).

impaired. The hyporresponsiveness to angiotensin II was observed in both endothelium-intact and endothelium-denuded aortas, further suggesting that the vascular dysfunction induced by early dyslipidemia takes place at the muscular layer of the hamster aorta. This finding also suggests that early dyslipidemia blunted the aortic generation of endothelial relaxing factors such as NO. In

fact, confocal microscopy assays confirmed that the bioavailability of eNOS-derived NO is decreased in the hamster aorta. This result is in agreement with previous findings showing impaired NO generation in early dyslipidemia (Ito et al., 2007; Poreba et al., 2009; Cavieres et al., 2014). Our functional results show that endothelial Nox-1 and Nox-4 in vascular smooth muscle cells generate contractile metabolites that are involved in angiotensin II-induced contraction in arteries from control hamsters. In fact, the primary ROS generated by Nox1 is O2
while Nox-4 mainly produces H2O2 as a result of SODmediated dismutation of O2
(Dikalov et al., 2008). Both O2
and H2O2 are signaling molecules that mediate the contraction in response to activation of AT1 receptors in vascular smooth muscle cells (Vanhoutte, 2001; Gao et al., 2003; Pernomian et al., 2012; Moreira et al., 2015). Interestingly, our functional study also revealed that early dyslipidemia impaired the generation of contractile metabolites by Nox-1 and Nox-4 in endothelial and smooth muscle cells, respectively. Since the hyporresponsiveness to angiotensin II in early dyslipidemia results from a muscular mechanism, we can suggest that impaired Nox-4-driven generation of H2O2 in the muscular layer accounts for such response. This suggestion is in line with the fact that early dyslipidemia reduced H2O2 levels in the hamster aorta. Despite the reduction on H2O2 levels, cholesterolemia reduced catalase activity and did not alter SOD activity or O2
levels in the hamster aorta. It is well described that H2O2 increases eNOS expression (Drummond et al., 2000; Cai et al., 2003; Laude et al., 2005) and activity (Thomas et al., 2002; Woods et al., 2005; Hu et al., 2008). Thus, the impaired eNOS-

P.C. Pereira et al. / European Journal of Pharmacology 781 (2016) 1–9

7

Fig. 7. H2O2 concentration in aortas from hamsters fed with control or cholesterolemic diets for 15 or 30 days. Data represent mean7 S.E.M. *Significantly different from control hamster aorta. P o0.05, one-way ANOVA followed by Bonferroni (n ¼7).

Fig. 5. Representative frames (A) and quantitative analysis (B) of 4,5-diaminofluorescein-2 triazole (DAF-2T) fluorescence intensity emitted by 4,5-diaminofluorescein-2-diacetate (DAF-2DA)-loaded aortic rings from hamsters fed with control or cholesterolemic diets for 15 or 30 days, pre-treated or not with L-NNA (eNOS inhibitor). Data represent mean 7 S.E.M. *Significantly different from nonpre-treated control hamster aorta. Po 0.05, one-way ANOVA followed by Bonferroni (n¼ 5).

Fig. 8. SOD activity in aortas from hamsters fed with control or cholesterolemic diets for 15 or 30 days. Data represent mean 7S.E.M. *Significantly different from control hamster aorta. P o 0.05, one-way ANOVA followed by Bonferroni (n ¼8).

Fig. 9. Catalase activity in aortas from hamsters fed with control or cholesterolemic diets for 15 or 30 days. Data represent mean 7S.E.M. *Significantly different from control hamster aorta. P o 0.05, one-way ANOVA followed by Bonferroni (n ¼9).

Fig. 6. Lucigenin chemiluminescence in aortas from hamsters fed with control or cholesterolemic diets for 15 or 30 days. Data represent mean 7 S.E.M. *Significantly different from control hamster aorta. P o 0.05, one-way ANOVA followed by Bonferroni (n¼ 6).

derived NO levels here described may result from the reduced aortic levels of H2O2. In fact, eNOS modulates catalase activity (Benjamin et al., 2009), whose impairment may be due to the decreased bioavailability of aortic eNOS-derived NO during early dyslipidemia. Taken together, our functional and biochemical data suggest a local auto-regulatory mechanism played by H2O2 on the muscular Nox-4-driven generation of H2O2 derived from O2
on

aortas of hamsters treated for 15 days with the cholesterolemic diet. In this sense, the impaired catalase activity would lead to a initial transient increase in the levels of O2
-derived H2O2, which will in turn inhibit its own generation by Nox-4 promoting a persistent reduction in H2O2 levels. Recently, Harraz et al. (2008) described a redox-dependent mechanism that regulates the activation of Rac1-dependent NAD(P)H oxidases, such as Nox-4 (Gorin et al., 2003; Mahadev et al., 2004; Hordijk, 2006). In such mechanism, local accumulation of H2O2induces the dissociation of SOD from the Rho-like small GTPase Rac1-GTP (Harraz et al., 2008). The binding of SOD inhibits the intrinsic GTPase activity from Rac1-GTP and keeps Rac1 in its active state, which is required for the activation of the assembling Nox complex (Hordijk, 2006).

8

P.C. Pereira et al. / European Journal of Pharmacology 781 (2016) 1–9

Thus, H2O2
mediated inhibition of SOD-Rac1 interaction leads to GTP hydrolysis by Rac1 and the formation of Rac1-GDP, which does not support the activation of Nox complex (Harraz et al., 2008). The contractile hyporresponsiveness to angiotensin II is reversed in aortas from hamsters treated with the cholesterolemic diet for 30 days. Our functional results suggest that the recovery of the vascular contractility induced by angiotensin II during the advanced phases of dyslipidemia is mediated by a muscular mechanism involving Nox-4-derived contractile metabolites. Interestingly, the contractile response induced by angiotensin II was restored in aortas from hamsters treated for 30 days with the cholesterolemic diet. This response was accompanied by the recovery of aortic catalase activity. However, impaired vascular SOD activity and increased lipid peroxidation were also detected, while aortic levels of H2O2 and eNOS-derived NO remained reduced. Indeed, advanced phases of dyslipidemia in hamsters are associated with decreased SOD activity (Zhao et al., 2014), which has been correlated to the increased deposition of oxidized LDL (oxLDL) in the vascular wall (Verreth et al., 2007). Upon SOD activity impairment, Nox-4-derived O2
generation increases while Nox-4-derived H2O2 levels persistently fall, further inhibiting H2O2
mediated auto-regulation of Nox-4. High levels of hydroxyl radical are directly generated from O2
by Fenton-like reactions upon the oxidation of NAD(P)H (Shi and Dalal, 1993). In turn, hydroxyl radical inhibits catalase activity (Davison et al., 1986) and induces the generation of vasoconstrictor endoperoxides, such as prostaglandin H2 (PGH2) (Vanhoutte, 2001), which contributes to the recovery of the contractile response induced by angiotensin II. The low levels of H2O2 combined with the high levels of O2
accounts for the reduced levels of eNOS-derived NO due to the low H2O2
mediated downregulation of eNOS (Drummond et al., 2000; Thomas et al., 2002; Cai et al., 2003; Laude et al., 2005; Woods et al., 2005; Hu et al., 2008) or the high O2
-mediated eNOS uncoupling (Silva et al., 2012).

5. Conclusions In summary, the major new finding of our study is that angiotensin II-induced contraction is reduced in aortas from hamsters in early phases of dyslipidemia (15 days-lasting cholesterolemic diet) due to the downregulation of the underlying NAD(P)H oxidase-mediated redox signaling. Paradoxically, the contractile response induced by angiotensin II is recovered by a compensatory mechanism that involves Nox-4-driven oxidative stress triggered by the advanced dyslipidemic stages (30 days-lasting cholesterolemic diet). The vascular hyporresponsiveness to angiotensin II may be elicited by an auto-inhibitory regulation of angiotensin II functionality during early dyslipidemia in attempt to reduce the effects of the upregulation of the vascular RAS, which is a typical event on the advanced stages of atherogenesis (Dietschy et al., 1993; Yang et al., 1998; Schieffer et al., 2000; Daugherty et al., 2004; Pernomian et al., 2015). In turn, the recovery of vascular angiotensin II functionality during the advanced phases of dyslipidemia occurs due to upregulation of a redox-pro-inflammatory pathway that may be predominantly involved in the progression of atherogenesis rather than in the restoration of vascular function. Taken together, these findings suggest that the early phase of dyslipidemia is the most favorable moment for effective atheroprotective therapeutic interventions. Role of the funding source This study was supported by funds and grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq,

Brazil, #142389/2011-0) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Brazil, # 2012/09019-3).

Conflict of interest statement The authors declare that there are no conflicts of interest.

Acknowledgements The authors thank Professor Dr. Carlos Henrique Tomich de Paula da Silva for providing language help and writing assistance.

References vAzumi, H., Inoue, N., Ohashi, Y., Terashima, M., Mori, T., Fujita, H., Awano, K., Kobayashi, K., Maeda, K., Hata, K., Shinke, T., Kobayashi, S., Hirata, K., Kawashima, S., Itabe, H., Hayashi, Y., Imajoh-Ohmi, S., Itoh, H., Yokoyama, M., 2002. Superoxide generation in directional coronary atherectomy specimens of patients with angina pectoris: Important Role of NAD(P)H oxidase. Arterioscler. Thromb. Vasc. Biol. 22, 1838–1844. vBeckman, J.S., Koppenol, W.H., 1996. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271, 1424–1437. vBenjamin, Y., Jin, Y., Sartoretto, J.L., Gladyshev, V.N., Michel, T., 2009. Endothelial nitric oxide synthase negatively regulates hydrogen peroxide-stimulated AMPactivated protein kinase in endothelial cells. PNAS 106, 17343–17348. vCai, H., Griendling, K.K., Harrison, D.G., 2003. The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases. Trends Pharmacol. Sci. 24, 471–478. vCavieres, V., Valdes, K., Moreno, B., Moore-Carrasco, R., Gonzalez, D.R., 2014. Vascular hypercontractility and endothelial dysfunction before development of atherosclerosis in moderate dyslipidemia: role for nitric oxide and interleukin6. Am. J. Cardiovasc. Dis. 11, 114–122. vChannon, K.M., 2002. Oxidative stress and coronary plaque stability. Arterioscler. Thromb. Vasc. Biol. 22, 1751–1752. vCôco, H., Pernomian, L., Marchi, K.C., Gomes, M.S., de Andrade, C.R., Ramalho, L.N. Z., Tirapelli, C.R., de Oliveira, A.M., 2016. Consequence of hyperhomocysteinemia on α1-adrenoceptor-mediated contraction in the rat corpus cavernosum: the role of reactive oxygen species. J. Pharm. Pharmacol. 68, 63–75. vDaugherty, A., Rateri, D.L., Lu, H., Inagami, T., Cassis, L.A., 2004. Hypercholesterolemia stimulates angiotensin peptide synthesis and contributes to atherosclerosis through the AT1A receptor. Circulation 110, 3849–3857. vDavison, A.J., Kettle, A.J., Fatur, D.J., 1986. Mechanism of the inhibition of catalase by ascorbate. Roles of active oxygen species, copper and semidehydroascorbate. J. Biol. Chem. 261, 1193–1200. vde Oliveira, J.C., Antonietto, C.R.K., Scalabrini, A.C., Marinho, T.S., Pernomian, L., Corrêa, J.W.N., Restini, C.B.A., 2012. Antioxidant protective effects of the resveratrol on the cardiac and vascular tissues from renal hypertensive rats. Open J. Med. Chem. 2, 61–71. vDietschy, J.M., Turley, S.D., Spady, D.K., 1993. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans. J. Lipid Res. 34, 1637–1659. vDikalov, S.I., Dikalova, A.E., Bikineyeva, A.T., Schmidt, H.H., Harrison, D.G., Griendling, K.K., 2008. Distinct roles of Nox1 and Nox4 in basal and angiotensin II-stimulated superoxide and hydrogen peroxide production. Free Radic. Biol. Med. 45, 1340–1351. vDillard, I., Matthan, N.R., Lichtenstein, A.H., 2010. Use of hamster as a model to study diet-induced atherosclerosis. Nutr. Metab. 7, 89. vDrummond, G., Cai, H., Davis, M.E., Ramasamy, S., Harrison, D.G., 2000. Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide. Circ. Res. 86, 347–354. vEder, K., Baffy, N., Falus, A., Fulop, A.K., 2009. The major inflammatory mediator interleukin-6 and obesity. Inflamm. Res. 58, 727–736. vGao, Y.-J., Hirota, S., Zhang, D.-W., Janssen, L.J., Lee, R.M.K.W., 2003. Mechanisms of hydrogen-peroxide-induced biphasic response in rat mesenteric artery. Br. J. Pharmacol. 138, 1085–1092. vGetz, G.S., Reardon, C.A., 2006. Diet and murine atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 26, 242–249. vGonzaga, N.A., Callera, G.E., Yogi, A., Mecawi, A.S., Antunes-Rodrigues, J., Queiroz, R.H., Touyz, R.M., Tirapelli, C.R., 2014. Acute ethanolintake induces mitogenactivatedprotein kinase activation, plateletderivedgrowth factor receptorphosphorylation, and oxidative stressin resistance arteries. J. Physiol. Biochem. 70, 509–523. vGorin, Y., Ricono, J.M., Kim, N.H., Bhandari, B., Choudhury, G.G., Abboud, H.E., 2003. Nox4 mediates angiotensin II-induced activation of Akt/protein kinase B in mesangial cells. Am. J. Physiol. Ren. Physiol. 285, F219–F229. vGregoire, F.M., Smas, C.M., SUL, H.S., 1998. Understanding of adipocyte differentiation. Physiol. Rev. 78, 783–806.

P.C. Pereira et al. / European Journal of Pharmacology 781 (2016) 1–9

vGriendling, K.K., Minieri, C.A., Ollerenshaw, J.D., Alexander, R.W., 1994. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ. Res. 74, 1141–1148. vHarraz, M.M., Marden, J.J., Zhou, W., Zhang, Y., Williams, A., Sharov, V.S., Nelson, K., Luo, M., Paulson, H., Schöneich, C., Engelhard, J.F., 2008. SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model. J. Clin. Investig. 118, 659–670. vHordijk, P.L., 2006. Regulation of NADPH oxidases. The role of Rac proteins. Circ. Res. 98, 453–562. vHu, Z., Chen, J., Wei, Q., Xia, Y., 2008. Bidirectional actions of hydrogen peroxide on endothelial nitric-oxide synthase phosphorylation and function: co-commitment and interplay of Akt and AMPK. J. Biol. Chem. 283, 25256–25263. vIto, K.M., Okayasu, M., Koshimoto, C., Shinohara, A., Asada, Y., Tsuchiya, K., Sakamoto, T., Ito, K., 2007. Impairment of endothelium-dependent relaxation of aortas and pulmonary arteries from spontaneously hyperlipidemic mice (Apodemussylvaticus). Vasc. Pharmacol. 47, 166–173. vKahlon, T.S., Chow, F.I., Irving, D.W., Sayre, R.N., 1996. Cholesterol response and foam cell formation in hamsters fed two levels of saturated fat and various levels of cholesterol. Nutr. Res. 16, 1353–1368. vKunsch, C., Medford, R.M., 1999. Oxidative stress as a regulator of gene expression in the vasculature. Circ. Res. 85, 753–766. vLaude, K., Cai, H., Fink, B., Hoch, N., Weber, D.S., McCann, L., Kojda, G., Fukai, T., Schmidt, H.H., Dikalov, S., Ramasamy, S., Gamez, G., Griendling, K.K., Harrison, D.G., 2005. Hemodynamic and biochemical adaptations to vascular smooth muscle overexpression of p22phox in mice. Am. J. Physiol. Heart Circ. Physiol. 288, H7–H12. vLibrandi, A.P.L., Chrysóstomo, T.N., Azzolini, A.E.C.S., Recchia, C.G.V., Uyemura, S.A., de Assis-Pandochi, A.I., 2007. Effect of the extract of the tamaring (Tamarindusindica) fruit on the complement system: studies in vitro and in hamsters submitted to a cholesterol-enriched diet. Food Chem. Toxicol. 45, 1487–1495. vLiu, G., Fan, L., Redinger, R., 1991. The association of hepatic apoprotein and lipid metabolism in hamsters and rats. Comp. Biochem. Physiol. 99A, 223–228. vMahadev, K., Motoshima, H., Wu, X., Ruddy, J.M., Arnold, R.S., Cheng, G., Lambeth, J.D., Goldstein, B.J., 2004. The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction. Mol. Cell. Biol. 24, 1844–1854. vMartinello, F., Soares, S.M., Franco, J.J., Santos, A.C., Sugohara, A., Garcia, S.B., Curti, C., Uyemura, S.A., 2006. Hypolipemic and antioxidant activities from Tamarindusindica L. pulp fruit extract in hypercholesterolemic hamsters. Food Chem. Toxicol. 44, 810–818. vMoreira, J.D., Pernomian, L., Gomes, M.S., Pernomian, L., Moreira, R.P., do Prado, A. F., da Silva, C.H., de Oliveira, A.M., 2015. Acute restraint stress increases carotid reactivity in type-I diabetic rats by enhancing Nox4/NADPH oxidase functionality. Eur. J. Pharmacol. 765, 503–516. vNistor, A., Bulla, A., Filip, D.A., Radu, A., 1987. The hyperlipidemic hamster as a model of experimental atherosclerosis. Atherosclerosis 68, 159–173. vPereira, A.C., Olivon, V.C., Pernomian, L., de Oliveira, A.M., 2014. Impairment of α1adrenoceptor-mediated calcium influx in contralateral carotids following balloon injury: beneficial effects of superoxide anions. Eur. J. Pharmacol. 723, 397–404. vPernomian, L., da Silva, C.H.T., 2015. Current basis for discovery and development of aryl hydrocarbon receptor antagonists for experimental and therapeutic use in atherosclerosis. Eur. J. Pharmacol. 764, 118–123. vPernomian, L., Gomes, M.S., Restini, C.B.A., Ramalho, L.N.Z., Tirapelli, C.R., Oliveira, A.M., 2012. The role of reactive oxygen species in the modulation of the contraction induced by angiotensin II in carotid artery from diabetic rat. Eur. J. Pharmacol. 678, 15–25. vPernomian, L., Pernomian, L., Restini, C.B.A., 2014a. Counter-regulatory effects played by the ACE-AngII-AT1 and ACE2-Ang-(1-7)-Mas axes on the reactive oxygen species-mediated control of vascular function: perspectives to pharmacological approaches in controlling vascular complications. VASA 43, 404–414. vPernomian, L., Gomes, M.S., Restini, C.B.A., de Oliveira, A.M., 2014b. Mas-mediated antioxidant effects restore the functionality of angiotensin converting enzyme 2-Angiotensin-(1-7)-Mas axis in diabetic rat carotid. Biom. Res. Int., 2014. http: //dx.doi.org/10.1155/2014.640329 (20 pages). vPernomian, L., do Prado, A.F., Gomes, M.S., Pernomian, L., da Silva, C.H.T., Gerlach, R.F., de Oliveira, A.M., 2015. MAS receptors mediate vasoprotective and atheroprotective effects of candesartan upon the recovery of vascular angiotensin-

9

converting enzyme 2-angiotensin-(1-7)-MAS axis functionality. Eur. J. Pharmacol. 764, 173–188. vPien, C.S., Davis, W.P., Marone, A.J., Foxall, T.L., 2002. Characterization of diet induced aortic atherosclerosis in Syrian F1B hamsters. J. Exp. Anim. Sci. 42, 65–83. vPoreba, R., Skoczynska, A., Gac, P., Poreba, M., Jedrychowska, I., Affelska-Jercha, A., Turczyn, B., Wojakowska, A., Oszmianski, J., Andrzejak, R., 2009. Drinking of chokeberry juice from the ecological farm Dzieciolowo and distensibility of brachial artery in men with mild hypercholesterolemia. Ann. Agric. Environ. Med. 16, 305–308. vRestini, C.B.A., Pereira, B.F.M., Pernomian, L., 2014. Protocols of confocal microscopy to study vascular dysfunction in Diabetes mellitus. In: Méndez-Vilas, A. (Ed.), Microscopy: Advances in Scientific Research and Education. Formatex, Badajoz, pp. 209–214. vSchieffer, B., Schieffer, E., Hilfiker-Kleiner, D., Hilfiker, A., Kovanen, P.T., Kaartinen, M., Nussberger, J., Harringer, W., Drexler, H., 2000. Expression of angiotensin II and interleukin 6 in human coronary atherosclerotic plaques: potential implications for inflammation and plaque instability. Circulation 101, 1372–1378. vSchiffrin, E.L., Park, J.B., Intengan, H.D., Touyz, R.M., 2000. Correction of arterial structure and endothelial dysfunction in human essential hypertension by the angiotensin receptor antagonist losartan. Circulation 101, 1653–1659. vShi, X., Dalal, N.S., 1993. Vanadate-mediated hydroxyl radical generation from superoxide radical in the presence of NADH: Haber-Weiss vs Fenton mechanism. Arch. Biochem. Biophys. 307, 336–341. vSilva, B.R., Pernomian, L., Bendhack, L.M., 2012. Contribution of oxidative stress to endothelial dysfunction in hypertension. Front. Physiol. 3, 441–446. vSimplicio, J.A., Pernomian, L., Simão, M.R., Carnio, E.C., Batalhão, M.E., Ambrosio, S. R., Tirapelli, C.R., 2014. Mechanisms underlying the vascular and hypotensive actions of the labdane ent-3-acetoxy-labda-8(17),13-dien-15-oic acid. Eur. J. Pharmacol. 726, 66–76. vStein, Y., Dabach, Y., Hollander, G., Stein, O., 1990. Cholesteryl ester transfer activity in hamster plasma: increase by fat and cholesterol rich diets. Biochim. Biophys. Acta 1042, 138–141. vThomas, S.R., Chen, K., Keaney Jr., J.F., 2002. Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide-3-kinase-dependent signaling pathway. J. Biol. Chem. 277, 6017–6024. vTsutsumi, K., Hagi, A., Inoue, Y., 2001. The relationship between plasma high density lipoprotein cholesterol levels and cholesteryl ester transfer protein activity in six species of healthy experimental animals. Biol. Pharm. Bull. 24, 579–581. vVanhoutte, P.M., 2001. Endothelium-derived free radicals: for worse and for better. J. Clin. Investig. 107, 23–24. vVerreth, W., De Keyzer, D., Davey, P.C., Geeraert, B., Mertens, A., Herregods, M.-C., Smith, G., Desajardins, F., Balligand, J.-L., Holvoet, P., 2007. Rosuvastatin restores superoxide dismutase expression and inhibits accumulation of oxidized LDL in the aortic arch of obese dyslipidemic mice. Br. J. Pharmacol. 151, 347–355. vWarnholtz, A., Nickenig, G., Schulz, E., Macharzina, R., Bräsen, J.H., Skatchkov, M., Heitzer, T., Stasch, J.P., Griendling, K.K., Harrison, D.G., Böhm, M., Meinertz, T., Münzel, T., 1999. Increased NADH-oxidase-mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the renin-angiotensin system. Circulation 99, 2027–2033. vWoods, A., Dickerson, K., Health, R., Hong, S.P., Momcilovic, M., Johnstone, S.R., Carlson, M., Carling, D., 2005. Ca2 þ/calmodulin-dependent protein kinase beta acts up-stream of AMP-activated protein kinase in mammalian cells. Cell Metab. 2, 21–33. vXiangdong, L., Yuanwu, L., Hua, Z., Liming, R., Qiuyan, L., Ning, L., 2011. Animal models for the atherosclerosis research: a review. Protein Cell 3, 189–201. vYang, B.C., Phillips, M.I., Mohuczy, D., Meng, H., Shen, L., Mehta, P., Mehta, J.L., 1998. Increased angiotensin II type 1 receptor expression in hypercholesterolemic atherosclerosis in rabbits. Arterioscler. Thromb. Vasc. Biol. 18, 1433–1439. vYogi, A., Callera, G.E., Hipólito, U.V., Silva, C.R., Touyz, R.M., Tirapelli, C.R., 2010. Ethanol-induced vasoconstriction is mediated via redox-sensitive cyclo-oxygenase-dependent mechanisms. Clin. Sci. 118, 657–668. vZhao, Y., Shu, P., Zhang, Y., Lin, L., Zhou, H., Xu, Z., Suo, D., Xie, A., Jin, X., 2014. Effect of Centella asiatica on oxidative stress and lipid metabolism in hyperlipidemic animal models. Oxidative Med. Cell. Longev. 2014. http://dx.doi.org/10.1155/ 2014/154295 7 pages.