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Nebivolol to attenuate the effects of hyper-homocysteinaemia in rats
Atherosclerosis 240 (2015) 33e39
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Nebivolol to attenuate the effects of hyper-homocysteinaemia in rats Cagdas Akgullu a, *, Mustafa Ahmet Huyut b, Murat Boyacioglu c, Ozay Gules¸ d, €r a Ufuk Eryilmaz a, Tolga Hekim b, Emir Dogan e, Cemil Zencir a, Hasan Güngo a
Department of Cardiology, Faculty of Medicine, Adnan Menderes University, Aydin, Turkey Department of Cardiology, Aydın City Hospital, Aydin, Turkey c Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Adnan Menderes University, Aydin, Turkey d Department of Histology and Embryology, Faculty of Veterinary Medicine, Adnan Menderes University, Aydin, Turkey e Department of Cardiology, Ada Tıp Hospital, Sakarya, Turkey b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 October 2014 Received in revised form 13 February 2015 Accepted 21 February 2015 Available online 28 February 2015
Objective: This study investigated the prophylactic effect of nebivolol against hyper-homocysteinaemia (hHcy) induced oxidative stress in brain, heart, liver and kidney tissues and histomorphometric changes in the thoracic aorta. Methods: Twenty-four adult male Wistar rats were divided into a control, nebivolol, hHcy and nebivolol þ hHcy group. hHcy was induced by oral administration of L-methionine (1 g/kg/day) for 28 days. 10 mg/kg/day nebivolol was administered orally for 28 days. Malondialdehyde (MDA) and glutathione (GSH) levels and catalase (CAT) and superoxide dismutase (SOD) activities in the tissues were determined. The total cross-sectional area (TCSA), luminal cross-sectional area (LCSA) and intima-media thickness (IMT) were measured in the thoracic aorta. Results: Homocysteine (Hcy) levels were lower in the nebivolol þ hHcy group than in the hHcy group. Nebivolol treatment significantly decreased high MDA levels in the brain, heart and liver tissues. The level of GSH was higher in the brain, heart and kidney tissues of the nebivolol þ hHcy group (P < 0.001). The activity of CAT increased only in the kidney tissue of the nebivolol þ hHcy group (P < 0.01), and the activity of SOD was significantly increased in all the tissues in this group. Increased TCSA and IMT in the nebivolol þ hHcy group were significantly decreased after nebivolol administration. The LCSA was significantly higher in the hHcy group than the control group, probably due to outward vascular remodelling. Conclusion: Nebivolol treatment may be useful in different clinical scenarios where hHcy affects physiopathological pathways. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Hyper-homocysteinaemia Nebivolol Oxidant/anti-oxidant parameters Rats Aorta
1. Introduction Homocysteine (Hcy) is a sulphur-containing amino acid. The metabolism of Hcy involves two pathways: remethylation to methionine, which requires folate and vitamin B12, and transsulphuration to cystathionine, which requires vitamin B6 [1]. Different defects at different levels of this process may lead to hyperhomocysteinemia (hHcy). Genetic diseases, acquired pathologies or poor dietary habits can cause hHcy [1]. Normally, the fasting plasma level of Hcy is between 5 and 15 mmol/L. A level of
* Corresponding author. Department of Cardiology, Medical Faculty, Adnan Menderes University, 090100 Aytepe, Aydin, Turkey. E-mail address: [email protected] (C. Akgullu). http://dx.doi.org/10.1016/j.atherosclerosis.2015.02.054 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
15e30 mmol/L is considered moderate, whereas levels of 30e100 mmol/L and 100e500 mmol/L are considered intermediate and severe hHcy, respectively [2]. The literature and the data about the relation between the homocysteine and atherosclerosis date back to 45 years. In 1969, depending on observations of vascular lesions among the children with hyperhomocysteinemia, Kilmer McCully proposed a hypothesis that homocysteine is atherogenic [3]. Since then numerous studies with a cumulative data have confirmed that an elevation of total plasma homocysteine (tHcy) is related to early atherosclerosis [4,5]. Today it is well known that hHcy elicits a cascade of events that injure the vascular wall [2]. These include chemical modification of lipoproteins, alterations of vascular structure, endothelial dysfunction, impairment of cell repair and proliferation of vascular smooth muscle cells [6,7]. Moreover, a rise in plasma Hcy levels was
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proposed to contribute to ischaemic changes and alter the structure and function of blood vessels through oxidative stress [2,8]. Oxidative stress appeared to give rise to vascular diseases and atherosclerosis via reactive oxygen species (ROS), with injury occurring when the critical balance between free radicals and the synthesis of anti-oxidant defence was altered [9]. Of interest, high levels of methionine or low levels of folate both of which contribute hHcy, seemed to have an adverse effect on the oxidant/anti-oxidant status of the liver and renal tissues and cause injury to these tissues [10,11]. Nebivolol is a racemic mixture of two stereoisomers: d-nebivolol and l-nebivolol and has high affinity and selectivity for b1adrenoceptor binding sites [12]. In addition to its b1 receptoreblocking and nitric oxid (NO)-releasing effects, nebivolol can substantially inhibit vascular oxidative stress [12]. A previous study demonstrated that nebivolol significantly decreased levels of 8-isoprostaglandin F2a (PGF2a) a marker of oxidative stress, in urine samples of healthy volunteers [13]. Moreover, nebivolol was shown to facilitate ROS scavenging in the rat aorta and to alleviate oxidative stress-induced impairment of endothelium-dependent vasorelaxation [14]. Besides, nebivolol was shown to prevent the development of atherosclerosis in cholesterol fed-rabbits [15]. There is a controversy in the literature about the usefulness of Hcy lowering therapy mainly with vitamin supplements to reduce the cardiovascular events [16e19]. Recent meta-analysis suggested that folic acid supplementation may improve endothelial dysfunction as assessed by flow-mediated vasodilation in the brachial artery in patients with coronary heart disease [18]. However, most of the analysis are currently in favour of no benefit from vitamin supplements to reverse the unfavourable effects of Hcy on the endothelium [16,17]. More effective agents are needed both to lower the serum Hcy levels and protect the endothelium from its harm. Nebivolol with its anti-hypertensive, anti-oxidant and NOreleasing effects may provide a good tool to reverse the unfavourable affects of Hcy on the endothelium which promotes atherosclerosis. To the best of our knowledge, there is no information available on the potentially preventive effects of nebivolol against the hHcy-induced damage in the wall of the aorta. Moreover, there are no data about the nebivolol to improve hHcyinduced oxidative stress in different rat tissues. The purpose of this study was to investigate the prophylactic effect of nebivolol against oxidative stress in brain, heart, liver and kidney tissues. Moreover, we aimed to demonstrate the potential prophylactic effect of nebivolol against hHcy-induced changes in the wall of the aorta. 2. Material and methods 2.1. Animals and experimental protocol Twenty-four healthy adult male Wistar rats (16 weeks old, weighing between 322 and 354 g) were used in the study. The animals were obtained from the Adnan Menderes University, Faculty of Medicine, Experimental Research Centre, Aydin, Turkey. They were placed in screen-bottomed stainless steel cages at 22e24 C in a room with a 12/12 h light/dark cycle. The rats were randomly divided into four groups (n ¼ 6 rats per group): a control group, nebivolol group, hHcy group and nebivolol þ hHcy group. They received a commercial rodent diet and had free access to tap water. After 15 days of acclimatisation, the rats were weighed. hHcy was induced with 1 g/kg/day of L-methionine (M9625 L-methionine reagent grade, 98% SigmaeAldrich (Gillingham, Dorset, UK), which was administered directly into the stomach via oral gavage in the mornings between 7:00 and 8:00 am [20]. Nebivolol
(Vasoxen 5 mg, Ibrahim Etem Ulugay, Istanbul, Turkey) was administered at a dose of 10 mg/kg/day via oral gavage. To prevent the interaction of nebivolol and methionine in the stomach, nebivolol was administered 6 h after the administration of methionine. The substances were administered to the rats by daily oral gavage for 28 days. The control animals received 0.9% NaCl solution in the same volumes as those applied to the nebivolol-treated rats by daily oral gavage for 28 days. L-methionine was dissolved in phosphate buffer (pH 7.4), and nebivolol was dissolved in 150 mM of distilled water. Serum samples were collected from the heart by cardiac puncture after the induction of anaesthesia with ketamine and xylasine (50 mg/kg and 5 mg/kg, respectively) at the end of the experiment. The animals were sacrificed by cervical dislocation, and the thoracic aorta was immediately removed for histomorphometric evaluation. To determine the oxidant/anti-oxidant status, malondialdehyde (MDA) and glutathione (GSH) levels and catalase (CAT) and superoxide dismutase (SOD) activities were determined in brain, heart, liver and kidney tissues. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experimental protocol was approved by the Animal Ethics Committee of the University of Adnan Menderes (2011/076). 2.2. Measurement of serum Hcy levels The concentration of Hcy in the serum samples was determined with a commercial ELISA test kit (CSB-E13376r, Cusabio Biotech Co., New Jersey, USA). 2.3. Biochemical analysis of tissues Dissected brain, heart, liver and kidney tissues were immediately rinsed in ice-cold phosphate-buffered saline. Tissues were homogenised (2000 rpm/min for 1 min, 1/10 w/v) using a Teflonglass stirrer (IKA Overhead Stirrer; IKA-Werke GmbH & Co. KG, Staufen, Germany) in a 10% 150 mM phosphate buffer (pH 7.4) in an ice bath. The homogenate was centrifuged (Hettich Zentrifugen, Mikro 200 R, Tuttlingen, Germany) at 6000 g for 10 min at 4 C. The supernatants were frozen at 80 C (Glacier Ultralow Temperature Freezer, Japan) until analysed and then used for determination of MDA and GSH levels and CAT and SOD activities. 2.4. Determination of the oxidant/anti-oxidant status of the tissues 2.4.1. MDA levels in the tissues The lipid peroxidation levels were determined according to the concentration of thiobarbituric acid reactive substances, and the amount of MDA produced was used as an index of lipid peroxidation. Absorbance was measured with a spectrophotometer at 532 nm. The concentration of MDA was calculated by the absorbance complex (absorbance coefficient ε ¼ 1.56 105/M/cm) and expressed as nmol/mg of tissue protein [21]. 2.4.2. GSH levels in the tissues The tissue GSH concentration was measured with a kinetic assay using the 5, 50 -dithiobis (2-nitrobenzoic acid) recycling method described by Tietze [22]. The results were compared with those of an aqueous standard solution of GSH (Sigma Chemical Co., St. Louis, Missouri, USA) and expressed as mg/g of tissue protein. Absorbance was spectrophotometrically monitored at 412 nm against reagent controls. 2.4.3. CAT activity in the tissues CAT activity was determined by measuring the decomposition of
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hydrogen peroxide at 240 nm spectrophotometrically, according to the method of Bergmeyer et al., and it was expressed as k/mg of tissue protein, where k is the first-order rate constant [23]. 2.4.4. SOD activity in the tissues SOD activity was determined according to the method of Sun et al., and the absorbance was measured at 560 nm with a spectrophotometer [24]. This method is based on the inhibition of nitro blue tetrazolium reduction using the xanthine:xanthine oxidase system as a superoxide generator. SOD activity was then measured by the degree of inhibition of this reaction. The results are shown as U/mg of tissue protein. The enzyme activity assays were performed in duplicate, and the results were averaged. The protein levels in the tissues were determined by the method described by Lowry et al. [25]. 2.5. Histomorphometric evaluation The thoracic aortas were removed in each animal group. The total cross-sectional area (TCSA), luminal cross-sectional area (LCSA) and intima media thickness (IMT) of the thoracic aorta were determined. The tissues were cleaned with phosphate-buffered saline and fixed in buffered 10% formalin solution, dehydrated in a graded series of ethanol, cleared in xylene and embedded in paraffin. The paraffin tissue blocks were cut at intervals of 100 mm and a thickness of 6 mm. The histological sections were placed on glass slides. Crossman's triple staining method [26] was used to measure the TCSA and LCSA, and the Orcein-Giemsa staining method [27] was applied for the identification of elastic laminae. Five serial sections were taken from each rat for each staining method. TCSA and LCSA were measured in five different sections. The IMT was measured in five random areas of each section. A Leica DC200 CCD camera (Germany) and Q-win standard image analysis software (Version 2.8) connected to a research microscope (Leica DMLB- Germany) were used to obtain histomorphometric data from the sections. 2.6. Statistical analysis All the analyses were performed with the Statistical Package for the Social Sciences (SPSS) software (Version 11.5). All the values are presented as the mean ± SEM. Differences were considered significant at P < 0.05. The KruskaleWallis analysis of variance (ANOVA) or a one-way ANOVA was used to compare the data among the groups, depending on whether the data were normally distributed. Post-hoc multiple comparisons were performed using the ManneWhitney U test, with Bonferroni correction or Duncan's test [28]. 3. Results There was no significant difference between the groups by means of weights (nebivolol group: 340.2 ± 3.07 g, hHcy group: 336.8 ± 4.08 g, nebivolol þ hHcy group: 334.7 ± 3.84 g, control group: 338.3 ± 2.3 g) (P > 0.05). The Hcy levels of the hHcy group were significantly higher than those of the other groups (P < 0.001) (Fig. 1). The Hcy level of the nebivolol þ hHcy group was lower than that of the hHcy group (12.8 ± 0.75 versus 37.13 ± 2.89 mmol/L) (P < 0.001). The Hcy level of the nebivolol group was slightly lower than that of the control group (5.18 ± 0.48 versus 7.36 ± 0.52 mmol/L) (P > 0.05). The influence of nebivolol on MDA and GSH levels and CAT and SOD activities in the brain, heart, liver and kidney tissues of the chronic hHcy-induced rats is shown in Fig. 2. In the brain tissue, the GSH level (P < 0.001) and SOD activity (P < 0.001) were higher and
Fig. 1. Effect of nebivolol on the serum homocysteine (Hcy) level in chronic hyperhomocysteinaemia (hHcy)-induced rats (n ¼ 6 in each group). a, b, c The different letters indicate statistically significant differences in the same column. ***P < 0.001; Hcy: homocysteine; hHcy: hyper-homocysteinaemia.
the MDA level was lower (P < 0.01) in the hHcy group than in the nebivolol þ hHcy and control groups. The activity of CAT was slightly higher in the nebivolol þ hHcy group compared to that in the hHcy group. In the heart tissue, the MDA level (P < 0.001) was lower, the GSH level (P < 0.001) was higher, and the SOD activity (P < 0.001) was higher in the nebivolol þ hHcy group as compared to the hHcy group. In the liver tissue, as compared with the other groups, the MDA level was higher and the SOD activity was lower in the hHcy group (P < 0.001 and P < 0.05, respectively). In the kidney tissue, the anti-oxidant parameters (GSH level and CAT and SOD activity) were significantly higher in the nebivolol þ hHcy group as compared to the hHcy group. The MDA level of the nebivolol þ hHcy group was lower than that of the hHcy group. However, the difference was not statistically significant (P > 0.05). The mean TCSA, LCSA and IMT values for the control and treatment groups (nebivolol and nebivolol þ hHcy groups) are given in Table 1. The TCSA, LCSA and IMT were significantly higher (P < 0.001) in the hHcy group than in the control group. The TCSA, LCSA and IMT were significantly lower in the nebivolol group (P < 0.001) than in the control group. The results of the histological analysis of the IMT sections are shown Fig. 3. There was no difference in the LCSA between the hHcy and nebivolol þ hHcy groups. However, the TCSA and IMT were significantly lower in the nebivolol þ hHcy group than in the hHcy group (P < 0.001).
4. Discussion In this study, we demonstrated that nebivolol may be useful in alleviating hHcy-induced oxidative stress in tissues, such as brain, heart, liver and kidney. We also showed that it may be useful in preventing hHcy-induced structural deterioration of the vascular wall of the aorta. To the best of our knowledge, this is the first report to demonstrate the potential usefulness of nebivolol treatment in alleviating hHcy. In this study, we used the experimental model of Cao et al. to produce hHcy [29]. We achieved intermediate hHcy in our model (37.13 ± 2.89 mmol/L) and demonstrated related changes in the wall of the aorta. Actually, hHcy has been shown to be related to endothelial damage and is accepted be an independent risk factor for the atherosclerosis. Of interest, a previous study showed that atherosclerotic lesions in the aortic root were larger in apolipoprotein E-deficient hyper-homocysteinaemic mice than in a control group [30]. Another study demonstrated that hHcy promoted low-
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Fig. 2. MDA (A) and GSH (B) levels and CAT (C) and SOD (D) activities in brain, heart, liver and kidney tissues of chronic hHcy-induced rats (n ¼ 6). Each value represents the mean ± SE. a, b, c, d The different letters indicate statistically significant differences. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001. hHcy: hyper-homocysteinaemia, MDA: malondialdehyde. GSH: glutathione, CAT: catalase, SOD: superoxide dismutase.
Table 1 The mean total cross sectional area (TCSA), lumen cross sectional area (LCSA) and intima-media thickness (IMT) values in the control and treatment groups (n ¼ 6 in each group). Parameters
Control
Nebivolol
hHcy
Nebivolol þ hHcy
TCSA (mm2) LCSA (mm2) IMT (mm)
1.94 ± 0.03 1.33 ± 0.02 81.63 ± 0.99
1.80 ± 0.04# 1.20 ± 0.05# 76.67 ± 1.38#
2.12 ± 0.07* 1.48 ± 0.08* 85.64 ± 0.94*
2.00 ± 0.03 1.46 ± 0.02* 81.07 ± 2.00
#
, * Denotes P < 0.001 compared with control data. hHcy: Hyperhomocysteinemia.
density lipoprotein oxidation and internalisation, which is thought to play an important role in the initial step of atherosclerosis [31]. One of the suggested mechanisms by which Hcy induces atherosclerosis is to promote the proliferation of vascular smooth muscle cells and endothelial dysfunction in an ROS-dependent manner [32]. In our study, the TCSA, LCSA and IMT were significantly higher in the aortas of the hHcy group than in the control group. An earlier study demonstrated that the intima of the descending aorta in rats fed a methionine-rich diet was thicker [33]. Ovechkin et al. showed that the aortic wall thickness increased in hyper-homocysteinaemic mice and that it was positively correlated with levels of plasma Hcy [34]. Moreover, Boyacioglu et al. recently demonstrated a significantly greater aortic diameter and thickness of aortic elastic laminae in hyper-homocysteinaemic rats [35]. In a previous study, hHcy was also associated with a greater carotid IMT [36]. Regarding Hcy's potential of damage, lots of clinical and experimental studies attempted to find a solution to lower Hcy serum levels and to improve its affects [16e19]. B-complex vitamins are
the mostly studied agents. The B-complex vitamins are required for the transformation, excretion, or for both steps in the total homocysteine levels metabolism pathway [16]. Supplementation with Bcomplex vitamins has been shown to reduce total Hcy levels [16]. However, clinical results of Hcy lowering with B-complex vitamins were generally disappointing except for the very few studies which were able to demonstrate improvement of endothelial function [16e19]. It should be noted that, the results of the existing clinical trials have demonstrated that reducing the Hcy level does not cause positive results in terms of reducing mortality risk of cardiovascular diseases [16,17,19]. Recently melatonin was suggested to lower Hcy levels in rats and genistein was suggested to attenuate the affects of Hcy on the endothelium in ovariectomised rats [6,7]. Oestradiol, vitamin C and Radix salviae miltiorrhizae (commonly known as “Danshen”) are the other molecules that were studied to attenuate the effects of Hcy in hHcy induced rat models [33,35,37]. In this particular study, we demonstrated that nebivolol attenuated hHcy. Nebivolol is the most selective b-1 receptor antagonist in clinical use [14]. Studies have shown that it causes vasorelaxation via a NO/Cyclic guanosine monophosphate (cGMP) dependent pathway in various vascular beds [38]. Another study demonstrated that nebivolol might provide protection against hydroxyl radical ($OH)-induced injury in heart tissue [39]. Akçay et al. previously demonstrated that nebivolol appeared to reduce MDA levels in patients with slow coronary flow [40]. Although the exact mechanism underlying the deterioration of hHcy is unknown, the stimulation of GSH production by nebivolol may play an important role. GSH is produced by the condensation of the amino acids glutamate and cysteine. GSH production requires cysteine as a substrate and when cysteine is required by an organism, Hcy is converted to
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Fig. 3. Image of the intima-media thickness (IMT) of the thoracic aortas in the control and experimental groups (n ¼ 6 in each group). The histological sections were stained with the Orcein-Giemsa method to identify elastic laminae. A) control group, B) nebivolol group, C) hHcy group D) nebivolol þ hHcy group. hHcy: hyper-homocysteinaemia, white arrows: elastic lamina of the aorta, L: lumen, CT: connective tissue (bar: 20 mm, 40).
cysteine via the trans-sulphuration pathway [1]. Moreover, we demonstrated that nebivolol significantly reduced the TCSA and IMT as compared to the control group. Of interest, Guerrero et al. previously showed that nebivolol treatment significantly reduced the aortic medial thickness and cross-sectional area in spontaneously hypertensive rats [41]. We also showed that both the TCSA and IMT were significantly lower in the nebivolol þ hHcy group as compared to the hHcy group, suggesting improving effects of nebivolol against hHcy. Moreover, there were no significant differences in the TCSA and IMT of the control and nebivolol þ hHcy groups, a data supporting the preventive role of nebivolol against affects of hHcy. In the present study, the LCSA was significantly higher in the hHcy and nebivolol þ hHcy group than in the control group. We think that this might be a consequence of vascular outward remodelling. Outward vascular remodelling during atherosclerosis compensates for the plaque growth and postpones the progression to flow-limiting stenosis. It is generally accepted and acknowledged that vascular remodelling is an important determinant in vascular pathologies [42]. An earlier study showed that Hcy was related to aortic aneurysms, which are known to be correlated with outward vascular remodelling [43]. In addition, nebivolol improved vascular remodelling [44]. We suggest that a compensatory increase might have occurred in the lumen of the aorta in response to the effect of hHcy. Nebivolol seems to improve vascular structural changes induced by hHcy, and its main action seems to be in the intima media. We suggest that the anti-oxidant effects of nebivolol may have contributed to these findings. Of note, De Groot et al. previously demonstrated that nebivolol alleviated ROS-induced impairment of endothelium-dependent vasorelaxation in the aorta [14]. We demonstrated a close relation between oxidative stress and hHcy by measuring SOD, MDA, CAT and GSH in brain, liver, kidney and heart tissues. In vitro studies and experimental models have associated hHcy with oxidative stress [11,45]. Previous studies
demonstrated a close relation between a high methionine/low folate diet and free radical generation in various tissues, such as the liver, renal cortex and heart [11]. Of interest, Yi et al. previously demonstrated that oxidative stress might play an important role in the renal damage caused by hHcy [46]. In our experimental model, we found that MDA levels increased in response to oral L-methionine administration, pointing to hHcyinduced oxidative stress in the brain, heart, liver and kidney tissues. We also showed that the administration of nebivolol significantly reduced the increased MDA levels. Decreased GSH activity may lead to breakdown in the ability of tissue to counteract fast generating superoxide anions (O2) or to protect the cells from reactive free radicals and peroxides [37]. GSH reductase mediates the reduction of oxidised glutathione to GSH. Nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) is an electron donor in this reaction and the NADPH oxidase enzyme plays a crucial role in this reaction by producing O2 and ROS [46]. Investigators reported that hHcy reduced GSH levels in endothelial cells or tissues [35,37]. Moreover, a previous study suggested that NADPH oxidase might play an important role in glomerular injury induced by hHcy [46]. In another study, the administration of L-Hcy to cultured rat mesangial cells markedly increased NADPH oxidase activity and caused structural changes, resulting in enhanced cell proliferation [47]. On the other hand, another study clearly demonstrated that nebivolol inhibited NADPH oxidase activity, prevented oxidative stress and attenuated endothelial dysfunction [48]. The effects of nebivolol on NADPH oxidase activity might have contributed to our findings. In our study, the GSH level of the tissues decreased significantly in the hHcy group. We also showed that nebivolol significantly prevented the hHcyinduced reduction of GSH levels in the kidney, heart and brain tissues. Moreover, we demonstrated that 4 weeks of concomitant nebivolol treatment with methionine administration significantly
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prevented hHcy induced deterioration of SOD activity in these tissues. There are some limitations of the study. The number of subjects were of borderline. Moreover, the design of our study is not sufficient to depict the underlying exact mechanism of nebivolol to reverse the affects of hHcy on the vascular structure. It may result as a consequence of nebivolol's direct effects or as a consequence of its effects on blood pressure, lipid profile or nitric oxid levels. These data are missing due to our study design and limited supply. These limitations restrict the significance of the study. 5. Conclusion In conclusion, in this study, nebivolol seemed to have an attenuating effect of the oxidative stress in brain, kidney, liver and heart tissues and improving structural changes of the aorta in a hHcy-induced rat model. Moreover, nebivolol appeared to decrease Hcy. Thus, nebivolol may be useful in clinical scenarios where hHcy affects physiopathological pathways. Further experimental and clinical studies are needed. Conflict of interest The authors of this manuscript declare that they do not have any potential conflict of interest. Acknowledgements Part of this study was supported by the University of Adnan Menderes Scientific Research Projects Commission (TPF-12010). References [1] J. Selhub, Homocysteine metabolism, Annu Rev. Nutr. 19 (1999) 217e246. [2] J.W. Cook, L.M. Taylor, S.L. Orloff, G.J. Landry, G.L. Moneta, J.M. Porter, Homocysteine and arterial disease-experimental mechanisms, Vasc. Pharmacol. 5 (2002) 293e300. [3] K.S. McCully, Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis, Am. J. Pathol. 56 (1969) 111e128. [4] A.G. Bostom, H. Silbershatz, I.H. Rosenberg, J. Selubh, R.B. D'Agostino, P.A. Wolf, et al., Nonfasting plasma total homocysteine levels and all cause and cardiovascular disease mortality in elderly Framingham men and women, Arch. Intern Med. 159 (1999) 1077e1080. [5] H. Refsum, P.M. Ueland, O. Nygard, S.E. Vollset, Homocysteine and cardiovascular disease, Annu Rev. Med. 49 (1998) 31e62. [6] E. Murawska-Cialowicz, L. Januszewska, J. Zuwala-Jagiello, et al., Melatonin decreases homocysteine level in blood of rats, J. Physiol. Pharmacol. 59 (2008) 717e729. [7] P. Zhen, Q. Zhao, D. Hou, et al., Genistein attenuates vascular endothelial impairment in ovariectomized hyperhomocysteinemic rats, J. Biomed. Biotechnol. (2012) 730462. [8] E. Osto, F. Cosentino, The role of oxidative stress in endothelial dysfunction and vascular inflammation, Nitric Oxide-Biol Ch 2 (2010) 705e754. [9] V. Victor, M. Rocha, E. Sola, C. Banuls, K. Garcia-Malpartida, A. HernandezMijares, Oxidative stress, endothelial dysfunction and atherosclerosis, Curr. Pharm. Des. 15 (2009) 2988e3002. [10] H. Yamada, N. Akahoshi, S. Kamata, et al., Methionine excess in diet induces acute lethal hepatitis in mice lacking cystathionine g-lyase, an animal model of cystathioninuria, Free Radic. Biol. Med. 52 (2012) 1716e1726. [11] M. Pravenec, V. Kozich, J. Krijt, et al., Folate deficiency is associated with oxidative stress, increased blood pressure, and insulin resistance in spontaneously hypertensive rats, Am J Hypertens 26 (2013) 135e140. [12] A. Kuroedov, F. Cosentino, T.F. Lüscher, Pharmacological mechanisms of clinically favorable properties of a selective beta1-adrenoceptor antagonist, nebivolol, Cardiovasc Drug Rev. 3 (2004) 155e168. [13] R. Troost, E. Schwedhelm, S. Rojczyk, et al., Nebivolol decreases systemic oxidative stress in healthy volunteers, Br. J. Clin. Pharmacol. 50 (2000) 377e379. [14] A.A. de Groot, M.J. Mathy, P.A. van Zwieten, S.L. Peters, Antioxidant activity of nebivolol in the rat aorta, J. Cardiovasc Pharmacol. 1 (2004) 148e153. [15] F. de Nigris, F.P. Mancini, M.L. Balestrieri, R. Byrns, C. Fiorito, S. WilliamsIgnarro, A. Palagiano, E. Crimi, L.J. Ignarro, C. Napoli, Therapeutic dose of nebivolol, a nitric oxide-releasing beta-blocker, reduces atherosclerosis in cholesterol-fed rabbits, Nitric Oxide 1 (2008) 57e63, http://dx.doi.org/ 10.1016/j.
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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Nebivolol to attenuate the effects of hyper-homocysteinaemia in rats Cagdas Akgullu a, *, Mustafa Ahmet Huyut b, Murat Boyacioglu c, Ozay Gules¸ d, €r a Ufuk Eryilmaz a, Tolga Hekim b, Emir Dogan e, Cemil Zencir a, Hasan Güngo a
Department of Cardiology, Faculty of Medicine, Adnan Menderes University, Aydin, Turkey Department of Cardiology, Aydın City Hospital, Aydin, Turkey c Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Adnan Menderes University, Aydin, Turkey d Department of Histology and Embryology, Faculty of Veterinary Medicine, Adnan Menderes University, Aydin, Turkey e Department of Cardiology, Ada Tıp Hospital, Sakarya, Turkey b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 October 2014 Received in revised form 13 February 2015 Accepted 21 February 2015 Available online 28 February 2015
Objective: This study investigated the prophylactic effect of nebivolol against hyper-homocysteinaemia (hHcy) induced oxidative stress in brain, heart, liver and kidney tissues and histomorphometric changes in the thoracic aorta. Methods: Twenty-four adult male Wistar rats were divided into a control, nebivolol, hHcy and nebivolol þ hHcy group. hHcy was induced by oral administration of L-methionine (1 g/kg/day) for 28 days. 10 mg/kg/day nebivolol was administered orally for 28 days. Malondialdehyde (MDA) and glutathione (GSH) levels and catalase (CAT) and superoxide dismutase (SOD) activities in the tissues were determined. The total cross-sectional area (TCSA), luminal cross-sectional area (LCSA) and intima-media thickness (IMT) were measured in the thoracic aorta. Results: Homocysteine (Hcy) levels were lower in the nebivolol þ hHcy group than in the hHcy group. Nebivolol treatment significantly decreased high MDA levels in the brain, heart and liver tissues. The level of GSH was higher in the brain, heart and kidney tissues of the nebivolol þ hHcy group (P < 0.001). The activity of CAT increased only in the kidney tissue of the nebivolol þ hHcy group (P < 0.01), and the activity of SOD was significantly increased in all the tissues in this group. Increased TCSA and IMT in the nebivolol þ hHcy group were significantly decreased after nebivolol administration. The LCSA was significantly higher in the hHcy group than the control group, probably due to outward vascular remodelling. Conclusion: Nebivolol treatment may be useful in different clinical scenarios where hHcy affects physiopathological pathways. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Hyper-homocysteinaemia Nebivolol Oxidant/anti-oxidant parameters Rats Aorta
1. Introduction Homocysteine (Hcy) is a sulphur-containing amino acid. The metabolism of Hcy involves two pathways: remethylation to methionine, which requires folate and vitamin B12, and transsulphuration to cystathionine, which requires vitamin B6 [1]. Different defects at different levels of this process may lead to hyperhomocysteinemia (hHcy). Genetic diseases, acquired pathologies or poor dietary habits can cause hHcy [1]. Normally, the fasting plasma level of Hcy is between 5 and 15 mmol/L. A level of
* Corresponding author. Department of Cardiology, Medical Faculty, Adnan Menderes University, 090100 Aytepe, Aydin, Turkey. E-mail address: [email protected] (C. Akgullu). http://dx.doi.org/10.1016/j.atherosclerosis.2015.02.054 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
15e30 mmol/L is considered moderate, whereas levels of 30e100 mmol/L and 100e500 mmol/L are considered intermediate and severe hHcy, respectively [2]. The literature and the data about the relation between the homocysteine and atherosclerosis date back to 45 years. In 1969, depending on observations of vascular lesions among the children with hyperhomocysteinemia, Kilmer McCully proposed a hypothesis that homocysteine is atherogenic [3]. Since then numerous studies with a cumulative data have confirmed that an elevation of total plasma homocysteine (tHcy) is related to early atherosclerosis [4,5]. Today it is well known that hHcy elicits a cascade of events that injure the vascular wall [2]. These include chemical modification of lipoproteins, alterations of vascular structure, endothelial dysfunction, impairment of cell repair and proliferation of vascular smooth muscle cells [6,7]. Moreover, a rise in plasma Hcy levels was
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proposed to contribute to ischaemic changes and alter the structure and function of blood vessels through oxidative stress [2,8]. Oxidative stress appeared to give rise to vascular diseases and atherosclerosis via reactive oxygen species (ROS), with injury occurring when the critical balance between free radicals and the synthesis of anti-oxidant defence was altered [9]. Of interest, high levels of methionine or low levels of folate both of which contribute hHcy, seemed to have an adverse effect on the oxidant/anti-oxidant status of the liver and renal tissues and cause injury to these tissues [10,11]. Nebivolol is a racemic mixture of two stereoisomers: d-nebivolol and l-nebivolol and has high affinity and selectivity for b1adrenoceptor binding sites [12]. In addition to its b1 receptoreblocking and nitric oxid (NO)-releasing effects, nebivolol can substantially inhibit vascular oxidative stress [12]. A previous study demonstrated that nebivolol significantly decreased levels of 8-isoprostaglandin F2a (PGF2a) a marker of oxidative stress, in urine samples of healthy volunteers [13]. Moreover, nebivolol was shown to facilitate ROS scavenging in the rat aorta and to alleviate oxidative stress-induced impairment of endothelium-dependent vasorelaxation [14]. Besides, nebivolol was shown to prevent the development of atherosclerosis in cholesterol fed-rabbits [15]. There is a controversy in the literature about the usefulness of Hcy lowering therapy mainly with vitamin supplements to reduce the cardiovascular events [16e19]. Recent meta-analysis suggested that folic acid supplementation may improve endothelial dysfunction as assessed by flow-mediated vasodilation in the brachial artery in patients with coronary heart disease [18]. However, most of the analysis are currently in favour of no benefit from vitamin supplements to reverse the unfavourable effects of Hcy on the endothelium [16,17]. More effective agents are needed both to lower the serum Hcy levels and protect the endothelium from its harm. Nebivolol with its anti-hypertensive, anti-oxidant and NOreleasing effects may provide a good tool to reverse the unfavourable affects of Hcy on the endothelium which promotes atherosclerosis. To the best of our knowledge, there is no information available on the potentially preventive effects of nebivolol against the hHcy-induced damage in the wall of the aorta. Moreover, there are no data about the nebivolol to improve hHcyinduced oxidative stress in different rat tissues. The purpose of this study was to investigate the prophylactic effect of nebivolol against oxidative stress in brain, heart, liver and kidney tissues. Moreover, we aimed to demonstrate the potential prophylactic effect of nebivolol against hHcy-induced changes in the wall of the aorta. 2. Material and methods 2.1. Animals and experimental protocol Twenty-four healthy adult male Wistar rats (16 weeks old, weighing between 322 and 354 g) were used in the study. The animals were obtained from the Adnan Menderes University, Faculty of Medicine, Experimental Research Centre, Aydin, Turkey. They were placed in screen-bottomed stainless steel cages at 22e24 C in a room with a 12/12 h light/dark cycle. The rats were randomly divided into four groups (n ¼ 6 rats per group): a control group, nebivolol group, hHcy group and nebivolol þ hHcy group. They received a commercial rodent diet and had free access to tap water. After 15 days of acclimatisation, the rats were weighed. hHcy was induced with 1 g/kg/day of L-methionine (M9625 L-methionine reagent grade, 98% SigmaeAldrich (Gillingham, Dorset, UK), which was administered directly into the stomach via oral gavage in the mornings between 7:00 and 8:00 am [20]. Nebivolol
(Vasoxen 5 mg, Ibrahim Etem Ulugay, Istanbul, Turkey) was administered at a dose of 10 mg/kg/day via oral gavage. To prevent the interaction of nebivolol and methionine in the stomach, nebivolol was administered 6 h after the administration of methionine. The substances were administered to the rats by daily oral gavage for 28 days. The control animals received 0.9% NaCl solution in the same volumes as those applied to the nebivolol-treated rats by daily oral gavage for 28 days. L-methionine was dissolved in phosphate buffer (pH 7.4), and nebivolol was dissolved in 150 mM of distilled water. Serum samples were collected from the heart by cardiac puncture after the induction of anaesthesia with ketamine and xylasine (50 mg/kg and 5 mg/kg, respectively) at the end of the experiment. The animals were sacrificed by cervical dislocation, and the thoracic aorta was immediately removed for histomorphometric evaluation. To determine the oxidant/anti-oxidant status, malondialdehyde (MDA) and glutathione (GSH) levels and catalase (CAT) and superoxide dismutase (SOD) activities were determined in brain, heart, liver and kidney tissues. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experimental protocol was approved by the Animal Ethics Committee of the University of Adnan Menderes (2011/076). 2.2. Measurement of serum Hcy levels The concentration of Hcy in the serum samples was determined with a commercial ELISA test kit (CSB-E13376r, Cusabio Biotech Co., New Jersey, USA). 2.3. Biochemical analysis of tissues Dissected brain, heart, liver and kidney tissues were immediately rinsed in ice-cold phosphate-buffered saline. Tissues were homogenised (2000 rpm/min for 1 min, 1/10 w/v) using a Teflonglass stirrer (IKA Overhead Stirrer; IKA-Werke GmbH & Co. KG, Staufen, Germany) in a 10% 150 mM phosphate buffer (pH 7.4) in an ice bath. The homogenate was centrifuged (Hettich Zentrifugen, Mikro 200 R, Tuttlingen, Germany) at 6000 g for 10 min at 4 C. The supernatants were frozen at 80 C (Glacier Ultralow Temperature Freezer, Japan) until analysed and then used for determination of MDA and GSH levels and CAT and SOD activities. 2.4. Determination of the oxidant/anti-oxidant status of the tissues 2.4.1. MDA levels in the tissues The lipid peroxidation levels were determined according to the concentration of thiobarbituric acid reactive substances, and the amount of MDA produced was used as an index of lipid peroxidation. Absorbance was measured with a spectrophotometer at 532 nm. The concentration of MDA was calculated by the absorbance complex (absorbance coefficient ε ¼ 1.56 105/M/cm) and expressed as nmol/mg of tissue protein [21]. 2.4.2. GSH levels in the tissues The tissue GSH concentration was measured with a kinetic assay using the 5, 50 -dithiobis (2-nitrobenzoic acid) recycling method described by Tietze [22]. The results were compared with those of an aqueous standard solution of GSH (Sigma Chemical Co., St. Louis, Missouri, USA) and expressed as mg/g of tissue protein. Absorbance was spectrophotometrically monitored at 412 nm against reagent controls. 2.4.3. CAT activity in the tissues CAT activity was determined by measuring the decomposition of
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hydrogen peroxide at 240 nm spectrophotometrically, according to the method of Bergmeyer et al., and it was expressed as k/mg of tissue protein, where k is the first-order rate constant [23]. 2.4.4. SOD activity in the tissues SOD activity was determined according to the method of Sun et al., and the absorbance was measured at 560 nm with a spectrophotometer [24]. This method is based on the inhibition of nitro blue tetrazolium reduction using the xanthine:xanthine oxidase system as a superoxide generator. SOD activity was then measured by the degree of inhibition of this reaction. The results are shown as U/mg of tissue protein. The enzyme activity assays were performed in duplicate, and the results were averaged. The protein levels in the tissues were determined by the method described by Lowry et al. [25]. 2.5. Histomorphometric evaluation The thoracic aortas were removed in each animal group. The total cross-sectional area (TCSA), luminal cross-sectional area (LCSA) and intima media thickness (IMT) of the thoracic aorta were determined. The tissues were cleaned with phosphate-buffered saline and fixed in buffered 10% formalin solution, dehydrated in a graded series of ethanol, cleared in xylene and embedded in paraffin. The paraffin tissue blocks were cut at intervals of 100 mm and a thickness of 6 mm. The histological sections were placed on glass slides. Crossman's triple staining method [26] was used to measure the TCSA and LCSA, and the Orcein-Giemsa staining method [27] was applied for the identification of elastic laminae. Five serial sections were taken from each rat for each staining method. TCSA and LCSA were measured in five different sections. The IMT was measured in five random areas of each section. A Leica DC200 CCD camera (Germany) and Q-win standard image analysis software (Version 2.8) connected to a research microscope (Leica DMLB- Germany) were used to obtain histomorphometric data from the sections. 2.6. Statistical analysis All the analyses were performed with the Statistical Package for the Social Sciences (SPSS) software (Version 11.5). All the values are presented as the mean ± SEM. Differences were considered significant at P < 0.05. The KruskaleWallis analysis of variance (ANOVA) or a one-way ANOVA was used to compare the data among the groups, depending on whether the data were normally distributed. Post-hoc multiple comparisons were performed using the ManneWhitney U test, with Bonferroni correction or Duncan's test [28]. 3. Results There was no significant difference between the groups by means of weights (nebivolol group: 340.2 ± 3.07 g, hHcy group: 336.8 ± 4.08 g, nebivolol þ hHcy group: 334.7 ± 3.84 g, control group: 338.3 ± 2.3 g) (P > 0.05). The Hcy levels of the hHcy group were significantly higher than those of the other groups (P < 0.001) (Fig. 1). The Hcy level of the nebivolol þ hHcy group was lower than that of the hHcy group (12.8 ± 0.75 versus 37.13 ± 2.89 mmol/L) (P < 0.001). The Hcy level of the nebivolol group was slightly lower than that of the control group (5.18 ± 0.48 versus 7.36 ± 0.52 mmol/L) (P > 0.05). The influence of nebivolol on MDA and GSH levels and CAT and SOD activities in the brain, heart, liver and kidney tissues of the chronic hHcy-induced rats is shown in Fig. 2. In the brain tissue, the GSH level (P < 0.001) and SOD activity (P < 0.001) were higher and
Fig. 1. Effect of nebivolol on the serum homocysteine (Hcy) level in chronic hyperhomocysteinaemia (hHcy)-induced rats (n ¼ 6 in each group). a, b, c The different letters indicate statistically significant differences in the same column. ***P < 0.001; Hcy: homocysteine; hHcy: hyper-homocysteinaemia.
the MDA level was lower (P < 0.01) in the hHcy group than in the nebivolol þ hHcy and control groups. The activity of CAT was slightly higher in the nebivolol þ hHcy group compared to that in the hHcy group. In the heart tissue, the MDA level (P < 0.001) was lower, the GSH level (P < 0.001) was higher, and the SOD activity (P < 0.001) was higher in the nebivolol þ hHcy group as compared to the hHcy group. In the liver tissue, as compared with the other groups, the MDA level was higher and the SOD activity was lower in the hHcy group (P < 0.001 and P < 0.05, respectively). In the kidney tissue, the anti-oxidant parameters (GSH level and CAT and SOD activity) were significantly higher in the nebivolol þ hHcy group as compared to the hHcy group. The MDA level of the nebivolol þ hHcy group was lower than that of the hHcy group. However, the difference was not statistically significant (P > 0.05). The mean TCSA, LCSA and IMT values for the control and treatment groups (nebivolol and nebivolol þ hHcy groups) are given in Table 1. The TCSA, LCSA and IMT were significantly higher (P < 0.001) in the hHcy group than in the control group. The TCSA, LCSA and IMT were significantly lower in the nebivolol group (P < 0.001) than in the control group. The results of the histological analysis of the IMT sections are shown Fig. 3. There was no difference in the LCSA between the hHcy and nebivolol þ hHcy groups. However, the TCSA and IMT were significantly lower in the nebivolol þ hHcy group than in the hHcy group (P < 0.001).
4. Discussion In this study, we demonstrated that nebivolol may be useful in alleviating hHcy-induced oxidative stress in tissues, such as brain, heart, liver and kidney. We also showed that it may be useful in preventing hHcy-induced structural deterioration of the vascular wall of the aorta. To the best of our knowledge, this is the first report to demonstrate the potential usefulness of nebivolol treatment in alleviating hHcy. In this study, we used the experimental model of Cao et al. to produce hHcy [29]. We achieved intermediate hHcy in our model (37.13 ± 2.89 mmol/L) and demonstrated related changes in the wall of the aorta. Actually, hHcy has been shown to be related to endothelial damage and is accepted be an independent risk factor for the atherosclerosis. Of interest, a previous study showed that atherosclerotic lesions in the aortic root were larger in apolipoprotein E-deficient hyper-homocysteinaemic mice than in a control group [30]. Another study demonstrated that hHcy promoted low-
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Fig. 2. MDA (A) and GSH (B) levels and CAT (C) and SOD (D) activities in brain, heart, liver and kidney tissues of chronic hHcy-induced rats (n ¼ 6). Each value represents the mean ± SE. a, b, c, d The different letters indicate statistically significant differences. NS, not significant. *P < 0.05, **P < 0.01, ***P < 0.001. hHcy: hyper-homocysteinaemia, MDA: malondialdehyde. GSH: glutathione, CAT: catalase, SOD: superoxide dismutase.
Table 1 The mean total cross sectional area (TCSA), lumen cross sectional area (LCSA) and intima-media thickness (IMT) values in the control and treatment groups (n ¼ 6 in each group). Parameters
Control
Nebivolol
hHcy
Nebivolol þ hHcy
TCSA (mm2) LCSA (mm2) IMT (mm)
1.94 ± 0.03 1.33 ± 0.02 81.63 ± 0.99
1.80 ± 0.04# 1.20 ± 0.05# 76.67 ± 1.38#
2.12 ± 0.07* 1.48 ± 0.08* 85.64 ± 0.94*
2.00 ± 0.03 1.46 ± 0.02* 81.07 ± 2.00
#
, * Denotes P < 0.001 compared with control data. hHcy: Hyperhomocysteinemia.
density lipoprotein oxidation and internalisation, which is thought to play an important role in the initial step of atherosclerosis [31]. One of the suggested mechanisms by which Hcy induces atherosclerosis is to promote the proliferation of vascular smooth muscle cells and endothelial dysfunction in an ROS-dependent manner [32]. In our study, the TCSA, LCSA and IMT were significantly higher in the aortas of the hHcy group than in the control group. An earlier study demonstrated that the intima of the descending aorta in rats fed a methionine-rich diet was thicker [33]. Ovechkin et al. showed that the aortic wall thickness increased in hyper-homocysteinaemic mice and that it was positively correlated with levels of plasma Hcy [34]. Moreover, Boyacioglu et al. recently demonstrated a significantly greater aortic diameter and thickness of aortic elastic laminae in hyper-homocysteinaemic rats [35]. In a previous study, hHcy was also associated with a greater carotid IMT [36]. Regarding Hcy's potential of damage, lots of clinical and experimental studies attempted to find a solution to lower Hcy serum levels and to improve its affects [16e19]. B-complex vitamins are
the mostly studied agents. The B-complex vitamins are required for the transformation, excretion, or for both steps in the total homocysteine levels metabolism pathway [16]. Supplementation with Bcomplex vitamins has been shown to reduce total Hcy levels [16]. However, clinical results of Hcy lowering with B-complex vitamins were generally disappointing except for the very few studies which were able to demonstrate improvement of endothelial function [16e19]. It should be noted that, the results of the existing clinical trials have demonstrated that reducing the Hcy level does not cause positive results in terms of reducing mortality risk of cardiovascular diseases [16,17,19]. Recently melatonin was suggested to lower Hcy levels in rats and genistein was suggested to attenuate the affects of Hcy on the endothelium in ovariectomised rats [6,7]. Oestradiol, vitamin C and Radix salviae miltiorrhizae (commonly known as “Danshen”) are the other molecules that were studied to attenuate the effects of Hcy in hHcy induced rat models [33,35,37]. In this particular study, we demonstrated that nebivolol attenuated hHcy. Nebivolol is the most selective b-1 receptor antagonist in clinical use [14]. Studies have shown that it causes vasorelaxation via a NO/Cyclic guanosine monophosphate (cGMP) dependent pathway in various vascular beds [38]. Another study demonstrated that nebivolol might provide protection against hydroxyl radical ($OH)-induced injury in heart tissue [39]. Akçay et al. previously demonstrated that nebivolol appeared to reduce MDA levels in patients with slow coronary flow [40]. Although the exact mechanism underlying the deterioration of hHcy is unknown, the stimulation of GSH production by nebivolol may play an important role. GSH is produced by the condensation of the amino acids glutamate and cysteine. GSH production requires cysteine as a substrate and when cysteine is required by an organism, Hcy is converted to
C. Akgullu et al. / Atherosclerosis 240 (2015) 33e39
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Fig. 3. Image of the intima-media thickness (IMT) of the thoracic aortas in the control and experimental groups (n ¼ 6 in each group). The histological sections were stained with the Orcein-Giemsa method to identify elastic laminae. A) control group, B) nebivolol group, C) hHcy group D) nebivolol þ hHcy group. hHcy: hyper-homocysteinaemia, white arrows: elastic lamina of the aorta, L: lumen, CT: connective tissue (bar: 20 mm, 40).
cysteine via the trans-sulphuration pathway [1]. Moreover, we demonstrated that nebivolol significantly reduced the TCSA and IMT as compared to the control group. Of interest, Guerrero et al. previously showed that nebivolol treatment significantly reduced the aortic medial thickness and cross-sectional area in spontaneously hypertensive rats [41]. We also showed that both the TCSA and IMT were significantly lower in the nebivolol þ hHcy group as compared to the hHcy group, suggesting improving effects of nebivolol against hHcy. Moreover, there were no significant differences in the TCSA and IMT of the control and nebivolol þ hHcy groups, a data supporting the preventive role of nebivolol against affects of hHcy. In the present study, the LCSA was significantly higher in the hHcy and nebivolol þ hHcy group than in the control group. We think that this might be a consequence of vascular outward remodelling. Outward vascular remodelling during atherosclerosis compensates for the plaque growth and postpones the progression to flow-limiting stenosis. It is generally accepted and acknowledged that vascular remodelling is an important determinant in vascular pathologies [42]. An earlier study showed that Hcy was related to aortic aneurysms, which are known to be correlated with outward vascular remodelling [43]. In addition, nebivolol improved vascular remodelling [44]. We suggest that a compensatory increase might have occurred in the lumen of the aorta in response to the effect of hHcy. Nebivolol seems to improve vascular structural changes induced by hHcy, and its main action seems to be in the intima media. We suggest that the anti-oxidant effects of nebivolol may have contributed to these findings. Of note, De Groot et al. previously demonstrated that nebivolol alleviated ROS-induced impairment of endothelium-dependent vasorelaxation in the aorta [14]. We demonstrated a close relation between oxidative stress and hHcy by measuring SOD, MDA, CAT and GSH in brain, liver, kidney and heart tissues. In vitro studies and experimental models have associated hHcy with oxidative stress [11,45]. Previous studies
demonstrated a close relation between a high methionine/low folate diet and free radical generation in various tissues, such as the liver, renal cortex and heart [11]. Of interest, Yi et al. previously demonstrated that oxidative stress might play an important role in the renal damage caused by hHcy [46]. In our experimental model, we found that MDA levels increased in response to oral L-methionine administration, pointing to hHcyinduced oxidative stress in the brain, heart, liver and kidney tissues. We also showed that the administration of nebivolol significantly reduced the increased MDA levels. Decreased GSH activity may lead to breakdown in the ability of tissue to counteract fast generating superoxide anions (O2) or to protect the cells from reactive free radicals and peroxides [37]. GSH reductase mediates the reduction of oxidised glutathione to GSH. Nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) is an electron donor in this reaction and the NADPH oxidase enzyme plays a crucial role in this reaction by producing O2 and ROS [46]. Investigators reported that hHcy reduced GSH levels in endothelial cells or tissues [35,37]. Moreover, a previous study suggested that NADPH oxidase might play an important role in glomerular injury induced by hHcy [46]. In another study, the administration of L-Hcy to cultured rat mesangial cells markedly increased NADPH oxidase activity and caused structural changes, resulting in enhanced cell proliferation [47]. On the other hand, another study clearly demonstrated that nebivolol inhibited NADPH oxidase activity, prevented oxidative stress and attenuated endothelial dysfunction [48]. The effects of nebivolol on NADPH oxidase activity might have contributed to our findings. In our study, the GSH level of the tissues decreased significantly in the hHcy group. We also showed that nebivolol significantly prevented the hHcyinduced reduction of GSH levels in the kidney, heart and brain tissues. Moreover, we demonstrated that 4 weeks of concomitant nebivolol treatment with methionine administration significantly
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prevented hHcy induced deterioration of SOD activity in these tissues. There are some limitations of the study. The number of subjects were of borderline. Moreover, the design of our study is not sufficient to depict the underlying exact mechanism of nebivolol to reverse the affects of hHcy on the vascular structure. It may result as a consequence of nebivolol's direct effects or as a consequence of its effects on blood pressure, lipid profile or nitric oxid levels. These data are missing due to our study design and limited supply. These limitations restrict the significance of the study. 5. Conclusion In conclusion, in this study, nebivolol seemed to have an attenuating effect of the oxidative stress in brain, kidney, liver and heart tissues and improving structural changes of the aorta in a hHcy-induced rat model. Moreover, nebivolol appeared to decrease Hcy. Thus, nebivolol may be useful in clinical scenarios where hHcy affects physiopathological pathways. Further experimental and clinical studies are needed. Conflict of interest The authors of this manuscript declare that they do not have any potential conflict of interest. Acknowledgements Part of this study was supported by the University of Adnan Menderes Scientific Research Projects Commission (TPF-12010). References [1] J. Selhub, Homocysteine metabolism, Annu Rev. Nutr. 19 (1999) 217e246. [2] J.W. Cook, L.M. Taylor, S.L. Orloff, G.J. Landry, G.L. Moneta, J.M. Porter, Homocysteine and arterial disease-experimental mechanisms, Vasc. Pharmacol. 5 (2002) 293e300. [3] K.S. McCully, Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis, Am. J. Pathol. 56 (1969) 111e128. [4] A.G. Bostom, H. Silbershatz, I.H. Rosenberg, J. Selubh, R.B. D'Agostino, P.A. Wolf, et al., Nonfasting plasma total homocysteine levels and all cause and cardiovascular disease mortality in elderly Framingham men and women, Arch. Intern Med. 159 (1999) 1077e1080. [5] H. Refsum, P.M. Ueland, O. Nygard, S.E. Vollset, Homocysteine and cardiovascular disease, Annu Rev. Med. 49 (1998) 31e62. [6] E. Murawska-Cialowicz, L. Januszewska, J. Zuwala-Jagiello, et al., Melatonin decreases homocysteine level in blood of rats, J. Physiol. Pharmacol. 59 (2008) 717e729. [7] P. Zhen, Q. Zhao, D. Hou, et al., Genistein attenuates vascular endothelial impairment in ovariectomized hyperhomocysteinemic rats, J. Biomed. Biotechnol. (2012) 730462. [8] E. Osto, F. Cosentino, The role of oxidative stress in endothelial dysfunction and vascular inflammation, Nitric Oxide-Biol Ch 2 (2010) 705e754. [9] V. Victor, M. Rocha, E. Sola, C. Banuls, K. Garcia-Malpartida, A. HernandezMijares, Oxidative stress, endothelial dysfunction and atherosclerosis, Curr. Pharm. Des. 15 (2009) 2988e3002. [10] H. Yamada, N. Akahoshi, S. Kamata, et al., Methionine excess in diet induces acute lethal hepatitis in mice lacking cystathionine g-lyase, an animal model of cystathioninuria, Free Radic. Biol. Med. 52 (2012) 1716e1726. [11] M. Pravenec, V. Kozich, J. Krijt, et al., Folate deficiency is associated with oxidative stress, increased blood pressure, and insulin resistance in spontaneously hypertensive rats, Am J Hypertens 26 (2013) 135e140. [12] A. Kuroedov, F. Cosentino, T.F. Lüscher, Pharmacological mechanisms of clinically favorable properties of a selective beta1-adrenoceptor antagonist, nebivolol, Cardiovasc Drug Rev. 3 (2004) 155e168. [13] R. Troost, E. Schwedhelm, S. Rojczyk, et al., Nebivolol decreases systemic oxidative stress in healthy volunteers, Br. J. Clin. Pharmacol. 50 (2000) 377e379. [14] A.A. de Groot, M.J. Mathy, P.A. van Zwieten, S.L. Peters, Antioxidant activity of nebivolol in the rat aorta, J. Cardiovasc Pharmacol. 1 (2004) 148e153. [15] F. de Nigris, F.P. Mancini, M.L. Balestrieri, R. Byrns, C. Fiorito, S. WilliamsIgnarro, A. Palagiano, E. Crimi, L.J. Ignarro, C. Napoli, Therapeutic dose of nebivolol, a nitric oxide-releasing beta-blocker, reduces atherosclerosis in cholesterol-fed rabbits, Nitric Oxide 1 (2008) 57e63, http://dx.doi.org/ 10.1016/j.
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