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The effects of celiprolol on serum concentrations of proinflammatory cytokines in hypertensive (SHR) and normotensive (WKY) rats
Pharmacological Reports 66 (2014) 68–73
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Original research article
The effects of celiprolol on serum concentrations of proinflammatory cytokines in hypertensive (SHR) and normotensive (WKY) rats Dariusz Andrzejczak *, Dorota Go´rska Department of Pharmacodynamics, Medical University of Lodz, Lodz, Poland
A R T I C L E I N F O
Article history: Received 9 January 2013 Received in revised form 30 July 2013 Accepted 13 August 2013 Available online 1 February 2014 Keywords: Celiprolol Proinflammatory cytokines Lipids SHR WKY
A B S T R A C T
Background: A growing body of evidence suggests that some cardiovascular drugs could modulate the level of proinflammatory cytokines. Therefore, the aim of the present study was to investigate whether celiprolol, a third generation b-adrenoceptor blocker, affects lipopolysaccharide (LPS)-induced serum concentrations of TNF-a, IL-1b, IL-6 in normotensive (WKY) and spontaneously hypertensive (SHR) rats. Methods: Celiprolol (150 mg kg 1) or vehicle was administered by gavage once daily for 21 days. Arterial blood pressure was measured in conscious rats, using the tail-cuff method. Serum concentrations of proinflammatory cytokines were measured with enzyme-linked immunosorbent assay kits. Additionally, plasma concentrations of total cholesterol, HDL-cholesterol and triglycerides were evaluated. Results: In normotensive WKY rats celiprolol did not affect heart rate, blood pressure, or the serum concentrations of triglycerides, total cholesterol or HDL-cholesterol. In hypertensive animals the drug decreased lipid parameters, increased diastolic and mean blood pressure after the first week of administration, and produced a small but significant decrease in heart rate after the first two weeks of the treatment. In both groups of animals, celiprolol decreased LPS-stimulated serum concentration of IL6 but did not affect levels of TNF-a and IL-1b. Conclusions: It is suggested that the IL-6-modulating properties of celiprolol could provide additional value to the therapeutic effectiveness of the drug in the treatment of hypertension. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Increased airway resistance, peripheral and coronary vasoconstriction, proatherosclerotic action, and increased insulin resistance are the most important side effects of conventional b-blockers, limiting their therapeutical usefulness [23,26]. A large body of clinical data has demonstrated that the contraindications of celiprolol, a selective b1-blocker endowed with b2-adrenomimetic activity, are markedly lower than those displayed by the first generation of b-adrenolytic drugs. For example, celiprolol is safer than conventional b-blockers in the treatment of asthmatic patients because of lower influence on the airway function [8,32]. Moreover, celiprolol does not produce venodilatation, and has negligible vasoconstricting effects on stenotic and normal coronary arteries. The vascular action of the drug is associated with the activation of b2-adrenoceptors, NO release from the endothelium, and weak
Abbreviations: IL, interleukin; LPS, lipopolysaccharide; SHR, spontaneously hypertensive rats; TNF-a, tumor necrosis factor alpha; WKY, Wistar-Kyoto rats. * Corresponding author. E-mail address: [email protected] (D. Andrzejczak).
blockade of a1-adrenoceptors [32,43]. Other desirable effects of celiprolol include an increase in insulin sensitivity in healthy volunteers and patients with an insulin-resistance state, and improvement of the serum lipid profile, i.e. a decrease in concentrations of triglycerides and LDL-cholesterol, and an increase in HDL-cholesterol [43]. Accumulating experimental evidence indicates the functional importance of an interplay between the autonomic nervous system and the immune system [15]. b-Adrenoceptors are expressed by various immune cells, such as lymphocytes, macrophages, neutrophils, eosinophils, and basophils. Stimulation of these receptors can lead to changes in the production of proinflammatory cytokines [42,52]. We have previously demonstrated that propranolol and atenolol, non-selective and cardioselective antagonists of b-adrenergic receptors, respectively, are endowed with immunomodulating properties [2,3]. This study analyzes the effects of celiprolol on lipopolysaccharide (LPS)stimulated levels of proinflammatory cytokines, namely tumor necrosis factor alpha (TNF-a), interleukin (IL)-1b and IL-6, in normotensive (Wistar-Kyoto; WKY) and spontaneously hypertensive (SHR) rats. In addition, the effects of celiprolol on heart rate, arterial blood pressure, and serum lipid profile were examined.
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2013.08.006
D. Andrzejczak, D. Go´rska / Pharmacological Reports 66 (2014) 68–73
tion was chosen according to Dredge et al. [13]. The blood was allowed to clot overnight at 4 8C, and then the samples were centrifuged for 20 min at 2000 g. The serum was removed and stored at 20 8C until used for biochemical measurements.
Materials and methods Animals and treatment The study was conducted on 12–14 weeks old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) male rats. The rats were housed in standard plastic cages, 10 animals per cage, at a constant temperature of 21 1 8C, and under an illumination cycle of 12 h light–dark (lights on between 7.00 and 19.00). The animals, with an initial body weight 280–310 g, had free access to standard food and tap water. In order to select the rats for experimentation, preliminary examinations were carried out at the end of the second and third weeks of their adaptation. The animals with high blood pressure fluctuations were excluded from the study. Experiments were conducted between 8 a.m. and 4 p.m. All experimental procedures were performed in accordance with the Polish governmental regulations concerning experiments on animals (Dz.U.05.33.289) and were approved by the Local Ethics Committee for Experimentation on Animals. Celiprolol (Celipres, Ranbaxy Laboratories Ltd., India; 150 mg kg 1 body weight) was suspended in 1% solution of methylcellulose (Sigma–Aldrich, Poznan, Poland) and administered by gavage at a volume of 2 ml kg 1 b.w. once daily for 3 weeks. Control rats received a 1% solution of methylcellulose (2 ml kg 1 b.w.). Measurements of arterial blood pressure were carried out after 7, 14, and 21 days of administration of celiprolol or vehicle. Preliminary studies showed no detectable levels of cytokines in the serum of SHR rats. Thus, in order to achieve a measurable cytokine level, 24 h after the last administration of celiprolol or vehicle, the rats received ip a small dose of LPS from Escherichia coli serotype 055:B5 (Sigma–Aldrich, Poznan, Poland; 0.1 mg kg 1 b.w. in 1 ml of saline kg 1 b.w.). After 2 h, the animals were anesthetized with ethyl ether, and the blood samples were collected by heart puncture. The time of blood sample collection after LPS administraTable 1 Effects of the repeated administration of celiprolol (150 mg kg rats. Time after drug administration (week)
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1
Arterial blood pressure and heart beat measurements Arterial blood pressure was measured in conscious rats by a manometer manufactured by LETICA (Panlab S.L., Spain), using a tail-cuff method as described in details by Go´rska and Andrzejczak [17]. Before the measurements, in order to calm the animals and dilate the tail blood vessels, the rats were placed inside a warming chamber (about 34 8C) for 30 min. The measurements of arterial blood pressure (systolic, diastolic and mean) were carried out at least three times for each animal, and the mean values of several successive measurements were used for further analysis. Changes in blood pressure were expressed as the percentage of baseline values. The heart rate was measured by the same apparatus automatically and was registered as beats per minute. Lipid profile determination Total serum cholesterol concentration was determined by the cholesterol oxidase method using a commercially available kit (Cholesterol CHOD PAP, Biolabo, Maizy, France) according to the manufacturer’s instructions. To measure HDL-cholesterol, low density lipoproteins, very low density lipoproteins, and chylomicrons were precipitated from serum samples by phosphotungstic acid and magnesium chloride. Following that, HDL-cholesterol was measured with the aid of commercially available kit (HDLcholesterol-PTA, Biolabo, Maizy, France). Triglycerides were measured using a commercially available kit (Triglycerides GPO Method, Biolabo, Maizy, France) according to the manufacturer’s instructions.
) on systolic (A), diastolic (B) and mean blood pressure (C) in normotensive (WKY) and hypertensive (SHR)
Mean changes in systolic blood pressure (% of initial values) SHR
WKY Vehicle (n = 10)
Celiprolol (n = 15)
Vehicle (n = 10)
Celiprolol (n = 15)
(A) 1 2 3
99.4 1.6 96.9 1.9 101.5 1.1
96.9 2.0 98.9 2.2 98.4 2.6
99.8 2.3 95.6 2.6 98.9 3.9
97.0 2.6 97.2 3.7 101.7 2.1
Time after drug administration (week)
Mean changes in diastolic blood pressure (% of initial values) WKY
SHR
Vehicle (n = 10)
Celiprolol (n = 15)
Vehicle (n = 10)
Celiprolol (n = 15)
(B) 1 2 3
97.8 4.4 103.1 1.4 101.6 3.6
105.5 4.4 108.8 3.6 109.2 5.7
97.1 3.7 102.6 3.6 98.3 3.7
110.6 3.9* 112.0 2.3* 102.6 2.3
Time after drug administration (week)
Mean changes in mean blood pressure (% of initial values) WKY
(C) 1 2 3
SHR
Vehicle (n = 10)
Celiprolol (n = 15)
Vehicle (n = 10)
Celiprolol (n = 15)
98.3 3.0 101.1 1.3 101.0 2.3
101.9 3.2 105.7 2.6 104.3 4.0
100.3 2.8 99.2 3.1 99.3 3.5
107.3 1.3* 105.4 2.1 102.3 1.1
Values are means SEM. * p < 0.05 in comparison with vehicle-treated control animals. Baseline values of blood pressure (mmHg) for WKY rats in vehicle-treated group: systolic 117.0 2.0; diastolic 84.6 1.7; mean 95.8 1.5. Baseline values of blood pressure (mmHg) for WKY rats in celiprolol-treated group: systolic 118.1 2.2; diastolic 85.1 1.8; mean 96.8 1.8. Baseline values of blood pressure (mmHg) for SHR in vehicle-treated group: systolic 237.3 3.2; diastolic 128.2 2.7; mean 165.2 3.0. Baseline values of blood pressure (mmHg) for SHR in celiprolol-treated group: systolic 235.6 2.4; diastolic 129.2 1.9; mean 164.3 2.0.
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Serum cytokine levels Serum TNF-a, IL-1b and IL-6 concentrations were measured in duplicates with a commercially available enzyme-linked immunosorbent assay kit (Quantikine, R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions. Analysis of data The results are expressed as the mean SEM values. The normality of distribution was checked by means of a combination of the Kolmogorov–Smirnov test and the Lilliefors test. The data were analyzed for statistical significance by one-way ANOVA followed by a post hoc Least Significant Differences (LSD) test, using StatSoft, Inc. (2010) STATISTICA version 9.1. If the data were not normally distributed, statistical evaluation was performed by using ANOVA (Kruskall–Wallis) and the Mann–Whitney U test. The statistical analysis of data presented in Table 2 was performed with the aid of a t-Test for Dependent Samples. Differences were considered statistically significant when p < 0.05. The initial values of arterial blood pressure were assumed as 100%. Results Blood pressure and heart rate In WKY rats, which were chosen as a control strain, the following values (in mmHg) of arterial blood pressure were found: systolic, 119.7 1.4; diastolic, 85.7 1.6; mean, 96.8 1.5 (n = 10 animals/group). The SHR selected for the study had initial arterial blood pressure (in mmHg): systolic, 235.7 2.9; diastolic, 129.8 1.7; mean, 164.6 1.7 (n = 15 animals/group). They were significantly higher than the corresponding values determined in WKY animals. SHR rats also had a significantly higher basal heart beat rate (332.0 1.9 beats/min; n = 15) compared to WKY rats (297.9 1.1 beats/min; n = 10). Celiprolol (150 mg kg 1 b.w.) did not affect blood pressure and heart rate in normotensive WKY rats (Tables 1 and 2). Administration of the drug to SHR rats resulted in a small, but statistically significant, increase in diastolic blood pressure, by 10% and 12% after the first and the second weeks, respectively, and mean blood pressure, by 7% after the first week (Table 1). Following one week of treatment with celiprolol, small but statistically significant decrease in heart rate of 5% was observed, and after two weeks this grew to 8% (Table 2). Lipid profile In WKY rats celiprolol did not modify serum concentrations of total cholesterol, HDL cholesterol, and triglycerides. On the other Table 2 Effects of the repeated administration of celiprolol (150 mg kg normotensive (WKY) and hypertensive (SHR) rats. Time after drug administration (week)
1 2 3
1
) on heart rate in
Fig. 1. Effects of the repeated administration of celiprolol (150 mg kg 1) on serum concentration of triglycerides, total cholesterol and HDL-cholesterol in WKY and SHR rats. Values are means SEM. *p < 0.05 in comparison with control group (vehicle-treated rats).
hand, in SHR rats, celiprolol significantly decreased all lipid parameters by 23–27% (Fig. 1). Serum cytokines As concentrations of TNF-a, IL-1b and IL-6 in the serum of SHR rats were found to be below the limits of detection in preliminary experiments, a small dose of LPS (0.1 mg kg 1 b.w.) was used in order to stimulate the production of cytokines to measurable levels. Celiprolol did not affect LPS-induced serum level of TNF-a Table 3 Effects of the repeated administration of celiprolol (150 mg kg 1) on serum concentration of TNF-a, IL-1b and IL-6 in normotensive (WKY) and hypertensive (SHR) rats. Serum concentration of cytokines (pg/ml)
Mean changes in heart rate (beats per min)
Vehicle (n = 10)
Celiprolol (n = 15)
TNF-a IL-1b IL-6
2325.3 122.2 138.2 27.3 7531.1 631.3
1900.7 203.3 121.3 25.9 3438.3 849.8*
Serum concentration of cytokines (pg/ml)
SHR rats
SHR
WKY Vehicle (n = 10)
Celiprolol (n = 15)
Vehicle (n = 10)
Celiprolol (n = 15)
296.7 1.5 295.8 1.4 296.5 1.0
298.5 2.3 300.8 2.4 301.2 2.1
333.3 2.2 329.8 3.8 328.8 3.1
315.2 3.0* 306.3 2.9* 329.0 4.3
Values are means SEM. * p < 0.05 in comparison with baseline value (‘‘0’’) in celiprolol-treated SHR rats. Baseline heart rate values in vehicle-treated WKY 297.9 1.1 beats/min and celiprolol-treated WKY 298.9 1.8. Baseline heart rate values in vehicle-treated SHR rats 332.0 1.9 beats/min and celiprolol-treated SHR 331.5 2.9.
WKY rats
TNF-a IL-1b IL-6
Vehicle (n = 10)
Celiprolol (n = 15)
2227.9 24.2 132.4 40.9 5404.4 596.8
2055.1 272.9 134.3 25.8 2579.1 605.0*
Values are means SEM. * p < 0.05 in comparison with control, vehicle-treated rats.
D. Andrzejczak, D. Go´rska / Pharmacological Reports 66 (2014) 68–73
and IL-1b in WKY and SHR rats. On the other hand, in both strains of animals, the drug decreased IL-6 serum level by approximately 54% (Table 3). Discussion Hypertension is a multifactorial disease with a very complex etiology. An accumulating body of experimental data suggests the involvement of the immune system and a low-grade inflammation process in the development of hypertension and in the cardiovascular complications associated with it [20,21]. Furthermore, ample evidence links proinflammatory cytokines with hypertension, but whether elevated levels of cytokines precede or follow the development of hypertension remains to be elucidated [41,46]. Taking into account the above facts, an additional anti-inflammatory activity of a drug used in the treatment of hypertension could be considered as its therapeutically important property. b-Adrenergic blockers are widely used in the treatment of various cardiovascular diseases, including hypertension. At present, little is known about the effects of this group of drugs on inflammatory mediators in hypertension. Nemati and coworkers [33] analyzed the effects of various hypotensive drugs, including atenolol, on LPS-induced IL-1b secretion from peripheral blood polymorphonuclear leukocytes isolated from normotensive individuals and from patients with essential hypertension. They found no significant effect of this selective b1-blocker on the secretion of cytokine by cells from hypertensive subjects. An eight-week treatment of hypertensive postmenopausal women with atenolol did not affect serum IL-6 level and slightly increased the level of TNF-a [37]. On the other hand, Madej et al. [28] demonstrated that treatment of hypertensive patients with bisoprolol decreased TNFa, IL-1b and IL-6 secretion from peripheral blood mononuclear cells. Using an animal model of hypertension, SHR rats, our previous studies have demonstrated that atenolol does not modify the LPS-induced elevation of TNF-a and IL-1b blood levels, while propranolol, a non-selective b-blocker, was found to reduce the stimulated IL-1b level [2,3]. Celiprolol is generally described as a third generation badrenoceptor antagonist. In addition to selective b1-adrenoceptor blocking properties, the drug has b2 partial agonist properties, exerts direct vasodilator effects, and does not depress the heart rate to the same extent as several other b-blockers. The therapeutic indication for the use of celiprolol includes mainly hypertension and angina complicated by impaired glucose tolerance or diabetes mellitus, peripheral vascular resistance and hyperlipidemia [14]. Data regarding cytokine-modulating properties of celiprolol are sparse. Hayashi et al. [22] observe that celiprolol (100 mg kg 1/day) improved endothelial function in Otsuka Long-Evans Tokushima Fatty diabetic rats. Furthermore, plasma concentrations of TNF-a were significantly elevated in vehicle- and atenolol-treated rats but not in animals receiving celiprolol. In another study, Tsubokou et al. [45] demonstrate that celiprolol decreases upregulated transforming growth factor-b1 (TGF-b1) expression in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Of note, this activity of celiprolol has been recently used in the treatment of vascular Ehlers-Danlos syndrome, where circulating TGF-b likely plays the role of an important pathogenic factor [5,7,24,35]. The results of the present study demonstrated, for the first time, effects of celiprolol on LPS-induced proinflammatory cytokines concentration in normotensive and hypertensive animals. Although the drug did not modify LPS-induced TNF-a and IL-1b serum levels in SHR and WKY rats, it potently decreased IL-6 level in both rat strains. It appears unlikely that the action of celiprolol on LPS-stimulated serum level of IL-6 is related to its partial b2sympathomimetic activity, as stimulation of b2-adrenoceptors
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with isoprenaline, a non-selective b-adrenoceptor agonist, prior to LPS administration resulted in a significant elevation of IL-6 plasma level in mice [42]. The action of celiprolol on IL-6 presumably did not also result from its b1-adrenolytic activity, as results from our laboratory have previously demonstrated that prolonged treatment of rats with atenolol, a selective b1-blocker, increases serum IL-6 levels [3]. IL-6 is a proinflammatory cytokine with pleiotropic activity [29]. A growing body of experimental evidence indicates that IL-6 plays an important role in mediating inflammatory and blood pressure response to angiotensin II. Thus, treatment of C57BL/6J mice with IL-6 increased vascular AT1 receptor expression, angiotensin II-induced vasoconstriction in aortic segments, enhanced vascular superoxide production and impaired endothelium-dependent vasodilatation [48]. During two weeks of angiotensin II infusion, the IL-6 knockout mice had significantly lower blood pressure than wild type animals. This observation led Lee et al. [25] to suggest that hypertension caused by angiotensin II and by a high-salt diet could result, to some extent, from the action of IL-6. Similar results were presented by Coles et al. [11]. Subcutaneous administration of angiotensin II (1.1 mg kg 1/day) to wild type mice evoked hypertension and cardiac hypertrophy. Such effects were not observed in IL-6 knockout mice. A study by Schrader et al. [39] on mice showed that IL-6 could be essential for angiotensin II-induced endothelial dysfunction, i.e. production of reactive oxygen species and reduction of endothelial nitric oxide synthase mRNA expression. In Sprague-Dawley rats, IL-6 caused a pattern of myocardial remodeling similar to that seen in hypertension [31]. It should be emphasized that an association between IL-6 levels and elevated blood pressure/hypertension [9,10], and increased risk of myocardial infarction [38] have been demonstrated in man. The two other studied proinflammatory cytokines, TNF-a and IL-1b, have been proven to exert negative effects on the endothelium. Thus, TNF-a could stimulate the angiotensinogenencoding gene [6] and limit the half-life of eNOS mRNA [51], whereas IL-1b has been shown to favor the development of atheromatous changes [27]. Increased IL-1b serum levels were found in hypertensive patients [12]. Recent studies suggest that TNF-a and IL-1b could act on the brain to increase blood pressure, heart rate, and sympathetic nerve activity [49]. Of note, etanercept, a TNF-a antagonist used mainly in autoimmune diseases, such as rheumatoid arthritis, was shown to prevent the development of hypertension in fructose-fed rats [44]. There are a few published reports on effects of b-blockers on serum levels of TNF-a and IL-1b. By analogy to celiprolol (present data), atenolol [3] and nebivolol [18], a b1-selective adrenergic blocker with additional vasodilating properties, did not modify LPS-stimulated levels of TNF-a and IL-1b in normotensive as well as in hypertensive rats. On the other hand, propranolol, a nonselective b-adrenoceptor antagonist, suppressed gene expression of TNF-a in murine viral myocarditis [47], and decreased LPSstimulated TNF-a and IL-1b levels in WKY and SHR rats [2]. After one and two weeks of celiprolol administration to SHR rats, a small (10–12%) but significant increase in diastolic blood pressure was observed. This effect could be only partially explained by the complex effect of the drug on the b1- and b2-adrenoceptors. AlvarezGuerra et al. [1] showed that celiprolol could antagonize, in a dosedependent manner, the hypotensive effects of salbutamol (a b2adrenoceptor agonist), and even inhibit its own hypotensive effect in male Sprague-Dawley rats. At present it might be speculated that the complex molecular mechanism of celiprolol action, loss of its selectivity for both antagonist and partial agonist effects [50], or increased peripheral resistance, consistent with the observed heart rate decrease, could contribute to the blood pressure changes in hypertensive rats described in this study.
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Celiprolol, as a third-generation b-blocker, has been reported to exert either neutral or beneficial effect on lipid profiles [19]. The drug usually raises HDL cholesterol levels and has a lowering effect on the total cholesterol [16,30,36,40]. In contrast to humans, there are only a few studies regarding the action of celiprolol on lipidogram in rats. Olbrich et al. [34] demonstrated that treatment of diabetic rats with celiprolol did not exert any significant changes on triglycerides or cholesterol levels. Balasubramaniam et al. [4] showed that celiprolol significantly increased HDL cholesterol in rats. In our studies, celiprolol did not affect lipid parameters in normotensive WKY rats, whereas in hypertensive SHR, it decreased serum levels of triglyceride, total cholesterol, and HDL cholesterol. In summary, our results demonstrate that the complex vascular action of celiprolol is accompanied by lowered LPS-stimulated production of the proinflammatory cytokine, IL-6. Furthermore, the drug was shown to have neutral (WKY rats) and beneficial (triglycerides and total cholesterol – SHR rats) effects on lipid parameters. It is suggested that the IL-6-modulating properties of celiprolol could provide an additional value to the therapeutic effectiveness of the drug in the treatment of hypertension. Conflict of interest
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20] [21] [22]
No conflict of interest. [23]
Funding [24]
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
The effects of celiprolol on serum concentrations of proinflammatory cytokines in hypertensive (SHR) and normotensive (WKY) rats Dariusz Andrzejczak *, Dorota Go´rska Department of Pharmacodynamics, Medical University of Lodz, Lodz, Poland
A R T I C L E I N F O
Article history: Received 9 January 2013 Received in revised form 30 July 2013 Accepted 13 August 2013 Available online 1 February 2014 Keywords: Celiprolol Proinflammatory cytokines Lipids SHR WKY
A B S T R A C T
Background: A growing body of evidence suggests that some cardiovascular drugs could modulate the level of proinflammatory cytokines. Therefore, the aim of the present study was to investigate whether celiprolol, a third generation b-adrenoceptor blocker, affects lipopolysaccharide (LPS)-induced serum concentrations of TNF-a, IL-1b, IL-6 in normotensive (WKY) and spontaneously hypertensive (SHR) rats. Methods: Celiprolol (150 mg kg 1) or vehicle was administered by gavage once daily for 21 days. Arterial blood pressure was measured in conscious rats, using the tail-cuff method. Serum concentrations of proinflammatory cytokines were measured with enzyme-linked immunosorbent assay kits. Additionally, plasma concentrations of total cholesterol, HDL-cholesterol and triglycerides were evaluated. Results: In normotensive WKY rats celiprolol did not affect heart rate, blood pressure, or the serum concentrations of triglycerides, total cholesterol or HDL-cholesterol. In hypertensive animals the drug decreased lipid parameters, increased diastolic and mean blood pressure after the first week of administration, and produced a small but significant decrease in heart rate after the first two weeks of the treatment. In both groups of animals, celiprolol decreased LPS-stimulated serum concentration of IL6 but did not affect levels of TNF-a and IL-1b. Conclusions: It is suggested that the IL-6-modulating properties of celiprolol could provide additional value to the therapeutic effectiveness of the drug in the treatment of hypertension. ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
Introduction Increased airway resistance, peripheral and coronary vasoconstriction, proatherosclerotic action, and increased insulin resistance are the most important side effects of conventional b-blockers, limiting their therapeutical usefulness [23,26]. A large body of clinical data has demonstrated that the contraindications of celiprolol, a selective b1-blocker endowed with b2-adrenomimetic activity, are markedly lower than those displayed by the first generation of b-adrenolytic drugs. For example, celiprolol is safer than conventional b-blockers in the treatment of asthmatic patients because of lower influence on the airway function [8,32]. Moreover, celiprolol does not produce venodilatation, and has negligible vasoconstricting effects on stenotic and normal coronary arteries. The vascular action of the drug is associated with the activation of b2-adrenoceptors, NO release from the endothelium, and weak
Abbreviations: IL, interleukin; LPS, lipopolysaccharide; SHR, spontaneously hypertensive rats; TNF-a, tumor necrosis factor alpha; WKY, Wistar-Kyoto rats. * Corresponding author. E-mail address: [email protected] (D. Andrzejczak).
blockade of a1-adrenoceptors [32,43]. Other desirable effects of celiprolol include an increase in insulin sensitivity in healthy volunteers and patients with an insulin-resistance state, and improvement of the serum lipid profile, i.e. a decrease in concentrations of triglycerides and LDL-cholesterol, and an increase in HDL-cholesterol [43]. Accumulating experimental evidence indicates the functional importance of an interplay between the autonomic nervous system and the immune system [15]. b-Adrenoceptors are expressed by various immune cells, such as lymphocytes, macrophages, neutrophils, eosinophils, and basophils. Stimulation of these receptors can lead to changes in the production of proinflammatory cytokines [42,52]. We have previously demonstrated that propranolol and atenolol, non-selective and cardioselective antagonists of b-adrenergic receptors, respectively, are endowed with immunomodulating properties [2,3]. This study analyzes the effects of celiprolol on lipopolysaccharide (LPS)stimulated levels of proinflammatory cytokines, namely tumor necrosis factor alpha (TNF-a), interleukin (IL)-1b and IL-6, in normotensive (Wistar-Kyoto; WKY) and spontaneously hypertensive (SHR) rats. In addition, the effects of celiprolol on heart rate, arterial blood pressure, and serum lipid profile were examined.
1734-1140/$ – see front matter ß 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.pharep.2013.08.006
D. Andrzejczak, D. Go´rska / Pharmacological Reports 66 (2014) 68–73
tion was chosen according to Dredge et al. [13]. The blood was allowed to clot overnight at 4 8C, and then the samples were centrifuged for 20 min at 2000 g. The serum was removed and stored at 20 8C until used for biochemical measurements.
Materials and methods Animals and treatment The study was conducted on 12–14 weeks old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) male rats. The rats were housed in standard plastic cages, 10 animals per cage, at a constant temperature of 21 1 8C, and under an illumination cycle of 12 h light–dark (lights on between 7.00 and 19.00). The animals, with an initial body weight 280–310 g, had free access to standard food and tap water. In order to select the rats for experimentation, preliminary examinations were carried out at the end of the second and third weeks of their adaptation. The animals with high blood pressure fluctuations were excluded from the study. Experiments were conducted between 8 a.m. and 4 p.m. All experimental procedures were performed in accordance with the Polish governmental regulations concerning experiments on animals (Dz.U.05.33.289) and were approved by the Local Ethics Committee for Experimentation on Animals. Celiprolol (Celipres, Ranbaxy Laboratories Ltd., India; 150 mg kg 1 body weight) was suspended in 1% solution of methylcellulose (Sigma–Aldrich, Poznan, Poland) and administered by gavage at a volume of 2 ml kg 1 b.w. once daily for 3 weeks. Control rats received a 1% solution of methylcellulose (2 ml kg 1 b.w.). Measurements of arterial blood pressure were carried out after 7, 14, and 21 days of administration of celiprolol or vehicle. Preliminary studies showed no detectable levels of cytokines in the serum of SHR rats. Thus, in order to achieve a measurable cytokine level, 24 h after the last administration of celiprolol or vehicle, the rats received ip a small dose of LPS from Escherichia coli serotype 055:B5 (Sigma–Aldrich, Poznan, Poland; 0.1 mg kg 1 b.w. in 1 ml of saline kg 1 b.w.). After 2 h, the animals were anesthetized with ethyl ether, and the blood samples were collected by heart puncture. The time of blood sample collection after LPS administraTable 1 Effects of the repeated administration of celiprolol (150 mg kg rats. Time after drug administration (week)
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1
Arterial blood pressure and heart beat measurements Arterial blood pressure was measured in conscious rats by a manometer manufactured by LETICA (Panlab S.L., Spain), using a tail-cuff method as described in details by Go´rska and Andrzejczak [17]. Before the measurements, in order to calm the animals and dilate the tail blood vessels, the rats were placed inside a warming chamber (about 34 8C) for 30 min. The measurements of arterial blood pressure (systolic, diastolic and mean) were carried out at least three times for each animal, and the mean values of several successive measurements were used for further analysis. Changes in blood pressure were expressed as the percentage of baseline values. The heart rate was measured by the same apparatus automatically and was registered as beats per minute. Lipid profile determination Total serum cholesterol concentration was determined by the cholesterol oxidase method using a commercially available kit (Cholesterol CHOD PAP, Biolabo, Maizy, France) according to the manufacturer’s instructions. To measure HDL-cholesterol, low density lipoproteins, very low density lipoproteins, and chylomicrons were precipitated from serum samples by phosphotungstic acid and magnesium chloride. Following that, HDL-cholesterol was measured with the aid of commercially available kit (HDLcholesterol-PTA, Biolabo, Maizy, France). Triglycerides were measured using a commercially available kit (Triglycerides GPO Method, Biolabo, Maizy, France) according to the manufacturer’s instructions.
) on systolic (A), diastolic (B) and mean blood pressure (C) in normotensive (WKY) and hypertensive (SHR)
Mean changes in systolic blood pressure (% of initial values) SHR
WKY Vehicle (n = 10)
Celiprolol (n = 15)
Vehicle (n = 10)
Celiprolol (n = 15)
(A) 1 2 3
99.4 1.6 96.9 1.9 101.5 1.1
96.9 2.0 98.9 2.2 98.4 2.6
99.8 2.3 95.6 2.6 98.9 3.9
97.0 2.6 97.2 3.7 101.7 2.1
Time after drug administration (week)
Mean changes in diastolic blood pressure (% of initial values) WKY
SHR
Vehicle (n = 10)
Celiprolol (n = 15)
Vehicle (n = 10)
Celiprolol (n = 15)
(B) 1 2 3
97.8 4.4 103.1 1.4 101.6 3.6
105.5 4.4 108.8 3.6 109.2 5.7
97.1 3.7 102.6 3.6 98.3 3.7
110.6 3.9* 112.0 2.3* 102.6 2.3
Time after drug administration (week)
Mean changes in mean blood pressure (% of initial values) WKY
(C) 1 2 3
SHR
Vehicle (n = 10)
Celiprolol (n = 15)
Vehicle (n = 10)
Celiprolol (n = 15)
98.3 3.0 101.1 1.3 101.0 2.3
101.9 3.2 105.7 2.6 104.3 4.0
100.3 2.8 99.2 3.1 99.3 3.5
107.3 1.3* 105.4 2.1 102.3 1.1
Values are means SEM. * p < 0.05 in comparison with vehicle-treated control animals. Baseline values of blood pressure (mmHg) for WKY rats in vehicle-treated group: systolic 117.0 2.0; diastolic 84.6 1.7; mean 95.8 1.5. Baseline values of blood pressure (mmHg) for WKY rats in celiprolol-treated group: systolic 118.1 2.2; diastolic 85.1 1.8; mean 96.8 1.8. Baseline values of blood pressure (mmHg) for SHR in vehicle-treated group: systolic 237.3 3.2; diastolic 128.2 2.7; mean 165.2 3.0. Baseline values of blood pressure (mmHg) for SHR in celiprolol-treated group: systolic 235.6 2.4; diastolic 129.2 1.9; mean 164.3 2.0.
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Serum cytokine levels Serum TNF-a, IL-1b and IL-6 concentrations were measured in duplicates with a commercially available enzyme-linked immunosorbent assay kit (Quantikine, R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions. Analysis of data The results are expressed as the mean SEM values. The normality of distribution was checked by means of a combination of the Kolmogorov–Smirnov test and the Lilliefors test. The data were analyzed for statistical significance by one-way ANOVA followed by a post hoc Least Significant Differences (LSD) test, using StatSoft, Inc. (2010) STATISTICA version 9.1. If the data were not normally distributed, statistical evaluation was performed by using ANOVA (Kruskall–Wallis) and the Mann–Whitney U test. The statistical analysis of data presented in Table 2 was performed with the aid of a t-Test for Dependent Samples. Differences were considered statistically significant when p < 0.05. The initial values of arterial blood pressure were assumed as 100%. Results Blood pressure and heart rate In WKY rats, which were chosen as a control strain, the following values (in mmHg) of arterial blood pressure were found: systolic, 119.7 1.4; diastolic, 85.7 1.6; mean, 96.8 1.5 (n = 10 animals/group). The SHR selected for the study had initial arterial blood pressure (in mmHg): systolic, 235.7 2.9; diastolic, 129.8 1.7; mean, 164.6 1.7 (n = 15 animals/group). They were significantly higher than the corresponding values determined in WKY animals. SHR rats also had a significantly higher basal heart beat rate (332.0 1.9 beats/min; n = 15) compared to WKY rats (297.9 1.1 beats/min; n = 10). Celiprolol (150 mg kg 1 b.w.) did not affect blood pressure and heart rate in normotensive WKY rats (Tables 1 and 2). Administration of the drug to SHR rats resulted in a small, but statistically significant, increase in diastolic blood pressure, by 10% and 12% after the first and the second weeks, respectively, and mean blood pressure, by 7% after the first week (Table 1). Following one week of treatment with celiprolol, small but statistically significant decrease in heart rate of 5% was observed, and after two weeks this grew to 8% (Table 2). Lipid profile In WKY rats celiprolol did not modify serum concentrations of total cholesterol, HDL cholesterol, and triglycerides. On the other Table 2 Effects of the repeated administration of celiprolol (150 mg kg normotensive (WKY) and hypertensive (SHR) rats. Time after drug administration (week)
1 2 3
1
) on heart rate in
Fig. 1. Effects of the repeated administration of celiprolol (150 mg kg 1) on serum concentration of triglycerides, total cholesterol and HDL-cholesterol in WKY and SHR rats. Values are means SEM. *p < 0.05 in comparison with control group (vehicle-treated rats).
hand, in SHR rats, celiprolol significantly decreased all lipid parameters by 23–27% (Fig. 1). Serum cytokines As concentrations of TNF-a, IL-1b and IL-6 in the serum of SHR rats were found to be below the limits of detection in preliminary experiments, a small dose of LPS (0.1 mg kg 1 b.w.) was used in order to stimulate the production of cytokines to measurable levels. Celiprolol did not affect LPS-induced serum level of TNF-a Table 3 Effects of the repeated administration of celiprolol (150 mg kg 1) on serum concentration of TNF-a, IL-1b and IL-6 in normotensive (WKY) and hypertensive (SHR) rats. Serum concentration of cytokines (pg/ml)
Mean changes in heart rate (beats per min)
Vehicle (n = 10)
Celiprolol (n = 15)
TNF-a IL-1b IL-6
2325.3 122.2 138.2 27.3 7531.1 631.3
1900.7 203.3 121.3 25.9 3438.3 849.8*
Serum concentration of cytokines (pg/ml)
SHR rats
SHR
WKY Vehicle (n = 10)
Celiprolol (n = 15)
Vehicle (n = 10)
Celiprolol (n = 15)
296.7 1.5 295.8 1.4 296.5 1.0
298.5 2.3 300.8 2.4 301.2 2.1
333.3 2.2 329.8 3.8 328.8 3.1
315.2 3.0* 306.3 2.9* 329.0 4.3
Values are means SEM. * p < 0.05 in comparison with baseline value (‘‘0’’) in celiprolol-treated SHR rats. Baseline heart rate values in vehicle-treated WKY 297.9 1.1 beats/min and celiprolol-treated WKY 298.9 1.8. Baseline heart rate values in vehicle-treated SHR rats 332.0 1.9 beats/min and celiprolol-treated SHR 331.5 2.9.
WKY rats
TNF-a IL-1b IL-6
Vehicle (n = 10)
Celiprolol (n = 15)
2227.9 24.2 132.4 40.9 5404.4 596.8
2055.1 272.9 134.3 25.8 2579.1 605.0*
Values are means SEM. * p < 0.05 in comparison with control, vehicle-treated rats.
D. Andrzejczak, D. Go´rska / Pharmacological Reports 66 (2014) 68–73
and IL-1b in WKY and SHR rats. On the other hand, in both strains of animals, the drug decreased IL-6 serum level by approximately 54% (Table 3). Discussion Hypertension is a multifactorial disease with a very complex etiology. An accumulating body of experimental data suggests the involvement of the immune system and a low-grade inflammation process in the development of hypertension and in the cardiovascular complications associated with it [20,21]. Furthermore, ample evidence links proinflammatory cytokines with hypertension, but whether elevated levels of cytokines precede or follow the development of hypertension remains to be elucidated [41,46]. Taking into account the above facts, an additional anti-inflammatory activity of a drug used in the treatment of hypertension could be considered as its therapeutically important property. b-Adrenergic blockers are widely used in the treatment of various cardiovascular diseases, including hypertension. At present, little is known about the effects of this group of drugs on inflammatory mediators in hypertension. Nemati and coworkers [33] analyzed the effects of various hypotensive drugs, including atenolol, on LPS-induced IL-1b secretion from peripheral blood polymorphonuclear leukocytes isolated from normotensive individuals and from patients with essential hypertension. They found no significant effect of this selective b1-blocker on the secretion of cytokine by cells from hypertensive subjects. An eight-week treatment of hypertensive postmenopausal women with atenolol did not affect serum IL-6 level and slightly increased the level of TNF-a [37]. On the other hand, Madej et al. [28] demonstrated that treatment of hypertensive patients with bisoprolol decreased TNFa, IL-1b and IL-6 secretion from peripheral blood mononuclear cells. Using an animal model of hypertension, SHR rats, our previous studies have demonstrated that atenolol does not modify the LPS-induced elevation of TNF-a and IL-1b blood levels, while propranolol, a non-selective b-blocker, was found to reduce the stimulated IL-1b level [2,3]. Celiprolol is generally described as a third generation badrenoceptor antagonist. In addition to selective b1-adrenoceptor blocking properties, the drug has b2 partial agonist properties, exerts direct vasodilator effects, and does not depress the heart rate to the same extent as several other b-blockers. The therapeutic indication for the use of celiprolol includes mainly hypertension and angina complicated by impaired glucose tolerance or diabetes mellitus, peripheral vascular resistance and hyperlipidemia [14]. Data regarding cytokine-modulating properties of celiprolol are sparse. Hayashi et al. [22] observe that celiprolol (100 mg kg 1/day) improved endothelial function in Otsuka Long-Evans Tokushima Fatty diabetic rats. Furthermore, plasma concentrations of TNF-a were significantly elevated in vehicle- and atenolol-treated rats but not in animals receiving celiprolol. In another study, Tsubokou et al. [45] demonstrate that celiprolol decreases upregulated transforming growth factor-b1 (TGF-b1) expression in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Of note, this activity of celiprolol has been recently used in the treatment of vascular Ehlers-Danlos syndrome, where circulating TGF-b likely plays the role of an important pathogenic factor [5,7,24,35]. The results of the present study demonstrated, for the first time, effects of celiprolol on LPS-induced proinflammatory cytokines concentration in normotensive and hypertensive animals. Although the drug did not modify LPS-induced TNF-a and IL-1b serum levels in SHR and WKY rats, it potently decreased IL-6 level in both rat strains. It appears unlikely that the action of celiprolol on LPS-stimulated serum level of IL-6 is related to its partial b2sympathomimetic activity, as stimulation of b2-adrenoceptors
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with isoprenaline, a non-selective b-adrenoceptor agonist, prior to LPS administration resulted in a significant elevation of IL-6 plasma level in mice [42]. The action of celiprolol on IL-6 presumably did not also result from its b1-adrenolytic activity, as results from our laboratory have previously demonstrated that prolonged treatment of rats with atenolol, a selective b1-blocker, increases serum IL-6 levels [3]. IL-6 is a proinflammatory cytokine with pleiotropic activity [29]. A growing body of experimental evidence indicates that IL-6 plays an important role in mediating inflammatory and blood pressure response to angiotensin II. Thus, treatment of C57BL/6J mice with IL-6 increased vascular AT1 receptor expression, angiotensin II-induced vasoconstriction in aortic segments, enhanced vascular superoxide production and impaired endothelium-dependent vasodilatation [48]. During two weeks of angiotensin II infusion, the IL-6 knockout mice had significantly lower blood pressure than wild type animals. This observation led Lee et al. [25] to suggest that hypertension caused by angiotensin II and by a high-salt diet could result, to some extent, from the action of IL-6. Similar results were presented by Coles et al. [11]. Subcutaneous administration of angiotensin II (1.1 mg kg 1/day) to wild type mice evoked hypertension and cardiac hypertrophy. Such effects were not observed in IL-6 knockout mice. A study by Schrader et al. [39] on mice showed that IL-6 could be essential for angiotensin II-induced endothelial dysfunction, i.e. production of reactive oxygen species and reduction of endothelial nitric oxide synthase mRNA expression. In Sprague-Dawley rats, IL-6 caused a pattern of myocardial remodeling similar to that seen in hypertension [31]. It should be emphasized that an association between IL-6 levels and elevated blood pressure/hypertension [9,10], and increased risk of myocardial infarction [38] have been demonstrated in man. The two other studied proinflammatory cytokines, TNF-a and IL-1b, have been proven to exert negative effects on the endothelium. Thus, TNF-a could stimulate the angiotensinogenencoding gene [6] and limit the half-life of eNOS mRNA [51], whereas IL-1b has been shown to favor the development of atheromatous changes [27]. Increased IL-1b serum levels were found in hypertensive patients [12]. Recent studies suggest that TNF-a and IL-1b could act on the brain to increase blood pressure, heart rate, and sympathetic nerve activity [49]. Of note, etanercept, a TNF-a antagonist used mainly in autoimmune diseases, such as rheumatoid arthritis, was shown to prevent the development of hypertension in fructose-fed rats [44]. There are a few published reports on effects of b-blockers on serum levels of TNF-a and IL-1b. By analogy to celiprolol (present data), atenolol [3] and nebivolol [18], a b1-selective adrenergic blocker with additional vasodilating properties, did not modify LPS-stimulated levels of TNF-a and IL-1b in normotensive as well as in hypertensive rats. On the other hand, propranolol, a nonselective b-adrenoceptor antagonist, suppressed gene expression of TNF-a in murine viral myocarditis [47], and decreased LPSstimulated TNF-a and IL-1b levels in WKY and SHR rats [2]. After one and two weeks of celiprolol administration to SHR rats, a small (10–12%) but significant increase in diastolic blood pressure was observed. This effect could be only partially explained by the complex effect of the drug on the b1- and b2-adrenoceptors. AlvarezGuerra et al. [1] showed that celiprolol could antagonize, in a dosedependent manner, the hypotensive effects of salbutamol (a b2adrenoceptor agonist), and even inhibit its own hypotensive effect in male Sprague-Dawley rats. At present it might be speculated that the complex molecular mechanism of celiprolol action, loss of its selectivity for both antagonist and partial agonist effects [50], or increased peripheral resistance, consistent with the observed heart rate decrease, could contribute to the blood pressure changes in hypertensive rats described in this study.
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Celiprolol, as a third-generation b-blocker, has been reported to exert either neutral or beneficial effect on lipid profiles [19]. The drug usually raises HDL cholesterol levels and has a lowering effect on the total cholesterol [16,30,36,40]. In contrast to humans, there are only a few studies regarding the action of celiprolol on lipidogram in rats. Olbrich et al. [34] demonstrated that treatment of diabetic rats with celiprolol did not exert any significant changes on triglycerides or cholesterol levels. Balasubramaniam et al. [4] showed that celiprolol significantly increased HDL cholesterol in rats. In our studies, celiprolol did not affect lipid parameters in normotensive WKY rats, whereas in hypertensive SHR, it decreased serum levels of triglyceride, total cholesterol, and HDL cholesterol. In summary, our results demonstrate that the complex vascular action of celiprolol is accompanied by lowered LPS-stimulated production of the proinflammatory cytokine, IL-6. Furthermore, the drug was shown to have neutral (WKY rats) and beneficial (triglycerides and total cholesterol – SHR rats) effects on lipid parameters. It is suggested that the IL-6-modulating properties of celiprolol could provide an additional value to the therapeutic effectiveness of the drug in the treatment of hypertension. Conflict of interest
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20] [21] [22]
No conflict of interest. [23]
Funding [24]
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