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Pathophysiology 18 (2011) 137–142

Amlodipine treatment decreases plasma and carotid artery tissuelevels of endothelin-1 in atherosclerotic rabbits

Bahman Rashidi a, Mostafa Mohammadi b,∗, Fariba Mirzaei b, Reza Badalzadeh c,Parham Reisi d

a Department of Anatomy and Histology, Isfahan University of Medical Sciences, Isfahan, Iranb Department of Physiology, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

c Tabriz Young Researcher Clubs, Tabriz Islamic Azad University, Tabriz, Irand Department of Physiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

Received 18 December 2009; accepted 10 May 2010

bstract

Alteration in transferring of calcium ions are seen in atherosclerotic cells and amlodipine can positively influence risk factors associatedith atherosclerosis, but all mechanisms are not known. Recent studies indicate that endothelin-1 (ET-1) contributes to the atheroma formation

nd progression of atherosclerosis. In this study, we have evaluated the effects of amlodipine treatment and/or high-cholesterol diet on bloodnd carotid artery tissue concentration of ET-1 in the atherosclerotic rabbits. Thirty six male New Zealand white rabbits were randomlyivided into four groups: normal-diet control (NC), normal-diet receiving amlodipine (NA), high-cholesterol diet (HC) and high-cholesteroliet receiving amlodipine (HA) groups. After 8 weeks all animals were anesthetized and blood or carotid tissue samples were colleted. Eighteeks of amlodipine treatment reduced significantly total cholesterol, LDL and TG in hypercholesterolemic (HA) group. Significant increase

n plasma HDL-C and decrease in TG were the main effects of amlodipine treatment on serum lipid profiles in the control group. The plasmand carotid tissue levels of ET-1 in HC group were significantly increased as compared with the NC group (p < 0.01). Amlodipine treatmentignificantly reduced ET-1 level in NA and HA rabbits (p < 0.01). Furthermore, high-cholesterol diet induced atherosclerotic lesions as showny the enhancement of endothelial cell diameter and accumulation of lipid droplets under endothelial cells. Amlodipine treatment reduced

therotic lesions in these rabbits. Amlodipine treatment reduced levels of total cholesterol, LDL and TG as well as plasma and carotid tissueevels of ET-1 in high lipid situation. We suggest that amlodipine treatment by reducing the ET-1 may contribute to reducing the progressionf atherosclerotic disease.

2010 Elsevier Ireland Ltd. All rights reserved.

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eywords: Atherosclerosis; Amlodipine; Endothelin-1; Rabbit

. Introduction

Atherosclerosis is the leading cause of mortality andorbidity in the developed world and most of developing

ountries [1]. Atherosclerosis is a complex process, and its possibly caused by high-fat diet and sedentary lifestyle

Abbreviations: ET-1, endothelin-1; LDL, low-density lipoprotein;DL, high-density lipoprotein; TG, triglyceride; HDL-C, high-density

ipoprotein-cholesterol; ECE, endothelin-converting enzyme; SMC, smoothuscle cells; CCBs, calcium channel blockers.∗ Corresponding author. Tel.: +98 311 7922414; fax: +98 311 6688597.

E-mail address: m.mohammadin@yahoo.com (M. Mohammadi).

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928-4680/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.pathophys.2010.05.003

2]. Hypercholesterolemia is one of the most important riskactors for atherosclerosis and cardiovascular disease [3].therosclerosis is a progressive structural and functional vas-

ular disorder that initiates molecular and cellular eventsriggered by endothelial dysfunction, resulting in decreaseditric oxide production, increased endothelin-1 [ET-1] pro-uction and cyclooxgenase activity and inflammation [4,5].

The 21-amino acid peptide ET-1 is produced by vascularndothelial cells from the 38-amino acid precursor peptide,

ig ET, by the endothelin-converting enzyme (ECE) [6] ET-. This most potent vasoconstrictive substance known today,xerts different biological activities in a large variety of car-iovascular diseases, including atherosclerosis [7]. Beside

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38 B. Rashidi et al. / Patho

ts vasoconstrictor effects, ET-1 contribute to cell prolifera-ion, thereby promoting vascular growth and atherogenesis6]. Furthermore, it has been shown that ET-1 is locallyroduced in the atherosclerotic intima by macrophages,ndothelial and smooth muscle cells [8,9]. Taken together,he in vitro observations suggest that ET-1, released in excessuring atherosclerosis, might contribute to the developmentf atherosclerotic lesions [10].

Increased circulating ET-1 levels have been reported inatients with advanced atherosclerosis and a positive corre-ation has been found between levels of ET-1 and thicknessf the intima-media complex in the common and internalarotid artery [10]; moreover, the increased venous levels ofT-1 are suggested to predict an increased cerebrovascularorbidity in patients with internal carotid stenosis [11]. The

tudies in men affected by atherosclerotic diseases agree withpossible role of ET-1 in the progression of carotid artery

therosclerosis [11].Calcium channel blockers [CCBs] have been suggested as

deterrent of cardiovascular disease and atherosclerosis, andheir anti-atherogenic effects have been described in patientsith coronary artery disease [12]. A variety of studies per-

ormed in humans and animals have indicated that calciumhannel blockers can influence the natural progression oftherosclerosis [13–15].

Amlodipine, a third generation calcium antagonist is aong-acting dihydropyridine calcium channel-blocking agenthat is lipophilic and contains a charged amino group and

lipid partition coefficient of about 1200. This reflects itsarked ability to penetrate to the cell membrane, and it can

nhibit calcium permeability in vascular smooth muscle cellsSMC) and reduce the atherosclerotic lesions [16]. How-ver, this effect could not be confirmed by others [17] andemains subject to controversy. In some animal studies, theffect has been indifferent [18] and the anti-atheroscleroticotential of CCBs and underlying mechanisms are underebate. Amlodipine can also positively influence risk factorshat are associated with atherosclerosis, but all the mecha-isms by which it might protect are not known. Previously, weave found that high-cholesterol regimen developed a seriesf histopathological alterations in the aortic artery such astheroma formation, rupture of intima and even calcificationf media in some loci of aortic tissue and amlodipine admin-stration improved all alterations [19]. Considering this, it isnteresting to evaluate the interaction of this regimen as wells amlodipine on other arteries including carotid artery tolarify whether the position or type of artery are determinantactors for similar pathological findings or there are variousehaviors among arteries in their responses.

Therefore, in this study we postulate that amlodipineould alter the progression of carotid artery atheroscle-

osis and therefore reduce the risk of events leading to

rogression of atherosclerosis. Therefore, the goal of thistudy was to study the effectiveness of amlodipine on thelood and carotid tissue levels of ET-1 and attaining aloser view of amlodipine as an anti-atherosclerotic role

is

gy 18 (2011) 137–142

ia effect on ET-1 in hypercholesterolemic New Zealandabbits.

. Materials and methods

.1. Animals and diet

Thirty six male New Zealand white rabbits (1.4 kg at theeginning, animal laboratory of Drug Applied Research Cen-er, Tabriz University of Medical Sciences, Tabriz, Iran) wereivided into four groups (n = 9 rabbits for each group): theormal-diet control group (NC), normal-diet group receiv-ng amlodipine (NA), high-cholesterol diet group (HC)nd high-cholesterol diet receiving amlodipine group (HA).he normal-diet groups were fed normal rabbit chow,hereas the high-cholesterol diet groups were fed with high-

holesterol diet in which 2% cholesterol powder (Merckompany, Germany) was added to normal food during 8-eek experiment. In addition, NA and HA groups receivedmg/kg/day amlodipine powder (Arya Company, Iran). Allnimals were housed in an environmentally controlled animaloom.

.2. Measurement of lipid profiles and endothelin-1evels

The rabbits were anesthetized at the end of experimentsy injecting ketamine (25 mg/kg, iv) and sodium pentobarbi-al (20 mg/kg, iv) via the margin ear vein. Blood samplesere drawn from the inferior vena cava and were stored

n tubes for the determination of lipid profile. Serum lipidrofile including total cholesterol, low-density lipoprotein-holesterol (LDL-C), high-density lipoprotein-cholesterolHDL-C), and triglyceride (TG) were determined by enzy-atic methods using an automatic analyzer (Abbott, Alcyon

00, USA). Other blood samples also were stored in tubesontaining EDTA (10 mmol/1 final concentration) on ice forhe determination of plasma endothelin-1 level. After cen-rifugation (15 min, 4 ◦C), 1 ml plasma was stored at 80 ◦Cnit for analyses. Plasma ET-1 was measured with specialit of ET-1 (Titer Zyme® EIA kit, No: 030806265). Foreasuring tissue ET-1 level, the common carotid artery was

mmediately isolated and then homogenized (homogeniz-ng solution: 20 mol/l HCl + 1 mol/l HCOOH). Thereafter,omogenized solution was centrifuged (10 min, 3000 rpm,◦C) and the light supernatant was taken and stored at80 ◦C units for analyses. Then tissue ET-1 was measuredith special kit (No: 030806265) after lyophilizing in (Christplphal4).

.3. Histological studies of blood vessels

Carotid artery tissue was immediately isolated and placedn formalin 10%. Briefly, after tissue processing steps, severalerial sections of blood vessel segments (5 �m thick) were

B. Rashidi et al. / Pathophysiology 18 (2011) 137–142 139

Table 1Serum lipid profile (in mg/dl) alterations in rabbits with normal-diet or cholesterol-diet whenever receiving amlodipine or not in a week long experiment.

Variable Groups

NC NA HC HA

Total cholesterol 49.13 ± 0.6 40.30 ± 0.80 860.30 ± 0.60*$ 524.50 ± 5.80*#$

LDL 7.23 ± 1.39 13.13 ± 0.20 722.00 ± 0.86*$ 451.43 ± 6.70*#$

HDL 14.00 ± 0.73 19.83 ± 0.54* 49.00 ± 0.63*$ 48.33 ± 0.95*$

TG 95.50 ± 1.70 81.00 ± 0.50* 466.60 ± 2.50*$ 138.60 ± 1.80*#$

HDL–LDL 2.47 ± 0.60 1.50 ± 0.05 0.07 ± 0.001*$ 0.11 ± 0.002*$

HDL-C 0.35 ± 0.02 0.40 ± 0.007* 0.06 ± 0.001*$ 0.09 ± 0.001*$

D < 0.05 were considered significant. *NC vs. NA, HC and HA; #HC vs. HA; $NAv ith amlodipine; HC, high-cholesterol diet control; HA, high-cholesterol diet witha

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Table 2Plasma and carotid tissue endothelin-1 (ET-1) alterations in rabbits withnormal-diet or cholesterol-diet whenever receiving amlodipine or not in aweek long experiment.

Groups Plasma ET-1 (pg/ml) Carotid tissue ET-1(pg/100 mg tissue)

NC 0.56 ± 0.01 1.59 ± 0.01NA 0.39 ± 0.01* 1.52 ± 0.04HC 0.80 ± 0.04* 33.10 ± 1.32*$

HA 0.60 ± 0.01$# 25.41 ± 1.13*$#

Data are expressed as mean ± SEM (n = 9 for each group). Differences ofp < 0.05 were considered significant. *NC vs. NA, HC and HA; #HC vs.H $

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ata are expressed as mean ± SEM (n = 9 for each group). Differences of ps. HC and HA. Abbreviations: NC, normal-diet control; NA, normal-diet wmlodipine.

tained by standard hematoxylin–eosin (H&E) and studiedy light microscopy.

.4. Statistical analysis

Data was expressed as mean ± SEM. Differences amongour groups were analyzed by one-way ANOVA and Tukeys post hoc test. A p-value of less than 0.05 was consideredtatistically significant.

. Results

Serum lipid profile. Our results clearly demonstrated thatweeks consumption of 2% cholesterol diet significantly

ncreased serum total cholesterol, HDL, LDL-C, and TGTable 1). These observations indicated that atherogeniciet indeed induced hypercholesterolemia in our experimen-al model. Table 1 shows that amlodipine administrationould significantly decrease these parameters. Althoughmlodipine treatment tended to enhance HDL/LDL andDL/cholesterol ratios in the HA group, these effects wereot statistically significant. The observed increase in plasmaDL-C and decrease in TG is considered to be the main effectf amlodipine treatment on serum lipid profiles in normal-iet control group. ET-1 level. The plasma level of ET-1n atherosclerotic (HC) group was significantly increased asompared with the NC group (p < 0.01). After treatment withmlodipine for 8 weeks, ET-1 level reduced significantly inormal (p < 0.01) and high-cholesterol diet rabbits (p < 0.01).igh-cholesterol diet increased significantly the tissue levelf ET-1 compared to those of the control (p < 0.01). Amlodip-ne administration reduced significantly the tissue levels ofT-1 in normal and high-cholesterol diet rabbits (p < 0.01)

Table 2).Histological findings. Eight weeks of 2% high-cholesterol

iet induced atherosclerotic lesions, thickening of the intima,nd enhancement of endothelial cells diameter with accumu-

ation of lipid droplets under endothelial cells in carotid arteryf all HC rabbits. According to our results in carotid group,oam cells observed in subendothelial layer led to partiallyhickening of this layer without atheroma formation or cal-

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A; NA vs. HC and HA. Abbreviations: NC, normal-diet control; NA,ormal-diet with amlodipine; HC, high-cholesterol diet control; HA, high-holesterol diet with amlodipine.

ification. Moreover, accumulation of lipid droplets underndothelial cells was seen. Integrity of vessel structure wasreserved and endothelial cells were flat. Foam cells wereeen as light cells containing lipids. It is clearly observed thatoam cells in subendothelial layers and thickness of intimaas increased. However, in amlodipine receiving group

educed atherotic injuries in high-cholesterol diet rabbits:oam cells were not observed or these were very sparse ando no increase in intima thickening was seen. The integrityetween endothelial cells was observed in carotid lumen ofmlodipine-treated rabbits without atheroma formation inistological sections.

All these changes indicate that amlodipine treatment wasffective in preventing atherosclerotic lesions. There were noesions in normal-diet control group or the normal-diet withmlodipine group (Figs. 1 and 2).

. Discussion

Our results indicated that 8 weeks consumption of 2%igh-cholesterol diet increased all serum cholesterol profileractions and induced formation of atherosclerotic lesions

ncluding the thickening of the intima and/or accumulationf lipid droplets under endothelial cells in carotid artery.oreover, the results of the present study revealed that the

ormation of ET-1 was significantly higher in the atheroscle-

140 B. Rashidi et al. / Pathophysiolo

Fig. 1. Standard hematoxylin–eosin (H&E) staining of carotid arteryfor the evaluation of the atherosclerotic lesions in 2% high-cholesteroldiet group. No lesions were observed in control group. Consumptionof 2% high-cholesterol diet induced atherosclerotic lesions that observedas enhancement of endothelial cells diameter with accumulation of lipiddroplets under endothelial cells that are seen as yellowish white (can seen inttt

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he tips of arrows). I: intima; M: media; A: adventitia. 660×. (For interpre-ation of the references to color in this figure legend, the reader is referredo the web version of the article.)

otic rabbits and amlodipine treatment could significantlyeduce it. Thus, our results support the hypothesis thatmlodipine altered the development of carotid artery lesions12].

The key finding of this report was that the third generationf dihydropyridine calcium antagonist, amlodipine is ableo inhibit progression of atherosclerotic plaque in our rab-

its. These changes are similar to those reported in rabbits,wine, monkeys and humans by using amlodipine [20]. Basedpon these studies, it appeared that CCBs would be mostffective if administered concomitantly with the atherogenic

ig. 2. Standard hematoxylin–eosin (H&E) staining of carotid artery for thevaluation of the atherosclerotic lesions in 2% high-cholesterol diet receivingmlodipine group. By amlodipine treatment these effects were observed:iameter of endothelial layer is reduced, and no or minimum accumulationf lipid droplets is seen under endothelial cells. I: intima; M: media; A:dventitia; EC: endothelial cell. 660×.

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gy 18 (2011) 137–142

timuli (i.e. cholesterol feeding). Since amlodipine is highlyipophilic, the drug can be rapidly absorbed in the atheromaf atherosclerotic lesions, accumulating locally, and it actsore effectively in atheromatous artery [10]. Because ofarked increase in calcium permeability in smooth muscle

ells during the development of atherosclerotic lesions, a roleor CCBs in the prevention of lesions would seem reason-ble. But many reports failed to confirm this effect and thetheroprotection role of CCBs was not established yet [15].he search for a CCB that might inhibit atherogenisis has

evealed a variety of interesting actions of this third gen-ration of dihydropyridine calcium antagonist, amlodipine.ecause excessive cell calcium transport contributes to manyellular changes in atherogenesis, it has been proposed thatalcium antagonists may be effective in slowing the progres-ion of atherosclerosis and heart diseases [20]. Although howxactly amlodipine improves atherosclerosis is still unclear,everal possible mechanisms of the anti-atherogen effects ofmlodipine have been proposed: recruitment of macrophages,ipid oxidation and proliferation of smooth muscle cells thatre calcium dependent and may be influenced by amlodipine21].

ET-1 contributes to vasoconstriction and cell proliferation,hereby promoting vascular growth and atherogenesis [22].T-1 may be an early marker and mediator of endothelialysfunction, leading to enhanced vasoconstrictor responsesnd contributing to the development of atherosclerotic lesions23]. Several observations have linked hypercholesterolemiaith the endothelin system and progression of atherosclero-

is [23]. Increased ET-1 level by high-cholesterol diet may bettributed to high levels of lipids and some lipoproteins (LDL)roduced by high-cholesterol diet. Since it has been recentlyeported that oxidized lipids can also induce endothelin-onverting enzyme-1 expression in human endothelial cells3], in our study, hypercholesterolemia produced by high-holesterol diet might contribute to enhanced ET-1 formationia increase of lipids and LDL. The main effect of amlodipines to inhibit Ca2+ influx, and oxidative modification of lipidould induce Ca2+ influx, and Ca2+ is closely associated withhe production of endothelin [24]. ET-1 via its chemoattrac-ant properties plays an important role in the recruitment ofells in the early stages of plaque development. ET-1 hasitogenic effects on smooth muscle cells and fibroblasts,

hus contributing to the fibroproliferative stage of the pro-ess. Its effect on fibroblasts and connective tissue formations also likely to play an important role in the stability of thetherosclerotic plaques [16].

It has been reported that local upregualtion of ET-1eceptor may play an important role in pathogenesis ofraft arteriosclerosis. Also it has been found that endothe-in receptor antagonist, bosentan could protect against thisathologic damage [25]. High level of endothelin in hyper-

holesterolemic rabbits suggests that the native circulatingipoproteins are important stimuli of ET-1 synthesis. It haseen found that ET-1 release has been stimulated by lipopro-ein in endothelial cells [26]. In patients with symptomatic

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therosclerotic vascular disease, plasma ET-1 concentrationsre higher than in normal subjects, and a significant correla-ion has been demonstrated between plasma ET-1 and numberf vascular sites exhibiting atherosclerosis [27]. Immunos-aining has shown reactivity for ET-1 in foam cells andn the intimal and medial smooth muscle cells of humanarotid atherosclerotic lesions [27]. Thus reduction of ET-level by amlodipine has potential to inhibit atheroscleroticlaques and diminish the lesions. Importantly, the increasedevel of ET-1 in carotid artery and plasma is closely linkedo the presence of enhancement of endothelial cells diam-ter with accumulation of lipid droplets under endothelialells that shows early stages of plaque evolution. Therefore,nhanced production of ET-1 may substantially contributeo cell growth and progress of atherosclerotic lesions. Weuggest that amlodipine treatment by reducing the ET-1 mayontribute to prevention and regression of atherosclerosis.lso these findings on carotid differ from the results of simi-

ar study on aortic artery [19] and this disparity may indicateifferent behaviors of these arteries under similar conditions,n which severe atheroma formation, tissue damage with cal-ification in media was observed in aorta but not in carotidrtery.

cknowledgment

This study was supported by funding from the Drugpplied Research Center, Tabriz University of Medical Sci-

nces, Tabriz, Iran. Authors acknowledge for master thisenter.

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