Calcium antagonists and atherosclerosis

European Heart Journal (1997) 18 {Supplement A), A80-A86

Calcium antagonists and atherosclerosis Experimental evidence A. L. Catapano Institute of Pharmacological Sciences, University of Milano and Centro per lo Studio delle Vasculopatie Periferiche, Ospedale Bassini, Milano, Italy

Accumulation of cholesterol and calcium is the hallmark of atherosclerosis. Ca2+ antagonists lessen the severity of experimentally induced atherosclerosis in a variety of animal models, including hypercholesterolaemic animals. They also reduce cholesterol accumulation in the arterial wall, but without affecting plasma lipids. This review briefly discusses in vitro and in vivo experimental studies on the

anti-atherosclerotic properties of Ca 2+ antagonists and outlines mechanisms by which these compounds affect cellular lipid metabolism. (Eur Heart J 1997; 18 (Suppl A): A80-A86)

Introduction

calcium antagonists reduce the spread of atherosclerotic lesions in rabbits fed a cholesterol-rich diet (Table 2). Recently, nifedipine was also shown to reduce atherosclerosis in the carotid artery of cholesterol-fed rhesus monkeys'71. Phenylalkylamine calcium antagonists, such as verapamil, have also been shown to be antiatherosclerotic as have diltiazem and flunarizine and a structurally unrelated compound, SIM 6080 (Table 2). However, not all calcium antagonists were shown to be effective (Table 2)'8~141. This may be

Table 1 Prototype and newer calcium antagonists Prototype

New generation

Verapamil

Anipamil Gallopamil Ronipamil Amlodipine Felodipine Darodipine Isradipine Lacidipine Lercanipine Nicardipine Nigludipine Niludipine Nivadipine Nimodipine Nisoldipine Nitrendipine Ryosidine

Nifedipine

In vivo studies Evidence suggests that calcium antagonists reduce the severity of experimental atherosclerosis in cholesterolfed animals without appreciably affecting plasma lipids. Many investigators have shown that dihydropyridine Correspondence: Prof. Alberico L. Catapano, Institute of Pharmacological Sciences, Via Balzaretti, 9, 20133 Milano, Italy. 0195-668X/97/OA0080+07 S18.00/0

Diltiazem Structurally unrelated

SIM 6080

© 1997 The European Society of Cardiology

Downloaded from eurheartj.oxfordjournals.org by guest on July 21, 2011

Calcium and cholesterol deposition is the hallmark of atherosclerotic lesions in the arterial wall1'1. Earlier experiments have examined whether inorganic compounds known to interfere with Ca 2+ fluxes could reduce the extent and gravity of experimentally induced atherosclerotic lesions. Studies on calcium-chelating agents and lanthanum, an inorganic calcium antagonist, showed that they effectively reduced the formation of atherosclerotic plaques'2'3'. Thus, an obvious extension of these findings was to study the anti-atherosclerotic properties of organic calcium antagonists as possible agents1'1. The formation of atherosclerotic plaques is believed to be multifactorial. However, cholesterol deposition, cellular proliferation and migration, increased cellular matrix, calcium overload and platelet aggregation are among the most common findings. All these processes are affected by calcium antagonists'2'61. In this review I shall briefly discuss the action of calcium antagonists (see Table 1 for list) on aortic lipid accumulation in experimentally induced atherosclerosis and identify the biochemical and cellular basis of this effect.

Key Words: Lipids, lipoprotein, LDL, LDL-receptor, cholesterol, ACAT.

Effect of calcium antagonists on experimental atherosclerosis Duration

Compound

AD AD AD AD AD AD AD AD AD AD AD

8 weeks 12 weeks 4 weeks 8 weeks 20 weeks 8 weeks 8 weeks 8 weeks 8 weeks 10 weeks 16 weeks

Nifedipine Nifedipine Nifedipine Nifedipine Nifedipine Nifedipine Nifedipine Nifedipine Nifedipine Nifedipine Nifedipine

12-6-15-6 m g . kg~ ' . day " ' 40 mg . animal " ' . day ~ ' 20 mg. k g " 1 . d a y " 1 0-625-1-25 m g . k g " 1 .day " ' 0-285 mg. k g " 1 . d a y " 1 2 mg . animal" ' . day " ' 40 mg . kg ~ ' . day." ' 40 mg . animal ~ ' . day " ' 80 mg. k g " 1 . d a y " 1 10 mg. k g " 1 . d a y " 1 40 mg . k g " ' . d a y " '

AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD

80 weeks 10 weeks 14 weeks 8 weeks 10 weeks 10 weeks 8 weeks 10 weeks 8 weeks 10 weeks 24 weeks 12 weeks 10 weeks 10 weeks 8 weeks 16 weeks 10 weeks 14 weeks

Nisoldipine Isradipine Nicardipine Nicardipine Nicardipine Nivadipine Lacidipine Verapamil Verapamil Verapamil Verapamil Verapamil Verapamil Verapamil Anipamil Diltiazem Diltiazem Diltiazem

20 mg . animal" ' . day " ' 1 mg . animal" ' . d a y " ' 60 mg . animal"' . day " ' 80 mg. k g " ' . d a y " 1 lOmg.kg"' .day"' 32 mg . kg " ' . day " ' 1, 3, l O m g . k g " ' . d a y " ' 40-400 mg . animal" ' . day " ' 16 mg , animal"' . d a y " ' 8 mg . k g " ' . d a y " ' +0-5 mg . k g " ' . day " ' 2 g/1000 ml of drinking water 2 g/1000 ml of drinking water 40 mg . animal" ' . d a y " ' +2 mg . k g " ' . day " ' 2 g/1000 ml of drinking water lOmg.kg"' .day"' 120 mg . animal"' . d a y " ' 103mg.kg"' .day"' 210mg . animal"' . day" '

Dose

Route

Atherosclerosis

Serum cholesterol

Refe

po po po po po po po po po sc po

Decreased Decreased Decreased Unchanged Unchanged Decreased Unchanged Unchanged Decreased Decreased Decreased

Unchanged Unchanged ND Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged

[4 [4 [4 [4 [4 [4 [4 [1 [5 [ [5

NR po po po sc sc po po po po + sc po po po + sc po NR po po po

Decreased Decreased Unchanged Decreased Decreased Decreased Decreased Decreased Unchanged Decreased Decreased Unchanged Unchanged Unchanged Decreased Unchanged Decreased Unchanged

Unchanged Unchanged Unchanged Decreased Unchanged Unchanged Unchanged Increased Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged

e heterozyg.)

ND


Method

[5 [5 [1 [5 [5 [5 [3 [1 [ [5 [5 [ [ [ [5 [5 [6 [1

Continued Duration

Compound

AD AD AD AD AD AD AD AD AD AD AD WHHL WHHL WHHL WHHL WHHL AD + BI BI BI BI BI AD + BI CI CI AD CI CI CI CI ES ES ES ES

10 weeks 10 weeks 50 days 10 weeks 10 weeks 60 days 10 weeks 8 weeks 10 weeks NR 24 months 20 months 6 months 17 weeks 17 weeks 6 months 4 weeks 14 days 14 days 14 days 25 days 8 months 21 days 21 days 8 weeks 21 days 21 days 21 days 21 days 4 weeks 4 weeks 4 weeks 4 weeks

Diltiazem Diltiazem Flunarizine Flunarizine Flordipine SIM 6080 Lanthanum Lanthanum Lanthanum Nifedipine Lanthanum Nifedipine Nifedipine (-)Anipamil (-)Anipamil Verapamil Nifedipine Isradipine Darodipine Nifedipine Verapamil Diltiazem Nifedipine Nivadipine Lacidipine Verapamil Diltiazem Diltiazem Flunarizine Nimodipine Verapamil Flunarizine Flunarizine

Dose

50 mg . animal ' . d a y " 1 30 mg . animal ' . d a y " ' 10 mg . k g " . d a y " ' 5-7 mg. k g " . d a y " ' 45 mg . kg " . d a y " ' 2mg. kg" .day"' 40 mg . kg " . d a y " ' 20 mg . kg " . d a y " ' 35 m g . k g " . d a y " ' NR 4 0 m g . k g " ' J day"' 40 mg. animal" ' . d a y " ' 40 mg. animal" ' . d a y " ' 1 mg . kg " ' .d a y " ' lOmg.kg"1 . day"' 46 mg . k g " ' . day "'+0-5 mg . kg " ' day" 40 mg . animal ~ ' . d a y " ' 0-25 mg. k g " 1 . d a y " ' 1 mg . kg " ' .day" 1 2 0 m g . k g " ' . day" 1 2 mg . kg " ' . d a y " ' 1 m g . k g " 1 . day"' 1 mg.kg"1 . day"' 001 mg. k g " 1 . d a y " ' 1, 3, l O m g . k g " ' . d a y " ' lOmg.kg"' . day"' lOOmg.kg" 1 . d a y " ' lOmg.kg"1 . day"' 5-7mgkg-'l day"' 9 0 m g . k g - ' . day"' 21-5 mg. kg" 1 . d a y " ' +2 m g . k g " ' day" 25mg.kg"' . day"' lOmg.kg"' . day"'

Route

Atherosclerosis

Serum cholesterol

'P ip po po po sc po po po NR po po po po po po + sc po sc sc po po po im im po im po im po po po + sc po po

Decreased Decreased Unchanged Unchanged Unchanged Decreased Decreased Decreased Unchanged Decreased Decreased Unchanged Unchanged Unchanged Decreased Unchanged Unchanged Decreased Decreased Decreased Decreased Decreased Decreased Decreased Decreased Unchanged Unchanged Decreased Unchanged Unchanged Decreased Decreased Decreased

Decreased Decreased Unchanged Unchanged Unchanged Unchanged Unchanged Unchanged Increased ND ND Unchanged Decreased Unchanged Unchanged Unchanged ND ND ND ND Unchanged Unchanged ND ND Unchanged ND Increased ND ND ND ND ND ND

ogenic diet; BI = balloon injury; Cl = cuff injury; ES=electrical stimulation; NR = not reported; ND = not determined; WHHL=Watanabe rabbit.

Refe


ous monkey ous monkey

Method

[6 [6 [6 [6

[2 [6 [6 [5 [6 [6 [4

[6 [6 [1 [6 [6 [6 [1 [6 [7 [7 [7 [3 [7 [7 [7 [7

[7 [

Calcium antagonists and atherosclerosis

The mechanism by which calcium antagonists exert their anti-atherosclerotic activity is not clear. One of the early stages in the pathogenesis of the atherosclerotic lesion is the accumulation of cholesterol in the arterial wall. Calcium antagonists reduce aortic cholesterol, but this effect is not mediated by a reduction in plasma lipid concentrations (Table 2), suggesting that calcium antagonists do not have a major impact on overall lipoprotein catabolism. However, several studies using cultured cells indicate that calcium antagonists modify cellular lipid metabolism in cells of the arterial wall. This effect apparently involves both cholesterol delivery to the cells, by affecting the receptor-mediated catabolism of lipoproteins, and the intracellular cycle of cholesterol.

In vitro studies Stein et a/.'171 first demonstrated that verapamil upregulates the expression of low-density lipoprotein (LDL) receptors, thus increasing binding and internalizing of LDL in cultured fibroblasts, arterial smooth muscle cells and endothelial cells. In our laboratory, we showed that of the calcium antagonists, only verapamillike compounds and diltiazem are effective in these cellular functions. This stimulatory effect involves a de novo synthesis of LDL receptors'17"191. Nifedipine-like compounds and flunarizine, however, are inactive'181. The effect of calcium antagonists on LDL degradation is multifaceted. Because of their basic nature, these compounds may target lysosomes and reduce enzyme activity in the organelles by raising the lysosomal pH'20]. This would offset the expected increase of LDL degradation owing to the calcium antagonistinduced receptor-mediated influx of lipoproteins into the cells. The total amount of LDL degradation by the cells

will depend on the fine tuning of the stimulatory activity of calcium antagonists on LDL uptake and the inhibitory effect on LDL degradation. Time of incubation, calcium antagonist concentration, and the type of cell tested may have a considerable affect on the results of intracellular lipoprotein degradation. Some workers suggested that inhibition of LDL degradation induces accumulation of intact LDL in human fibroblasts and smooth muscle cells'21'221. This mechanism, and not stimulation of LDL-receptor activity, would be responsible for the increased uptake of LDL by cells'221. This hypothesis, however, is not supported by experimental evidence. First, calcium antagonists induce both LDL internalization and LDL binding in the absence of an exogenous cholesterol supply in cultured cells owing to increased synthesis of the LDL receptor. Second, stimulation of LDL uptake is detectable in both the presence and absence of lipoprotein degradation by calcium antagonists'6'17'22>23). Although accumulation of intact LDL alone cannot explain the effect of calcium antagonists on LDL receptor activity'241, a review of the currently available data suggests that a basic group needs to be present on the calcium antagonist molecule to modulate LDL receptor expression. In our laboratories we have shown that the dihydropyridine derivative amlodipine, which has basic properties similar to verapamil, effectively stimulates LDL receptor expression in human fibroblasts'231. Nifedipine, which is not a basic drug, is inactive'181. This observation also suggests that the voltage-dependent calcium channel blocking activity of these compounds is not involved in LDL receptor stimulation. This is further supported by the concept that several of the cell lines used in these studies lack voltage dependent Ca 2+ channels and by the observation that enantiomers of lercanidipine are both equally effective (Bernini et al., personal communication). Although calcium antagonist concentration in plasma is lower than the concentration of the same molecules required for an in vitro effect in LDL-receptor expression, the distribution volumes of several calcium antagonists suggest that these molecules accumulate in tissues. Concentrations likely to exert an effect on lipoprotein receptors may be achieved in the aortic wall. Data from our laboratory suggest that this is the case for some calcium antagonists'251. Calcium antagonist induction of LDL receptormediated catabolism may reduce the period in which LDL resides in the extracellular fluid, where LDL undergoes modifications that may lead to the formation of 'atherogenic' lipoproteins'261. Moreover, increased LDL uptake promotes the transfer of LDL cholesteryl ester from extracellular interstitium to an intracellular catabolic compartment. Verapamil affects lipoprotein catabolism in macrophages by reducing LDL and acetyl-LDL degradation and increasing the cellular uptake of these lipoproteins'27'281. Calcium antagonists also interfere with cholesteryl ester hydrolysis and reesterification, thus Eur Heart J, Vol. 18, Suppl A 1997


because: (1) in some studies the induction of atherosclerotic lesions was only mild to moderate, and possibly insufficient to detect any protective effect; (2) plasma and tissue concentrations of the drugs were not determined and therefore drug concentrations may have been ineffective. This is supported by Blumlein and coworkers'151,who pointed out that verapamil reduced atherosclerotic lesions only in rabbits with detectable plasma levels of the drug, but not in animals in which the concentrations were negligible. Another factor may have been the timing of drug administration: it appears that the drug is effective if it is administered concomitantly with the atherogenic stimuli (i.e. cholesterol feeding), but if the lesions have already begun to form, calcium antagonists usually have little or no effect'161. This observation may also explain the ineffectiveness of Ca 2+ antagonists in homozygous Watanabe rabbits, an animal model for familial hypercholesterolaemia (Table 21521). Recently, however, Fischer Hansen et al. (Table 2|67]) showed that ( - )Anipamil retards atherosclerotic development in the thoracic aorta of young Watanabe rabbits, further stressing the necessity for early treatment.

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Partial inhibition of lysosomal lipoprotein degradation and cholesteryl ester hydrolysis by basic calcium antagonists, including amlodipine, may therefore be regarded as beneficial rather than as a possible pro-atherogenic side effect. In fact, these compounds may modulate excessive release of free cholesterol not only to the cytoplasmic compartment (as previously discussed) but also to the plasma membrane'3'1 where cholesterol may be toxic[32]. The non-hydrolyzed cholesteryl ester, retained in the cells because of lysosomal inhibition by calcium antagonists, is eventually eliminated. This hypothesis is supported by data on smooth muscle cells where verapamil induces an initial (2 to 7 days) increase in cellular cholesteryl ester content that, at longer incubation times (18 to 35 days), is followed by a net decrease as compared with control cells1331. Some investigators have also suggested that reduction of lysosomal cholesteryl ester hydrolysis may increase the scavenging activity of monocytes/ macrophages, thus improving their ability to remove cholesteryl ester from the arterial wall'27'281. Whether this finding should be regarded as beneficial depends on the Eur Heart J, Vol. 18, Suppl A 1997

ability of lipid-laden macrophages to leave the arterial wall[34]. Another possible lipid-related cellular mechanism involved in the anti-atherogenic potential of dihydropyridine calcium antagonists has been identified by Etingin and Hajjar'351 in rabbit smooth muscle cells. They demonstrated that nifedipine decreases cholesterol and cholesteryl ester in lipid-loaded smooth muscle cells, which presumably results from activation of cellular cholesteryl ester hydrolysis. Experiments on aortic tissue from patients treated with nifedipine or diltiazem confirmed these findings1351. In addition, nifedipine reduces the cholesterol content of macrophages by inducing cholesterol efflux'361. Finally a number of Ca 2+ antagonists have been shown to interfere with smooth muscle cell migration and proliferation in vitro, and possibly in vivo137"421 thus indicating that this pathway, leading to neointimal accumulation of smooth muscle cells, can be controlled by calcium antagonists. In conclusion, calcium antagonists may reduce cholesterol accumulation in the arterial wall, thus displaying anti-atherosclerotic properties by directly affecting cellular lipid metabolism. Depending on the calcium antagonist used, the mechanism(s) underlying this activity may differ. Whether these in vitro effects of calcium antagonists on cellular lipid metabolism are relevant to the in vivo condition deserves further investigation. The experimental work of the author reported in this study was supported, in part by a C.N.R Grant (Progetto Finalizzato Invecchiamento, Publication No. 963670) and a Research Grant of the European Community (PL931790). Miss Maddalena Marazzini typed the manuscript.

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affecting the intracellular deposition of cholesteryl ester. Daugherty et al.l29] showed that nifedipine, BAY K 8644 (a dihydropyridine calcium agonist) and verapamil analogues, but not diltiazem or flunarizine, inhibit the /? very low density lipoprotein-induced cholesterol esterification in rabbit macrophages. These investigators concluded that these effects are independent of calcium entry blockade. Verapamil has two main effects on cholesteryl ester metabolism in macrophages1281. First, similar to its action in LDL apoprotein degradation, verapamil inhibits cholesteryl ester hydrolysis in lysosomes, resulting in accumulation of esterified sterol. Second, verapamil reduces cholesterol esterification by the acyl-coenzyme A cholesterol acyltransferase (ACAT) by inhibiting the delivery of cholesterol to the esterification site. This effect is independent of inhibition of lysosomal cholesteryl ester hydrolysis. A reduction in ACAT activity would reduce cholesteryl ester deposition in the cells and favour cholesterol efflux. The effect of verapamil on cellular cholesteryl ester content results from the balance of these two actions. Although the aforementioned activities have opposite consequences on cellular cholesteryl ester content, both reduce the delivery of cholesterol to the cholesteryl ester cycle operating in the cytoplasm of macrophages, where esterified and free cholesterol are continuously hydrolyzed and reesterified[30]. This 'futile' cycle uses acyl-coenzyme A derivatives in the reesterification reaction, thereby inducing continuous waste of adenosine triphosphate[30]. Because verapamil reduces the delivery of cholesterol to the cycle it is possible that this drug exerts a 'protective effect' on cells by reducing adenosine triphosphate depletion. Such a mechanism, if present in atherosclerotic lesions, may be of relevance in reducing foam cell necrosis in poorly oxygenated tissue. Recent data from Bernini et al. (personal communication) suggest that some Ca 2+ antagonists may directly modulate ACAT activity.

Calcium antagonists and atherosclerosis

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