Cardiovascular Drugs and Therapy 4: 1015-1020, 1990 @ Kluwer Academic Publishers, Boston. Printed in U.S.A.
Antiatherogenic Effects of Calcium-Channel Blockers: Possible Mechanisms of Action Philip D. Henry Baylor College of Medicine, Houston, TX, USA
Summary. Calcium-channel blockers (Ca blockers), such as nifedipine, verapamil, diltiazem, flunarizine, and their respective derivatives, have been reported to suppress the formation of arterial lesions in animals fed atherogenic diets. The fact that structurally unrelated Ca blockers exert similar antiatherogenic effects may suggest that the drugs act by a calcium-channel-dependent mechanism. However, in cell culture experiments in which putative antiatherosclerotic effects were observed only in the presence of a very high drug concentration (> 10 IxM), calcium-channel-independent mechanisms are likely. It does not appear that Ca blockers act predominantly by altering coronary risk factors such as arterial pressure or hypercholesterolemia. On the other hand, current evidence is accumulating that Ca blockers may act by suppressing chemotaxis and the proliferation of cells involved in lesion formation. Recent reports indicate that relatively low concentrations (< 1 pxM) of nifedipine may promote the release of cholesterol from fat-laden smooth cells and macrophages. Controlled clinical trials are needed to determine whether Ca blockers have utility in the prevention of the progression of atherosclerosis in humans. Key Words. atherosclerosis,.nifedipine, verapamil, diltiazem
Calcium-channel blockers with disparate chemical structure and pharmacologic profiles have been shown to retard the progression of atherosclerois in different animal species (Tables 1 and 2). Although antiatherosclerotic actions of these drugs have been extensively investigated with both in-vivo and in-vitro techniques, their mechanism(s) remains unclear (Table 3). In this paper, we briefly review effects of calcium blockers that could account Ibr their antiatheroselerotie activity.
Effects on Hemodynamics Atheroma formation and localization depends upon hemodynamic factors, which play an important role in determining the distribution of atheromatous lesions in the vasculature (Schettler et al., 1983). Arterial hyr pertension tends to accelerate atherogenesis, whereas a low endovascular pressure, as occurs physiologically in the pulmonary arterial circulation, helps to prevent lesion formation. Studies on the effects of calcium
blockers on diet-induced atherosclerosis have been performed mostly in cholesterol-fed rabbits (Henry, 1988). Bretherton et al. (1977) demonstrated in cholesterol-fed rabbits that hypertension aggravated atherosclerosis, but the effects of pharmacologically induced arterial hypotension with hydralazine did not conclusively attenuate arterial disease. Nevertheless, since calcium blockers act as hypotensive agents, their effects on atherogenesis could reflect a reduction in arterial pressure. In our initial report on the antiatherogenic effects of nifedipine in cholesterol-fed rabbits, diurnal monitoring of arterial pressure demonstrated only minor, short-lasting reductions in pressure (-10 mmHg for < 120 minutes) (Henry and Bentley, 1981). Blumlein et al. (1984) concluded that the antiatherogenic action of verapamil in cholesterolfed rabbits was not mediated by an effect on arterial pressure. In our study with low-dose isradipine (PN 200110, < 0.3 mg/day/kg), the calcium blocker suppressed atherogenesis without a measurable reduction in arterial pressure (Habib et al., 1986). These results do not support the view that calcium blockers exert antiatherogenic effects predominantly by lowering arterial pressure. Calcium blockers could theoretically exert protective fluid mechanical effects that are not directly related to systemic arterial resistance regulation, such as effects on secondary flow patterns (Schettler et al., 1983) o1" arterial wall perfusion (Heistad and A~'mstrong, 1986).
Effects on Platelets Platelets have been invoked to play a major role in the development of atherosclerotic lesions, and aspirin has been demonstrated to suppress diet-induced atherosclerosis in monkeys (Ross, 1986; Pick et al., 1979).
Address for correspondence and reprint requests: Philip D. Henry, MD, Professor of Medicine, Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Suite 513E, Houston, TX 77030, USA.
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Table 1. Calcium-channel blockers reported to e.rert autiatheroge~tic effect in iMact a~timals
Table 2. Species in which calcium-channel blockers suppressed tn'ogressio~ qf atherosclerosis
I. Nifedipinefand derivatives (dihydropyridine derivatives) Nifedipine (Henry, 1981) Nieardipine (Willis, 1985) Isradipine (Habib, 1986) Nisoldipine (Fronek, 1988) Nilvadipine (Nomoto, 1987) Nimodipine (Betz, 1988) II. Verapamil and derivatives ("phenylalkylamines") Verapamil (Rouleau, 1983) Anipamil (Catapano, 1988) III. Diltiazem (Ginsburg, 1983) IV. Flunarizine (Betz, 1985)
Rabbit (Henry, 1981; Rouleau, 1983; Ginsbm-g, 1983 and others) Rat (Handley, 1986) Rhesus monkey (Lichtor, 1989) Humans (Liehtlen, 1990; Waters, in press 1990)
Therefore, calcium-channel blockers could exert antiatherosclerotic effects by acting on platelets. Studies on the effects of calcium blockers on platelets, however, have been somewhat conflicting (Henry and Perez, 1984). In general, it seems that inhibition of aggn'egation in vitro with verapamil and diltiazem occurs only at very high drug concentrations (10-100 IxM), and dihydropyridine derivatives may be completely inactive (Henry and Perez, 1984; MacIntyre and Shaw, 1983). Since platelets are devoid of voltagedependent calcium channels (Pannochia et al., 1987), it is possible that some of the calcium-channel blockers act by nonspecific mechanisms such as local aesthetic effects (Feinstein et al., 1976) or blockade of cyclic AMP phosphodiasterase activity (Moore et al., 1985). In addition, verapamil may act by binding to platelet alpha.,-adrenergic receptors (Jones et al., 1985). In a number of reports, calcium-channel blockers have been shown to block platelet aggregation in vivo (Jones et al., 1985; Molinari et al., 1987). Unfortunately, in several studies appropriate control experiments with non-calcium-blocking vasodilators were not performed, and it is unclear to what extent the putative effects on platelets reflected indirect microcirculatory events. Platelets may undergo changes as they pass through hypoxic or ischemic circulatory beds as a result of local releases of aggTegating substances such as catacholamines. Vasodilation and partial correction of such microcirculatory abnormalities may help to prevent platelet alterations in vivo.
Effects on Circulating Lipoproteins Worldwide use of different calcium blockers in humans has failed to reveal appreciable effects of these drugs on circulating lipids and lipoproteins (Lardinois and Neuman, 1988). In the vast majority of experimental studies, dihydropyridine calcium blockers, verapamil, and diltiazem did not suppress hyperlipidemic re-
sponses to high fat diets (Henry, 1988). Ohata et al. (1984), however, observed that nicardipine produced substantial reductions in LDL cholesterol and elevations in HDL.~ cholesterol levels in fat-fed rats. Willis et al. (1985) noted a reduction in cholesterol concentration in the very low density fraction (d < 1.006 g/ml) in cholesterol-fed rabbits treated with nicardipine. In one study (Sugano et al., 1986), diltiazem reduced the total and LDL cholesterol concentrations in cholesterol-fed rabbits, but in other studies with rabbits this drug had no effect on serum lipoproteins (Ginsburg et al., 1983; Naito et al., 1984; Diccianni et al., 1987). It should be pointed out that in most studies on the effects of calcium blockers on atherogenesis, detailed analyses of lipoproteins or plasma lipids were not performed. Therefore, it cannot be ruled out that these drugs act on some atherogenic lipids (lipoproteins) such as postprandial renmants, Lp(a), or modified (oxidized) lipoproteins.
Effects on Endothelium Endothelial injury (o1"more generally altered behavior of endothelial cells toward blood cell or plasma components) has been postulated to play a major role in initiating tile formation of atheromatous lesions (Ross, 1986). One endothelial function that has received considerable attention in atherosclerosis research is endothelial permeability to plasma macromolecules (lipoprotein complexes). Tedgui et al. (1987) observed that in isolated rabbit aortas exposed to a solution containing a high potassium concentration (80m mM, KC1), increased permeation of albumin across the endothelial barrier was blocked by nicardipine but not by verapamil. McDonagh and Roberts (1986) observed that nisoldipine prevented capillary macromolecular leakage in rat hearts subjected to ischemia and reperfusion. Betz and collaborators (Betz et al., 1985; Betz, 1988; Strohschneider and Betz, 1989) evoked in rabbits endothelial hyperpermeability of the carotid artery by implanting electrodes across the artery and passing intermittently low currents through it. Treatment of the rabbits with flunarizine, anipamil (a verapamil derivative), or nimodipine prevented the permeation of a macromolecular marker (horseradish
Anti-Atherogenic Mechanisms
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Table 3. Experime~ttal methods applied.lbr the demonstratim~ qf rmliatherosclerolic effects q/calcium-cha,~tel blockers I. Experiments with intact animals A. Morphometric and biochemical characterization of atheromatous lesions in fat-fed animals (Henry, 1981; Willis, 1985; Stender, 1986; Strohschneider 1989) B. Repair response of arteries to local injury induced with a balloon catheter or with electric stimulation (Handley, 1986; Jackson, 1988; Betz, 1988) C. Endothelial permeability to injected macromolecularmarkers (Strohschneider, 1989; Betz, 1989) D. Measurement of risk factors: plasma lipoprotein levels, arterial pressure (Ohata, 1984: Willis, 1985; Sugano, 1986:Bretherton, 1977) II. Experiments with isolated arteries A. Arterial reactivity to endothelium-dependent and -independent agonists (Henry, 1980; Habib, 1986; Bossaller, 1987) B. Endothelial permeability of perfused artery (Tedt,mi, 1987) C. Calcium flux studies (Striekberger, 1988) III. Experiments with cells in culture A. B. C. D. E.
Lipoprotein uptake (smooth muscle, macrophage) (Stein, 19s5: Corsini, 1,q86:Stein, 1987: Daugherty, 1987) Release of lipid from fat-laden cells (smooth muscle, macrophage) (Etingin, 1985; Schmitz, 1988) Chemotaxis (Boyden chamber, i.e., migration across Millipore filters) (Nakao, 198::I:Nomoto, 1987) Cellular proliferation (DNA accumulation) (Nilsson, 1985; Stein, 19871 Production of matrix proteins ("tibrosis") (Weinstein, 1987)
peroxidase, MW 40,000) through the endothelial bartier. Endothelium regulates vascular tone by releasing endothelimn-del)endent relaxation factor (EDRF) and other vasoactive agents. We have shown that atherosclerosis in cholesterol-fed rabbits (Habib et al., 1986) and humans (Bossaller et al., 1987) is associated with an impairment of endothelium-dependent relaxation. Habib et al. (1986) reported that treatment of cholesterol-fed rabbits with a dihydropyridine calcium blocker preserved endothelium-dependent arterial relaxation. Collectively these observations are compatible with the notion that calcium-channel blockers might exert direct effects on injured endothelium.
Effects on U p t a k e a n d D e g r a d a t i o n o f L i p o p r o t e i n s b y A r t e r i a l Cells A halhnark of atherosclerotic lesions is the accumulation of intracellular lipid and the formation of foam cells. Since endocytotic processes underlying internalization of lipoproteins are calcium dependent, one might envision that calcium blockers could interfere with calcium-dependent lipoprotein uptake. Stein et al. (1985) showed that cultured bovine aortic endothelial cells and human skin fibroblasts after exposure to 10-50 IxM verapamil unexpectedly acquire an increased binding and uptake capacity for LDL, a phenomenon ascribed to drug-induced increases in the abundance of LDL receptors. They proposed that in-
creased endocytotic removal of cholesterol esters from the aortic interstitium might exert antiatherosclerotic effects. In a subsequent study, the same group of workers cultm'ed rabbit aortic smooth muscle for up to 5 weeks in the presence of a hypercholesterolemic plasma fl'actions (density < 1.019 g/ml) (Stein et al., 1987). The cholesterol-rich fractions (~3-VLDLs) evoked increased DNA and cholesterol ester accumulation per culture dish. Addition of 50 ~m verapamil suppressed DNA accumulation, a response thought to represent antiproliferative activity. During the first week, the drug increased, but later decreased, cholesteryl ester accumulation. Corsini et al. (1986) likewise observed that 10 txM verapamil acutely increased specific binding and internalization of LDL by human skin fibroblasts in culture. In contrast to the acute increases in cholesteryl ester deposition described by Stein et al. (1987) and Corsini et al. (1986), Daugherty et al. (1987) found acute decreases in eholesteryl ester accumulation in cultured alveolar rabbit macrophages exposed to 0.1-10 txm verapamil. In cholesterol-fed rabbits, Stender et al. (1986) observed that accumulation of cholesteryl esters in aorta was suppressed after treatment with verapamil (18 mg/kg.day). Variable acute effects of very high concentrations of verapamil on cultured cells indicate that results in vitro should be interpreted cautiously in the context of chronic pharmacologic actions in intact animals. Etingin and Hajjar (1985) reported that aortic smooth muscle cultured from fat-fed rabbits were depleted of their cholesteryl ester content by exposure
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to 0.3 ~xM nifedipine. Effects on the lipid-laden cells were ascribed to stimulation of cyclic-AMP-dependent cholesteryl e~ter hydrolase activity in lysosomes. In acetyl-LDL-laden mouse peritoneal macrophages in culture, Schmitz et al. (1988) observed that 2 ~xM nifedipine stimulated cholesterol release from the cells. The drug induced the formation of lysosomederived "lamellar bodies," which were secreted as cholesterol-rich particles in the culture medium in the absence of HDL as a cholesterol acceptor. The authors described an additional mechanism of cholesterol excretion via lamellar bodies derived fi'om intracellular lipid droplets that fused u4th HDL-containing endosomes. The release of HDL-associated cholesterol depended upon the availability of extracellular HDL. The action of nifedipine oll cholesterol efflux described by Schmitz et al. (1988) has features in common with those characterized previously by Etingin and Hajjar (1985). Cholesteryl ester accumulation in cultured vascular smooth muscle has been reported to be dosedependently inhibited by exposure to isradipine (10100 txM) (Weinstein and Heider, 1987). Using even higher drug concentrations of nifedipine (up to 146 ~M), Saito et al. (1986) suppressed cholesterol synthesis in rabbit aortic smooth muscle in culture.
Effects on Chemotaxis Nakao et al. (1983) found that 12-HETE, a lipoxygenase product of arachidonic acid, had potent chemoattractant effects on rat aortic smooth muscle in culture. They demonstrated that nicardipine (10 :~ to 10 -r' M) inhibited HETE-associated cell mign'ation across Millipore filters. In similar experiments, Nomoto et al. (1987) stimulated the mign'ation of rat aortic smooth muscle cells in culture in response to zymosanactivated exudate and showed that nilvadipine (10 '-' to 10 -~ M) exerted potent inhibitory effects on migration. Inhibitory effects in the studies by Nakao and Nomoto occurred at seemingly very low drug concentrations (pico- to nanomolar), but it is unclear whether the Millipore filter across which mign'ation occm'red accumulated the lipophilic drugs from the aqueous medimn.
Effects on Cell Proliferation Betz and collaborators (Betz et al., 1985; Betz, 1988; Strohschneider and Betz, 1989) showed that rabbit carotid arteries subjected to repeated electric stimulation with implanted electrodes underwent an intimal proliferation of smooth muscle beneath the anode. If the rabbits were also given a cholesterol-rich diet, proliferative plaques became typical atheromas.
Treatment of the rabbits during the periods of electric stimulation with different calcium blockers (flunarizine, nimodipine, anipamil) suppressed the proliferative responses. Handley et al. (1986) produced intimal proliferative lesions in rat carotid arteries by damaging the vessels with a balloon catheter. Treatment of the rats with isradipine reduced the average lesion cross-sectional area by 44%. Jackson et al. (1988) injured the rabbit aorta with a balloon catheter and quantified the intimal proliferative response by motphometry and incorporation of :~H-thymidine into DNA. In rabbits treated with nifedipine (10 mg/ kg.day), there was a 39% reduction in lesion crosssectional area 2 weeks after injury and a 53% reduction in thymidine labeling of the intima-media. Antiproliferative effects of calcium blockers have also been assessed in cell culture systems. Nilsson et al. (1985) showed in cultured rat aortic smooth muscle cells that 1 jxM nifedipine slowed the rate of transformation of the cells fi'om a contractile to a "synthetic" phenotype and inhibited the initiation of DNA synthesis as well as cell proliferation. The inhibitory effect on DNA synthesis was seen in cells stimulated with whole serum or with platelet-derived growth factor. Weinstein and Heider (1987) found that 50 jxM isradipine inhibited in monkey aortic smooth muscle cells in culture the incorporation of :~aS-sulfate into glycosaminoglycans, ~4C-proline into collagen, and :~Htryptophan into cell total protein. Results were interpreted to represent inhibition of the biosynthesis of matrix proteins, probably independent of an effect on calcium channels. Orekhov et al. (1987) cultured intimal cells ("modified smooth muscle cells") fl'om human aortic lesions and observed that verapamil (5-50 ~xM)inhibited :~H-thymidine incorporation into cellular DNA, intracellular deposition of lipid, and extracellular deposition of collagen. Similarly, Stein et al. (1987) showed that 50 txM verapamil inhibited DNA synthesis by cultured rabbit and bovine aortic smooth muscle cells, as mentioned above.
Clinical Implications All studies on the treatment of atherosclerosis in animals fed high-cholesterol diets have focused oll the formation or regression of early foam-cell-rich lesions. Occlusive atherosclerotic lesions, as seen in patients undergoing diagnostic workup for symptomatic coronary disease, are rich in fibrous tissue, extracellular lipid, and thrombotic material (Roberts, 1977; Haudenschild and Backa, personal communication), components that could be completely refi'actory to suppressive mechanisms of calcium blockers. Accordingly, clinical trials on antiatherosclerotic effects of
Anti-Atherogenie Mechanisms
calcium blockers will have to concentrate not on advanced lesions (stenoses), but more on new lesion formation. Unfortunately, new lesions may produce little or no angiographieally detectable luminal distortions (Glagov et al., 1987), and new techniques, such as endovaseular ultrasound imaging, will have to be applied to assess new lesion formation in the clinical setting.
References Betz E. The effect of calcium antagonists on intimal cell proliferation in atherogenesis. A ~ . N Y Acod Sci 1988;522:399-410. Betz E, Hammerle H, Strohschneider T. Inhibition of smooth muscle cell proliferation and endothelial permeability with flunarizine in vitro and in experimental atheromas. Rex Exp Med 1985; 185:;:125-340. Blumlein SL, Sievers R, Kidd P, Parmley WW. Mechanism of protection fi-om atheroselerosis by verapamil in the cholesterol-fed rabbit. Am J Cardiol 1984;54:884-889. Bossaller C, Habib GB, Yamamoto H, et al. Impaired muscarinic endothelium-dependent relaxation and cyclic g-aanosine 5'-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta. J Clin Invest 1987;79:170-i74. Bretherton KN, Day A J, Skinner SL. Hypertension-accelerated atherogenesis in cholesterol-fed rabbits. Atherosclerosis 1977; 27:79-87. Catapano AL, Maggd FM, Cicerano U. The antiatherosclerotic effect of anipamil in cholesterol-fed rabbits. A ~ N Y Acad Sci 1988;522:519-521. Cm'sini A, Fumagalli R, Paoletti R. Calcium antagonists and low density lipoproteins metabolism by human fibroblasts and by human hepatoma cell line HEP G2. lqmrm.e Res ('.ram 1986:18:1-16. Daugherty A, Rateri DL, Sehonfeld G, Sot)el BE. Inhibition of choleteryl ester deposition in macrophages by calcium entry blockers: An effect dissociable fi'om calcium entry blockade. [:h" J P h . r m . c o l 1987;91:113-118. Diccianni MB, Cardin AD, Britt AL, et al. Effect of a sustained release formulation of diltiazem on the development of atherosclerosis in cholesterol-fed rabbits...lH~erosch, r.sis 1987;65: 199-205. Etingin OR, Hajjar DP. Nifedil)ine increases cholesteryl ester hydrolytic activity in lipid-laden rabbit arterial smooth muscle cells. J Clin Invest 1985;75:1554-1558. Feinstein MB, Fiekers .J, Fraser C. An analysis of the mechanism of local anesthetic inhibition of platelet aggregation and secretion, l'harmucol s I) Thor 1976;215-228. Fronek K. Effect of nisoldipine on diet-induced atherosclerosis in rabbits. Am~ N Y Ae.d Sci 1988;522:525-526. Ginsburg R, Davis K, Bristow MR, et al. Calcium antagonists suppress atherogenesis in aorta but n(,t in the intranmral coronary arteries of cholesterol-fed rabbits. Lab lnv(,sl 1983;49: 154-158. Glagov S, Weisenberg E, Zarins CK, et al. Compensatory enlargement of human atherosclerotic coronary arteries. N Eugl J Meal 1987;316:1371-1375. Habib J B, Bossaler C, Wells S, et al. Preservation of endothelium-dependent vascular relaxation in cholesterol-fed rabbit by treatment with the calcium blocker PN 200110. ('ire Rex 1981-;;58:305-309. Handley DA, Van Valen RG, Melden MK, Saunders RN. Suppression of rat carotid lesion development by the calcium channel blocker PN 200-110. Am J Pathol 1986;124:88-93.
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Haudenschild, Backa. Personal communication. Heistad DD, Armstrong ML. Blood flow through vasa vasorum of cm'onary arteries in atherosclerotic monkeys. Arteriosclerosis 1986;6:::126-::I:31. Henry PD. Calcium antagonists as antiatherogenic agents. Ann N Y Acad Sci 1988;522:411-419. Henry PD, Bentley KI. Suppression of atherogenesis in cholesterol-fed rabbit treated with nifedipine. J Clb~ Invest 1981 ;68:1::166--1369. Henry PD, Perez JE. Clinical pharmacolo~, of calcium antagonists. In: Conti CR, Brest AN, eds. Cardiac drug therap!l. Philadelphia: F.A. Davis, 1984:93-109. Henry PD, Yokoyama M. Supersensitivity of atherosclerotic rabbit aorta to ergonovine. Mediation by a serotonergic mechanism. J Clbl Invest 1980;66:306-313. Jackson L J, Bush RC, Bowyer DE. Inhibitory effect of calcium antagonists on balloon catheter-induced arterial smooth muscle cell proliferation and lesion size. Atherosclerosis 1988;69: 115-192. Jones CR, Pasanishi F, Elliott HL, Reid JL. Effects of verapamil and nisoldipine on human platelets: In vivo and in vitro studies. Br J Clbl Phurmae 1985;20:191-196. Lardinois CK, Neuman SL. The effects of antihypertensive agents on serum lipids and lipoproteins. Arch Intern Med 1988; 148:1980-1288. Lichtlen PR, Hugenholtz PG, Rafflenbuel W, et al. Retardation of angiogq'aphic progression of coronary disease by nffedipine. Lain'el 1990;3:35:1109-1113. Liehtor T, Davis HR, Vesselinoviteh D, et al. Suppression of atherogenesis nifedipine in the cholesterol-fed rhesus monkey. App Pathol 1989;7:8-18. McDonagh PF, Roberts DJ. Prevention of transcoronm'y macromolecular leakage after ischemia-reperfusion by the calcium entry blocker nisoldipine. Cite Rcs 1986:58:127-136. Mclntyre DE, Shaw AM. Phospholipid-induced human platelet activation: Effects of calcium channel blockers and calcium chelators. Thromb Rcs 1983;31:833-844. Molinari A, Guarneri L, Pacel E, et al. Mouse antithrombotic assay: The effects of Ca' " channel blockers are plateletindependent. J Ptmrm.e.I E,rp Ther 1987;240:623-627. Moore JB, Fuller BL, Falotico R, Toman EL. Inhibition of rabbit platelet phosphodiesterase activity and aggq'egation by calcium channel blockers. Thromb Res 1985;40:401-411. Naito M, Kuzuya F, Asai K, et al. Ineffectiveness of Ca e ' antagonists nicardipine and diltiazem on experimental atherosclerosis in cholesterol-fed rabbits. Angi.lm.l!l 1984;35:622-627. Nakao J, Ito H, Ooyama T, et al. Calcium dependency of aortic smooth nmscle cell nfigration induced by 12-L-hydroxy5,8,10,14-eicosatetraenoic acid. Alhcr.sclerosis 1983;46:309319. Nilsson J, Sjolund M, Palmberg L, et al. The calcium antagonist nifedipine inhibits arterial smooth nmscle cell proliferation. Alheroseh,r~sis 1985;58:109-122. Nomoto A, Hirosumi J, Sekiguchi C, et al. Antiatherogenic activity of FR34235 (Nilvadipine) a new potent calcium antagonist. Atheroseh'r~sis 1987;64:255-261. Ohata I, Sakamoto N, Nagano K, Maeno H. Low density lipoprotein-lowering and high density lipoprotein-elevating effects of nicardipine in rats. Bim'hem Phurmuc 1984;33:21992205. Orekhov AN, Tertov VV, Khashimov IC4., et al. Evidence of antiatherosclerotic action of verapamil fl'om direct effects on arterial cells. Am J Cardiol 1987;59:495-496. Pannocchia A, Praloran N, Arduino C, et al. Absence of ( - ) (:~H)desmethoxyverapamil binding sites on human platelets
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and lack of evidence for voltage-dependent calcium channels. E u r J Pharmaeol 1987:142:83-91. Pick R, Chediak J. Glick G. Aspirin inhibits development of coronary atherosclerosis in cynomolgus monkeys (Macacafc~scicularis) fed an atherogenic diet. J CIb~ I~eest 1979;63:158162. Roberts WC. Coronary heart disease: A review of abnormalities observed in the coronary arteries. Cardioeasc Med 1977;2:2949. Ross R. The pathogenesis of atherosclerosis--an update. N E~(,ll J ,lied 1986;314:488-500. Rouleau JL, Parmley WW, Stevens J, et al. Verapamil suppresses atherosclerosis in cholesterol-fed rabbits. J A m Co!l Cardiol 1983;1:1453-1460. Saito Y, Fujiyama Y, Shirai K, Yoshida S. Effect of nifedipine on lipid metabolism is smooth muscle cells. In: Liehtlen PR, ed. Proceedings qf the 61h h~term~tional Adalat S!lmposium. New therap!! q/" ischem ic hea ~q disease a ~d h !lperte~sion. Amsterdam: Excerpta Mediea, 1986:479-483. Schettler G, Nerem RM, Sehmid-Sehonbein H. et al. In: Fluid d!mamic,s as a Iocalizi~lg ,lactor qt alherosclerosis. Berlin: Springer-Verlag, 1983:1-226. Schmitz G, Robenek H, et al. C a ' ' antagonists and ACAT inhibitors promote cholesterol efflux fi'om maerophages by different mechanisms. Arteriosclerosis 1988;8:46-56. Stein O, Halperin G, Stein Y. Long-term effects of verapamil on aortic smooth muscle cells cultured in the presence of hypercholesterolemic serum. Arteriosclerosis 1987;7:585-592.
Stein O, Leitersdorf E, Stein Y. VerapamiI enhauces receptormediated endocytosis of low density lipoproteins by aortic cells in culture. Arteriosclerosis 1985;5:35-44. Steuder S, Ravn H, Haugegaard M, Kjeldsen K. Effect of verapamil on accumulation of fl'ee and esterified cholesterol in the thoracic aorta of cholesterol-fed rabbits. Atheroselerosis 1986:61:15-23. Strickberger SA, Russek LN, Phair RD. Evidence for increased aortic plasma membrane calcium transport caused by experimental atherosclerosis in rabbits. Circ Res 1988;62:75-80. $trohschneider T, Betz E. Densitometrie measurement of increased endothelial permeability in arteriosclerotic plaques and inhbition of permeability under the influence of two calcium antagonists. Atherosclerosis 1989;75:135-144. Sugano M, Nakashima Y, Matsushima T, et al. Suppression of atheroselerosis in cholesterol-fed rabbits by diltiazem injection. Arterioseh'rosis 1986:6:237-241. Tedgui A, Chiton B, Curmi P, et al. Effect of nicardipine and verapamil on in vitro albumin transport in rabbit thoracic aorta. Arteriosclerosis 1987;7:80-87. Weinstein DB, Heider JG. Antiatherogenic properties of calcium antagonists. A m J Cardiol 1987;59:163B-172B. Willis AL, Nagel B, Churchill V, et al. Antiatherosclerotic effects of nicardipine and nifedipine in cholesterol-fed rabbits. Arteriosclerosis 1985;5:250-255.
Antiatherogenic Effects of Calcium-Channel Blockers: Possible Mechanisms of Action Philip D. Henry Baylor College of Medicine, Houston, TX, USA
Summary. Calcium-channel blockers (Ca blockers), such as nifedipine, verapamil, diltiazem, flunarizine, and their respective derivatives, have been reported to suppress the formation of arterial lesions in animals fed atherogenic diets. The fact that structurally unrelated Ca blockers exert similar antiatherogenic effects may suggest that the drugs act by a calcium-channel-dependent mechanism. However, in cell culture experiments in which putative antiatherosclerotic effects were observed only in the presence of a very high drug concentration (> 10 IxM), calcium-channel-independent mechanisms are likely. It does not appear that Ca blockers act predominantly by altering coronary risk factors such as arterial pressure or hypercholesterolemia. On the other hand, current evidence is accumulating that Ca blockers may act by suppressing chemotaxis and the proliferation of cells involved in lesion formation. Recent reports indicate that relatively low concentrations (< 1 pxM) of nifedipine may promote the release of cholesterol from fat-laden smooth cells and macrophages. Controlled clinical trials are needed to determine whether Ca blockers have utility in the prevention of the progression of atherosclerosis in humans. Key Words. atherosclerosis,.nifedipine, verapamil, diltiazem
Calcium-channel blockers with disparate chemical structure and pharmacologic profiles have been shown to retard the progression of atherosclerois in different animal species (Tables 1 and 2). Although antiatherosclerotic actions of these drugs have been extensively investigated with both in-vivo and in-vitro techniques, their mechanism(s) remains unclear (Table 3). In this paper, we briefly review effects of calcium blockers that could account Ibr their antiatheroselerotie activity.
Effects on Hemodynamics Atheroma formation and localization depends upon hemodynamic factors, which play an important role in determining the distribution of atheromatous lesions in the vasculature (Schettler et al., 1983). Arterial hyr pertension tends to accelerate atherogenesis, whereas a low endovascular pressure, as occurs physiologically in the pulmonary arterial circulation, helps to prevent lesion formation. Studies on the effects of calcium
blockers on diet-induced atherosclerosis have been performed mostly in cholesterol-fed rabbits (Henry, 1988). Bretherton et al. (1977) demonstrated in cholesterol-fed rabbits that hypertension aggravated atherosclerosis, but the effects of pharmacologically induced arterial hypotension with hydralazine did not conclusively attenuate arterial disease. Nevertheless, since calcium blockers act as hypotensive agents, their effects on atherogenesis could reflect a reduction in arterial pressure. In our initial report on the antiatherogenic effects of nifedipine in cholesterol-fed rabbits, diurnal monitoring of arterial pressure demonstrated only minor, short-lasting reductions in pressure (-10 mmHg for < 120 minutes) (Henry and Bentley, 1981). Blumlein et al. (1984) concluded that the antiatherogenic action of verapamil in cholesterolfed rabbits was not mediated by an effect on arterial pressure. In our study with low-dose isradipine (PN 200110, < 0.3 mg/day/kg), the calcium blocker suppressed atherogenesis without a measurable reduction in arterial pressure (Habib et al., 1986). These results do not support the view that calcium blockers exert antiatherogenic effects predominantly by lowering arterial pressure. Calcium blockers could theoretically exert protective fluid mechanical effects that are not directly related to systemic arterial resistance regulation, such as effects on secondary flow patterns (Schettler et al., 1983) o1" arterial wall perfusion (Heistad and A~'mstrong, 1986).
Effects on Platelets Platelets have been invoked to play a major role in the development of atherosclerotic lesions, and aspirin has been demonstrated to suppress diet-induced atherosclerosis in monkeys (Ross, 1986; Pick et al., 1979).
Address for correspondence and reprint requests: Philip D. Henry, MD, Professor of Medicine, Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Suite 513E, Houston, TX 77030, USA.
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Table 1. Calcium-channel blockers reported to e.rert autiatheroge~tic effect in iMact a~timals
Table 2. Species in which calcium-channel blockers suppressed tn'ogressio~ qf atherosclerosis
I. Nifedipinefand derivatives (dihydropyridine derivatives) Nifedipine (Henry, 1981) Nieardipine (Willis, 1985) Isradipine (Habib, 1986) Nisoldipine (Fronek, 1988) Nilvadipine (Nomoto, 1987) Nimodipine (Betz, 1988) II. Verapamil and derivatives ("phenylalkylamines") Verapamil (Rouleau, 1983) Anipamil (Catapano, 1988) III. Diltiazem (Ginsburg, 1983) IV. Flunarizine (Betz, 1985)
Rabbit (Henry, 1981; Rouleau, 1983; Ginsbm-g, 1983 and others) Rat (Handley, 1986) Rhesus monkey (Lichtor, 1989) Humans (Liehtlen, 1990; Waters, in press 1990)
Therefore, calcium-channel blockers could exert antiatherosclerotic effects by acting on platelets. Studies on the effects of calcium blockers on platelets, however, have been somewhat conflicting (Henry and Perez, 1984). In general, it seems that inhibition of aggn'egation in vitro with verapamil and diltiazem occurs only at very high drug concentrations (10-100 IxM), and dihydropyridine derivatives may be completely inactive (Henry and Perez, 1984; MacIntyre and Shaw, 1983). Since platelets are devoid of voltagedependent calcium channels (Pannochia et al., 1987), it is possible that some of the calcium-channel blockers act by nonspecific mechanisms such as local aesthetic effects (Feinstein et al., 1976) or blockade of cyclic AMP phosphodiasterase activity (Moore et al., 1985). In addition, verapamil may act by binding to platelet alpha.,-adrenergic receptors (Jones et al., 1985). In a number of reports, calcium-channel blockers have been shown to block platelet aggregation in vivo (Jones et al., 1985; Molinari et al., 1987). Unfortunately, in several studies appropriate control experiments with non-calcium-blocking vasodilators were not performed, and it is unclear to what extent the putative effects on platelets reflected indirect microcirculatory events. Platelets may undergo changes as they pass through hypoxic or ischemic circulatory beds as a result of local releases of aggTegating substances such as catacholamines. Vasodilation and partial correction of such microcirculatory abnormalities may help to prevent platelet alterations in vivo.
Effects on Circulating Lipoproteins Worldwide use of different calcium blockers in humans has failed to reveal appreciable effects of these drugs on circulating lipids and lipoproteins (Lardinois and Neuman, 1988). In the vast majority of experimental studies, dihydropyridine calcium blockers, verapamil, and diltiazem did not suppress hyperlipidemic re-
sponses to high fat diets (Henry, 1988). Ohata et al. (1984), however, observed that nicardipine produced substantial reductions in LDL cholesterol and elevations in HDL.~ cholesterol levels in fat-fed rats. Willis et al. (1985) noted a reduction in cholesterol concentration in the very low density fraction (d < 1.006 g/ml) in cholesterol-fed rabbits treated with nicardipine. In one study (Sugano et al., 1986), diltiazem reduced the total and LDL cholesterol concentrations in cholesterol-fed rabbits, but in other studies with rabbits this drug had no effect on serum lipoproteins (Ginsburg et al., 1983; Naito et al., 1984; Diccianni et al., 1987). It should be pointed out that in most studies on the effects of calcium blockers on atherogenesis, detailed analyses of lipoproteins or plasma lipids were not performed. Therefore, it cannot be ruled out that these drugs act on some atherogenic lipids (lipoproteins) such as postprandial renmants, Lp(a), or modified (oxidized) lipoproteins.
Effects on Endothelium Endothelial injury (o1"more generally altered behavior of endothelial cells toward blood cell or plasma components) has been postulated to play a major role in initiating tile formation of atheromatous lesions (Ross, 1986). One endothelial function that has received considerable attention in atherosclerosis research is endothelial permeability to plasma macromolecules (lipoprotein complexes). Tedgui et al. (1987) observed that in isolated rabbit aortas exposed to a solution containing a high potassium concentration (80m mM, KC1), increased permeation of albumin across the endothelial barrier was blocked by nicardipine but not by verapamil. McDonagh and Roberts (1986) observed that nisoldipine prevented capillary macromolecular leakage in rat hearts subjected to ischemia and reperfusion. Betz and collaborators (Betz et al., 1985; Betz, 1988; Strohschneider and Betz, 1989) evoked in rabbits endothelial hyperpermeability of the carotid artery by implanting electrodes across the artery and passing intermittently low currents through it. Treatment of the rabbits with flunarizine, anipamil (a verapamil derivative), or nimodipine prevented the permeation of a macromolecular marker (horseradish
Anti-Atherogenic Mechanisms
1017
Table 3. Experime~ttal methods applied.lbr the demonstratim~ qf rmliatherosclerolic effects q/calcium-cha,~tel blockers I. Experiments with intact animals A. Morphometric and biochemical characterization of atheromatous lesions in fat-fed animals (Henry, 1981; Willis, 1985; Stender, 1986; Strohschneider 1989) B. Repair response of arteries to local injury induced with a balloon catheter or with electric stimulation (Handley, 1986; Jackson, 1988; Betz, 1988) C. Endothelial permeability to injected macromolecularmarkers (Strohschneider, 1989; Betz, 1989) D. Measurement of risk factors: plasma lipoprotein levels, arterial pressure (Ohata, 1984: Willis, 1985; Sugano, 1986:Bretherton, 1977) II. Experiments with isolated arteries A. Arterial reactivity to endothelium-dependent and -independent agonists (Henry, 1980; Habib, 1986; Bossaller, 1987) B. Endothelial permeability of perfused artery (Tedt,mi, 1987) C. Calcium flux studies (Striekberger, 1988) III. Experiments with cells in culture A. B. C. D. E.
Lipoprotein uptake (smooth muscle, macrophage) (Stein, 19s5: Corsini, 1,q86:Stein, 1987: Daugherty, 1987) Release of lipid from fat-laden cells (smooth muscle, macrophage) (Etingin, 1985; Schmitz, 1988) Chemotaxis (Boyden chamber, i.e., migration across Millipore filters) (Nakao, 198::I:Nomoto, 1987) Cellular proliferation (DNA accumulation) (Nilsson, 1985; Stein, 19871 Production of matrix proteins ("tibrosis") (Weinstein, 1987)
peroxidase, MW 40,000) through the endothelial bartier. Endothelium regulates vascular tone by releasing endothelimn-del)endent relaxation factor (EDRF) and other vasoactive agents. We have shown that atherosclerosis in cholesterol-fed rabbits (Habib et al., 1986) and humans (Bossaller et al., 1987) is associated with an impairment of endothelium-dependent relaxation. Habib et al. (1986) reported that treatment of cholesterol-fed rabbits with a dihydropyridine calcium blocker preserved endothelium-dependent arterial relaxation. Collectively these observations are compatible with the notion that calcium-channel blockers might exert direct effects on injured endothelium.
Effects on U p t a k e a n d D e g r a d a t i o n o f L i p o p r o t e i n s b y A r t e r i a l Cells A halhnark of atherosclerotic lesions is the accumulation of intracellular lipid and the formation of foam cells. Since endocytotic processes underlying internalization of lipoproteins are calcium dependent, one might envision that calcium blockers could interfere with calcium-dependent lipoprotein uptake. Stein et al. (1985) showed that cultured bovine aortic endothelial cells and human skin fibroblasts after exposure to 10-50 IxM verapamil unexpectedly acquire an increased binding and uptake capacity for LDL, a phenomenon ascribed to drug-induced increases in the abundance of LDL receptors. They proposed that in-
creased endocytotic removal of cholesterol esters from the aortic interstitium might exert antiatherosclerotic effects. In a subsequent study, the same group of workers cultm'ed rabbit aortic smooth muscle for up to 5 weeks in the presence of a hypercholesterolemic plasma fl'actions (density < 1.019 g/ml) (Stein et al., 1987). The cholesterol-rich fractions (~3-VLDLs) evoked increased DNA and cholesterol ester accumulation per culture dish. Addition of 50 ~m verapamil suppressed DNA accumulation, a response thought to represent antiproliferative activity. During the first week, the drug increased, but later decreased, cholesteryl ester accumulation. Corsini et al. (1986) likewise observed that 10 txM verapamil acutely increased specific binding and internalization of LDL by human skin fibroblasts in culture. In contrast to the acute increases in cholesteryl ester deposition described by Stein et al. (1987) and Corsini et al. (1986), Daugherty et al. (1987) found acute decreases in eholesteryl ester accumulation in cultured alveolar rabbit macrophages exposed to 0.1-10 txm verapamil. In cholesterol-fed rabbits, Stender et al. (1986) observed that accumulation of cholesteryl esters in aorta was suppressed after treatment with verapamil (18 mg/kg.day). Variable acute effects of very high concentrations of verapamil on cultured cells indicate that results in vitro should be interpreted cautiously in the context of chronic pharmacologic actions in intact animals. Etingin and Hajjar (1985) reported that aortic smooth muscle cultured from fat-fed rabbits were depleted of their cholesteryl ester content by exposure
1018
Hem'!!
to 0.3 ~xM nifedipine. Effects on the lipid-laden cells were ascribed to stimulation of cyclic-AMP-dependent cholesteryl e~ter hydrolase activity in lysosomes. In acetyl-LDL-laden mouse peritoneal macrophages in culture, Schmitz et al. (1988) observed that 2 ~xM nifedipine stimulated cholesterol release from the cells. The drug induced the formation of lysosomederived "lamellar bodies," which were secreted as cholesterol-rich particles in the culture medium in the absence of HDL as a cholesterol acceptor. The authors described an additional mechanism of cholesterol excretion via lamellar bodies derived fi'om intracellular lipid droplets that fused u4th HDL-containing endosomes. The release of HDL-associated cholesterol depended upon the availability of extracellular HDL. The action of nifedipine oll cholesterol efflux described by Schmitz et al. (1988) has features in common with those characterized previously by Etingin and Hajjar (1985). Cholesteryl ester accumulation in cultured vascular smooth muscle has been reported to be dosedependently inhibited by exposure to isradipine (10100 txM) (Weinstein and Heider, 1987). Using even higher drug concentrations of nifedipine (up to 146 ~M), Saito et al. (1986) suppressed cholesterol synthesis in rabbit aortic smooth muscle in culture.
Effects on Chemotaxis Nakao et al. (1983) found that 12-HETE, a lipoxygenase product of arachidonic acid, had potent chemoattractant effects on rat aortic smooth muscle in culture. They demonstrated that nicardipine (10 :~ to 10 -r' M) inhibited HETE-associated cell mign'ation across Millipore filters. In similar experiments, Nomoto et al. (1987) stimulated the mign'ation of rat aortic smooth muscle cells in culture in response to zymosanactivated exudate and showed that nilvadipine (10 '-' to 10 -~ M) exerted potent inhibitory effects on migration. Inhibitory effects in the studies by Nakao and Nomoto occurred at seemingly very low drug concentrations (pico- to nanomolar), but it is unclear whether the Millipore filter across which mign'ation occm'red accumulated the lipophilic drugs from the aqueous medimn.
Effects on Cell Proliferation Betz and collaborators (Betz et al., 1985; Betz, 1988; Strohschneider and Betz, 1989) showed that rabbit carotid arteries subjected to repeated electric stimulation with implanted electrodes underwent an intimal proliferation of smooth muscle beneath the anode. If the rabbits were also given a cholesterol-rich diet, proliferative plaques became typical atheromas.
Treatment of the rabbits during the periods of electric stimulation with different calcium blockers (flunarizine, nimodipine, anipamil) suppressed the proliferative responses. Handley et al. (1986) produced intimal proliferative lesions in rat carotid arteries by damaging the vessels with a balloon catheter. Treatment of the rats with isradipine reduced the average lesion cross-sectional area by 44%. Jackson et al. (1988) injured the rabbit aorta with a balloon catheter and quantified the intimal proliferative response by motphometry and incorporation of :~H-thymidine into DNA. In rabbits treated with nifedipine (10 mg/ kg.day), there was a 39% reduction in lesion crosssectional area 2 weeks after injury and a 53% reduction in thymidine labeling of the intima-media. Antiproliferative effects of calcium blockers have also been assessed in cell culture systems. Nilsson et al. (1985) showed in cultured rat aortic smooth muscle cells that 1 jxM nifedipine slowed the rate of transformation of the cells fi'om a contractile to a "synthetic" phenotype and inhibited the initiation of DNA synthesis as well as cell proliferation. The inhibitory effect on DNA synthesis was seen in cells stimulated with whole serum or with platelet-derived growth factor. Weinstein and Heider (1987) found that 50 jxM isradipine inhibited in monkey aortic smooth muscle cells in culture the incorporation of :~aS-sulfate into glycosaminoglycans, ~4C-proline into collagen, and :~Htryptophan into cell total protein. Results were interpreted to represent inhibition of the biosynthesis of matrix proteins, probably independent of an effect on calcium channels. Orekhov et al. (1987) cultured intimal cells ("modified smooth muscle cells") fl'om human aortic lesions and observed that verapamil (5-50 ~xM)inhibited :~H-thymidine incorporation into cellular DNA, intracellular deposition of lipid, and extracellular deposition of collagen. Similarly, Stein et al. (1987) showed that 50 txM verapamil inhibited DNA synthesis by cultured rabbit and bovine aortic smooth muscle cells, as mentioned above.
Clinical Implications All studies on the treatment of atherosclerosis in animals fed high-cholesterol diets have focused oll the formation or regression of early foam-cell-rich lesions. Occlusive atherosclerotic lesions, as seen in patients undergoing diagnostic workup for symptomatic coronary disease, are rich in fibrous tissue, extracellular lipid, and thrombotic material (Roberts, 1977; Haudenschild and Backa, personal communication), components that could be completely refi'actory to suppressive mechanisms of calcium blockers. Accordingly, clinical trials on antiatherosclerotic effects of
Anti-Atherogenie Mechanisms
calcium blockers will have to concentrate not on advanced lesions (stenoses), but more on new lesion formation. Unfortunately, new lesions may produce little or no angiographieally detectable luminal distortions (Glagov et al., 1987), and new techniques, such as endovaseular ultrasound imaging, will have to be applied to assess new lesion formation in the clinical setting.
References Betz E. The effect of calcium antagonists on intimal cell proliferation in atherogenesis. A ~ . N Y Acod Sci 1988;522:399-410. Betz E, Hammerle H, Strohschneider T. Inhibition of smooth muscle cell proliferation and endothelial permeability with flunarizine in vitro and in experimental atheromas. Rex Exp Med 1985; 185:;:125-340. Blumlein SL, Sievers R, Kidd P, Parmley WW. Mechanism of protection fi-om atheroselerosis by verapamil in the cholesterol-fed rabbit. Am J Cardiol 1984;54:884-889. Bossaller C, Habib GB, Yamamoto H, et al. Impaired muscarinic endothelium-dependent relaxation and cyclic g-aanosine 5'-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta. J Clin Invest 1987;79:170-i74. Bretherton KN, Day A J, Skinner SL. Hypertension-accelerated atherogenesis in cholesterol-fed rabbits. Atherosclerosis 1977; 27:79-87. Catapano AL, Maggd FM, Cicerano U. The antiatherosclerotic effect of anipamil in cholesterol-fed rabbits. A ~ N Y Acad Sci 1988;522:519-521. Cm'sini A, Fumagalli R, Paoletti R. Calcium antagonists and low density lipoproteins metabolism by human fibroblasts and by human hepatoma cell line HEP G2. lqmrm.e Res ('.ram 1986:18:1-16. Daugherty A, Rateri DL, Sehonfeld G, Sot)el BE. Inhibition of choleteryl ester deposition in macrophages by calcium entry blockers: An effect dissociable fi'om calcium entry blockade. [:h" J P h . r m . c o l 1987;91:113-118. Diccianni MB, Cardin AD, Britt AL, et al. Effect of a sustained release formulation of diltiazem on the development of atherosclerosis in cholesterol-fed rabbits...lH~erosch, r.sis 1987;65: 199-205. Etingin OR, Hajjar DP. Nifedil)ine increases cholesteryl ester hydrolytic activity in lipid-laden rabbit arterial smooth muscle cells. J Clin Invest 1985;75:1554-1558. Feinstein MB, Fiekers .J, Fraser C. An analysis of the mechanism of local anesthetic inhibition of platelet aggregation and secretion, l'harmucol s I) Thor 1976;215-228. Fronek K. Effect of nisoldipine on diet-induced atherosclerosis in rabbits. Am~ N Y Ae.d Sci 1988;522:525-526. Ginsburg R, Davis K, Bristow MR, et al. Calcium antagonists suppress atherogenesis in aorta but n(,t in the intranmral coronary arteries of cholesterol-fed rabbits. Lab lnv(,sl 1983;49: 154-158. Glagov S, Weisenberg E, Zarins CK, et al. Compensatory enlargement of human atherosclerotic coronary arteries. N Eugl J Meal 1987;316:1371-1375. Habib J B, Bossaler C, Wells S, et al. Preservation of endothelium-dependent vascular relaxation in cholesterol-fed rabbit by treatment with the calcium blocker PN 200110. ('ire Rex 1981-;;58:305-309. Handley DA, Van Valen RG, Melden MK, Saunders RN. Suppression of rat carotid lesion development by the calcium channel blocker PN 200-110. Am J Pathol 1986;124:88-93.
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Haudenschild, Backa. Personal communication. Heistad DD, Armstrong ML. Blood flow through vasa vasorum of cm'onary arteries in atherosclerotic monkeys. Arteriosclerosis 1986;6:::126-::I:31. Henry PD. Calcium antagonists as antiatherogenic agents. Ann N Y Acad Sci 1988;522:411-419. Henry PD, Bentley KI. Suppression of atherogenesis in cholesterol-fed rabbit treated with nifedipine. J Clb~ Invest 1981 ;68:1::166--1369. Henry PD, Perez JE. Clinical pharmacolo~, of calcium antagonists. In: Conti CR, Brest AN, eds. Cardiac drug therap!l. Philadelphia: F.A. Davis, 1984:93-109. Henry PD, Yokoyama M. Supersensitivity of atherosclerotic rabbit aorta to ergonovine. Mediation by a serotonergic mechanism. J Clbl Invest 1980;66:306-313. Jackson L J, Bush RC, Bowyer DE. Inhibitory effect of calcium antagonists on balloon catheter-induced arterial smooth muscle cell proliferation and lesion size. Atherosclerosis 1988;69: 115-192. Jones CR, Pasanishi F, Elliott HL, Reid JL. Effects of verapamil and nisoldipine on human platelets: In vivo and in vitro studies. Br J Clbl Phurmae 1985;20:191-196. Lardinois CK, Neuman SL. The effects of antihypertensive agents on serum lipids and lipoproteins. Arch Intern Med 1988; 148:1980-1288. Lichtlen PR, Hugenholtz PG, Rafflenbuel W, et al. Retardation of angiogq'aphic progression of coronary disease by nffedipine. Lain'el 1990;3:35:1109-1113. Liehtor T, Davis HR, Vesselinoviteh D, et al. Suppression of atherogenesis nifedipine in the cholesterol-fed rhesus monkey. App Pathol 1989;7:8-18. McDonagh PF, Roberts DJ. Prevention of transcoronm'y macromolecular leakage after ischemia-reperfusion by the calcium entry blocker nisoldipine. Cite Rcs 1986:58:127-136. Mclntyre DE, Shaw AM. Phospholipid-induced human platelet activation: Effects of calcium channel blockers and calcium chelators. Thromb Rcs 1983;31:833-844. Molinari A, Guarneri L, Pacel E, et al. Mouse antithrombotic assay: The effects of Ca' " channel blockers are plateletindependent. J Ptmrm.e.I E,rp Ther 1987;240:623-627. Moore JB, Fuller BL, Falotico R, Toman EL. Inhibition of rabbit platelet phosphodiesterase activity and aggq'egation by calcium channel blockers. Thromb Res 1985;40:401-411. Naito M, Kuzuya F, Asai K, et al. Ineffectiveness of Ca e ' antagonists nicardipine and diltiazem on experimental atherosclerosis in cholesterol-fed rabbits. Angi.lm.l!l 1984;35:622-627. Nakao J, Ito H, Ooyama T, et al. Calcium dependency of aortic smooth nmscle cell nfigration induced by 12-L-hydroxy5,8,10,14-eicosatetraenoic acid. Alhcr.sclerosis 1983;46:309319. Nilsson J, Sjolund M, Palmberg L, et al. The calcium antagonist nifedipine inhibits arterial smooth nmscle cell proliferation. Alheroseh,r~sis 1985;58:109-122. Nomoto A, Hirosumi J, Sekiguchi C, et al. Antiatherogenic activity of FR34235 (Nilvadipine) a new potent calcium antagonist. Atheroseh'r~sis 1987;64:255-261. Ohata I, Sakamoto N, Nagano K, Maeno H. Low density lipoprotein-lowering and high density lipoprotein-elevating effects of nicardipine in rats. Bim'hem Phurmuc 1984;33:21992205. Orekhov AN, Tertov VV, Khashimov IC4., et al. Evidence of antiatherosclerotic action of verapamil fl'om direct effects on arterial cells. Am J Cardiol 1987;59:495-496. Pannocchia A, Praloran N, Arduino C, et al. Absence of ( - ) (:~H)desmethoxyverapamil binding sites on human platelets
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Stein O, Leitersdorf E, Stein Y. VerapamiI enhauces receptormediated endocytosis of low density lipoproteins by aortic cells in culture. Arteriosclerosis 1985;5:35-44. Steuder S, Ravn H, Haugegaard M, Kjeldsen K. Effect of verapamil on accumulation of fl'ee and esterified cholesterol in the thoracic aorta of cholesterol-fed rabbits. Atheroselerosis 1986:61:15-23. Strickberger SA, Russek LN, Phair RD. Evidence for increased aortic plasma membrane calcium transport caused by experimental atherosclerosis in rabbits. Circ Res 1988;62:75-80. $trohschneider T, Betz E. Densitometrie measurement of increased endothelial permeability in arteriosclerotic plaques and inhbition of permeability under the influence of two calcium antagonists. Atherosclerosis 1989;75:135-144. Sugano M, Nakashima Y, Matsushima T, et al. Suppression of atheroselerosis in cholesterol-fed rabbits by diltiazem injection. Arterioseh'rosis 1986:6:237-241. Tedgui A, Chiton B, Curmi P, et al. Effect of nicardipine and verapamil on in vitro albumin transport in rabbit thoracic aorta. Arteriosclerosis 1987;7:80-87. Weinstein DB, Heider JG. Antiatherogenic properties of calcium antagonists. A m J Cardiol 1987;59:163B-172B. Willis AL, Nagel B, Churchill V, et al. Antiatherosclerotic effects of nicardipine and nifedipine in cholesterol-fed rabbits. Arteriosclerosis 1985;5:250-255.
