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Unexplored Territories With Calcium Channel Blockers: Potential For The Future

Atheroprotection with Amlodipine: Cells to Lesions and the PREVENT Trial

Tulenko, Thomas N.; Brown, Jeffrey; Laury-Kleintop, Lisa; Khan, Mark*; Walter, Mary F.; Mason, R. Preston

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Journal of Cardiovascular Pharmacology: 1999 - Volume 33 - Issue - p S17-S22
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Abstract

Calcium channel blockers (CCBs) have added scope to the potential for pharmacologic treatment of cardiovascular disease because they have the ability to act both as antihypertensive and as antianginal agents (1). Although the basis for their molecular interaction with membrane calcium channels is not entirely clear, it is apparent that they inhibit calcium uptake through L-type calcium channels, which are abundant on cardiac and vascular smooth muscle cells. By blocking calcium channels, they decrease membrane calcium permeability, reduce cytosolic calcium levels, and suppress the excitatory state in these tissues. After pharmaceutical development, the newer agents in this class have improved vascular selectivity and can mediate a decrease in peripheral vascular resistance (and therefore arterial pressure). There have been significant improvements in optimal dose-taking and dose release since the adverse effects of the earlier short-acting CCBs. In the coronary vasculature, they dilate large epicardial arteries without producing coronary steal. Between 1980 and 1989, the inhibition of progression of atherosclerotic lesions by a variety of CCBs in several animal models caused considerable excitement (2-6). A wide range of CCBs were used in these studies, including verapamil, diltiazem, nifedipine, and nisoldipine. However, early enthusiasm decreased when several reports appeared that were unable to duplicate the atheroprotective effects of these same agents (6,7). In retrospect, this loss of interest may have been premature because careful review of the literature indicates that inhibition of calcium permeability appears in a majority of studies to have selective and proven atheroprotective features (8). In animal studies, if CCBs are administered before or simultaneously with initiation of cholesterol feeding, calcium channel blockade was shown to inhibit the development of new lesions but to have little effect on established lesions (1,4,9,10). This finding was essentially confirmed in human trials, such as the INTACT-1 study (4), but because of the relatively short duration of these studies, inhibition of new lesion formation in symptomatic patients is difficult to confirm statistically. First- and second-generation CCB differences were seen which led to further experimentation with the advent of a newer generation of CCBs. There has therefore been a resurgence in interest in using these agents for atheroprotection. The individual CCB used may influence the exact mechanism that underlies the protective effect. In particular, amlodipine, a charged dihydropyridine with marked lipophilicity, has recently demonstrated promise in atheroprotection (8,11-13). Here we describe novel actions of amlodipine that suggest that some of its atheroprotective activity may result directly from its physical effect on the membrane bilayer of arterial wall cells. This effect is relevant to the cell biology of atherogenesis because one of the potential cellular pathways central to the development of atherosclerotic lesions involves a fundamental disturbance in the membranes of arterial smooth muscle cells (SMCs) and endothelial cells (ECs) (14-16). To examine the specific effects of CCBs on atherosclerosis, we focus here on the PREVENT trial, which examines this atheroprotective potential in patients with ischemic heart disease.

ATHEROGENESIS

The lipid hypothesis of atherogenesis is well established (17-19). Hypercholesterolemia has been firmly identified as a risk factor for heart disease, primarily from seminal findings from Framingham and similar studies (17,18,20). However, the mechanistic link between hypercholesterolemia and plaque development is only poorly understood. The atherogenic dyslipidemias are largely characterized by elevated LDL levels and/or suppressed high-density lipoprotein (HDL) levels, or by defects that alter the biologic activity of these two lipoproteins. These dyslipidemias lead to excessive and augmented forward cholesterol transport to the peripheral cells by cholesterol-rich LDL particles or to a decrease in reverse cholesterol transport out of the cells by HDL. The atherogenicity of LDL appears to come from the prolonged retention of LDL particles in the vessel wall (21). Once retained there, various "normal" processes, including oxidation and LDL-cellular interactions, can occur for prolonged periods of time.

Much attention has focused on oxidized LDL in the origin of ischemic heart disease (22). Although the evidence for oxidative stress in the cellular events of atherogenesis is compelling, most of it comes from cell culture and animal model studies. In humans, antioxidants have largely failed to provide convincing evidence that the oxidative pathway is paramount in linking hypercholesterolemia to heart disease (23-26). On balance, oxidative events, although perhaps not the single causal agent initiating atherogenesis, are likely to be only one of several events capable of initiating atherogenesis at the cellular level.

Another factor that may be of importance in initiating atherogenesis is the cholesterol molecule itself, i.e., unesterified cholesterol. Ischemic heart disease correlates directly with serum levels of the cholesterol-rich LDL particle, and inversely with serum levels of the cholesterol acceptor particle, HDL. The role of cholesterol in heart disease permeates the epidemiologic, lipoprotein, and lay literature but has received less attention in the vascular biology research community. Our laboratory has found that cholesterol enrichment of either SMCs or ECs induces phenotypic modulation in these cells, shifting them towards the atherogenic pathway (14,15,27-29). Moreover, the mechanism of the cellular effects of excess cholesterol appears to reside largely in the cell membranes, possibly in the cell plasma membrane, where cholesterol is known to concentrate (30). We have proposed that excess membrane cholesterol in SMCs and ECs actually constitutes a "membrane lesion" (14) that may satisfy, in part, the response-to-injury hypothesis originally proposed by Ross and co-workers (19,31,32).

Arterial wall cells become enriched with cholesterol in the very early period of atherogenesis in vivo, i.e., within days after the initiation of cholesterol feeding (27,33). Cholesterol accumulates in the membranes of these cells, including the plasma membrane, where it alters the membrane bilayer composition, organization, and structure (14,16). In SMCs, changes in the cell membrane are accompanied by a number of alterations in membrane function. These include an increase in membrane calcium permeability and an increase in cytosolic calcium levels (34). Table 1 lists some of the alterations in SMC membrane and cell function after cholesterol enrichment in vivo and in vitro. The membrane alterations actually precede the development of fatty-streak lesions in experimental dietary atherosclerosis (14) and are therefore consistent with a causal effect in lesion initiation.

TABLE 1
TABLE 1:
Phenotypic changes observed in SMCs after cholesterol enrichment in vivo by dietary cholesterol feeding for 10 weeks or in vitro by incubation of cholesterol-rich liposomes for 48 h

Whether cholesterol directly causes these alterations in SMC function in atherosclerotic vessels was explored using normal aortic SMCs isolated in culture, where cholesterol enrichment could be studied as a single, isolated, and independent variable. Identical changes in membrane function were found in SMCs exposed to cholesterol in culture and in cells freshly isolated from cholesterol-fed atherosclerotic animals (Table 1). In culture, these alterations occurred after only 48 h of cholesterol exposure, suggesting rapid cellular modulation. On the basis of these studies, we have hypothesized that excess membrane cholesterol in SMCs constitutes a membrane injury or lesion that contributes to early atherogenic signals (14-16,33,34).

EFFECTS OF CCBs ON ATHEROSCLEROSIS

Because calcium regulation may be at or near the core of the cellular alterations in SMCs that accompany the development of dietary atherosclerosis, we became interested in the ability of various calcium antagonists to inhibit the cellular alterations that occur with cholesterol enrichment of SMCs. We studied a variety of calcium antagonists and found amlodipine effectively blocked the cholesterol-induced increase in SMC calcium permeability, with an IC50 of approximately 10−9 M. Furthermore, at 10−12 M, a concentration which had essentially no effect on membrane calcium permeability (Fig. 1)(15), amlodipine decreased cholesterol-induced SMC proliferation. This finding is consistent with the suggestion that amlodipine has actions in SMCs unrelated to inhibition of membrane calcium channels. Moreover, because calcium permeability is elevated in SMCs of atherosclerotic arteries, we examined the ability of amlodipine to inhibit this augmentation. We found amlodipine did not affect the augmented calcium permeability in atherosclerosis (33). Similar results were obtained with other calcium antagonists, including verapamil, diltiazem, and nifedipine, as well as the inorganic calcium antagonists Ni+ and La3+. This resistance of SMCs to a wide variety of calcium antagonists during atherogenesis suggests the increased calcium permeability is representative of a nonspecific calcium leak pathway. Although this finding suggests amlodipine would not affect the development of atherosclerotic lesions, experimental data have demonstrated that amlodipine does have clear antiatherosclerotic activity in animal models. Nayler (8) has shown amlodipine effectively inhibits lesion development in cholesterol-fed rabbits (Fig. 2), and Kramsch and Sharma (12) have similar data in a monkey model. Therefore, if amlodipine does not block calcium permeability in atherosclerotic arteries, the question of how it inhibits lesion progression remains.

FIG. 1
FIG. 1:
The effects of amlodipine on cholesterol-induced SMC proliferation and calcium influx in SMCs. Amlodipine inhibits the cholesterol-induced increase in calcium permeability in cultured SMCs with an IC50 ≈ 1 nM. However, amlodipine completely inhibits cholesterol-induced proliferation in cultured SMCs at 1 pM. This is consistent with the hypothesis that the mechanism for the antiproliferative effects of amlodipine in this model is mediated by a process independent of its calcium channel blocking properties. Data represent the mean ± SEM (n ≥ 4). From Tulenko et al. (15).
FIG. 2
FIG. 2:
Effects of amlodipine on progression of new lesions in rabbits fed cholesterol for 6 weeks. Dose-dependent inhibition by amlodipine on the progression of fatty-streak lesions in New Zealand rabbits fed cholesterol (2%) for 6 weeks. Amlodipine was administered at 1 mg/kg/day and was initiated before cholesterol feeding and maintained for 6 weeks (*p < 0.01; **p < 0.001; n = 6). From Nayler (8).

Clues to the mechanism of the antiatherosclerotic actions of amlodipine may come from recent studies on its action on membrane bilayers. Amlodipine is profoundly lipophilic (Kp ≈ 104) and readily partitions into the cell membrane (35). We examined the potential of amlodipine to alter the organization of the membrane bilayer using small-angle x-ray diffraction analysis (15). Amlodipine restored bilayer structure (width) in SMC membranes obtained from the atherosclerotic rabbit aorta (Table 2).

TABLE 2
TABLE 2:
The effects of amlodipine (10−9 m) on membrane bilayer structure in SMCsa

Hence, this calcium antagonist appears to have a unique action on membrane bilayers independent of its action on calcium channels. In fact, we proposed, aside from its calcium channel blocking properties, amlodipine has a separate and distinct pharmacology based on its ability to reorder membrane bilayers. This action may account for its antiproliferative activity in cholesterol-enriched SMCs (Fig. 1). If this finding is confirmed, amlodipine would represent a compound for a new pharmacologic class, one whose activity is based on the ability to reorder membrane lipid bilayer defects (15).

Amlodipine's lipid interactions may also be related to its potent antioxidant effects. In model membranes enriched with polyunsaturated fatty acids, we found amlodipine inhibits lipid peroxidation in the nanomolar dose range (Table 3). This effect is significantly more potent than observed with other calcium antagonists, including verapamil. Moreover, consistent with its antioxidant action in vitro, Kramsch and Sharma (12) reported amlodipine-treated monkeys had significantly reduced levels of oxidized LDL compared with untreated animals. These data describe another effect of amlodipine on membranes that may explain its actions in preserving vascular function.

TABLE 3
TABLE 3:
Inhibition of lipid peroxide formation in calcium-free phosphatidylcholine vesicles by amlodipinea

We have also examined the ability of amlodipine to inhibit intimal hyperplasia after angioplasty in hypercholesterolemic rabbits. We observed a marked inhibition of intimal hyperplasia, as reflected by a decrease in the intimal: medial ratio (Table 4).

TABLE 4
TABLE 4:
Inhibition of intimal hyperplasia after angioplasty in hypercholesterolemic rabbitsa

In summary, amlodipine has a complex pharmacology. The molecular mechanisms responsible for the atheroprotective actions of amlodipine may involve its calcium channel blocking actions, membrane-modifying properties, antioxidant effects, and/or a combination of these actions.

THE PREVENT TRIAL

There is growing evidence amlodipine has atheroprotective activity in a cell culture model of atherosclerosis (16), in a rabbit model of atherosclerosis (8) and restenosis (Table 4), and in a subhuman primate model of atherosclerosis (11,12). It is important to confirm these experimental observations in clinical trials. Therefore, the Prospective Randomized Evaluation of the Vascular Effects of Norvasc Trial (PREVENT) has been launched.

The PREVENT trial was designed to determine whether the vascular actions of amlodipine described above hold true in humans with atherosclerotic disease. The objective of the PREVENT trial is to evaluate the effectiveness of amlodipine relative to placebo in preventing the development and progression of atherosclerotic lesions in coronary and carotid arteries of patients with coronary artery disease (CAD) and to correlate the rates of progression in these two vascular sites.

PREVENT is a multicenter, double-blind, placebo-controlled trial involving approximately 830 patients of either gender (n = 415 per treatment group) aged 30-80 years. At entry, each patient undergoes quantitative coronary angiography to assess the severity of coronary lesions and B-mode ultrasound to assess the severity of carotid disease and carotid arterial compliance. B-mode ultrasound will be performed at 6-month intervals throughout the study and quantitative coronary angiography will be repeated at the conclusion of the 3-year treatment period. The effects of amlodipine on restenosis after angioplasty will be examined in a substudy of patients who require angioplasty during the course of the main study (36).

The primary hypothesis of PREVENT is that amlodipine reduces the progression of early atherosclerotic lesions (5-20% stenosis) in patients with CAD. The measured end point of this arm is the change in the average minimal diameter of the early atherosclerotic lesions. The two secondary hypotheses of the study are that amlodipine reduces the rate of progression of all visible coronary lesions stratified according to their severity (i.e., less than 5% stenosis, 20-50% stenosis, and greater than 50% stenosis) and that amlodipine reduces the rate of progression of atherosclerosis in the carotid arteries, as measured by the slope of the maximum intimal:medial thickness of the carotid artery averaged over time. Categorical end points in each patient include no change, progression of lesions, regression of lesions, or a mixture of these. In the angioplasty patients, the end point is the presence or absence of restenosis of the ballooned segment. Other study parameters include the following: (a) hospitalization for unstable angina; (b) need for angioplasty, atherectomy, and/or coronary artery bypass; (c) incidence of stroke or myocardial infarction; (d) cardiac arrest; (e) death related to a cardiovascular event; (f) hospitalization for new-onset heart failure; (g) combined cardiovascular events; and (h) total mortality.

All patients are instructed to maintain a Phase I American Heart Association (AHA) diet. Patients whose plasma LDL cholesterol levels are above 130 mg/dl, despite appropriate lipid-altering therapy and/or adherence to the Phase I diet, are instructed to follow the AHA Phase II diet. All reasonable attempts are made to maintain LDL cholesterol levels below the recommended goal of 160 mg/dl by dietary and/or pharmacologic means throughout the study.

Patients who require additional therapy, beyond amlodipine and lipid regulation, for control of hypertension and/or angina are permitted therapy instituted with protocol-permitted medications. However, the use of other calcium antagonists and of angiotensin-converting enzyme (ACE) inhibitors is strongly discouraged. Concomitant therapy with calcium antagonists and ACE inhibitors or discontinuation from the study, for reasons other than an adverse experience, occurs only after consultation with and approval from the Data Coordinating Center (an independent overseeing arm of the University of Michigan).

In summary, evidence from cell studies and animal models is consistent with the hypothesis that certain CCBs have antiatherosclerotic actions. These may result from the complex pharmacologic activity of the CCBs and may or may not be related to classical calcium channel blockade. The PREVENT Trial will test this hypothesis in humans with confirmed CAD. To date, 825 patients have been recruited for this study, and the last patient completed the 3-year follow-up by the end of 1997. We look forward to the results of this important trial in 1999.

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Section Description

Official Satellite Symposium for the XVIIth Congress of the European Society of Cardiology, The National Exhibition Centre, Birmingham, United Kingdom August 28, 1996

Keywords:

Amlodipine; Angioplasty; Atherosclerosis; Calcium channel blockers; Cholesterol; Dihydropyridines; Hyperplasia

© 1999 Lippincott Williams & Wilkins, Inc.