Calcium plays a pivotal role in a wide spectrum of physiologic events, ranging from a structure-stabilizing effect (e.g., in bone) to signal transduction (e.g., in excitation-contraction coupling, neurotransmitter release, excitation-secretion coupling, cell growth, and cell motility) (1). These calcium-mediated signal transduction events invariably involve the interaction of ionized calcium with specific proteins, resulting in conformational changes in specific proteins that subsequently precipitate a cascade of protein-protein interactions. Although calcium ions are essential for these events, their excessive availability or failure to maintain intracellular homeostasis inevitably results in cell death and tissue necrosis (2).
The calcium channel blockers were developed as therapeutic agents because of their ability to restrict calcium ion movement across the cell membrane through the specific voltage-activated, calcium-selective channels present in a variety of excitable cells, including smooth muscle cells (3). The rationale behind the introduction of these agents involved, among other factors, the idea that reducing calcium ion entry by this route would reduce the likelihood of "calcium overloading" and hence of cell death and tissue necrosis (2,3). In practice, drugs of this type are now widely used in clinical medicine in the treatment of patients with a variety of vascular disorders, including angina pectoris (4) and hypertension (5). Of current interest is the possibility that these drugs can be used as agents to slow the progression of atherosclerosis (6). Before discussing the basic data relevant to this possibility, it may be useful to consider briefly the events involved in lesion formation.
THE ETIOLOGY OF AN ATHEROSCLEROTIC LESION
Naturally occurring atherosclerosis is a progressive and complex disease, the end point of which is the ruptured fibrotic and calcified plaque seen at autopsy (7,8). Early events include elevated plasma levels of low-density lipoproteins (LDL) or abnormally low levels of high-density lipoproteins (HDL), enhanced endothelial cell membrane permeability, altered surface adhesion properties of endothelial cells, increased stickiness of monocytes and platelets (which results in their adherence to the endothelial cell surface), and oxidation of LDL. These events are followed by smooth muscle cell proliferation and migration, excess matrix and collagen synthesis, and finally by calcification, fibrosis, and necrosis (8,9). Ionized calcium is required for many of these events (Table 1), including the release of various growth factors (Table 2). In addition, calcium plays a key role in two of the major risk factors for atherosclerosis, hypertension and increased coronary tone, which occur in angina and vasospastic angina and predispose towards plaque formation and rupture.
CALCIUM CHELATORS AND ANTIATHEROGENIC AGENTS
Experiments that preceded investigations into the efficacy of calcium channel blockers as antiatherogenic agents included those using calcium-chelating agents such as ethylenediaminetetra-acetic acid (10), thiophone (11), chondroitin sulfate A (11), and diphosphoric acid (12). These agents were shown to reduce the incidence of atherosclerosis in cholesterol loading-induced models of atheroma. Although these experiments have little if any clinical relevance, they paved the way for subsequent investigations in which calcium channel blockers, both inorganic (13) and organic (6,14), were used.
LABORATORY MODELS USED TO INVESTIGATE THE ANTIATHEROGENIC ACTIVITY OF CALCIUM CHANNEL BLOCKERS
These models have been described in detail elsewhere (15) and therefore need only be summarized here. They can be subdivided into three major groups: intact animal models, isolated cell preparations, and subcellular fractions.
Intact animal models include those in which the diet has been deliberately manipulated to produce atheromatous lesions. For example, atherosclerosis is easily produced in rabbits by loading their diet with cholesterol. The resultant lesions, which develop predominantly in the aortic arch and thoracic aorta, resemble the thick fatty streaks found in the early stages of human atherosclerotic lesions, without the complications of fibrosis, hemorrhage, ulceration, and thrombosis that are associated with the fully developed lesion in human coronary vasculature (7). Cholesterol-fed rabbits therefore provide a useful laboratory model for the study of potentially beneficial antiatherogenic agents.
Cholesterol-rich diets also promote the development of atherosclerotic lesions in other animals. For example, pigeons develop lesions similar to those found in humans when fed an excessively cholesterol-rich diet (16). Diet-induced atherosclerosis is also relatively easy to produce in rhesus and cynomolgus monkeys, in which the distribution and pathology of the lesion is remarkably similar to that observed in humans (16).
In more recent studies, mechanical injury has been superimposed on cholesterol loading in attempts to exacerbate lesion formation. For example, the carotid artery has been cuffed in cholesterol-fed rabbits (17). Not all intact models of atheroma formation depend on cholesterol loading. Some investigators have used balloon inflation or abrasion by a nylon filament to trigger "lesion" development in otherwise normal arteries (18). Other models rely on the use of calcinizing regimens, as in the ingestion of high doses of vitamin D by rats (19). However, in the latter case, the resultant lesions are more akin to those triggered by "calcium overloading," and therefore their relevance as models for naturally occurring atheroma is more remote than that of lesions caused by excessive cholesterol intake.
The recent trend in experiments aimed at studying the antiatherosclerotic potential of therapeutic agents has extended to the use of both cellular and subcellular preparations, in contrast to intact animal models. Isolated aortic smooth muscle cells (20,21), isolated platelets (22), isolated myocytes (23), and isolated membrane preparations (24) are some of the cell preparations now being used for this purpose. The trend towards the use of such preparations is not altogether surprising, given the involvement of these organelles in the etiology of lesion formation. For example, monocyte infiltration into the subendothelial space, smooth muscle cell migration and proliferation, and platelet aggregation are all events that contribute significantly to lesion development and growth (8), as does increased membrane permeability (25). The latter possibility is a consequence of increased cholesterol availability.
LABORATORY EVIDENCE RELATING TO THE ANTIATHEROSCLEROTIC ACTIVITY OF CALCIUM CHANNEL BLOCKERS
Antiatherosclerotic activity of calcium channel blockers in the cholesterol-fed rabbit model
There is abundant evidence in support of the hypothesis that organic calcium channel blockers slow the progression of atherosclerosis in this cholesterol-loaded laboratory model of the disease. The earliest reported study is that of Henry and Bentley (14), in which nifedipine was shown to reduce lesion formation in cholesterol-fed rabbits at a dose level that was below that needed to lower blood pressure in the test model. Hence, it was established that the effectiveness of this particular dihydropyridine-based calcium channel blocker was not directly dependent on its blood pressure-lowering activity. The initial findings of Henry and Bentley were confirmed several years later by Willis et al. (26) and again by Nayler and Panagiotopoulos (27), the latter investigators reporting that the antiatherosclerotic activity of this calcium channel blocker was dose-dependent and that its efficacy need not be accompanied by any significant change in the plasma lipid profile. Habib and colleagues (28) extended our knowledge in this field by establishing that the antiatherosclerotic potential of calcium channel blockers was not peculiar to nifedipine. In the cholesterol-fed rabbit model, they demonstrated a similar effect for another dihydropyridine-based calcium channel blocker, isradipine. At about the same time, nicardipine was found to be equally effective (26).
Some of the earlier studies relating to the antiatherosclerotic effect of dihydropyridine-based calcium blockers are sometimes criticized on the basis of the relatively high doses of the compounds that were used. For example, Henry and Bentley used doses of 15-17 mg/kg/day in their early studies with nifedipine (14). Although this regimen resulted in a 64% reduction in lesion area in the cholesterol-fed rabbit model, the dose level is considerably in excess of that used clinically. The same criticism can be applied to many of the other early studies, including the studies of Willis et al. (26), who used doses of up to 80 mg/kg/day of nicardipine.
Criticism on the basis of dose levels, however, cannot be leveled at the study of Nayler and colleagues (27) who, using a dose level of only 1 mg/kg/day of nifedipine given orally, obtained a 25.8% reduction in lesion formation in the abdominal aorta of cholesterol-fed rabbits over a treatment period of 6 weeks, with a similar reduction in lesions in the thoracic aorta.
Early studies relating to the antiatherogenic potential of the calcium channel blockers in the cholesterol-fed rabbit model were not restricted to the use of the dihydropyridine-based compounds. Verapamil (29) and diltiazem (30) were widely and effectively used. Their efficacy established that the antiatherosclerotic effect of the calcium channel blockers was a class effect and was not dependent on the dihydropyridine-based chemistry of the compounds used in the earlier studies. At the same time, many of the studies with verapamil and diltiazem confirmed the independence of the antiatherosclerotic effect of the calcium channel blockers from any blood pressure-lowering effect.
The more recently developed long-acting calcium channel blockers have also been evaluated for antiatherogenic activity in the cholesterol-fed rabbit model. For example, amlodipine is a dihydropyridine-based calcium channel blocker which, by virtue of its own chemistry, is long acting (31). In contrast to nifedipine, its first-generation prototype, amlodipine is a charged molecule. Moreover, it is extremely lipophilic and has been shown to partition into the membrane bilayer, where it assumes a time-averaged position similar to that occupied by cholesterol (32). Because of its pharmacodynamic and pharmacokinetic properties, amlodipine has been regarded as the prototype of a third generation of calcium channel blockers (33). With the inherent advantage of its long duration of action, the presence of antiatherosclerotic activity would be advantageous. In a series of experiments using cholesterol-fed rabbits, Nayler has shown that amlodipine reduces atherosclerotic lesion formation in a dose-dependent and sustained manner without altering plasma cholesterol levels but while reducing aortic calcium (15,34). Effective dose levels of amlodipine were 1 mg and 5 mg/kg/day, given orally. Data relating to the antiatherosclerotic activity of amlodipine in the cholesterol-fed rabbit model are given in Table 3.
Antiatherosclerotic effect of calcium channel blockers in butter-fed monkeys
Because the lesions that form in rabbits are early lesions resembling the fatty-streak stage of human adult lesions and because the distribution of these lesions differs from that found in humans (15), it was necessary to establish an alternative model for the study of this disease. For this reason, several investigators, including Kramsch (35), have turned to the monkey model. The study by Kramsch lasted for 18 months, during which time the monkeys were fed a butter-rich atherogenic diet. The amlodipine-treated monkeys had a plasma concentration of 16.7-35.6 ng/ml 24 h after dosing. One hundred per cent of the animals receiving the atherogenic diet developed moderate to severe atherosclerosis, whereas almost 50% of the animals receiving concomitant amlodipine developed no lesions at all.
Antiatherosclerotic activity of calcium channel blockers in other animal models
Another model that has been used to investigate the antiatherogenic activity of the calcium channel blockers is that in which the carotid artery of cholesterol-fed rabbits is cuffed (17). In this model, nilvadipine, another dihydropyridine-based calcium channel blocker, has been shown to possess antiatherogenic activity.
Calcium channel blockers have also been shown to be effective antiatherogenic agents even when the lesion is triggered by mechanical damage to the intimal surface caused by balloon inflation. Amlodipine, for example, is effective under these conditions (25). On the basis of the data presented here, it can be concluded that the calcium channel blockers, as a class, slow the development of experimentally induced atherosclerotic lesions (Table 4). Such an effect is independent of any blood pressure-lowering effect and can be demonstrated in a variety of intact animal models.
Evidence of the antiatherogenic mechanisms of calcium antagonists as revealed by studies using cellular and subcellular preparations
Whereas there is abundant evidence showing that the calcium channel blockers as a class exhibit antiatherogenic activity in experimental models of the disease (Table 4), evidence relating to the mechanisms that are involved is only now beginning to emerge. The mechanisms identified as being sensitive to calcium channel blockers are listed in Table 5.
In a variety of experimental models, the calcium channel blockers have been shown unequivocally to reduce atherosclerotic lesion formation. This activity is expressed independently of the chemistry of the calcium channel blocker and without any dependence on a blood pressure-lowering effect. Although the primary action of calcium channel blockers is to inhibit calcium ion entry through the voltage-activated, calcium-selective transmembrane channels, their antiatherosclerotic activity probably involves many additional properties of these compounds. Some of the mechanisms that appear to be involved include an inhibitory effect on monocyte adhesion, slowed release of growth factors, slowed smooth muscle cell proliferation and migration, preservation of endothelial function, slowed platelet aggregation, protection against lipid peroxidation, maintenance of membrane permeability in the presence of elevated cholesterol levels, slowed cholesterol uptake by macrophages, and slowed calcium uptake. Only future investigations will determine which of these possibilities provides the key to determining why calcium channel blockers slow the development of atherogenic lesions in animal models of the disease (Table 4) and in naturally occurring atherosclerosis in humans (6,45).
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Official Satellite Symposium for the XVIIth Congress of the European Society of Cardiology, The National Exhibition Centre, Birmingham, United Kingdom August 28, 1996