© 1995 American Heart Association, Inc.
Articles |
Mibefradil Prevents Neointima Formation After Vascular Injury in Rats
Possible Role of the Blockade of the T-Type Voltage-Operated Calcium Channel
From the Pharma Division, Preclinical Research, Clinical Research and Development (R.S., F.R.B.), F. Hoffmann–La Roche Ltd, Basel, Switzerland.
Correspondence to R. Schmitt, Clinical Research and Development, F. Hoffmann–La Roche Ltd, CH-4002 Basel, Switzerland.
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Abstract |
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Abstract Mibefradil is a novel calcium antagonist that is selective for the T-type voltage-operated calcium channel rather than the L type. Because T-type calcium channels are present on rapidly proliferating cells and mediate the increase of intracellular calcium induced by some growth factors, such as platelet-derived growth factor, we hypothesized that the blockade of T channels could prevent the excessive smooth muscle cell proliferation that occurs in conditions such as vascular injury. To test this hypothesis, we evaluated in rats the effects of mibefradil (which blocks both L- and T-type channels) on neointima formation after vascular injury, and we compared them with those of equihypotensive doses of amlodipine and verapamil (which block only L-type channels). Mibefradil (30 mg/kg) decreased the area of neointima formed 14 days after balloon injury by 54% (P<.001). In contrast, neither verapamil nor amlodipine had an effect despite a blood pressure reduction at least equal to that of mibefradil. The in vivo effect of mibefradil was indeed an inhibition of smooth muscle cell proliferation, as shown by thymidine incorporation experiments. This antiproliferative effect of mibefradil was also observed in vitro in smooth muscle cells stimulated by fetal calf serum. In this condition also, verapamil was ineffective. We conclude that in rats mibefradil has a potent antiproliferative effect on smooth muscle cells after vascular injury. This effect might be due to blockade of voltage-operated T channels.
Key Words: calcium channel antagonist • vascular injury • rats • cell proliferation, smooth muscle • mibefradil
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Introduction |
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Recent electrophysiological studies have shown that L- and T-type voltage-operated calcium channels exist in SMCs.1 2 The T channels have a lower conductance than the L channels and are activated transiently and at low voltage.3 These T-type calcium channels are present in rapidly proliferating cells, such as fibroblasts.4 5 T channels have also been shown to mediate the sustained increase of intracellular calcium induced by angiotensin II,6 endothelin,7 and PDGF.8 However, up to now, no pathophysiological role has been attributed to these channels because no drug has been able to selectively block these channels or been usable in vivo.
Recently, mibefradil, which is a new nondihydropyridine calcium antagonist,9 10 11 12 has been shown to block T channels selectively.13 In SMCs, mibefradil is about 10 times more potent in blocking T channels than L channels.13 In contrast, other available calcium antagonists such as nifedipine are known not to be able to block T channels at biologically relevant concentrations.14
The goal of the present study was to test the hypothesis that T channels could play a role in the vascular response to injury. For this purpose, we used a previously described rat model, in which injury is induced by ballooning the carotid artery.15 16 17 In this model, we compared the effects of mibefradil (which is known to block T channels) with those of amlodipine and verapamil (which are classic blockers of the voltage-operated L channels).
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Methods |
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In Vivo Experiments
Experiments were performed in male Wistar-Kyoto rats with body weights of 280 to 340 g. Blood pressure was measured by the tail-cuff method18 3 to 4 hours after drug administration by gavage.
Balloon Injury of the Carotid Artery and Processing of the Isolated
Vessels
Rats were anesthetized using 40 mg/kg pentobarbital IP
(Vetanarcol, Veterinaria). A midline incision was made in the neck, and
the distal left common carotid artery was exposed. A small cut was then
made in the external carotid, and an embolectomy catheter (Fogarty 2F,
Edwards Laboratories) was passed through the common carotid artery into
the aortic arch. The balloon was inflated with water until slight
resistance was met to expand the common carotid artery and then slowly
twisted back to the carotid bifurcation. This procedure was repeated
three times to achieve complete denudation of the
endothelium in the common artery, including a
mild-to-moderate injury of the inner layers of the media. Afterward the
balloon was removed, the external carotid ligated, and the wound
closed.
After 14 days, the animals were anesthetized and perfusion-fixed as described,19 with the modification that 2.5% glutaraldehyde and a 90 mm Hg perfusion pressure were used. The ballooned left carotid artery and the right carotid artery (control) were isolated, placed in fixative (2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer) for 6 hours, then incubated in 0.05 mol/L KMnO4 and 0.1 mol/L cacodylate buffer (pH 7.4) for 1 hour, dehydrated through a gradient of alcohol and propylene oxide, and finally embedded in Epon 812. The arteries were cut into six segments. Semithin sections (1 µm) of the two middle segments were cut with a microtome and stained with toluidine blue and basic fuchsin. Computer-assisted morphometry was performed using the software program DIASYS I (Heinz Meyer) and an IBM AT-03 computer. Arterial cross sections were recorded with a color video camera mounted on a light microscope at a magnification of x220. The images were transferred to a monitor, and the exact boundaries of the neointima and media of the vessels were determined.
To study the extent of SMC replication in the neointima,
proliferating cells in the vascular wall were labeled with
[3H]thymidine for 24 hours before perfusion on day 9.
This time point was chosen because 9 days after ballooning,
neointimal proliferation is likely to be in full progress.
Before fixation (17, 9, and 1 hour), the animals were given
[3H]thymidine by intraperitoneal
injection (0.5 mCi/kg; 6.7 Ci mmol/L, New England Nuclear). Assuming
that the S phase of SMCs takes about 8 hours, the three-dose regimen
should have resulted in labeling of all SMCs undergoing DNA synthesis
within the 24-hour period prior to fixation. After fixation and cutting
of the Epon 812–embedded arteries (1 µm), the sections were dipped
in a 1:1 mixture of water and K5 nuclear research emulsion (Ilford) and
stored in light-tight boxes for 2 weeks at 4°C. The sections were
developed with Kodak D19 developer and subsequently stained with
toluidine blue and basic fuchsin. Morphometric analysis was
then performed. When autoradiographically processed, cells
that proliferate during the 24-hour labeling period exhibit black
grains over the nucleus. Cells were considered to be positively labeled
if the number of grains per nucleus was 
3. All cells in the medial
and neointimal cell layers were counted. The system
provided the user with the variables for the media and
neointima: number of unlabeled cells, number of labeled
cells, and proliferation index as a percentage (number of labeled cells
divided by number of total cells multiplied by 100).
Study Design
In the first experiments, the effects of three doses of
mibefradil (3, 10, and 30 mg/kg per day) were evaluated. Mibefradil was
given once a day by oral gavage starting 1 day before ballooning and
for 14 days thereafter.
In the second set of experiments, mibefradil (30 mg/kg per day) was compared with amlodipine (30 mg/kg per day) and verapamil (100 mg/kg per day). Preliminary experiments using telemetric measurements of arterial pressure have shown that these doses give similar hypotensive effects for 24 hours after dosing.
In all cases, morphology measurements were performed 14 days after ballooning (except the thymidine incorporation experiment).
In Vitro Experiments
Preparation of Vascular SMCs
Vascular SMCs were isolated from normotensive Wistar-Kyoto rat
aortas by enzymatic digestion as previously described.20
The aorta was cleaned extensively by dissection to remove all
connective tissue and then minced prior to enzymatic treatment. Cells
were grown in DMEM supplemented with 20% FCS, glutamine, pyruvate, and
antibiotics (GIBCO). The cultures were kept in a 38°C humidified
incubator with an atmosphere of 95% air and 5% CO2. The
cells became confluent 10 days after inoculation. The primary cells
were passaged by brief exposure to 0.05% trypsin and 0.02% EDTA in
Puck's saline A and transferred into a new dish in DMEM/10% FCS. The
so-established SMCs were allowed to grow to passage 3 to 8. Viability
of cultured cells was verified by trypan blue exclusion throughout the
experiment.
Cell Proliferation Assay
Cells in passage 5 were placed into 24-well culture plates
(Costar, 3524) and suspended in DMEM, 10% FCS, glutamate, pyruvate,
and antibiotics.
Twenty-four hours later, the drugs (or solvent, if in the control group) were added to the wells. During the incubation phase, cells remained adherent as assured by microscopic examination. At defined times the cells were trypsinized to achieve a single cell suspension and then counted using a Coulter counter. Experiments were performed in triplicate for each time point given. Phase-contrast microscopy was used to inspect the dishes for evidence of cell detachment or change in cell morphology throughout the experiments. Viability of the cells was tested routinely using FDA. The cells were first counted with phase contrast, and then the fluorescent cells were counted again 5 minutes after addition of 10 µg/mL FDA in PBS to the cultures in the same image using an appropriate filter.
Thymidine Assay Incorporation
Effects of increasing concentrations (10-8 to
10-5 mol/L) of mibefradil and verapamil on
cell proliferation and thymidine incorporation were evaluated.
Cells in passage 8 were placed into 24-well culture plates (Costar, 3524) and suspended in DMEM/10% FCS. After 48 hours the cells were growth-arrested for 72 hours by serum deprivation using DMEM supplemented with 0.5% FCS. Subsequently, the nutrient-deficient medium was exchanged for normal culture medium together with the indicated drugs, and incubation was continued for another 24 hours. During the last 2 hours of incubation 1 µCi/mL of [methyl-3H]thymidine (2.00 Ci/mmol, Du Pont) was added to the cells. Thereafter, the radioactive medium was aspirated and each well processed as follows: one wash with PBS, methanol fixation for 2x 5 minutes (1 mL/well), followed by a wash with 5% ice-cold trichloroacetic acid (1 mL/well) for 2x 10 minutes and a final rinse with water. The precipitated material was dissolved in 0.3N NaOH (0.75 mL/well) and mixed with scintillation liquid (Hionic-Fluor, Packard Instrument Co Inc). The incorporation of [3H]thymidine into acid-insoluble material was determined using a Betamatic Liquid Scintillation Counter (Kontron) and expressed as disintegrations per minute. Experiments were performed in triplicate cultures for each data point.
Statistical Analysis
Results are presented as mean±SEM. Differences between
groups in the balloon injury experiments were tested by one-way ANOVA.
Blood pressure was statistically evaluated only after 2 weeks of
treatment. For the in vitro culture experiment, a two-way ANOVA taking
into account effects of time and treatment was performed. If the
overall ANOVA indicated statistical significance, differences in group
means were assessed by Dunnett's multiple comparison test. A value of
P<.05 was considered to be statistically significant.
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Results |
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In Vivo Experiments
Mibefradil dose-dependently prevented the formation of neointima (Table 1
). This effect was
significant with 10 and 30 mg/kg of mibefradil that decreased the area
of neointima by 31% and 48%, respectively. Mibefradil
also dose-dependently decreased systolic arterial blood
pressure (Table 1). In contrast with mibefradil, neither
verapamil nor amlodipine prevented neointima
formation (Table 2). This was not due to too low doses
of amlodipine and verapamil, since these two drugs
decreased systolic arterial blood pressure at least as much
as mibefradil (Fig 1).
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Autoradiographically processed cross sections of ballooned arteries of mibefradil-treated animals showed significantly fewer total neointimal cells on day 9 (275±35 mibefradil vs 423±29 control, P<.01) and fewer labeled cells in the neointima after treatment with mibefradil (56±8 vs 125±10, P<.001). Therefore, the proliferation index, defined as the number of labeled cells divided by the number of total cells multiplied by 100, tended to be lower after treatment with mibefradil compared with the control group (22±3% vs 30±3%, P=.06).
Within the medial cell layer, both in the control group and in the
mibefradil-treated group, far fewer cells were labeled compared with
the neointima, indicating that at 9 days after injury
medial proliferative response to injury is nearly complete. The number
of unlabeled cells and the number of total cells were slightly lower
after treatment with mibefradil, but the proliferation index was not
different from controls (Table 3). In the uninjured
carotid arteries, the proliferation index was less than 0.05%.
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The morphometric data revealed a significant reduction in
neointima formation (55%, P<.01 compared with
control group) and a significantly smaller media in treated animals
(Table 3
).
In Vitro Experiments
Rat SMCs grown in the presence of 10% FCS showed a fivefold
increase in cell number after 3 days in culture. On day 3, mibefradil
decreased the growth by 50% (P<.001) compared with control
cultures, whereas verapamil had no effect (Fig 2). Both mibefradil and verapamil had direct
toxic effects on the cells at high concentrations (10-4
mol/L). However, both drugs had no direct toxic effects at doses of
10-5 mol/L.
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The decrease of proliferation induced by mibefradil was paralleled
by a decrease of thymidine incorporation (Fig 3). In
contrast, verapamil did not change thymidine
incorporation.
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Discussion |
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Our results show that mibefradil has strong antiproliferative effects on SMCs both in vitro and in vivo. Interestingly, neither amlodipine nor verapamil had such an effect. Since mibefradil has, in comparison with amlodipine and verapamil, the additional property to block voltage-operated T-type calcium channels,13 these results suggest that T channels might play an important role in the proliferative response of SMCs after vascular injury.
The effect of mibefradil was to decrease SMC proliferation, as shown by
the association of a decrease in neointima and a decreased
number of cells labeled with thymidine. The decrease in
neointima formation induced by mibefradil was dose related
and reached 
54% with the highest dose tested (30 mg/kg). The
antiproliferative effect of mibefradil was also observed in vitro for
concentrations higher than 10-6 mol/L. Such concentrations
are well within the therapeutic range of those observed in clinical
trials.21 In addition, mibefradil has been shown
experimentally to be more concentrated in tissues than in plasma (H.R.
Wiltshire, personal communication). Therefore, it is likely that
concentrations as high as 10-5 mol/L can be obtained in
several organs of patients treated with the recommended dose of
mibefradil. In vitro, electrophysiological studies
have also shown that the pharmacologically active doses on SMCs are
between 10-6 and 10-5 mol/L.
The slight decrease of the media in unballooned arteries is unlikely to be due to a direct toxic effect of mibefradil but is rather related to the decrease in arterial blood pressure. A similar effect seems to have occurred with verapamil. In addition, chronic (1 year's duration) treatment with doses as high as 40 mg/kg per day of mibefradil did not induce a toxic vascular effect (data not shown).
Interestingly, in vivo neither amlodipine nor verapamil had such an antiproliferative effect, which suggests that the effect of mibefradil is unlikely to be due to a blockade of the L-voltage–operated calcium channel. These negative results were not due to a too low dosage, because arterial blood pressure was markedly decreased with both verapamil and amlodipine. In fact, both drugs decreased arterial blood pressure slightly more than did mibefradil.
This result confirms one of our previous studies, in which verapamil also had no significant antiproliferative effect.22 To our knowledge, effects of amlodipine on vascular injury have not been reported. In contrast, in a similar model, another dihydropyridine-type calcium antagonist (isradipine) decreased neointima formation.23 However, the effect was smaller than what we observed. In another study in rats, verapamil and nifedipine had an effect on thymidine incorporation in the neointima, but no quantitative morphometry was performed.24 In rabbits, a decrease of 39% of neointima formation was observed with nifedipine.24
The fact that mibefradil had an antiproliferative effect in contrast with amlodipine and verapamil suggests that the blockade of T channels could play a significant role. In SMCs, mibefradil is a selective blocker of T channels in contrast with dihydropyridine-type calcium antagonists such as nisoldipine.13 These T channels seem to mediate the increase of intracellular calcium induced by factors such as angiotensin II,6 endothelin,7 and PDGF.8 It has even been shown that blockade of T channels with nordihydroguaiaretic acid could prevent the proliferative effect of PDGF.8
In addition, the density of T channels seems to increase when cells start to proliferate or grow. This has been shown very well in cardiac myocytes from hearts with hypertrophy25 or cardiomyopathy.26 Interestingly, T channels have a higher density in neonatal or fetal cells,27 and it is known that myocytes from hypertrophied hearts tend to recover a neonatal phenotype.28
To our knowledge, the present study is one of the first showing a pathophysiological role for T channels. In sinus node cells, it has been suggested that T channels could play a pacemaker role,29 30 but in SMCs no precise function has been attributed to T channels. This is mainly explained by the absence of a specific blocker. Mibefradil is easy to use in vivo and is a potent blocker of T channels. Therefore, this drug allows in vivo testing of the role of T channels.
Mibefradil is presently being tested in humans for the treatment of
hypertension and angina pectoris.2 The therapeutically
active plasma concentration observed in humans (10-6
mol/L) is compatible with the antiproliferative effect of mibefradil
observed in the present study. However, drugs such as
angiotensin-converting enzyme inhibitors that
are active in the rat balloon injury model22 have been
shown to be inactive in a clinical restenosis
trial.31 Therefore, further preclinical studies are needed
to evaluate the antiproliferative effects of mibefradil in other
species and under other experimental conditions before extrapolating
the present findings to the clinical environment.
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Selected Abbreviations and Acronyms |
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Received February 10, 1995; accepted May 10, 1995.
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