Collagens are key components of the extracellular framework of arterial media and major constituents of human atherosclerotic plaques (1,2). Matrix metalloproteinases (MMPs) specialize in resorption of extracellular matrix components, especially collagens (3). The physiologic activity of MMPs is inhibited by specific tissue inhibitors of metalloproteinases (TIMPs). MMP-1, also called interstitial metalloproteinase, is a member of MMP family and is characterized by its distinctive ability to degrade collagen types I and III (4), which are most abundant in atherosclerotic plaques.
In human atherosclerosis, unstable atherosclerotic plaque rupture is an important event that triggers acute coronary syndrome. Plaque rupture is frequently correlated with loss of the extracellular matrix at certain locations, often in the shoulder areas of the plaque. Focal destruction of the extracellular matrix renders the plaque less resistant to mechanical stresses imposed during systole and therefore vulnerable to rupture. Recent findings have revealed that MMP activity is increased in the vulnerable region of plaques and contributes to the weakening of plaque caps by degrading extracellular collagens (5-8).
Calcium channel blockers have been demonstrated to reduce the severity of experimentally induced atherosclerosis (9), and some clinical trials suggested that they retard or impede the progression of atherosclerosis in humans (10,11). However, Furberg et al. (12) recently reported that the short-acting calcium channel blocker nifedipine, when used for the treatment of hypertension or in patients after myocardial infarction, paradoxically increases the rate of heart attack and mortality, creating controversy concerning the safety and efficacy of this drug class. Although the advantages of the long-acting calcium channel blocker amlodipine to prevent coronary atherosclerosis is under investigation (13), a PRAISW-1 trial has shown a substantial beneficial effect in a subgroup of patients with dilated cardiomyopathy (14). In contrast to nifedipine, amlodipine has been shown also to act by L-type channel-independent mechanisms such as stimulation of nitric oxide (NO) production by endothelial cells (ECs) (15,16). If calcium channel blockers affect MMP activity, they could influence the plaque stability and disease progression of coronary artery diseases. We investigated whether the calcium channel blockers amlodipine and nifedipine modulate MMP-1 expression in human vascular ECs.
Amlodipine and nifedipine were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). They were dissolved in dimethylsulfoxide (DMSO) at a concentration of 10−2M. Recombinant human interleukin-1β (IL-1β) was kindly provided by Otsuka Pharmaceutical Co. (Tokushima, Japan). Fetal calf serum (FCS) was purchased from Cell Culture Laboratories (Cleveland, OH, U.S.A.). Bovine serum albumin (BSA) and endothelial cell growth factor supplement (ECGS) were purchased from Collaborative Research (Bedford, MA, U.S.A.).
Human EC culture
Primary ECs were harvested from human umbilical cord veins treated with 0.1% collagenase, as described elsewhere (17). The ECs were grown on 5% gelatin-precoated 60-mm culture dishes in M199 containing 20% heat-inactivated FCS, 1% penicillin/streptomycin solution, glutamine (2 mM), HEPES (15 mM) and ECGS (60 μg/ml). Confluent monolayers of ECs (1.2 × 105 cells) between passages two and four on 24-cell plates in FCS-free, 0.1% BSA-containing medium were used for the experiments.
Assay for MMP-1 and TIMP-1 levels
The MMP-1 and TIMP-1 concentrations of the culture media were determined using respective enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions (Amersham International plc, Buckinghamshire, U.K.). The lower limits of detection of MMP-1 and TIMP-1 were 6.25 and 3.13 ng/ml, respectively. After washing, cells were dissolved in 0.2 ml of 1% sodium dodecyl sulfate and used for protein assay (Bio-Rad protein assay kit, Hercules, CA, U.S.A.) with bovine serum albumin as a standard. MMP-1 and TIMP-1 levels were corrected by protein measurement, and data are shown as μg/mg protein.
Assay for collagenolytic activity
Collagenolytic activity, which reflects MMP-1 activity as well as MMP-8 and MMP-13 activity, was measured by fluorescent-labeled collagen digestion (Yagai Co., Yamagata, Japan). Fluorescent-labeled collagen tubes (50 μg/50 μl), after adding 50 μl neutralized fluids (pH 7.5), were mixed with 100-μl samples or with double-diluted neutralized fluids as the blank and the total. Samples and the blank were incubated at 37°C for 3 h with 10 μl aminophenyl mercuric acetate (2.4 mg/ml), while the total was treated at 100°C for 1 min. After the reaction was stopped by adding 200 μl stop solution, the fluorescence in the supernatants was measured at 520 nm with excitation at 495 nm using a fluorescence spectrometer (Nihon Bunko Corp., Tokyo, Japan). Collagenolytic activity was calculated according to the equation applied in the protocol.
All values were expressed as mean ± SEM of four samples, which represented at least three separate experiments. The significance of the difference was determined using one-way analysis of variance (ANOVA) combined with Scheffé's test. Differences of p < 0.05 were considered significant.
Effects of calcium channel blockers on MMP-1 and TIMP-1 levels
We initially investigated the dose-response effect of the calcium channel blockers amlodipine and nifedipine on MMP-1 levels in conditioned media of ECs. Incubation of ECs with these agents for 48 h showed no significant effect on basal MMP-1 levels (data not shown). The addition of IL-1β (10 ng/ml) for 48 h significantly increased MMP-1 levels of ECs. Amlodipine significantly decreased these IL-1β-induced MMP-1 levels at 10−5M, whereas nifedipine showed no significant effect (Fig. 1).
The ECs expressed low levels of TIMP-1. Amlodipine and nifedipine showed no significant effect on basal TIMP-1 levels (data not shown). As shown in Fig. 2, the addition of IL-1β for 48 h significantly increased TIMP-1 levels, and amlodipine, but not nifedipine, slightly decreased TIMP-1 levels of IL-1β-stimulated ECs.
Effects of calcium channel blockers on collagenolytic activity
We then investigated the effect of the calcium channel blockers on collagenolytic activity in conditioned media of IL-1β-stimulated ECs. As shown in Fig. 3, the addition of amlodipine for 48 h dose-dependently reduced collagenolytic activity, whereas nifedipine showed no significant effect.
The extracellular matrix molecules are primarily responsible for maintaining the mechanical integrity of the atherosclerotic fibrous cap. These matrix components may be degraded by MMPs, which can be produced by ECs as well as by macrophages and vascular smooth muscle cells. Previously Singhal et al. (18) reported that amlodipine decreased MMP-2 activity in rat glomerular mesangial cells, but no studies have examined the effects of calcium channel blockers on MMP expression in ECs. Our findings revealed that amlodipine, but not nifedipine, significantly decreased MMP-1 expression in human vascular ECs.
To assess the role of the active MMP-1, it also is important to consider the production of TIMP-1. TIMP-1 is synthesized by most types of cells, including ECs, and acts against all members of MMPs but has a particular affinity for MMP-1. Production of TIMP-1 as well as MMP-1 has been shown to be induced by cytokines such as IL-1β (4,19). The decrease in IL-1β-induced MMP-1 expression by amlodipine does not necessarily lead to decreased matrix degradation, because TIMP-1 levels also were slightly decreased by amlodipine. Therefore we measured collagenolytic activity, which reflects MMP-1 activity as well as MMP-8 and MMP-13, and found that collagenolytic activity also was significantly suppressed by amlodipine, not by nifedipine.
It is unknown why amlodipine alone decreased MMP-1 expression in ECs. Calcium channel blockers may act by L-type channel-independent mechanisms as well as by L-type channel-dependent mechanisms. Recently Stepien et al. (15) reported that amlodipine and nifedipine inhibited DNA synthesis in rat vascular smooth muscle cells, but the calcium channel agonist Bay K 8644 effectively antagonized only the effect of nifedipine, but not that of amlodipine. They speculated that in addition to its L-type channel inhibition, amlodipine inhibited other intracellular signaling pathways. On the other hand, Zhang and Hintze (16) investigated the effects of calcium channel blockers on NO production from canine coronary microvessels, and found that nifedipine did not increase NO production, whereas in marked contrast, amlodipine caused a dose-dependent increase in NO production. The ECs have not been shown to possess L-type calcium channels (20). Thus, we speculated that some unknown mechanisms other than L-type channel inhibition are involved in the inhibitory effect of amlodipine on MMP-1 expression.
Previously, Mason et al. (21) investigated membrane antioxidant effects of calcium channel blockers and found that amlodipine significantly inhibited lipid peroxide formation, whereas other representative calcium channel blockers, felodipine, verapamil, and diltiazem, showed no effect. They speculated that the chemical structure of amlodipine contributes to distinct biophysical membrane interactions that lead to the potent lipid antioxidant effect, independent of calcium channel modulation. This antioxidant effect of amlodipine may be related to its inhibitory effect on MMP-1 expression.
Some findings suggest that MMP activity contributes to the destruction of connective tissues in the atherosclerotic lesion, leading to surface disruption. By using in situ hybridization and immunohistochemistry, several researchers have detected the expression of several MMPs including MMP-1 at the borders of the lipid core adjacent to fibrous caps and shoulder areas (22-24). Galis et al. (25) reported that MMP-1, undetectable in the endothelium of nonatherosclerotic arteries, was expressed in the endothelium covering atherosclerotic plaques. These observations suggest that matrix degeneration exceeds the synthesis at certain locations in some atheromas, predisposing them to plaque rupture. In addition, increased MMP-1 activity in atherosclerotic plaques may allow the migration and proliferation of vascular smooth muscle cells from the media into the intima, where they form the major components of stenotic lesions.
In atherosclerotic lesions, fibrillar collagen types I and III form 90% of the total collagen (1,2). The accumulation of collagen is influenced by its de novo synthesis and deposition, and degradation of existing collagen by MMP-1 (26). We found that amlodipine inhibits IL-1β-induced MMP-1 expression in human ECs. The concentration (10−5M) required to mediate the observed effects was at least 10-fold higher than the therapeutic serum concentrations (10−7-10−6M) reported for amlodipine. However, as amlodipine has a high membrane partition coefficient and a slow rate of dissociation (27), local accumulation into the membrane may be high and in the micromolar range; this concentration may induce significant inhibition of MMP-1 expression.
Acknowledgment: We thank Toshiko Kambe for her technical assistance. This study was supported by a research grant for Cardiovascular Diseases (10C-1) from the Ministry of Health and Welfare, a research grant (10670675) from the Ministry of Education, Science, Sports and Culture, and the Takeda Medical Foundation.
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