Background

Neutrophils, in concert with proinflammatory cytokines, play an important role in the progression of atherosclerosis. Calcium channel blockers are commonly used in the treatment of hypertension, and their pleiotropic effects, other than the lowering of blood pressure, have been recently recognized.

Methods

We studied the effects of various calcium channel blockers (amlodipine, nicardipine, cilnidipine, benidipine, efonidipine, nifedipine, azelnidipine, verapamil, and diltiazem; each being used at 5 and 10 mol/l) on superoxide (O₂^-) release, migration, and signaling pathways in human neutrophils stimulated by granulocyte–macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor- (TNF-).

Results

GM-CSF-induced O₂^- release was suppressed by amlodipine, nicardipine, and cilnidipine, whereas TNF--induced O₂^- release was suppressed by amlodipine, nicardipine, cilnidipine, benidipine, efonidipine, nifedipine, and azelnidipine. TNF--induced phosphorylation of extracellular signal–regulated kinase (ERK) and Akt, but not p38 mitogen-activated protein kinase (MAPK), was attenuated by nicardipine, cilnidipine, benidipine, efonidipine, and azelnidipine. By contrast, GM-CSF-induced phosphorylation of ERK, p38, and Akt was affected by none of the blockers. GM-CSF-induced neutrophil migration was also suppressed by amlodipine and nicardipine, but not by azelnidipine, when these blockers were assessed for their effect on neutrophil migration.

Conclusions

These findings suggest that (i) some calcium channel blockers can suppress cytokine-induced neutrophil activation, leading to possible prevention of the progression of atherosclerosis; and (ii) that activation of the ERK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways, induced by TNF- but not by GM-CSF, is selectively affected by some blockers.

Methods

Reagents. Recombinant human GM-CSF and TNF- produced by Escherichia coli were provided by Schering-Plough (Osaka, Japan) and Dainippon Pharmaceutical (Osaka, Japan), respectively. The specific activity of TNF- was 3 10⁶ U/mg protein. Phorbol myristate acetate, cytochrome c and superoxide dismutase were purchased from Sigma (St Louis, MO). Ficoll and the enhanced chemiluminescence Western blotting system were purchased from Amersham Pharmacia Biotech (Buckinghamshire, England). Conray was purchased from Mallinckrodt (St Louis, MO). Rabbit polyclonal antibodies against ERK1/2, Thr²⁰²/Tyr²⁰⁴-phosphorylated ERK1/2, p38 MAPK, Thr¹⁸⁰/Tyr¹⁸²-phosphorylated p38 MAPK, Akt, and Ser⁴⁷³-Phosphorylated Akt were purchased from Cell Signaling Technology (Beverly, MA). Amlodipine, nicardipine, cilnidipine, benidipine, efonidipine, and nifedipine were provided by Ajinomoto (Tokyo, Japan). Azelnidipine, verapamil, and diltiazem were provided by Sankyo (Tokyo, Japan), Eisai (Tokyo, Japan) and Tanabe (Osaka, Japan), respectively. All the calcium channel blockers were highly purified preparations and, except for nifedipine and diltiazem, they were racemic mixtures. The calcium channel blockers were dissolved in dimethylsulfoxide.

Preparation of cells. Human peripheral blood neutrophils were prepared from healthy adult donors as described previously,¹⁸ using dextran sedimentation, centrifugation with Conray-Ficoll, and hypotonic lysis of contaminating erythrocytes. Neutrophil fractions contained >98% neutrophils. The neutrophils were suspended in Hanks' balanced salt solution containing 10 mmol/l N-2-hydroxyethyl-piperazine-N'-2-ethane-sulfonic acid (pH 7.4).

Determination of O ₂^- release. O₂^- was assayed by superoxide dismutase–inhibitable reduction of ferricytochrome c as described previously.^18,19 The cell suspension in Hanks' balanced salt solution was added to each well of a 48-well plate containing 100 mol/l ferricytochrome c with or without superoxide dismutase (200 U/ml) to obtain a final volume of 0.2 ml. The well surface was precoated with fetal calf serum to prevent direct stimulation of neutrophils by adherence to the plastic surface. The final cell concentration was 3 10⁵ cells/0.2 ml. When required, cells were pretreated with various calcium channel blockers (5 and 10 mol/l) for 20 min at 37 °C. After incubation with GM-CSF (5 ng/ml), TNF- (100 U/ml), or phorbol myristate acetate (100 ng/ml) for 2 h at 37 °C, the reduction of ferricytochrome c was measured at 550 nm with a reference wavelength at 540 nm. Cell viability was determined using the trypan blue exclusion test, and it was confirmed that the calcium channel blockers used were not toxic to neutrophils.

Time-lapse recording and analysis of cell migration. Cells (3.5 10⁵/ml) suspended in Hanks' balanced salt solution were placed in a glass-bottom dish (MatTek, Ashland, MA), the surface of which was precoated with fetal calf serum to prevent spontaneous neutrophil adherence to the glass surface. The cell suspensions were kept at 37 °C, a warmer being placed under the dish. Cells were monitored under a microscope (Olympus, Tokyo, Japan) with a 40 phase-contrast objective lens, and the videotape recording was performed as described previously.²² The videotape recording was converted to digital imaging for analysis. The migration distance and speed were determined using an image analysis software MOVE-TR/2D (Library, Tokyo, Japan). The center of the cell body was traced every 10 s with the software in order to determine the migration distance during the period required. The migration speed was determined by measuring the distances that cells traveled during each 5-min period. When required, the cells were pretreated with various calcium channel blockers for 20 min at 37 °C before stimulation with GM-CSF.

Western blotting. Cells suspended in Hanks' balanced salt solution were stimulated with GM-CSF (5 ng/ml) or TNF- (100 U/ml) for 10 min at 37 °C.¹⁸ When required, the cells were pretreated with various calcium channel blockers (10 mol/l) for 20 min at 37 °C. The reactions were terminated by the addition of trichloroacetic acid. The final trichloroacetic acid concentration was 10%. After 1 h of incubation on ice, the cells were centrifuged, and the pellet was washed with acetone containing 10 mmol/l dithiothreitol. The pellet was resuspended in 1.3 sample buffer (4% sodium dodecyl sulfate, 20% glycerol, 10% mercaptoethanol, and a trace amount of bromophenol blue dye in 125 mmol/l Tris-HCl, pH 6.8), sonicated, heated at 100 °C for 5 min, and frozen at -20 °C until use. Samples were subjected to 10% sodium dodecyl sulfate gel electrophoresis. After electrophoresis, proteins were electrophoretically transferred from the gel onto a nitrocellulose membrane in a buffer containing 100 mmol/l Tris, 192 mmol/l glycine, and 20% methanol at 2 mA/cm² for 1.5 h at 25 °C. Residual binding sites on the membrane were blocked by incubation of the membrane in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 and 5% nonfat dry milk for 2 h at 25 °C. The blots were washed in Tris-buffered saline containing 0.1% Tween 20 and then incubated with appropriate antibody overnight at 4 °C. After washing with Tris-buffered saline containing 0.1% Tween 20, the membrane was incubated with anti-rabbit IgG-antibody conjugated with horseradish peroxidase, and the antibody complexes were visualized with the enhanced chemiluminescence detection system, in accordance with the directions of the manufacturer.

Statistical analysis. An ANOVA followed by a multiple comparison test or Student's t- test was used for determining statistical significance.

Results

GM-CSF- or TNF--induced O₂^- release in neutrophils is suppressed by calcium channel blockers

In the initial experiment, we studied the effect of amlodipine on GM-CSF- or TNF--induced O₂^- release in neutrophils. Neutrophils were pretreated with various concentrations (1–10 mol/l) of amlodipine for 20 min, and thereafter stimulated with GM-CSF or TNF- for 2 h. As shown in Figure 1, GM-CSF- or TNF--induced O₂^- release was suppressed by amlodipine in a dose-dependent manner. GM-CSF- or TNF--induced O₂^- release was significantly suppressed at 2.5 mol/l of amlodipine, and was almost completely abolished at 10 mol/l of amlodipine. GM-CSF- or TNF--induced O₂^- release was also suppressed by nicardipine, another calcium channel blocker, in a dose-dependent manner (Figure 1). GM-CSF-induced O₂^- release was significantly suppressed at 5 mol/l of nicardipine, whereas TNF--induced O₂^- release was significantly suppressed at 1 mol/l of nicardipine. GM-CSF- or TNF--induced O₂^- release was markedly suppressed at 10 mol/l of nicardipine. Neutrophil O₂^- release induced by GM-CSF and TNF- showed similar sensitivities to the suppressive effect of amlodipine. On the other hand, the suppressive effect of nicardipine on TNF--induced O₂^- release was significantly greater (P < 0.01) than that on GM-CSF-induced O₂^- release, as assessed at each concentration of nicardipine (1, 2.5, 5, and 10 mol/l) (Figure 1). These findings indicate that neutrophil response induced by TNF- is more sensitive to nicardipine, but not amlodipine, as compared with that induced by GM-CSF. Because a significant suppressive effect was observed at 5 and 10 mol/l of amlodipine or nicardipine, the comparative study with various calcium channel blockers was performed using 5 and 10 mol/l of each blocker.

Figure 1.

Effects of (a) amlodipine and (b) nicardipine on O₂^- release in neutrophils stimulated by granulocyte–macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor- (TNF-). Neutrophils were pretreated with or without the indicated concentrations of amlodipine or nicardipine for 20 min at 37 °C, and thereafter stimulated with GM-CSF (5 ng/ml) or TNF- (100 U/ml) for 2 h at 37 °C. In this experiment, the amounts of O₂^- release in control cells stimulated by GM-CSF and TNF- were 5.1 0.8 and 6.9 1.3 nmol/2 h/3 10⁵ cells, respectively. The data are expressed as mean values SD of three independent experiments. Significantly inhibited by amlodipine or nicardipine (*P < 0.01).

Full figure and legend (13K)

For the comparative study, neutrophils were pretreated with various calcium channel blockers (5 or 10 mol/l) for 20 min, and thereafter stimulated with GM-CSF or TNF- for 2 h. As shown in Figure 2, GM-CSF-induced O₂^- release was significantly suppressed by amlodipine, nicardipine, and cilnidipine, whereas it was unaffected by the other calcium channel blockers (benidipine, efonidipine, nifedipine, azelnidipine, verapamil, and diltiazem) at the concentrations used. The potency of these blockers to suppress GM-CSF-induced O₂^- release was amlodipine > nicardipine > cilnidipine. The suppressive effect of 10 mol/l of amlodipine was significantly (P < 0.05) greater than that of 10 mol/l of nicardipine, which, in turn, was significantly (P < 0.05) greater than that of 10 mol/l of cilnidipine (Figure 2).

Figure 2.

Effects of various calcium channel blockers on O₂^- release in neutrophils stimulated by (a) granulocyte–macrophage colony-stimulating factor (GM-CSF), (b) tumor necrosis factor- (TNF-), or (c) phorbol myristate acetate (PMA). Neutrophils were pretreated with or without the indicated concentrations (5 or 10 mol/l) of various calcium channel blockers for 20 min at 37 °C, and thereafter stimulated with GM-CSF (5 ng/ml), TNF- (100 U/ml), or PMA (100 ng/ml) for 2 h at 37 °C. In this experiment, the amounts of O₂^- release in control cells stimulated by GM-CSF, TNF-, and PMA were 4.8 0.9, 7.3 1.7, and 10.9 1.4 nmol/2 h/3 10⁵ cells, respectively. The data are expressed as mean values SD of five independent experiments. Significantly inhibited by each calcium channel blocker (*P < 0.01, **P < 0.05).

Full figure and legend (35K)

TNF--induced O₂^- release was significantly suppressed by amlodipine, nicardipine, cilnidipine, benidipine, efonidipine, nifedipine, and azelnidipine, whereas it was unaffected by verapamil and diltiazem. The potency of these blockers to suppress TNF--induced O₂^- release was amlodipine > nicardipine > cilnidipine, benidipine, efonidipine, nifedipine, and azelnidipine (Figure 2). The suppressive effect of 10 mol/l of amlodipine was significantly (P < 0.05) greater than that of 10 mol/l of nicardipine, which, in turn, was significantly (P < 0.05) greater than that of 10 mol/l of cilnidipine, benidipine, efonidipine, nifedipine, or azelnidipine. The suppressive effect of nicardipine, cilnidipine, benidipine, efonidipine, nifedipine, or azelnidipine on TNF--induced O₂^- release was significantly greater (P < 0.01) than that on GM-CSF-induced O₂^- release when the suppressive effect was assessed at the given concentration (10 mol/l) (Figure 2). These findings indicate that the neutrophil response induced by TNF- is more sensitive to these blockers than that induced by GM-CSF.

In contrast to cytokine-induced O₂^- release, phorbol myristate acetate–induced O₂^- release was unaffected by any of the blockers. These findings suggest that some calcium channel blockers specifically suppress neutrophil O₂^- release induced by proinflammatory cytokines, and that the suppressive effect of calcium channel blockers on cytokine-induced O₂^- release may be physiologically relevant (Figure 2).

GM-CSF-induced neutrophil migration is suppressed by calcium channel blockers

The time-lapse recording showed that GM-CSF, like granulocyte colony-stimulating factor,²² induced neutrophil migration (Figure 3). Active migration was detected within 5 min and was sustained for >50 min after GM-CSF stimulation. Using this method, the effect of amlodipine, nicardipine, and azelnidipine on GM-CSF-induced neutrophil migration was examined. As shown in Figure 3, GM-CSF-induced neutrophil migration was suppressed by amlodipine and nicardipine, but not by azelnidipine. Neutrophils displayed little migration in response to TNF- stimulation. Therefore the effect of calcium channel blockers on TNF--induced neutrophil migration was not further examined.

Figure 3.

Effects of amlodipine, nicardipine, and azelnidipine on granulocyte–macrophage colony-stimulating factor (GM-CSF)-induced neutrophil migration. Neutrophils were pretreated with or without amlodipine (10 mol/l), nicardipine (10 mol/l) or azelnidipine (10 mol/l) for 20 min at 37 °C, and thereafter simulated with GM-CSF (5 ng/ml). (a) The tracks of the moving neutrophils at 20–25 min after GM-CSF stimulation. The images shown are representative of three independent experiments. (b) The migration speed determined at 20–25 min after GM-CSF stimulation. Essentially, identical results were obtained at other time points as well. The data are expressed as mean values SEM (n = 50–70 cells). Significantly inhibited by amlodipine or nicardipine (*P < 0.01, **P < 0.05).

Full figure and legend (72K)

Effects of calcium channel blockers on GM-CSF- or TNF--induced phosphorylation of ERK, p38, and Akt

Our previous studies have shown that ERK, p38, and PI3K/Akt play a crucial role in the activation of human neutrophils by cytokines (GM-CSF and TNF-),^18,21 thereby raising the possibility that calcium channel blockers might suppress cytokine-induced O₂^- release or migration by preventing activation of these signaling pathways. As shown in Figure 4, GM-CSF-induced phosphorylation of ERK, p38, and Akt was unaffected by amlodipine, nicardipine, and azelnidipine when the effect was assessed using the broad concentrations of these blockers. By contrast, TNF--induced phosphorylation of ERK and Akt, but not p38, was attenuated by nicardipine and azelnidipine, but not by amlodipine, in a dose-dependent manner (Figure 5). Because TNF--induced phosphorylation of ERK and Akt was found to be significantly attenuated by nicardipine and azelnidipine at a concentration of 10 mol/l, the effects of the other calcium channel blockers on GM-CSF- or TNF--induced phosphorylation of ERK, p38, and Akt were assessed using 10 mol/l of each blocker. As shown in Figure 6, GM-CSF-induced phosphorylation of ERK, p38, and Akt was unaffected by any of the blockers. On the other hand, TNF--induced phosphorylation of ERK and Akt, but not p38, was attenuated by nicardipine, cilnidipine, benidipine, efonidipine, and azelnidipine.

Figure 4.

Effects of amlodipine, nicardipine, and azelnidipine on granulocyte–macrophage colony-stimulating factor (GM-CSF)-induced phosphorylation of (a) extracellular signal–regulated kinase (ERK), (b) p38, and (c) Akt. Neutrophils were pretreated with or without the indicated concentrations of amlodipine, nicardipine, or azelnidipine for 20 min at 37 °C, and thereafter stimulated with GM-CSF (5 ng/ml) for 10 min at 37 °C. Immunoblotting was performed using antibodies against ERK, p38, and Akt, and the phosphorylated form of each protein. The results shown are representative of three independent experiments (upper panel of each set). The densitometric data from three independent experiments are expressed as mean values SD. The ratio of phosphorylated protein to total protein band intensity was calculated, and the ratio of cells stimulated with GM-CSF in the absence of any blocker is expressed as 1 (lower panel of each set).

Full figure and legend (64K)

Figure 5.

Effects of amlodipine, nicardipine, and azelnidipine on tumor necrosis factor- (TNF-)-induced phosphorylation of (a) extracellular signal–regulated kinase (ERK), (b) p38, and (c) Akt. Neutrophils were pretreated with or without the indicated concentrations of amlodipine, nicardipine, or azelnidipine for 20 min at 37 °C, and thereafter stimulated with TNF -(100 U/ml) for 10 min at 37 °C. Immunoblotting was performed using antibodies against ERK, p38, and Akt, and the phosphorylated form of each protein. The results shown are representative of three independent experiments (upper panel of each set). The densitometric data from three independent experiments are expressed as mean values SD. The ratio of phosphorylated protein to total protein band intensity was calculated, and the ratio of cells stimulated with TNF- in the absence of any blocker is expressed as 1 (lower panel of each set). Significantly inhibited by nicardipine or azelnidipine (*P < 0.05, **P < 0.01).

Full figure and legend (55K)

Figure 6.

Effects of various calcium channel blockers on (a) granulocyte–macrophage colony-stimulating factor (GM-CSF)- or (b) tumor necrosis factor- (TNF-)-induced phosphorylation of extracellular signal–regulated kinase (ERK), p38, and Akt. Neutrophils were pretreated with or without various calcium channel blockers (10 mol/l of each blocker) for 20 min at 37 °C, and thereafter stimulated with GM-CSF (5 ng/ml) or TNF- (100 U/ml) for 10 min at 37 °C. Immunoblotting was performed using antibodies against ERK, p38, and Akt, and the phosphorylated form of each protein. The results shown are representative of three independent experiments.

Full figure and legend (78K)

Discussion

The present experiments show that some calcium channel blockers suppress O₂^- release in human neutrophils stimulated by GM-CSF or TNF- at relatively low concentrations (1–10 mol/l). The plasma levels of both cytokines are reported to be elevated in hypertensive patients, and TNF- is synthesized in the atherosclerotic plaque.^11,12,23 In addition, neutrophil responses (O₂^- release, and phosphorylation of ERK and Akt) induced by TNF- are more sensitive to most blockers than are those induced by GM-CSF. These findings suggest that suppression of cytokine-induced neutrophil activation by some calcium channel blockers may be physiologically relevant, and may provide a favorable effect on the prevention of atherosclerosis progression in hypertensive patients who are treated with these specific blockers. The preferential effect of some blockers on TNF--induced neutrophil activation may also favor their use in preventing cardiovascular disease in hypertensive patients suffering from chronic inflammatory diseases such as rheumatoid arthritis and ulcerative colitis, in which TNF- plays a critical role in the progression of the disease.^24,25

GM-CSF-induced O₂^- release was suppressed by amlodipine, nicardipine, and cilnidipine, but was not affected by the other blockers studied. GM-CSF-induced neutrophil migration was suppressed by amlodipine and nicardipine, but not affected by azelnidipine. These findings indicate that these calcium channel blockers may suppress activation of distinct neutrophil functions induced by GM-CSF. Although these blockers (amlodipine, nicardipine, and cilnidipine) suppressed GM-CSF-induced neutrophil O₂^- release or migration, they did not affect GM-CSF-induced phosphorylation of ERK, p38, and Akt, all of which play an important role in GM-CSF-induced O₂^- release or actin reorganization.^{18,19,20,21,22} These findings suggest that these blockers affect the molecules downstream of ERK, p38 or Akt. Another possibility is that these blockers affect the pathway independent of the ERK, p38, or Akt pathway.

By contrast, TNF--induced phosphorylation of ERK and Akt, but not p38, was attenuated by nicardipine, cilnidipine, benidipine, efonidipine, and azelnidipine. TNF--induced O₂^- release was also suppressed by these blockers. These findings suggest that these blockers (nicardipine, cilnidipine, benidipine, efonidipine, and azelnidipine) may suppress TNF--induced O₂^- release, in part, by preventing activation of ERK and PI3K/Akt pathways. It is of interest that these blockers attenuate phosphorylation of ERK and Akt induced by stimulation with TNF-, but not with GM-CSF. These findings suggest that TNF- and GM-CSF use different signaling pathways in inducing phosphorylation of ERK and Akt, and that the pathway utilized by TNF-, but not the one used by GM-CSF, may be specifically affected by these calcium channel blockers. In any case, these findings indicate that these blockers differentially regulate the signaling pathways utilized by TNF- and GM-CSF. It has been reported that activation of ERK in various types of cells, including neutrophils, is dependent on PI3K, according to the stimuli used.²⁶ In fact, our previous study shows that TNF--induced phosphorylation of ERK and MAPK/ERK kinase (an upstream kinase of ERK), but not p38, in neutrophils is partially attenuated by wortmannin (a PI3K inhibitor).²¹ These findings, taken together with the observed attenuation of TNF--induced phosphorylation of ERK and Akt, but not p38, by some blockers, suggest that TNF--induced PI3K activation might be primarily prevented by these blockers.

Among the calcium channel blockers studied, amlodipine was the most potent in suppressing GM-CSF- and TNF--induced O₂^- release. However, it failed to attenuate GM-CSF- and TNF--induced phosphorylation of ERK, p38, and Akt. In addition, neutrophil O₂^- release induced by GM-CSF and TNF- showed similar sensitivities to amlodipine. These findings suggest that amlodipine suppresses GM-CSF- and TNF--induced neutrophil responses by affecting the same targets, but the nature of these targets remains to be determined. Nifedipine, like amlodipine, did not attenuate TNF--induced phosphorylation of ERK, p38, and Akt despite the strong inhibition of O₂^- release induced by TNF-. These findings suggest that amlodipine and nifedipine affect the molecules downstream of ERK, p38, or Akt to suppress TNF-- or GM-CSF-induced O₂^- release. Another possibility is that these blockers affect the pathway independent of the ERK, p38, or Akt pathways. Similar findings are obtained regarding the effect of serine protease inhibitors on cytokine-induced neutrophil activation; i.e., serine protease inhibitors suppress GM-CSF- or TNF--induced O₂^- release and adherence in human neutrophils without affecting phosphorylation of ERK and p38.²⁷

It is conceivable that the suppressive effect of calcium channel blockers on neutrophil functions may be independent of calcium channel blocking, given that voltage-dependent calcium channels are not expressed on neutrophils, and intracellular calcium concentration is not increased in neutrophils stimulated with GM-CSF or TNF-.^17,28 Amlodipine is extremely lipophilic, and is reported to be accumulated in the plasma membrane, leading to potent anti-oxidative effect on membrane lipids, apparently independent of calcium channel modulation activity.^1,7 Many calcium channel blockers, including some of the blockers used in this study, are racemic mixtures, and the biological effects of these blockers appear to be dependent on distinct enantiomers. In fact, the R⁺enantiomer, but not S-enantiomer, of amlodipine causes the production of NO in canine coronary microvessels, whereas the calcium channel blocking activity is localized in the S-enantiomer.¹ These findings taken together suggest that the suppressive effect of certain calcium channel blockers on neutrophil functions may be ascribed to distinct enantiomers. It has been reported that phorbol myristate acetate– or N-formyl-methionyl-leucyl-phenylalanine-induced O₂^- release in human neutrophils is suppressed by certain calcium channel blockers, including nicardipine, verapamil, and diltiazem, at relatively high concentrations, IC₅₀ being at least >50 mol/l.^29,30,31 Such a high concentration of calcium channel blockers could exert non-specific effects on neutrophil functions.

Both activated neutrophils and proinflammatory cytokines such as GM-CSF and TNF- are implicated in the pathogenesis of atherosclerosis and its progression.¹³ In this study, we show that some calcium channel blockers can suppress cytokine-induced neutrophil activation, and that activation of the ERK and PI3K/Akt pathways induced by TNF-, but not by GM-CSF, is selectively affected by some blockers. It is plausible that suppression of cytokine-induced neutrophil activation by some calcium channel blockers may have a favorable effect on preventing atherosclerosis progression in hypertensive patients who are treated with these specific blockers.

DISCLOSURE

The authors declared no conflict of interest.

References

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Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research, Japan, and Osaka City University Research Foundation.