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【摘要】
Objectives- Acyclic retinoid (ACR) is a synthetic retinoid with a high safety profile that has been pursued with high expectations for therapeutic use in prevention (recurrence) and treatment of malignancies. With the objective of addressing the therapeutic potential in the cardiovasculature, namely neointima formation, effects of ACR on neointima formation and the involved mechanisms were investigated.
Methods and Results- ACR was administered to cuff-injured mice which showed inhibition of neointima formation. Investigation of involved mechanisms at the cellular and molecular levels showed that ACR induces apoptosis of neointimal cells and this to be mediated by selective induction of retinoic-acid receptor ß (RARß) which shows growth inhibitory and proapoptotic effects on smooth muscle cells.
Conclusion- We show that ACR inhibits neointima formation by inducing RARß which in turn inhibits cell growth and induces apoptosis. The retinoid, ACR, may be potentially exploitable for treatment and prevention of neointima formation.
Acyclic retinoid (ACR) is a synthetic retinoid with a high safety profile that has been pursued with high expectations for therapeutic use in prevention (recurrence) and treatment of malignancies. With the objective of addressing the therapeutic potential in the cardiovasculature, namely neointima formation, effects of ACR on neointima formation and the involved mechanisms were investigated.
【关键词】 smooth muscle cell cell growth retinoid
Introduction
Retinoids are natural and synthetic derivatives of vitamin A which modulate growth, apoptosis, and differentiation of cells. 1 Many retinoids have been pursued from therapeutic standpoints with the aim of chemotherapy of cancer, leukemia, and prevention of atherosclerosis. 1 The biological effects of retinoids are mainly mediated by their receptors, retinoic-acid receptor (RAR) and retinoic-X receptor (RXR). 2 RAR interacts with both all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9cRA), and has 3 isoforms (, ß, and ). 2
Among the retinoid receptors, RARß plays a critical role in mediating the effects of retinoid on cell growth and tumor inhibition in various cancer cells. 3,4 RARß expression diminishes with development of esophageal squamous cell carcinoma. 4 Induction of RARß by ATRA and overexpression of RARß also play important roles in mediating the growth-inhibitory effects of ATRA in human lung squamous cell carcinoma. 3 These findings indicate that modulation of expression of RARß critically regulates cell growth inhibition. The role of RARß in vascular SMCs (smooth muscle cells), however, has not been previously addressed.
Acyclic retinoid (ACR) is a novel synthetic RAR-selective retinoid (supplemental Table I) which has been shown to inhibit the growth of hepatoma cells in vivo and in vitro. 5 ACR has been shown to inhibit the recurrence of hepato-cellular carcinoma with oral administration in clinical trials, and has a high safety profile causing less side effects than other retinoids. 6 Recently, ACR has been shown to preferentially act on and to elicit biological effects mainly through RARß. 7,8 At the cellular level, ACR induces apoptosis in hepatoma cells. 9,10 ACR is therefore well-defined and safe, and is thus an attractive retinoid for therapeutic use. However, its effects on vascular remodeling have not been studied.
In the present study, we investigated whether ACR shows effects on neointima formation. We also addressed the mechanism of action of ACR in the vasculature, namely through effects on expression of RARs in SMCs with a focus on the biological role of RARß. We show that expression of RARß as induced by ACR exerts inhibitory effects on vascular remodeling through regulation of SMCs. We further show that induction of RARß expression stimulates apoptosis. Collectively, we show that ACR may be a promising therapeutic agent against vascular disease, namely neointima formation.
Materials and Methods
Murine Cuff Injury Analysis
Nine-week-old male C57BL6/N mice (Oriental Yeast) underwent cuff-placement surgery in the right femoral artery as described. 11,12 ACR and ATRA were emulsified in soybean oil. ACR, ATRA, and soybean oil (as vehicle control) were administered from 1 week before the procedure up to 5 weeks after the procedure. Agents were administered orally with a stomach tube (n=40; 10 from the vehicle control group, 10 from the ATRA group, 10 from the ACR low group, and 10 from the ACR high group). Animals were euthanized after the administration period and perfused first with phosphate-buffered saline (PBS) and then with neutral buffered formalin. Thereafter, the cuffed artery was removed. The segment of the artery was stained by Elastica van Gieson staining as described. 11 We assessed areas of the neointima around the cuff by morphometric analysis as detailed previously. 12 Experiments were reproduced in at least 3 other occasions.
Please see supplement, available online at http://atvb.ahajournals.org, for additional Materials and Methods.
Results
ACR Inhibits Vascular Remodeling
We first investigated the effects of ACR on vascular remodeling by the cuff-injury model. We administered ACR, ATRA (as control), or soybean oil (as vehicle control) to wild-type mice with cuffed femoral arteries. After the administration period, we assessed the neointima by histological methods.
In the groups treated with ACR, area of the neointima around the cuff was attenuated as compared with those in the groups treated with ATRA or vehicle control. ACR attenuated formation of the neointima in a dose-dependent manner, with those administered 200 mg/kg showing greater effect than those administered 100 mg/kg. Area of the neointima was composed mainly of SMCs and SMC-like cells as shown by SM -actin staining ( Figure 1 A). To assess the effect on the neointima in a quantitative manner, the intima/media ratio, which is an analytic parameter of the neointima, was used. The intima/media ratio of the groups administered ACR was less than that of the ATRA-administered group or the vehicle control group; of note there was no difference in the medial area among groups ( Figure 1 B). ACR therefore attenuated vascular remodeling by reduction of neointimal SMCs in a dose-dependent manner.
Figure 1. A, Effect of ACR, ATRA, and vehicle control (Cntl) on the formation of neointima in a murine cuffed artery model. Representative cross sections with Elastica van Gieson staining of the cuffed artery. The medial thickness was consistent in all groups. The upper part of the figure shows low-powered views (a to d) and the middle figure shows high-powered views (e to h). Representative cross sections with SM -actin staining same as above (i to l). ACR low, 100 mg/kg; ACR high, 200 mg/kg. Scale bars 50 µm (a to d), 10 µm (e to h), 25 µm (i to l). B, Intima/media ratio and media area ratio in the cuffed artery model. Note that the intima/media ratio significantly decreased under administration of 100 mg/kg and 200 mg/kg of ACR, and media area is similar among the 4 groups. ** P <0.01. All above experiments were reproduced on at least 3 other occasions.
It is noteworthy that although ATRA has been reported to attenuate neointima formation in the vascular injury model, the effects of synthetic retinoids compared with ATRA on vascular remodeling have not been studied in vivo. Moreover, retinoid activity was assessed by measuring serum alkaline phosphatase (ALP) which is a measure of effects on bone resorption. 13 The relative levels of ALP when treated with 10 mg/kg of ATRA were at levels similar to that of ACR at 100 mg/kg, which were the dosages used for direct comparison in the present study (data not shown). Dosage of ACR at 100 mg/kg was therefore more potent than that of ATRA at 10 mg/kg.
Further, we examined adverse effects of retinoid administration. There was no significant difference in body weight gain or bone fracture during the experimental period among the groups administered ACR, ATRA, or vehicle control. Moreover, abnormal blood chemistry such as liver dysfunction or hyperlipidemia, which is known to be associated with overdose of vitamin A, 13 was not seen (data not shown). Therefore, our results were likely attributable to the primary effects of the retinoid rather than secondary effects through adverse effects.
ACR Induces Apoptosis in Neointimal Cells
We next examined whether the effect of ACR on inhibition of neointima is attributable to ACR-induced apoptosis. For this, we performed terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining on cuff-injury samples. TUNEL index in the neointima was significantly higher in the ACR-administered (100 mg/kg) group than in the ATRA-administered group or the vehicle control group. TUNEL index for the ATRA group showed marginal values, and there was no significant difference between the ATRA and vehicle control groups ( Figure 2A and 2 B).
Figure 2. A, TUNEL staining in cuffed artery specimens obtained from mice at 35 days after cuff placement. The arrows denote TUNEL-positive cells. Scale bar=10 µm. B, TUNEL index (TUNEL-positive cells/total number of cells) of ACR (100 mg/kg) and vehicle control (Cntl). ** P <0.01. C, TUNEL staining of rat SMCs treated with ACR (10 -6 mol/L) or vehicle (DMSO, Cntl) for 24 hours. The right upper and lower panels show phase contrast micrographs, and the left upper and lower panels show confocal-microscopic images. The left and right panels show the same view. D, TUNEL index of ACR (10 -6 mol/L), ATRA (10 -6 mol/L), or vehicle (DMSO, Cntl). ** P <0.01. E, DNA fragmentation assay using rat SMCs cultured with ACR or ATRA. The absorbance values representing DNA fragmentation at 0 hours were divided by that at 24 hours. ** P <0.01. F, Growth inhibition of SMCs and endothelial cells (EC) under ACR or ATRA treatment for 24 hours. The number of cells treated with retinoids for 0 hours was considered 100%. Results were expressed as percentage of growth with 100% representing untreated cells. Percentage of cells after retinoid treatment was determined. The solid line (filled circles and open rectangles) shows growth of SMCs, and the dotted line (filled triangles and open diamonds) shows growth of bovine aortic endothelial cells (BAECs). ** P <0.01. All above experiments were reproduced in at least 3 other occasions.
Next, to test whether ACR shows effects on cell proliferation, we immunostained for proliferating cell nuclear antigen (PCNA) as an established marker of cell proliferation. 14 Costaining with TUNEL was not possible because of difference in reagents and procedure, but consecutive samples were used. These studies showed that the PCNA index (PCNA positive nuclei/total nuclei) was not affected by ACR administration (supplemental Figure IA and IB). These results indicate that ACR induced apoptosis of neointimal SMCs in vivo and may lead to inhibition of progression of neointima formation by apoptosis and that effects on apoptosis rather than effects on cell growth regulation was the main effect of ACR in neointima formation. Therefore, the effect of inhibition of neointima formation correlated with the effect of ACR on neointimal apoptosis.
Cellular Apoptosis Is Induced by ACR
We then confirmed the effect of ACR on cellular apoptosis in vitro. First, SMCs were treated with ACR or ATRA then examined by TUNEL staining using confocal immunofluorescence microscopy. Cells treated with ACR (1 µmol/L) showed significant increase in TUNEL positive cells than in cells with administration of vehicle (dimethylsulfoxide minimum ; Figure 2 C). The TUNEL index was consistently higher in the groups administered ACR than for ATRA or vehicle (DMSO) ( Figure 2 D). ACR thus induced cellular apoptosis.
To next confirm the dose-dependency of apoptotic effects of ACR on SMCs, cells were treated with ACR or ATRA from 10 -8 mol/L to 10 -5 mol/L for 0 or 24 hours and analyzed by DNA fragmentation assay. DNA fragmentation ratio for treatment with 10 -7 mol/L of ACR was 1.5-fold higher than that of treatment by vehicle (DMSO) alone ( Figure 2 E, lanes 1 and 3). Additionally, that at 10 -6 mol/L of ACR was 2-fold higher, and that at 10 -5 mol/L of ACR was 3-fold higher as compared with treatment by vehicle (DMSO; lanes 1, 4, and 5). Furthermore, direct comparison of effects of ATRA and ACR on apoptosis by DNA fragmentation assay showed that ACR markedly induces apoptosis in a dose-dependent manner as compared with the dose-dependent yet very marginal increases as induced by ATRA (lanes 2 to 5). Therefore, ACR induced apoptosis of SMCs in a dose-dependent manner.
Furthermore, to compare effects between ACR and ATRA on growth inhibition, SMCs were treated with ACR or ATRA from 10 -8 mol/L to 10 -5 mol/L for 24 hours. Growth inhibition of SMCs treated with ACR 10 -8 mol/L was greater than that treated with ATRA or vehicle (DMSO; Figure 2 F), and was dose-dependent up to 10 -5 mol/L (lanes 2 to 5). On the other hand, ACR mildly inhibited cell growth of endothelial cells, which was not significantly different from that when treated with ATRA (lanes 2 to 5). These results indicated that the growth inhibitory effect of ACR is cell-type dependent with preferential effects on SMCs. ACR, therefore, showed more potent effects on apoptotic SMCs growth inhibition as compared with ATRA.
ACR Induces Expression of RARß in SMCs
ACR has been shown to preferentially act on RARß and to elicit biological effects including apoptosis mainly through RARß. 7,8 RARß, however, shows only marginal expression in SMCs. 15 We therefore hypothesized that the effects of ACR on vascular cells may be mediated by effects on RARß.
We examined effects of ACR on the expression levels of RAR, RARß, and RAR mRNA by RT-polymerase chain reaction (PCR) analysis and real-time PCR analysis. In samples treated with ACR, a marked increase in RARß was seen starting at 6 hours after the addition of ACR which increased up to 12 and 24 hours. This effect was both dose-dependent and time-dependent, as 10 µmol/L showed stronger effects as 1 µmol/L, and as effects were more prominent after longer treatment. In contrast, no changes in RAR or RAR were seen ( Figure 3 A, supplemental Figure IC to IE). Furthermore, RARß was expressed in endothelial cells, but importantly, it was not induced by ACR as seen in SMCs (supplemental Figure IIA). ACR therefore induced expression of RARß in SMCs.
Figure 3. A, Temporal profile of mRNA expression of RAR isoforms in response to ACR treatment. 18S was used as internal control. B, Temporal profile of mRNA expression of RARß in response to ACR or ATRA administration. 18S was used as internal control. C, Temporal profile of RARß protein levels in response to ACR or ATRA administration. GAPDH was used to control the amount of protein. Note that ACR induced RARß more rapidly than ATRA. All above experiments were reproduced on at least 3 other occasions.
We next compared effects of ACR and ATRA on RARß expression. ATRA is known to marginally induce expression of RARß in cultured SMCs. 15 ACR induced RARß expression in a more potent manner than ATRA in both dose-dependent and time-dependent manners at both the mRNA and protein levels ( Figure 3B and 3 C). ACR, therefore, potently induced expression of RARß.
ACR Increases Retinoic Acid Response Element (RARE)-Dependent Transcriptional Activity Through RARß
ATRA has been shown to induce endogenous expression of RARß in cultured SMCs 15 by acting on the retinoic acid responsive element (RARE) of the RARß promoter. 16 However, it remains to be clarified whether RARß expression would also be affected by treatment of SMCs with retinoids other than ATRA, namely ACR. To next examine whether ACR preferentially regulates RAR-dependent transcription through RARß, reporter assays were done using a reporter harboring a RARE or a retinoic acid non-responsive element (non-RARE [DR-1]) with expression of similar amounts of each RAR isoform and ATRA, ACR, or absence of ligand. Combination of ACR with RARß transfection stimulated RARE reporter activity higher than with RAR or RAR by approximately 2.2-fold ( Figure 4 A, lanes 6, 9, and 12). ACR did not stimulate the non-RARE (DR-1) promoter (supplemental Figure IIB). ACR therefore preferentially stimulates RARE-dependent transcription of the RARß promoter. These findings, coupled with the fact that ACR selectively induces expression of RARß in SMCs ( Figure 3A to 3 C), suggest that the biological effects of ACR in SMCs were mediated through RARß.
Figure 4. A, RARE promoter activity transfected with empty or RAR isoforms under ACR (1 µmol/L) or ATRA (1 µmol/L) administration and in the absence of ligand in C2/2 cells. ß-Galactosidase was used to normalize for transfection efficiency. ACR activated RARE promoter activity greatest in cells expressing RARß. Note that ACR also increased RARE promoter activity marginally in cells expressing RAR or RAR, however not nearly to the same extent as in the presence of RARß with ACR. * P <0.05. B, DNA fragmentation ratio in C2/2 cells overexpressing the different RAR isoforms. The absorbance values representing DNA fragmentation at 0 hours were divided by that at 24 hours. Note that there was a significant increase for RARß expression. Dose-dependent expression of RARs was shown by Western blot. * P <0.05; ** P <0.01. C, Effects of siRNA against RARß using rat SMCs treated with ACR to induce RARß then assessed by Western blot using antibodies against RAR isoforms. GAPDH was used to control the amount of protein. siEGFP, siRNA against EGFP; siRARß, siRNA against RARß. D, DNA fragmentation ratio in SMCs treated with ACR (1 µmol/L) and RARß siRNA. The absorbance values representing DNA fragmentation at 0 hours were divided by that at 24 hours. Note that there was a significant decrease in apoptosis for RARß siRNA induction. ** P <0.01. All above experiments were reproduced on at least 3 other occasions.
RARß Induces Apoptosis of Vascular SMCs
To determine whether the induction of apoptosis on SMCs by ACR is attributable to RARß induction, we next examined whether RARß induces apoptosis. DNA fragmentation assay in cells transfected with the RARs showed marginally increased DNA fragmentation ratio by approximately 1.5-fold for RAR and RAR ( Figure 4 B, lanes 4 to 12). Importantly, cells transfected with RARß showed a markedly greater dose-dependent increase by approximately 2.5-fold (lanes 8 and 9). These results show that expression of RARß induces apoptosis in SMCs.
To confirm that RARß mediates apoptosis, we used an RNA interference (RNAi) knockdown approach to examine the requirement of RARß in mediating this response. Specific effects of the constructs and protocols as not to induce apoptosis were further established by use of the negative control construct against enhanced green fluorescent protein (EGFP). 17 We first established conditions under which RARß could be specifically knocked down by siRNA transfer. The specificity of the siRNA construct against RARß was confirmed by lack of effect on related factors, RAR and RAR ( Figure 4 C).
We next examined whether RARß siRNA could inhibit apoptosis. siRNA was transfected into rat SMCs treated with ACR. After 24 hours incubation, cells were analyzed by DNA fragmentation assay. Under conditions in which ACR induced apoptosis, RARß siRNA, but not control siRNA, inhibited apoptosis of SMCs treated with ACR in a dose-dependent manner ( Figure 4 D). These findings confirm that RARß mediates cell growth as regulated by ACR.
ACR Increases RARß Expression in Neointimal Cells
As the above results showed that ACR inhibits neointima formation, that ACR is associated with increased apoptosis, furthermore that ACR induces RARß, and finally that RARß induces apoptosis, then it would be expected that RARß expression would be increased in the neointima of the cuff-injury model.
Immunohistochemistry of RARß and double immunostaining of RARß and SM -actin (smooth muscle -actin) to show that RARß expressing cells are indeed SMCs were done using the cuffed artery samples treated with ACR (100 mg/kg) or vehicle control. We found that RARß expression in the neointima was increased in the samples of the ACR-administered (100 mg/kg) group (supplemental Figure III). ACR therefore increased RARß in the neointima of injured vessels.
Discussion
ACR Inhibits Neointima Formation by Inhibiting Cell Growth and Inducing Apoptosis
In the present study, we showed that ACR inhibits neointima formation after vascular injury. Of the retinoids, ATRA, in particular, has been shown to inhibit neointima formation in the rat balloon injury model. 18,19 Our findings add to these previous studies by demonstrating that the retinoid derivative, ACR, also shows inhibitory effects on vascular remodeling through the cuff-injury model. Importantly, ACR has a unique high safety profile with lower frequency of adverse effects as compared with conventional retinoids, which is thought to be attributable to its unique acyclic structure. 6 Interestingly, ACR also shows preventive effects on carcinogenesis (eg, recurrence) that distinguishes it from ATRA, which shows effects on tumor size reduction similar to ACR. 9 With promising expectations for therapeutic application of ACR because of this property, trials have been begun in liver cancer. 5,9 Our present findings suggest that ACR may also show therapeutic (preventive) potential for vascular remodeling.
To understand the underlying effects of ACR in vascular remodeling, we investigated its cellular and biochemical mechanisms of action. ATRA has been shown to inhibit growth of SMCs and migration from the media to the intima, and to restore their phenotype in vascular lesions. 20 We found that ACR can inhibit growth of SMCs similar to ATRA, but also that ACR induces apoptosis of neointimal cells. ATRA is known to induce cell cycle arrest, 21 and also to induce differentiation of SMCs, 22 but effects on apoptosis in vivo remain unclear. Likely a result of this additional pathway to induce apoptosis, ACR reduces neointima formation more potently than ATRA. These results suggest that exposure to ACR influences the response of vascular SMCs in vascular injury.
An important finding of the present study is that in contrast to most published studies on therapeutic intervention against neointimal formation which are centered on acute (first 24 hours) to subacute effects (up to 2 weeks) and thus leave in question their long-term therapeutic effects as would be necessary in the clinical setting, ACR uniquely was able to induce as well as maintain apoptosis of neointimal cells at least up to 5 weeks after injury, which suggests that it is a viable therapeutic agent for prolonged use. Our results therefore suggest that the mechanisms of action of ACR in inhibition of neointima formation are likely also attributable to inhibition of cell growth and stimulation of apoptosis.
RARß Is a Molecular Target of ACR, and Upregulation of RARß by ACR Leads to Apoptosis of SMCs
SMCs express all 3 RAR isoforms, but there is very little expression of RARß. 15 The role of RARß in the vasculature is poorly known other than that ATRA can marginally induce RARß in SMCs. 15 ACR, however, has been shown to preferentially act on RARß. 7,8 We therefore sought to understand the role of RARß in vascular remodeling. Our results suggest ACR may exert inhibitory effects on vascular remodeling at least in part through upregulation of RARß in SMCs. Increased inducibility of RARß in SMCs may be a critical determinant of increased apoptosis by ACR as compared with ATRA.
There are a number of cells which show endogenous expression of RARß (eg, endothelial cells, esophageal cells) 23,24 where its physiological function seems to be regulation of senescence, 25 but it seems that induction of RARß in nonexpressed cells, such as in cancer cells (eg, lung carcinoma cells) 25 in addition to SMCs as shown in the present article, has been shown to induce apoptosis which is consistent with its role as a potential tumor suppressor. One report which addressed the acute apoptotic effects of vascular injury (eg, less than 4 hours after injury) documented decreased expression of the apoptotic regulator, bcl-X, not only in the neointima but also in the luminal region of the media. 26 This modulation of bcl-X expression was coupled with increased TUNEL-positive nuclei and thus apoptosis in medial SMCs on the luminal side. 26 The different effects on luminal SMCs was thought to be attributable to increased mechanical stress on the luminal side. 26 In the present study, medial SMCs showed a different response as compared with neointimal SMCs in terms of the functional expression of RARß. That is, in contrast to neointimal SMCs which showed induction of RARß which was associated with apoptosis, medial SMCs showed greater expression on the adventitial side as compared with the luminal side, but did not manifest apoptosis. We envision that mechanical stress on the luminal side affected expression of RARß within the media. However, we do note that the medial area was not decreased nor showed apoptosis despite expression of RARß, which is consistent with previous reports which have documented the lack of medial SMC apoptosis at later stages. 27,28 Further investigation will be necessary to understand possible cell-type specific effects (ie, medial SMCs being more resistant to apoptosis) as well as stage-specific effects (ie, late as compared with acute effects) in addition to the physiological and pathological roles of RARß in the vasculature.
To our knowledge, very little is known of the molecular actions of ACR on apoptosis other than an observation that ACR induces apoptosis of hepatoma cells through tissue transglutaminase. 9 Further, the molecular actions of RARß on apoptosis have also been poorly addressed, other than a single report on apoptotic activity in oral cancer cells. 29 ACR has also been shown to selectively act on RARß in hepatoma cells. 7 It is noteworthy that tissue transglutaminase seems to be a common downstream proapoptotic pathway for ACR and ATRA, although the precise role of ATRA in apoptosis still remains controversial. Apoptotic effects are presently thought to be selective for synthetic and not natural retinoids and to be ascribed to intracellular target proteins which are independent of nuclear receptor functions. 30 Further, both ACR and ATRA can induce RARß through RARE-dependent transcription. It is therefore difficult to clearly delineate a specific pathway whereby the apoptotic actions of ACR are selectively mediated through RARß, given the presence of crosstalk as well as likely hitherto unknown independent pathways, but as a similar pathway of ACR mediated apoptosis through RARß has been documented in hepatoma cells, it is tempting to speculate that this common pathway dominantly functions to selectively induce apoptosis at least in these cell types including neointima cells and hepatoma cells.
In summary, ACR leads to growth inhibition and apoptotic induction in SMCs. Additionally, ACR, which exerts its actions through RARß, induces RARß which in turn induces apoptosis in SMCs. This regulatory pathway through activation of the RARß pathway by pharmaceutical intervention may be potentially exploitable for therapeutic purposes. Taken together, ACR may be a promising agent for therapeutic inhibition of neointima formation.
Acknowledgments
Disclosures
None.
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作者单位：Departments of Cardiovascular Medicine (N.K., T.S., K.A., T.M., N.T., Y.M., D.S., R.N.), Clinical Bioinformatics (T.S.), and Gastroenterology (T.I.), The University of Tokyo, and Pharmaceutical Research Laboratories (N.I., N.S.), Nikken Chemicals Co, Saitama, Japan.