Introduction

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PTCA is a highly successful approach for the acute treatment of ischemic heart disease, improving the blood flow through coronary arteries compromised by atherosclerotic disease (Gruentzig et al., 1979; Bittl, 1996). A major unsolved complication of PTCA is the occurrence of restenosis 3 to 6 months after angioplasty in 40% to 50% of the patients (Liu et al., 1989). This process of vascular narrowing is the subject of intensive research, but the molecular basis of its pathophysiology is still poorly understood.

Studies on animal models and human tissue samples have suggested that medial smooth muscle migration and proliferation, intimal SMC proliferation and extracellular matrix deposition occur in the process of neointimal formation (Isner et al., 1994; Schwartz et al., 1995). Therefore, it has been suggested that inhibition of these processes may reduce the vascular pathogenesis related to restenosis, and thus several agents have been tested for their ability to prevent restenosis. Recent reports on clinical trials have shown that tranilast, an antiallergic and antifibrotic agent (Ukai et al., 1993; Yamada et al., 1995), has a significant effect on preventing restenosis after the PTCA procedure and coronary stenting in Japan (Ueda et al., 1995; Hsu et al., 1996; Tamai et al., 1996). In in vitro experiments, tranilast was shown to have potent inhibitory effects on migration and proliferation of and collagen synthesis by vascular SMCs (Tanaka et al., 1994; Miyazawa et al., 1995; Fukuyama et al., 1996).

In this present communication, we report that the new drug TAS-301 [3-bis (4-methoxyphenyl)methylene-2-indolinone] (fig. 1) displayed a much more potent effect on neointimal formation than tranilast in the rat balloon injury model in terms of dosage and maximum efficiency and propose a possible mechanism of TAS-301 action based on the results of in vitro and in vivo experiments on the migration and proliferation of vascular SMCs.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Chemical structure of TAS-301.

	Materials and Methods

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Male Sprague-Dawley rats (Clea Japan Inc., Tokyo, Japan), 13 to 15 weeks old, were used in this study. These animals were housed in constant temperature facilities and given standard lab chow and water ad libitum.

All experiments were carried out according to protocols approved by the Institutional Animal Care and Use Committee.

Surgical Procedures

Balloon catheter denudation of the carotid artery endothelium was performed according to the method described by Clowes et al. (1983b). Briefly, rats were anesthetized with a gas mixture of N₂O/O₂ (70:30) containing 2% halothane. Then, after a median incision of the abdominal skin had been made, we carefully dissected the right iliac artery. The right iliac artery was cannulated with a 2F balloon catheter (embolectomy catheter arterial balloon, Medical Technology Transfer, Channel Islands, England), which was then inflated with saline and passed four times up and down the left common carotid artery to produce a distending, deendothelializing injury. The iliac artery was ligated after removal of the catheter, and the abdominal wound was closed. TAS-301 or tranilast, suspended in 0.5% hydroxypropyl methylcellulose solution, was administered orally by gavage once a day from 2 hr after the denudation to the day before removal of the artery for evaluation.

Intimal Thickening

On the 14th day after the balloon injury, the rats were anesthetized with ether so as to avoid any stress to the animals and then perfused transcardially with saline, followed by 10% buffered formalin. Next, the left carotid artery (length from aortic arch to bifurcation) was removed, postfixed and embedded in paraffin. Then, 3-µm-thick artery sections (six sections for each artery) were cut and stained with hematoxylin and eosin. The cross-sectional areas of the intima and the media on photographs were measured by use of a digital analyzer (Digitalizer, Wacom, Tokyo, Japan). The average of the ratio of the intimal area to the medial area in each artery was determined. Experimental groups were as follows: Vehicle (n = 9), TAS-301 (3, 10, 30 and 100 mg/kg, n = 9) and tranilast (100 and 300 mg/kg, n = 9). We omitted the data on two rats (one in TAS-301 100 mg/kg group and one in tranilast 100 mg/kg group) from the evaluation because of death due to faulty oral administration.

In vivo migration assay. The method to quantify SMC migration into the intima after balloon injury was performed according to the method described by Bendeck et al. (1994). Briefly, 4 days after the balloon injury, rats treated with the drugs as described below were anesthetized with ether and then perfused transcardially with saline, followed by 4% buffered paraformaldehyde. Next, the left carotid artery was removed and postfixed. The middle of the denuded left common carotid artery was cut lengthwise and pinned intimal side up onto a Teflon plate. The arteries were rinsed in PBS and then placed in 0.3% hydrogen peroxide in cold methanol to block endogenous peroxidase activity. Then the arteries were incubated for 30 min at room temperature in 10% normal horse serum in PBS and subsequently with monoclonal antibody against human nuclei and chromosomes (MAB 1276, 1:100) overnight at 4°C. Next, a biotinylated anti-mouse IgG and avidin-biotinylated horseradish peroxidase (Elite ABC) kit were used according to the supplier's recommendations (Vector Laboratories, Burlingame, CA), after which the sections were immersed in 0.1% 3,3^'-diaminobenzidine in 50 mM Tris-HCl containing 0.02% H₂O₂ (pH 7.6). After staining, the tissues were placed intimal side up on glass slides.

In vivo proliferation assay. On the 4th and 8th days after the balloon injury, drug-treated rats were anesthetized with ether and then perfused transcardially with saline, followed by 4% buffered paraformaldehyde. Next, the left carotid artery was removed, postfixed and embedded in paraffin. Then 3-µm-thick artery sections (five or six sections in each artery) were prepared. Deparaffinized sections were boiled in 10 mM citrate buffer solution (pH 6.0) twice for 5 min to unmask PCNA antigen. After that, the arteries were rinsed in PBS and then placed in 0.3% hydrogen peroxide in cold methanol. Then the arteries were incubated for 60 min at room temperature in 10% normal horse serum in PBS and then with biotinylated anti-PCNA monoclonal antibody (PCNA15, 1:25) overnight at 4°C. An avidin-biotinylated horseradish peroxidase (Elite ABC) kit was used according to the supplier's recommendations (Vector), after which the sections were immersed into 0.1% 3,3'-diaminobenzidine in a 50 mM Tris-HCl solution containing 0.02% H₂O₂ (pH 7.5). The sections were counterstained with hematoxylin to ensure identification of all nuclei. The number of labeled nuclei per section was counted, and the labeling index [labeled nuclei/total nuclei × 100 (%)] was calculated. Experimental groups were as follows: vehicle (n = 8), TAS-301 (100 mg/kg, n = 8) and tranilast (300 mg/kg, n = 8).

Cell Culture

SMCs were prepared from the thoracic aorta of 12- to 15-week-old Sprague-Dawley rats (Clea Japan) by the explantation method as previously described (Fischer-Dzoga et al., 1973). Early passages of SMCs (4-10 passages) were maintained at 37°C in 5% CO₂/95% air in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 units/ml of penicillin and 100 mg/ml of streptomycin.

In vitro migration assay. The migration of SMCs was assayed by a modified Boyden's chamber method (Bilato et al., 1995). The wells were subsequently fitted with a Chemotaxicel filter (polycarbonate filter, 8-µm pores; Kurabo, Osaka, Japan) that had been coated overnight with 100 µg/ml type I collagen. An SMC suspension (3 × 10⁵ cells/ml) in serum-free DMEM containing 0.1% BSA was pretreated or not with TAS-301 or tranilast for 2 hr and then loaded into the upper wells (200 µl). The chemoattractant, PDGF-BB (0.3 ng/ml), IGF-1 (1 ng/ml) or HB-EGF (1 ng/ml) was first diluted in serum-free DMEM containing 0.1% BSA in the presence or absence of TAS-301 or tranilast. Chemoattractants (600 µl) were loaded into the lower wells. The chamber was incubated at 37°C in 5% CO₂/95% air. After a 5-hr incubation, nonmigrating SMCs on the upper surface were removed with cotton swabs. The filters were then fixed in methanol and stained with Giemsa staining solution. The number of SMCs per 4 HPFs (×100) that had migrated to the lower surface of the filter was then determined microscopically (n = 6 for TAS-301 group, n = 5 for tranilast group).

In vitro proliferation assay. Cell proliferation was determined by the incorporation of BrdU by quiescent cells as described previously (Magaud et al., 1988; Marrero et al. 1997). SMCs were seeded at 1 × 10⁴ cells/well in 96-well plates in DMEM containing 10% FBS. Two days after the seeding, their growth was arrested for 3 days in a serum-free DMEM containing 5 µg/ml insulin, 5 µg/ml transferrin and 5 ng/ml sodium selenium (ITS). Then, the DMEM/ITS was removed, and serum-free DMEM containing 0.1% BSA with or without TAS-301 or tranilast was added to the quiescent cells 2 hr before treatment with the growth factor (i.e., bFGF 0.1 ng/ml). At 24 hr after stimulation, BrdU (10 µM) was added to the cells; 24 hr later, the cells were fixed. An ELISA was used according to the supplier's recommendations (Amersham, Buckinghamshire, England) to detect and to quantify the incorporated BrdU (n = 6). The drugs were present during the entire experiment.

Materials

TAS-301 (fig. 1) was synthesized by Taiho Pharmaceutical (Saitama, Japan). Tranilast was purchased from Shiratori Pharmaceutical (Chiba, Japan).

The following reagents (with their source in parentheses) were used: PDGF-AA, PDGF-BB and IGF-1 (Life Technologies, Grand Island, NY), bFGF (Pepro Tech EC, London, England), HB-EGF (Sigma Chemical, St. Louis, MO), rat tail type-I collagen (Upstate Biotechnology, Lake Placid, NY), biotinylated anti-PCNA monoclonal antibody (Caltag Laboratories, San Francisco, CA) and mouse anti-human nuclei and chromosomes (Chemicon International, Temecula, CA).

Statistical Analysis of Data

Data were expressed as the mean ± S.D. Multiple comparisons with vehicle were tested by Dunnett's multiple comparison test. Statistically significant differences between two groups were calculated by (two-tailed) Aspin-Welch t test (McArdle, 1987). Comparison within the drug treatment groups was done by (two-tailed) Aspin-Welch t test, with the Bonferroni multiple-comparison adjustment. Differences with P < .05 were considered to be significant. Seven to nine of experiments were performed for in vivo experiments and five to six for in vitro experiments were performed for statistical comparisons.

	Results

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Effects of TAS-301 and Tranilast on Intimal Thickening

Obvious intimal thickening was observed 14 days after vascular injury induced by the balloon catheter in vehicle-treated control rats. This intimal formation started to become obvious 8 days after the injury. The ratio of neointimal area to medial area (I/M ratio) was 0.57 ± 0.17 and 1.49 ± 0.43 (mean ± S.D.) at 8 and 14 days, respectively, after the balloon injury.

Typical light micrographs of carotid arteries from normal rats and those of denuded arteries from vehicle-, TAS-301- and tranilast-treated rats at 14 days after balloon injury are shown in figure 2. The oral administration of TAS-301 reduced the neointimal thickening and I/M ratio 14 days after injury in a dose-dependent manner (3-100 mg/kg, fig. 3). In particular, TAS-301 at doses of 10, 30 and 100 mg/kg significantly reduced the I/M ratio by 33.0%, 45.9% and 56.1%, respectively (P < .01). On the other hand, tranilast only tended to lower the I/M ratio at a dose of 100 mg/kg and significantly reduced it, by 28.0%, at a dose of 300 mg/kg (P < .05, fig. 3). The inhibitory effect by TAS-301 at the dose of 100 mg/kg was significantly greater than that by tranilast at the dose of 300 mg/kg (P < .05).

View larger version (90K): [in this window] [in a new window]   Fig. 2.   Typical photomicrographs of cross sections of rat carotid artery 14 days after balloon injury (a-c) and normal carotid artery (d). a, Vehicle-treated rat. b, TAS-301 (100 mg/kg)-treated rat. c, Tranilast (300 mg/kg)-treated rat. d, Normal vessel. Note the potent reduction in neointimal thickness and area in the TAS-301-treated rat.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Effects of TAS-301 and tranilast on neointimal thickening 14 days after balloon catheter injury to the rat common carotid artery. Data are expresed as medial area, intimal area and the ratio of the intimal area to the medial area (mean ± S.D., n = 8-9). *P < .05, **P < .01, Dunnett's multiple analysis. P < .05, comparing TAS-301 treatment groups (10, 30 and 100 mg/kg) with tranilast treatment group (300 mg/kg) by Aspin-Welch t test, with the Bonferroni multiple comparison adjustment. Note the potent inhibitory effect of TAS-301 on neointimal formation.

In vivo migration assay. There are no cells in the intima of the normal rat carotid artery. However, cells in the intima were observed and countable 4 days after the balloon injury, as previously reported (Jackson et al., 1993). The number of intimal cells was 95 ± 15 cells/mm² in vehicle-treated control rats (mean ± S.D.). The effects of TAS-301 and tranilast on the number of intimal cells are shown in figure 4. The treatment with TAS-301 significantly reduced the number of cells in the intima. The level of inhibition by TAS-301 was 17.9%, 30.5% and 47.4% at a dose of 10, 30 and 100 mg/kg, respectively (P < .01). On the other hand, tranilast showed weak but significant reduction, by 20.0%, at a dose of 300 mg/kg (P < .05). The inhibitory effect of TAS-301 at the dose of 100 mg/kg on migration of cells into the intima was significantly greater than that of tranilast at the dose of 300 mg/kg (P < .01).

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Effects of TAS-301 and tranilast on the intima cells 4 days after the balloon injury. Data show the number of cells that migrated onto the lumen surface and were immunoreactive with antibody against nuclei and chromosomes (mean ± S.D., n = 7 or 8). **P < .01, Dunnett's multiple analysis. P < .05, Aspin-Welch t test vs. vehicle. $$P < .01, comparing TAS-301-treatment groups (10, 30 and 100 mg/kg) with tranilast treatment group (300 mg/kg) by Aspin-Welch t test, with the Bonferroni multiple comparison adjustment. Note the potent inhibitory effect of TAS-301 on migration of cells into the intima.

In vivo proliferation assay. The cells immunoreactive with anti-PCNA antibody (PCNA-positive cells) in the medial layer started to be observed 2 days after the balloon injury (data not shown) and were obvious 4 days after injury in vehicle-treated control rats. The labeling index at that time was calculated to be 20.6 ± 2.2% (mean ± S.D.). These data are comparable with those of a previous report (Clowes et al., 1983a). The effects of TAS-301 and tranilast on the labeling index are shown in table 1. The treatment with TAS-301 at a dose of 100 mg/kg significantly reduced the labeling index in the media at 4 days (P < .01). Furthermore, the treatment with tranilast at a dose of 300 mg/kg also significantly reduced the labeling index there (P < .01). The inhibitory effect of TAS-301 was almost the same as that of tranilast. At 8 days after the injury, the number of PCNA-positive cells in the intima was 433 ± 152 (mean ± S.D.), representing >70% of intimal SMCs in the vehicle-treated control group. These data are also comparable with previous findings (Clowes et al., 1983a). TAS-301 treatment at a dose of 100 mg/kg significantly reduced the labeling index in the intima (table 1, P < .01), as did the treatment with tranilast at a dose of 300 mg/kg (table 1, P < .01). Again, the inhibitory effects of the two drugs were comparable.

In vitro migration assay. PDGF-BB, HB-EGF and IGF-1 produced a dose-dependent increase in the migration of rat SMCs with a submaximal effect obtained at a dose of 0.3, 1 and 1 ng/ml, respectively (data not shown). The number of SMCs that were induced to migrate over a 5-hr period by these growth factors reached ~150 cells/4 HPFs for PDGF-BB and IGF-1 and ~200 cells/4 HPFs for HB-EGF. The effects of TAS-301 on migration induced by these growth factors are shown in figure 5. PDGF-AA, a well-accepted inhibitor of migration, at a dose of 10 ng/ml, reduced the cell migration elicited by all of chemoattractants tested by ~50%. Under the same conditions, the treatment with TAS-301 reduced, in a dose-dependent manner (0.3-3 µM), the migration of cells induced by all of the growth factors tested and with almost the same potency at 3 µM (percent inhibition: 55.2%, 60.5% and 62.7% for PDGF-BB-, IGF-1- and HB-EGF-induced migration, respectively). The effects of tranilast measured in the same type of experiment are also shown in figure 5. Tranilast also caused a significant reduction in PDGF-BB- and IGF-1-induced migration at doses of 100 and 300 µM, and its inhibitory effect tended to reach maximum at a dose of 100 µM. However, tranilast did not inhibit HB-EGF-induced migration, even at a dose of 300 µM.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Effect of TAS-301 and tranilast on the number of SMCs that migrated in the modified Boyden chamber assay. a, PDGF-BB (0.3 ng/ml)-induced migration. b, IGF-1 (1 ng/ml)-induced migration. c, HB-EGF (1 ng/ml)-induced migration. Data show the number of SMCs induced to migrate by growth factors (mean ± S.D., n = 6 for TAS-301, n = 5 for tranilast). *P < .05, **P < .01, Dunnett's multiple analysis. Note the potent inhibitory effect of TAS-301 on migration elicited by all chemoattractants.

In vitro proliferation assay. The effects of TAS-301 and tranilast on the proliferation of rat SMCs in vitro are shown in figure 6. Pronounced BrdU incorporation by the cells, an index of DNA synthesis (O.D. change, 1.37 ± 0.26), was induced by the treatment with bFGF (mean ± S.D.). TAS-301 reduced bFGF-induced BrdU incorporation dose-dependently (1 - 10 µM) and significantly inhibited it at doses of 3 and 10 µM, by 42.9% (P < .01) and 72.2% (P < .01), respectively. The release of LDH, a marker of cell injury, was not detected after treatment with TAS-301 at a dose of 10 µM. Treatment with tranilast at a dose of 300 µM showed potent inhibition of bFGF-induced BrdU incorporation to a level below that of the nonstimulated control. Because tranilast at the dose of 300 µM induced LDH release into the medium (data not shown), its inhibitory effect might have been due to cytotoxicity. However, tranilast at a dose of 100 µM also significantly inhibited BrdU incorporation induced by bFGF (51.4% inhibition, P < .05) but caused no LDH release.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   Effects of TAS-301 and tranilast on bFGF-induced BrdU incorporation into medial SMCs. Data show BrdU incorporation (change in O.D.) induced by bFGF treatment (mean ± S.D., n = 6). *P < .05, **P < .01, Dunnett's multiple analysis. Note the potent inhibitory effect of TAS-301 and tranilast on BrdU incorporation into the cells.

	Discussion

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The present results demonstrate that TAS-301 inhibited neointimal thickening after balloon catheter injury to the rat common carotid artery by inhibiting both the migration and proliferation processes of SMCs.

A large number of pharmacological trials have examined whether systematically administered pharmacological agents reduce the risk of angiographic restenosis. Despite encouraging results in animal models, no systemic pharmacological agent has been shown conclusively to produce a clinically worthwhile reduction in restenosis after PTCA (Franklin and Faxon, 1993; Moliterno and Topol, 1995). A first explanation may be interspecies differences, and a second may be limitation of successful clinical outcome due to low doses or inadequate duration of therapy. Very high doses of drugs have been necessary to inhibit restenosis in experimental models because they had not been originally developed with the goal of preventing restenosis after PTCA. Furthermore, the vascular geometric remodeling may, at least in part, contribute to the restenosis after angioplasty in addition to the intimal hyperplasia.

Recently, tranilast, which was originally developed for the treatment of allergy and keloid formation (Ukai et al., 1993; Yamada et al., 1995), has been successful in terms of reducing both experimental intimal thickening and clinical angiographic restenosis (Fukuyama et al., 1996; Hsu. et al., 1996; Tamai et al., 1996). These successful clinical data on restenosis of tranilast may be attributable to the unexpected and powerful inhibitory effects on SMCs migration and proliferation and collagen synthesis by SMCs and not on allergic reactions (Tanaka et al., 1994; Miyazawa et al., 1995).

Given the experimental and clinical success of tranilast, a series of compounds were synthesized and screened for their ability to inhibit both SMC migration and proliferation. As a result of this process, TAS-301 was identified as a potential inhibitor of restenosis. In addition, we examined the effect of this drug on intimal thickening after balloon injury in comparison with that of tranilast.

The most common characteristic response of blood vessels to injury is the formation of a neointima. This neointimal formation in response to balloon injury is related to balloon inflation pressure (Indolfi et al., 1995). In our study, the I/M ratio was almost 1.5 by 14 days after the balloon injury; and this high degree of thickness of the neointima might have been the result of the high inflation pressure of ballooning and consequent severe damage to the vessel wall. Under this experimental condition, TAS-301 significantly inhibited the neointimal formation even at a dose of 10 mg/kg, and the inhibitory effect of the drug increased dose dependently, up to >50% inhibition at a dose of 100 mg/kg. This potency of TAS-301 was much greater than that of tranilast at the point of effective doses and the maximum efficiency.

Several growth factors are believed to be involved in the processes of intimal thickening, particularly bFGF and PDGF.

It has been reported that PDGF increased SMC mobilization in the in vitro Boyden chamber system, and other growth factors, such as IGF-1 and HB-EGF, are also potent chemoattractants for SMCs (Khorsandi et al., 1992; Higashiyama et al., 1993; Raines and Ross, 1993; Bornfeldt et al., 1994)

The signal transduction pathways associated with directed migration of SMCs have been well elucidated. Actin filament disassembly and assembly, modulated by levels of phosphatidylinositol bisphosphate and calcium, are crucial and common steps for directed cell migration in a gradient of chemoattractant (Stossel, 1993; Bornfeldt et al., 1994, 1995). Cell migration is also modulated by integrin-dependent adhesion to the extracellular matrix (Skinner et al., 1994; Abedi et al., 1995).

TAS-301 inhibited the migration of SMCs through a type I collagen-coated filter in the case of all chemoattractants we used as inducers. These inhibitory effects were dose dependent and significant. Furthermore, TAS-301 showed a potent inhibitory effect on the cell migration into the intima in a dose-dependent manner at the same dose ranges that lowered the I/M ratio. However, tranilast also reduced cell migration in vitro induced by PDGF and IGF-1 but not that caused by HB-EGF; and tranilast showed a weak but significant inhibitory effect on the migration of SMCs in the in vivo assay. These specific inhibitory effects of tranilast on the migration in the in vitro assay might account for the weak inhibitory effect on migration of medial SMCs into intima and on consequent intimal thickening.

Jackson et al. (1993) showed the contribution of platelets, which produce PDGF, to the migration of SMCs into the intima. We tested the effect of TAS-301 on the aggregation of rat platelets induced by several factors, such as thrombin, ADP and collagen. Because TAS-301 at a dose of 30 µM had only a weak, but not significant, inhibitory effect on such aggregation (data not shown), we speculate that the inhibitory effect of TAS-301 on the migration process in vivo may be due to a direct effect on the SMCs, not to an indirect one of inhibiting platelet aggregation.

Furthermore, it has been reported that bFGF could play an important role in the regulation of SMC proliferation after balloon catheter injury (Lindner et al., 1991; Olson et al., 1992). In our in vitro study, bFGF increased BrdU incorporation into cultured SMCs as reported above, and TAS-301 showed a potent and dose-dependent inhibitory effect on this bFGF-induced BrdU incorporation. Furthermore, in vivo treatment with TAS-301 significantly reduced the number of PCNA-positive cells and labeling index in the media and the intima 4 and 8 days, respectively, after denudation. These results in vivo confirmed those of the in vitro proliferation assay. This inhibitory effect of TAS-301 on proliferation of the medial SMCs might augment the inhibitory effect of TAS-301 on the migration of medial SMCs into the intima.

Protein tyrosine kinases are critical components of signaling pathways that control cell proliferation. The tyrosine phosphorylation of bFGF receptor and subsequent tyrosine phosphorylation of mitogen-activated protein kinases (ERK1 and ERK2) are involved in the proliferation processes in SMCs triggered by growth factors (Segar and Krebs, 1995). Recently, Mohammadi et al. (1997) showed that the oxindole structure, as found in TAS-301, did have a high affinity for the adenine binding site in protein tyrosine kinases. Further investigations are needed to clarify the effect of TAS-301 on protein tyrosine kinases stimulated by growth factors.

The present results clearly indicate that TAS-301 inhibited both cell migration and proliferation in the in vivo experiments. Recently, Martin et al. (1996) indicated that simultaneous application of neutralizing antibody against PDGF and that against bFGF induced a much more potent reduction in neointimal formation than did the application of each alone. This finding strongly indicated that it is necessary to block both potent mitogens and potent chemoattractants for the remarkable reduction in neointimal hyperplasia. This report supports the potent effect of TAS-301 found in the present study and allows us to expect the possibility of TAS-301 for prevention of intimal hyperplasia after PTCA procedures. Recent clinical intravascular ultrasound studies showed that vascular geometric remodeling, in addition to the intimal hyperplasia, is one of the contributors to the restenosis after angioplasty (Luo et al., 1996). However, balloon injury to the rat common carotid artery resulted in the vascular narrowing by the intimal hyperplasia, not by vascular remodeling. Further investigation on the effect of TAS-301 on vascular remodeling is needed to predict the clinical efficacy of TAS-301.

In summary, we have shown that newly synthesized TAS-301, designed for targeting restenosis after angioplasty, reduced the neointimal formation after balloon catheter injury to the rat common carotid artery. The results of our in vitro experiments suggest that the inhibitory effect of TAS-301 observed in vivo is due to the inhibition of both SMC migration and proliferation. Furthermore, these inhibitory effects of TAS-301 were much more potent than those of tranilast in the points of effective doses and maximum efficiency. This higher potent activity of TAS-301 over that of tranilast suggest the need to examine the therapeutic usefulness of this drug in clinical trials.