FK409, a new nitric-oxide donor, suppresses smooth muscle proliferation in the rat model of balloon angioplasty

ATHEROSCLEROSIS AtherosclerosisI 17 (1995)97- 106

FK409, a new nitric-oxide donor, suppresses smooth muscle proliferation in the rat model of balloon angioplasty Jiro Seki*, Mie Nishio, Yasuko Kato, Yukio Motoyama, Keizo Yoshida Deportment of Pharmacology,

New Drug Research Laboratories, Fujisawa Pharmaceutical Yodogawa-ku, Osaka 532, Japan

Co. Ltd., 2-l-6

Kashima,

Received30 November1994;revision received7 March 1995;accepted13 March 1995

Abstract The effect of FK409, a new nitric-oxide (NO) donor, on neointimal formation of rat carotid arteries following balloon injury was studied. The intimal thickening at 14 days was strongly suppressedby twice daily administration of FK409 at 10 mg/kg from 2 days before to 13 days after injury. The neointima area and neointima/media ratio were decreased by 48.0% (P < 0.01) and 38.5% (P < 0.01) respectively, compared with control. On the other hand, isosorbide dinitrate (ISDN), a classical nitro-vasodilator, did not suppress intimal thickening even at 100 mg/kg twice a day. An in vivo 5-bromo-2’-deoxyuridine (BrdU) uptake study revealed that FK409 inhibited the proliferative response of smooth muscle cells (SMC) in media at early stage of injury. In fact, the neointimal formation at 14 days was inhibited by the short term administration of FK409 only from the day of injury to 4 days after at 10 mg/kg twice a day. In cultured rat SMC, FK409 (l-10 pmol/l) markedly enhanced intracellular c-GMP and inhibited the proliferation in 10% FBS-containing medium. These results suggest that FK409 suppresses intimal thickening following balloon injury of the rat carotid artery by inhibition of SMC proliferation. Keywords: Smooth muscle cells; Intimal thickening; Angioplasty; Anti-proliferative

effect; Nitric oxide donor; Cyclic

GMP

1. Introduction

Restenosis after successful PTCA is the major clinical problem limiting the long term efficacy of

* Corresponding author, Department of Pharmacology, New Drug ResearchLaboratories, Fujisawa Pharmaceutical Co. Ltd., 2-l-6 Kashima, Yodogawa-ku,Osaka 532, Japan. Tel.: + 81 6 390 1144, Fax: + 81 6 304 1192

this procedure. Restenosis occurs in 30%-40% of patients within 3 to 6 months after the procedure [l]. Attempts to reduce the incidence of restenosis by pharmacological interventions or modified procedures have been either unsuccessful or equivocal ]2]. The mechanism of restenosis remains unclear, but aberrant smooth muscle cell (SMC) proliferation at the site of vascular injury by balloon inflation plays a major role in intimal hyperplasia [3,4].

002l-9150/95/$09.50 0 1995ElsevierScienceIreland Ltd. All rights reserved SSDI 0021-9150(95)05563-C

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In 1980, Furchgott and Zawadzki [5] demonstrated that vascular relaxation induced by acetylcholine was dependent on the release of a labile factor from functionally intact endothelial cells, and called it endothelium-derived relaxing factor (EDRF). EDRF has been identified as nitric oxide (NO) or a substance closely related to it such as nitrosothiol [6]. In addition to effects on vascular tone, EDRF/NO influences platelet adhesion [7], platelet aggregation [8] and neutrophil adhesion [9]. The action of EDRF/NO appears to be mediated by the stimulation of soluble guanylate cyclase and the generation of cGMP [lo]. C-GMP elevating vasodilators, such as sodium nitroprusside, atria1 natriuretic peptide, and 8bromo-cGMP have been shown to inhibit mitogenesis and proliferation of cultured SMC and mesangial cells in vitro [ 11- 131. These results suggestthat EDRF/NO might also inhibit cellular proliferative responses. In fact, L-arginine, a precursor of NO, was shown to suppress intimal hyperplasia after balloon injury in rats [14] and rabbits [ 151.However, the anti-proliferative effect of EDRF/NO or cGMP elevating agents in vivo is still controversial [ 161. Endothelial cells might be damaged by balloon inflation during PTCA and the level of EDRF/ NO at the site of injury will decrease. This decrease in the level of EDRF/NO might trigger the proliferative response of SMC and cause the intima1 hyperplasia of the injured vessel. Therefore, we postulated that the supply of exogenous NO might suppress the proliferative response of SMC. FK409, ( + )-(E)-4-ethyl-2-hydroxyimino-5-nitro-3-hexenamide, is a structurally unique orally active nitro-vasodilator with anti-platelet activity obtained from fermentation products [17]. The vasorelaxant effect of FK409 is due to activation of soluble guanylate cyclase and a resultant increase in intracellular c-GMP similar to other nitro-vasodilators or EDRF/NO [ 18-201. Recently, Kita et al. have found that FK409 released NO spontaneously from the compound itself [21]. In this paper, we evaluated the effect of this compound and another orally active nitro-vasodilator, isosorbide dinitrate (ISDN), on the proliferative response of SMC in the rat model of balloon angioplasty.

I I7 (1995) 97- 106

2. Materials

and methods

2.1. Materials FK409, ISDN and captopril were synthesized by Fujisawa Pharmaceutical Co. (Osaka, Japan). The following reagents were obtained from the indicated sources: BrdU, BSA and ADP (Sigma Chemical Co., St. Louis, MO), anti-BrdU monoclonal antibody (Becton-Dickinson, Mountain View, CA), peroxidase-labeled goat anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA), orcein (Merck, Darmstadt, Germany), sodium pentobarbital (Abott Laboratories, North Chicago, IL), cGMP radioimmunoassay kit (Amersham, Amersham, UK), 3-isobutyl-1-methylxanthine (Nacalai, Kyoto, Japan), fetal bovine serum (FBS; Gibco Laboratories, Grand Island, NY), collagen (HormonChemie, Mtinchen, Germany). 2.2. Vascular injury models A vascular injury model was performed as described by Clowes et al. [22]. Male, 3-month-old Sprague-Dawley rats (SLC, Shizuoka, Japan) were anesthetized using intraperitoneal administration of sodium pentobarbital, 50 mg/kg. The left common carotid and external carotid arteries were exposed through a midline incision in the neck. The left carotid artery was denuded of endothelium by intraluminal passageof a Fogarty 2F balloon catheter (Baxter Healthcare Corp., Santa Ana, CA) introduced through the external carotid artery. The catheter was passed three times with the balloon sufficiently inflated with saline to generate slight resistance. The external carotid was ligated after removal of the catheter and the wound was closed. 2.3. EfSecton intimal thickening 14 days after balloon injury, animals were anesthetized and perfusion-fixed with 10% buffered formalin (pH 7.4) at 100 mmHg after infusion of saline containing heparin (20 U/ml, Shimizu Pharmaceutical Co., Shizuoka, Japan) via a cannula placed in the left ventricle. The left carotid arteries were then removed and placed in lOohbuffered formalin for further fixation. The central part of excised vessels was embedded in paraffin and 3

J. Seki et al. / Atherosclerosis

pm cross-sections were prepared. Cross-sections were stained with orcein. The cross-sectional areas of neointima and media were measured blind with an image analyzer (LUZEX 2D, Nikon, Tokyo, Japan). Two sections per animal were analysed. FK409 was dissolved in 0.5% methylcellulose solution and administered orally by gavage twice daily (once in the morning and once in the evening) from 2 days before to 13 days after or from 0 day (the day of operation) to 4 days after balloon injury. ISDN was suspended in 0.5% methylcellulose and orally administered twice daily from 2 days before to 13 days after injury. Captopril, used as a positive control drug, was mixed with normal food (MF; Oriental Yeast, Tokyo, Japan) and given from 6 days before up to 14 days after balloon injury in such a way that the average daily dose was 100 mg/kg. 2.4. Effect on in vivo BrdU uptake

Three or seven days after balloon injury, animals were killed by overdose of sodium pentobarbital. BrdU, a thymidine analogue, was given intraperitoneally at 25 mg/kg to animals at 17, 9 and 1 h before sacrifice [23]. The left carotid arteries were excised and fixed with 10% buffered formalin by overnight immersion at 4°C in the dark. The central part of excised vessels was embedded in paraffin and 3 pm cross-sections were prepared. Immunohistochemical detection of BrdU labeled nuclei was performed using anti-BrdU mouse monoclonal antibody and peroxidase-labeled anti-mouse IgG (goat). One section was stained with haematoxylin and eosin for counting total nuclei. The number of labeled nuclei per section was counted and labeling index (labeled nuclei/total nuclei) was calculated. FK409 was administered twice a day from 0 day (once before balloon injury) to 3 days after injury when sacrificed at day 3, or from 4 days to 7 days after injury when sacrificed at day 7. The last dosing was done 6 h before sacrifice. 2.5. Cell culture

Rat SMC were isolated from the neointima of balloon injured rat carotid arteries by an explant

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method [24,25]. The left carotid artery was denuded of endothelium as described above. After 14 days, the intimal layer was stripped from the excised artery using a fine forceps under magnification. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. Cells were used for the experiments between the fourth and fifth passages. 2.4. Cell proliferation

assay

Cells were plated into 35 mm dishes (Corning Glass Inc., Corning, NY) at 1 x 10’ cells/well and cultured in DMEM containing 10% FBS for 24 h. The culture medium was then removed and replaced with the same medium containing FK409 or ISDN. After 24 h, culture medium was exchanged with fresh medium containing the drug. After another 24 h, cells were washed with a calcium and magnesium free PBS (137 mmol/l NaCl, 8.1 mmol/l NaH,PO,, 2.7 mmol/l KCl, 1.5 mmol/l KH2P0,, pH 7.4) and harvested with trypsin-EDTA solution (0.05% trypsin, 0.02% EDTA, Gibco Laboratories). Counts were performed with a coulter counter (Coulter Electronics Inc., Northwell Drive, UK) immediately after the cell harvest. 2.7. Determination

of cGMP

For the assay of cGMP formation in cultured rat SMC, the cells in 35 mm dishes were incubated in balanced salt solution containing 20 mmol/l HEPES at pH 7.4, 130 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l MgCl,, 1.5 mmol/l CaCl,, 10 mmol/l glucose, and 0.1% BSA for 15 min in the presence of 0.5 mmol/l 3-isobutyl- 1-methylxanthine (IBMX). The cells were then stimulated by FK409 or ISDN in the same medium for 15 min. Cellular cGMP was extracted with 0.1 N HCl [12] and determined by radioimmunoassay after acetylation according to the manufacturer’s instructions. 2.8. Statistical analysis

All values are expressed as mean + S.E.M. Data were analyzed with Dunnet’s t-test or Student’s t-test. Differences were considered to be significant at P < 0.05.

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Table 1 Effects of FK409, isosorbide dinitrate (ISDN), and captopril on neointimal formation in rat model of vascular injury by balloon catheter Drug

Vehicle FK409 Vehicle FK409 Vehicle ISDN ISDN Vehicle Captopril

Dose

1 mg/kg x 2 lOmg/kgx2 10 mg/kg x 2 100 mg/kg x 2 100 mg/kg per day

Period

n

Area of neointima (mm’)

Neointima/media W)

- 2-13 days - 2-13 days - 2-13 days - 2-13 days - 2-13 days -2-13 days -2-13 days - 6- 14 days - 6- 14 days

14 11 17 19 8 6 6 12 10

0.189 & 0.023 0.138 f 0.020 0.171 * 0.019 0.089 + 0.009** 0.172 + 0.022 0.143 & 0.017 0.152 + 0.022 0.178 + 0.021 0.099 * 0.021*

96 k 7.8 78k 11.3 91 + 8.6 56 * 5.7* 98 + 13.6 87 & 6.6 93 + 16.4 90 + 10.2 62 + 12.6*

FK409 and ISDN were dissolved or suspendedin 0.5% methylcellulose solution and orally administered by gavage. Captopril was mixed with food. Values are mean + S.E.M. Significant difference from vehicle group at *P < 0.05 and **P < 0.01 (Student’s t-test).

3. Results 3.1. Effect on intimal thickening Obvious intimal thickening was observed 14 days after vascular injury by balloon catheter in vehicle-treated control rats. The area of the neointima was almost the same as that of the media. The effect of FK409, ISDN and captopril on neointimal formation is shown in Table 1. In this experiment, captopril was used as a positive control drug, since angiotensin converting enzyme inhibitor such as cilazapril [26,27] has been shown to be a strong inhibitor of intimal thickening in this model. In fact, captopril at the dose of 100 mg/kg per day from 6 days before to 14 days after balloon injury significantly suppressed neointimal formation. The neointimal area and neointima/ media ratio were decreasedby 44.4% (P < 0.05) and 31.1% (P < 0.05), respectively, compared with vehicle-treated controls. FK409 also strongly inhibited intimal thickening at 10 mg/kg twice a day when administered 2 days before to 13 days after balloon injury. The neointimal area and neointima/media ratio were decreased by 48.0% (P < 0.01) and 38.5% (P < 0.01) respectively. Typical light micrographs of carotid arteries from vehicle- and FK409-treated animals at 14 days after balloon injury are shown in Fig. 1. FK409 did not affect body weight, food intake or gross behavior. The neointimal area was also decreased

by 27.0% at 1 mg/kg twice a day, but this was not statistically significant. On the other hand, ISDN, another orally active nitro-vasodilator, did not suppress neointimal formation at doses of 10 mg/kg or 100 mg/kg twice a day from 2 days before to 13 days after balloon injury. 3.2. Efect on in vivo BrdU uptake Intimal thickening after vascular injury by balloon catheter in rats is thought to be due to aberrant proliferation of SMC derived from the medial layer [22,28]. In this model, the proliferation of SMC reaches a maximum at 2-3 days in media and at 7 days in intima after balloon injury [28]. Therefore, we investigated the effect of FK409 on DNA synthesis of medial SMC at 3 days and of intimal SMC at 7 days after balloon injury using an in vivo BrdU uptake method. In order to test the specific effect of this drug on the proliferation of intimal SMC, the drug administration was started at 4 days after injury when sacrifice was at day 7. In uninjured normal carotid arteries, BrdU positive nuclei in the medial SMC were barely detectable (data not shown). However, at 3 days after injury 50.4 f 10.6 (n = 7) nuclei per section in the medial SMC of vehicle-treated animals were positively stained by anti-BrdU monoclonal antibody. The labeling index was calculated as

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Fig. 1. Representative light micrographs of injured carotid arteries of vehicle-treated (left) and FK409-treated (right) rats at 14 days. FK409 was administered 10 mg/kg twice a day from 2 days before to 13 days after balloon injury.

12.3 f 2.0%. On the other hand, the number of BrdU positive nuclei in the media of FK409 treated animals was only 22.9 + 4.9 (n = 7) per section (P < 0.01). The medial area was significantly smaller in the FK409-treated group (Table 2). At 7 days after injury, over 80% of intimal SMC were labeled in both groups. The number of BrdU positive nuclei per section was 142 + 10.2 (n = 7) in vehicle-treated animals and 109 + 11.3 (n = 7) in FK409-treated animals. There was no significant difference between them. 3.3. EJ2cl on intimal thickening (short term administration of FK409) Since FK409 inhibited the proliferative response of medial SMC at early stage of balloon injury, we examined the effect of short term administration of this drug. The drug was administered only from the day of operation to 4 days after injury, and the intimal thickening was measured at 14 days. As a

result, the area of neointima was significantly decreasedby 28% even by the short term administration of FK409 at 10 mg/kg twice a day (0.162 k 0.012 mm’ (n = 8) in vehicle-treated animals and 0.117 + 0.011 mm2 (n = 7) in FK409-treated animals, P < 0.05). 3.4. EfSecton the proli@ration and cGMP level in cultured rut SMC The effect of FK409 and ISDN on the proliferation of cultured rat SMC in 10% FBS containing medium is shown in Table 3. FK409 significantly inhibited the proliferation of SMC at 1 and 10 pmol/l. In this experiment, no significant lactate dehydrogenase release by FK409 was observed. On the other hand, ISDN did not inhibit the cell proliferation even at 100 pmol/l. The effect of these two drugs on intracellular cGMP level in SMC is shown in Fig. 2. FK409 (0.1~ 10 pmol/l) markedly enhanced cGMP level

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Table 2 Effect of FK409 on in vivo BrdU uptake to medial SMC of carotid artery at 3 days after balloon injury Drug

Vehicle FK409

Dose

n

I I

10 mg/kg x 2

BrdU uptake

Medial area (mm’)

Labeled nuclei/section

Labeling index (%)

50.4 f 10.6 22.9 _+4.9**

12.3+ 2.0 7.8 + 1.4

0.224 + 0.020 0.169 + 0.009*

FK409 was administered from the day of operation (once before balloon injury) to the day of sacrifice. BrdU was given intraperitoneally at 25 mg/kg at 17, 9, and 1 h before sacrifice. Immunohistochemical detection of BrdU was performed using anti-BrdU mouse monoclonal antibody and peroxidase-labeled anti mouse IgG (goat). Values are mean k S.E.M. Significant difference from vehicle group at *P < 0.05 and **P < 0.01 (Student’s t-test).

dose dependently, while ISDN did not show a significant effect at 100 pmol/l. In these experiments, we used rat SMC derived from neointima of carotid arteries. We also tested the effect of FK409 on the proliferation and cGMP level in medial SMC from rat aorta, since medial SMC seemed to be more sensitive to this drug in vivo; however, the results were almost the same (data not shown). 4. Discussion

In this report, we found that FK409 inhibited in vivo SMC proliferation induced by endothelial denudation with a balloon catheter in rats. FK409 is a structurally unique orally active nitro-va-

sodilator with anti-platelet activity obtained from fermentation products [ 171.The pharmacological activity of this compound has been shown to be closely related to the increase in intracellular cGMP level [18-201. FK409 activates soluble guanylate cyclase, and the activating effect is completely inhibited by oxyhemoglobin [29]. Recently, Kita et al. reported that FK409 released NO spontaneously from the compound itself [21]. They also found the accumulation of nitrite/nitrate, metabolites of NO, in urine, and an increase in cGMP level in plasma after oral administration of FK409 [21]. These results indicate that FK409 functions as an NO donor both in vitro and in vivo. 500,

Table 3 Effects of FK409 and ISDN on the proliferation of cultured rat SMC in vitro Drug Control FK409 Control ISDN

Dose Otmol/U 0.1 1 10 10 100

II 5 5 5 5 5 5 5

Cell number ( x 105/well) 10.2+ 0.21 10.1+ 0.23 8.84 + 0.12** 6.40 k 0.13** 8.58 + 0.26 8.54 + 0.07 8.87 f 0.27

Cells in 10% FBS-DME were seededin 35 mm dishes at 1 x 10s cells. Drug was added at 24 h and 48 h after seeding. Cells were harvested at 72 h with trypsin-EDTA solution and counted with a Coulter counter. Values are mean + S.E.M. Significant difference from control group at **P ~0.01 (Dunnet’s t-test).

1

.t

1

400. p %8

300

(I.

5 g

200.

8

ioomm

O---

Cont

0.1

1 FKbO9

10

Cont

100 MnoVL) ISDN

Fig. 2. Effects of FK409 and ISDN on intracellular cGMP level in cultured rat SMC. Cells were incubated with FK409 or ISDN for 15 min in the presence of 0.5 mM IBMX. Cellular cGMP was extracted with 0.1 N HCl and measured by RIA after acetylation. Cont. represents vehicle-treated control. Values are mean f S.E.M. (n = 5). **Significant difference from control group at P < 0.01 (Dunnet’s t-test).

J. Seki et al. / Atherosclerosis

The neointimal area was decreasedby 48.0% (P < 0.01) by the oral administration of FK409 at 10 mg/kg twice a day (20 mg/kg per day) from 2 days before to 13 days after balloon injury, compared with vehicle-treated control. This degree of suppression by FK409 was almost as potent as that of 100 mg/kg per day of captopril, which is confirmed to be a potent inhibitor of intimal thickening in this model. The neointimal formation was also decreased at the dose of 1 mg/kg twice a day (2 mg/kg per day) but this was not statistically significant. FK409 is now developing as an anti-angina1 drug at phase II stage in Japan. The clinical dose was assumed to be 40 mg x 3 per day (120 mg/day; about 2 mg/kg per day) [30]. Therefore, the effective dose of FK409 in the rat balloon injury model is about 10 times higher than the clinical dose. Since ACE inhibitors and many different types of drugs which showed positive results in this rat model showed disappointing activity on clinical restenosis after PTCA, we cannot extrapolate the result to the clinical situation. FK409 significantly inhibited in vivo BrdU uptake to medial SMC at 3 days after balloon injury. The medial area was also decreased by FK409 (Table 2). However, this drug did not show a significant effect on the proliferation of intimal SMC at 7 days after injury. These results agree with the recent report by Taguchi et al. [14]. They found the potentiation of medial SMC proliferation by N-nitro-L-arginine (NAME), an NO synthetase inhibitor. They also reported that the proliferation of intimal SMC was not affected by NAME. These results suggest that the anti-proliferative effect of FK409 or NO might be related to the phenotype of SMC. The contractile form of SMC in media seemsto be more sensitive to NO than the synthetic form of SMC in intima. The proliferative response of medial SMC can be divided into two cellular events. One is the phenotypic change of SMC from contractile form to synthetic form [31], and the other is the DNA synthesis of synthetic type SMC. Therefore, FK409 or NO might inhibit the phenotypic change of SMC. Recently, basic fibroblast growth factor (bFGF) has been proven to be a main mediator for the early proliferative response of medial SMC after balloon injury [32]. FK409 or

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NO might selectively affect the expression of bFGF or inhibit the proliferative response induced by bFGF like heparin [33]. These studies are now under investigation. If these mechanisms of action have a major role for suppressing neointimal formation at 14 days, short term administration of this drug only at the very early stage of injury might be enough for its effect. Therefore, we investigated the effect of short term administration of this drug. In fact, the neointimal formation at 14 days after injury was significantly suppressedby twice daily administration of FK409 only from the day of balloon injury to 4 days after at 10 mg/kg. The mechanism for the inhibition of SMC proliferation in vivo is not clear. Oral administration of FK409 at 10 mg/kg gave a transient but strong hypotension. Mean arterial pressure was decreased by 40%- 50% immediately after administration and recovered to predosing level within 1 h. Such a hypotensive stresswould be expected to stimulate the hypothalamic-pituitary-adrenal axes with consequent large elevation in cortisol levels which could presumably inhibit the proliferative response [34]. However, Nifedipine, a potent calcium antagonist, did not inhibit neointimal formation in this model even at a high enough dose to cause strong hypotension (data not shown). Therefore, the anti-proliferative effect of FK409 in vivo seems not to be due to its pronounced hypotensive action. FK409 inhibited the proliferation of cultured rat SMC at 1 and 10 pmol/l. Since no significant lactate dehydrogenase release was observed, the effect is not due to the cytotoxicity of this drug. At these doses, this drug markedly enhanced the intracellular cGMP level. These results indicate that the inhibitory effect on SMC proliferation by FK409 in vitro is closely related to the activation of guanylate cyclase. Recently, we found a significant increase in plasma cGMP level after oral administration of FK409 at 10 mg/kg [21]. Therefore, the anti-proliferative effect of this drug in vivo might also be cGMP-mediated. The measurement of cGMP level in injured vessels is now under investigation. However, the concentration needed for the significant inhibition of proliferation in vitro is higher than the maximum plasma

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concentration ( * 160 rig/ml = 0.76 pmol/l, unpublished result) after oral administration of FK409 at 10 mg/kg in rats. This difference may be due to the different level of activation of SMC to proliferate. Indeed, in the in vitro assay the proliferation of SMC is stimulated near maximal by 10% FBS and consequently may be less sensitive to inhibition by FK409. Another possibility is related to the mechanism for NO donation of this drug. In isolated arteries, FK409 shows vasorelaxant and cGMP elevating effect even at 10 or 1 nmol/l [19]. Thus, cells other than SMC may play a role in the activity of FK409 at very low concentration. Conversion of FK409 to NO or NOlike substances was shown to be partly spontaneous [21]; however, some biotransformation of FK409 might occur in tissue or in vivo like nitroglycerin [35]. In addition, a specific supersensitivity to nitrovasodilators in vascular vessels after inhibition of NO synthesis by the endothelial removal [36] might also be involved in the strong anti-proliferative effect of FK409 in vivo. FK409 inhibits platelet aggregation in vitro [17]. Recently, we have found that oral administration of this compound at 10 mg/kg inhibited the thrombus formation in the extracorporeal shunt model in rats [37]. Since the thrombus formation in this model is proven to be primarily platelet dependent [38], the result indicates that the oral dosing of FK409 at 10 mg/kg can attenuate platelet function in vivo. It has been well established that platelets have an important role in intimal hyperplasia. In fact, Fingerle et al. [23] have shown that intimal lesion formation was suppressed by treatment of anti-platelet antibody in this rat model. In addition, platelet-derived growth factor, primarily released from the aggregated platelets, has been shown to be essential for the migration of SMC from media to intima [39]. Therefore, FK409 might suppress neointimal formation by the inhibition of SMC function through its anti-platelet activity. However, Aspirin did not suppress intimal thickening in this model (data not shown). Recently, Langford et al. reported that platelet activation in patients undergoing PTCA in the presence of Aspirin treatment was prevented by infusion of an NO donor, S-nitrosoglutathione [40]. Thus, platelet inhibition by

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NO donors might be more potent than conventional anti-platelet agents. On the other hand, ISDN, a classical nitrate, showed no effect even at 100 mg/kg twice a day in the rat balloon injury model. This drug did not inhibit proliferation of cultured SMC up to 100 pmol/l. The vasorelaxant effect of ISDN is also due to activation of soluble guanylate cyclase and a resultant increase in intracellular cGMP [41]. However, ISDN differs from FK409 in two respects. Firstly, the vasodilatory activity of ISDN is weaker than that of FK409. In isolated rat aorta contracted with norepinephrine, these compounds induced a concentration-dependent relaxation; however, the ED50 values of FK409 and ISDN are 1.O and 310 nmol/l, respectively [21]. Thus, ISDN is about 300 times less potent than FK409 in terms of vasorelaxation of isolated artery in the rat. Oral administration of ISDN at 10 mg/kg did not show significant change in blood pressure and cGMP level in plasma [21]. Gang et al. reported the anti-proliferative action of ISDN in cultured rat SMC [l 11.However, they used a millimolar concentration of ISDN for the assay. The anti-platelet effect of ISDN has been reported also [42,43]; however, the effective dose is also at the millimolar level. Secondly, the mechanism for NO-generation from the compounds is different. FK409 can release NO or NO-like substances spontaneously [21], while ISDN requires metabolic bioconversion by reduced thiol groups for generation of NO [44], like nitroglycerin. Thiol group dependency for the NO-generation is thought to be closely related to the tolerance of nitro-vasodilators [45]. Actually, ISDN has been shown to develop the tolerance of activity, while FK409 scarcely develops self-tolerance and cross-tolerance to nitroglycerin [19]. The tolerance for ISDN might attenuate the effect of this drug on neointimal formation in the rat balloon injury model. In summary, we found that FK409, a spontaneous NO donor, suppressedintimal thickening in the rat balloon injury model. This drug inhibited the proliferative response of SMC in media at the early stage of injury. These results suggest that NO/EDRF may function as a modulator of intima1 hyperplasia in vivo.

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