Shintaro Nishio

Cytoprotective effect of TRK-100, a prostacyclin analogue, against chemical injuries in cultured human vascular endothelial cells

We studied the cytoprotective effect of TRK-100, a chemically stable analogue of prostacyclin (PGI2), in the cultured human endothelial cells from umbilical vein. TRK-100 (10 and 100 nM) stimulated significantly proliferation of endothelial cells but did not affect PGI2 production in endothelial cells. Exposure of cultured endothelial cells to homocysteine (2.5 mM) or glucose (50 mM) caused concentration-dependent cytotoxicity, as evidenced by a decrease in number of viable cells. When endothelial cells were treated with TRK-100 simultaneously or prior to, but not after, exposure to injury substances, decreases in viable cell were significantly suppressed. The protective effect of TRK-100 against homocysteine-induced cytotoxicity also appeared in endothelial cells treated with acetylsalicylic acid, suggesting that endogenous PGI2 did not involve in the protective effect of TRK-100.

Enhancement by beraprost sodium, a stable analogue of prostacyclin, in thrombomodulin expression on membrane surface of cultured vascular endothelial cells via increase in cyclic AMP level

Prostacyclin and beraprost sodium (beraprost), a stable analogue of prostacyclin, increased cyclic AMP (cAMP) levels of cultured human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner. The elevation of cAMP by beraprost was sustained longer than that by prostacyclin. The expression of thrombomodulin (TM) on membrane surface of HUVEC was enhanced by beraprost and prostacyclin, and the persistence of the increase in TM expression by beraprost was greater than prostacyclin. Dibutyryl cAMP (db-cAMP) mimicked the effects of beraprost and 3-isobutyl-1-methylxanthine enhanced the effects. Beraprost, prostacyclin and db-cAMP also effectively blocked the interleukin-1- and tumor necrosis factor-induced depression of TM expression substantially. These results suggest that TM expression is positively regulated by cAMP in HUVEC, and that beraprost may be potentially effective for reducing thrombotic events through the mechanism which initiates the stimulation of cAMP/TM system in vascular endothelial cells.

Protective effect of beraprost sodium, a stable prostacyclin analogue, in development of monocrotaline-induced pulmonary hypertension.

Experimental pulmonary hypertension (PH) was induced by a single injection of monocrotaline (MCT), a pyrrolizidine alkaloid extracted from Crotalaria spectabilis. The effect of beraprost sodium, a stable prostacyclin analogue, on the development of MCT-induced PH in rats was studied. Chronic administration of beraprost sodium at a dose of 30 micrograms/kg/day initiated on the same day as MCT injection decreased the degree of PH determined by weight ratio of right ventricular free wall to that of left ventricle plus septum depending on the duration of administration. Although the injection of prostaglandin E1 (PGE1) at a dose of 200 micrograms/kg/day initiated 1 week after MCT injection did not decrease the degree of PH significantly, beraprost sodium administration at doses of 30 and 100 micrograms/kg/day decreased the degree of PH significantly. The cytoprotective effect of beraprost sodium against endothelial cell (EC) damage is believed to be involved in inhibiting development of PH in MCT-injected rats. The amounts of cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) produced by alveolar macrophages decreased in accordance with the inhibiting effect of beraprost sodium on development of PH, indicating that beraprost sodium inhibited the development of PH in MCT-injected rats not only through its effect of vasodilation and anti-platelet aggregation in pulmonary circulation but also through its antiinflammatory effects.

Anti-Inflammatory Effects of Beraprost Sodium, a Stable Analogue of PGI2, and Its Mechanisms

We examined whether beraprost sodium (beraprost), a stable analogue of PGI2, has an anti-inflammatory effect on the permeability barrier through endothelial cells in vivo. The injection of collagen (5 micrograms/head) plus epinephrine (0.6 microgram/head) showed time-dependently the increased Evans blue dye leakage of the lung in mice for 60 min. Beraprost significantly suppressed this leakage dose-dependently (control; 11.26 +/- 1.64 micrograms/lung, beraprost 10 micrograms/kg; 7.49 +/- 1.36 micrograms/lung, 30 micrograms/kg; 5.33 +/- 0.71 micrograms/lung, 100 micrograms/kg; 5.52 +/- 0.79 micrograms/lung). Pulmonary thromboembolism-induced Evans blue dye leakage was also reduced significantly by aspirin (5 mg/kg), but PGE1 (170 micrograms/kg) showed a tendency to potentiate the edematogenic response. One week after the injection of same dosage of collagen plus epinephrine in mice, pulmonary thromboembolism showed the increase of wet-to-dry weight ratio of the lung (normal; 3.84 +/- 0.01, control; 3.96 +/- 0.04) and right ventricular hypertrophy (normal; 28.2 +/- 0.9%, control; 32.3 +/- 0.9%) compared to normal mice. Beraprost significantly suppressed lung edema and hypertrophy dose-dependently, and over 30 micrograms/kg/day of beraprost, the effects were statistically significant (beraprost 30 micrograms/kg/day; 3.85 +/- 0.02 and 27.8 +/- 1.4%, 100 micrograms/kg/day; 3.85 +/- 0.02 and 27.3 +/- 1.1%). Beraprost significantly reduced 5-hydroxytryptamine (5-HT; 17 nmol/paw)-induced rat paw edema dose-dependently (5-HT alone; 100%, beraprost 10(-13) mol/paw; 91.19 +/- 2.22%, 10(-12) mol/paw; 85.79 +/- 4.85%, 10(-11) mol/paw; 78.49 +/- 3.95%). 5-HT-induced edema was also suppressed significantly by the co-injection of (-)-isoproterenol (10(-12) mol/paw), but PGE1 (10(-11) mol/paw) significantly potentiated the edematogenic response. From these results, we propose that the anti-inflammatory effect of beraprost may be contributed, in part, to the permeability barrier through end othelial cells in vivo.

Inhibition of restenosis by beraprost sodium (a prostaglandin I2 analogue) in the atherosclerotic rabbit artery after angioplasty

We examined the effect of beraprost sodium (EPS), a stable prostaglandin I2 (PGI2) analogue, on re-stenosis after balloon angioplasty in the atherosclerotic artery in rabbits. Regional atherosclerosis was induced in the femoral artery of New Zealand white rabbits by balloon deendothelialization and 2% cholesterol diet. After establishment of atheroma in the femoral artery, angioplasty was performed. In all, 65 rabbits were assigned to the following six subcutaneous drug treatment groups: control group (n = 13, saline 0.25 ml/kg); BPS low-dose group (n = 11, BPS 50 μg/kg twice daily); BPS high-dose group (n = 12, BPS 100 μg/kg twice daily); 2-day BPS high-dose group (n = 11, BPS 100 μg/kg twice daily for 2 days after angioplasty); aspirin (ASA) group (n = 10, ASA 30 mg once daily); and BPS + ASA group (n = 8, BPS 50 μg/kg twice daily plus ASA 30 mg once daily). Administration of each drug was started 30 min before balloon angioplasty and was continued until 4 weeks thereafter, except in the 2-day BPS high-dose group. Re-examination 4 weeks after the angioplasty showed significant (p < 0.05) preservation of the luminal diameter in the BPS high-dose and 2-day BPS high-dose groups (1.30 ± 0.15 and 1.25 ± 0.09 mm, respectively) as compared with that in the control group (0.83 ± 0.10 mm); however, the luminal diameter in the BPS low-dose, ASA, and BPS + ASA groups (0.94 ±0.18, 1.06 ± 0.11, and 1.05 ±0.15 mm, respectively) was not significantly different from that in the control group.

Positive inotropic and chronotropic effects of beraprost sodium, a stable analogue of prostacyclin, in isolated guinea pig myocardium

1. Inotropic and chronotropic effects of beraprost sodium (beraprost), a chemically stable prostacyclin analogue, were examined in isolated myocardial preparations of guinea pigs. 2. In the left atria, 10(-9) - 10(-7) M beraprost had no significant effect on the contractile force, but 10(-6) and 10(-5) M produced a concentration-dependent positive inotropic response. This effect was antagonized by S-145, a potent and selective thromboxane A2 (TXA2) receptor antagonist, but not by propranolol. 3. Beraprost, from 10(-9) to 10(-5) M, had no significant inotropic effect in the right ventricular papillary muscles. 4. In the right atria, 10(-9) and 10(-8) M beraprost had no significant effect, but 10(-7) - 10(-5) M caused an increase in beating rate; this effect was not affected by S-145. 5. The present study demonstrates that beraprost has positive inotropic and chronotropic effects at high doses on isolated guinea pig atria. The inotropic effect may be mediated by the TXA2 receptor, but some mechanism other than the TXA2 receptor is responsible for the chronotropic effect.

Glycoprotein Ia/IIa-mediated activation-dependent platelet adhesion to collagen

In the presence of anti-glycoprotein (GP) IIb/IIIa antibody at the concentration which completely inhibit platelet aggregation, ADP (0.5-3 microM) increased platelet adhesion to collagen in a concentration-dependent manner under static conditions when platelet-rich plasma (PRP) was used for the assay instead of washed platelets. This was also supported by the results of scanning electron microscopic analyses. The ADP-induced platelet adhesion to collagen was inhibited by PGI2 or beraprost, a stable analogue of PGI2, in a concentration-dependent manner (1-10 ng/ml). These findings suggested the presence of activation-dependent platelet adhesion to collagen. ADP-induced platelet adhesion to collagen was almost completely inhibited by anti-GPIa/IIa and anti-GPIIa antibodies. In the present study, we provide the first direct evidence that the activation-dependent platelet adhesion to collagen is induced by ADP and that GPIa/IIa also plays an important role in the mechanisms of this adhesion.

Cytoprotective Action of Beraprost Sodium against Peroxide-Induced Damage in Vascular Endothelial Cells

Using a peroxide-injured endothelial cell model, beraprost sodium (beraprost) was tested in relation to the action to protect against the damage of vascular endothelial cells employing the viability of the cells and change in lipid peroxides as the indicators. At a concentration of 1-3 mumol/l or higher, beraprost significantly inhibited the decrease in viability of the cells and the increase in lipid peroxides level caused by t-butyl hydroperoxide or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid. When similar tests were conducted with prostaglandin I2, its inhibiting action was equal to or slightly weaker than that of beraprost unlike PGE1 and PGD2. This action of beraprost in inhibiting cell damage caused by peroxides suggests that beraprost may be useful for protecting cells from damage due to ischemic diseases.

Effects of TRK-100, a stable prostacyclin analogue, on regulation of cyclic AMP metabolism in platelets

TRK-100 is a chemically stable analogue of prostacyclin and effective in inhibiting platelet aggregation when orally administered in experimental animals. In the present study we compared the potency of TRK-100 with those of PGI2 and PGE1 to cause an activation of adenylate cyclase activity in rat and human platelet membranes. TRK-100 was half as effective as PGI2, and 10 times more effective than PGE1 in both platelet membranes. TRK-100 also induced an activation of phosphodiesterase activity when directly added to intact platelets probably as a feedback mechanism of intracellular cAMP level like PGI2 did. TRK-100 would mimic PGI2 in the regulation of cAMP metabolism.

Beraprost sodium protects occlusion/reperfusion injury in the dog by inhibition of neutrophil migration.

1. The effects of beraprost sodium (beraprost) on myocardial infarct size in an anesthetized open-chest canine model of regional myocardial ischemia and reperfusion were investigated. 2. Administration of beraprost (300 ng/kg/min, intravenously) to dogs 45 min after left circumflex coronary artery occlusion until 105 min after reperfusion resulted in a significant reduction in infarct size. 3. The values of control and beraprost treated infarct size expressed as a percentage of the total left ventricle were 15 +/- 3% and 4 +/- 2%, respectively. 4. Reperfusion arrhythmia, plasma creatine phosphokinase (CK) and lactate dehydrogenase (LDH) level were significantly suppressed by treatment with beraprost. 5. By histological examination, beraprost proved to reduce neutrophil migration in the ischemic myocardium after 5 h reperfusion. 6. Therefore, it is suggested that the cytoprotective effect of beraprost during myocardial ischemia and reperfusion may be the consequence of the inhibition of neutrophil migration.