Goro Miyakoda

Cilostazol reduces brain lesion induced by focal cerebral ischemia in rats--an MRI study.

To investigate the effects of cilostazol on the hemispheric ischemic lesion, we monitored the apparent diffusion coefficient (ADC) and T2 images by MRI techniques in comparison with histology at the terminal of and after 24-h reperfusion following 2-h occlusion of middle cerebral artery (MCA). The ADC values of tissue water and T2-weighted images were quantified by high field magnetic resonance. No significant difference was observed by ADC image among vehicle and cilostazol treatment groups when measured during MCA occlusion. Oral treatment with cilostazol 30 mg/kg two times at 5 min and 4 h significantly suppressed the hemispheric lesion area and volumes when detected by ADC, T2 images and histology, but 3 and 10 mg/kg cilostazol were without effect. Cilostazol (30 mg/kg) significantly reduced the increased cerebral water content at the ischemic hemisphere compared with vehicle group. In line with these results, the neurological deteriorations were much improved in the cilostazol-treated group. Taken together, it is concluded that post-treatment with cilostazol exerts a potent protective effect against cerebral infarct size by reducing the cytotoxic edema.

Cilostazol, a phosphodiesterase inhibitor, attenuates photothrombotic focal ischemic brain injury in hypertensive rats

The aim of this study was to evaluate and compare the effects of anti-platelet agents with different modes of action (cilostazol, aspirin, and clopidogrel) on brain infarction produced by photothrombotic middle-cerebral-artery (MCA) occlusion in male, spontaneously hypertensive rats. Cerebral blood flow (CBF) was measured with laser-Doppler flowmetry in the penumbral cortex. Infarct size was evaluated 24 h after MCA occlusion. The effects of these drugs on infarct size were examined by pretreatment of rats undergoing MCA occlusion. Pretreatment with cilostazol (100 mg/kg) significantly reduced infarct size. In contrast, aspirin (10 mg/kg) and clopidogrel (3 mg/kg) failed to mitigate infarct size, regardless of their apparent inhibitory effects on platelet aggregation. Post-treatment with cilostazol also significantly attenuated the infarct size, associated with improved CBF in the penumbral region. In support of this effect, cilostazol increased nitric oxide (NO) production and prostaglandin-I2 (PGI2) release in cultured human brain microvascular endothelial cells. Cilostazol-induced NO production and PGI2 release were completely abolished by an NO synthase inhibitor and aspirin, respectively. These findings show that cilostazol reduced brain infarct size due to an improvement in penumbral CBF possibly in association with increased endothelial NO and PGI2 production.

Cilostazol inhibits platelet–leukocyte interaction by suppression of platelet activation

The influence of three anti-platelet drugs, cilostazol, aspirin, and tirofiban, was investigated on platelet–leukocyte interaction by flow cytometry. When platelets and leukocytes were pre-incubated with anti-platelet drugs and stimulated by thrombin or collagen, cilostazol was found to inhibit platelet adhesion to monocytes and polymorphonuclear cells (PMNs). Similar effects were observed with anti-CD62P antibody, while aspirin and tirofiban did not appear to interfere with interaction between platelets and leukocytes. In the platelets pre-incubated with anti-platelet drugs, cilostazol significantly reduced CD62P expression and GPIIb/IIIa activation on platelet surface stimulated by thrombin or collagen. Aspirin inhibited CD62P expression and GPIIb/IIIa activation induced by collagen, but not thrombin. Tirofiban significantly blocked GPIIb/IIIa activation induced with both, and weakly inhibited CD62P expression induced by collagen. When added after stimulation of platelets, cilostazol again significantly inhibited CD62P expression and GPIIb/IIIa activation, although to a lesser extent than in the pre-incubation study. Aspirin hardly inhibited CD62P expression or GPIIb/IIIa activation, while tirofiban strongly blocked GPIIb/IIIa activation induced by thrombin or collagen, but had little effects on CD62P expression. In conclusion, our results suggest that cilostazol inhibits platelet–leukocyte interaction by reducing CD62P expression on the platelet surface.

Cilostazol inhibits modified low-density lipoprotein uptake and foam cell formation in mouse peritoneal macrophages

Internalization of modified low-density lipoprotein (LDL) via macrophage scavenger receptors (e.g. scavenger receptor A and CD36) is thought to play a crucial role in the development of atherosclerotic lesions. Cilostazol, an antiplatelet agent with selective phosphodiesterase 3 inhibitory action, has been reported to ameliorate atherosclerosis in mouse models. However, the effect of cilostazol on modified LDL uptake in macrophages is not known. Thus, we investigated the effect of cilostazol on LDL uptake in mouse peritoneal macrophages (MPM). Cilostazol significantly inhibited oxidized and acetylated LDL uptake in MPM, while cyclic AMP (cAMP)-elevating agents, db-cAMP and other phosphodiesterase 3 or 4 inhibitors, did not inhibit the uptake. Cilostazol did not change cytosolic cAMP levels in MPM, and a protein kinase A (PKA) inhibitor did not influence the inhibitory effects of cilostazol. Cilostazol decreased scavenger receptor A but not CD36 expression. Moreover, cilostazol significantly inhibited foam cell formation, which was represented by an increase in esterified cholesterol content. In conclusion, cilostazol significantly inhibits the uptake of modified LDL and foam cell formation in mouse peritoneal macrophages, and the inhibitory effect of cilostazol can be induced in a cAMP- and PKA-independent manner.

Pranidipine, a new 1, 4-dihydropyridine calcium channel blocker, enhances cyclic GMP-independent nitric oxide-induced relaxation of the rat aorta

Pranidipine, a new calcium channel modulator, prolonged endothelium-dependent relaxation induced by acetylcholine in a aortic ring preparation, contracted with prostaglandin F2α. This action was not shared by amlodipine. The effect was not modified by indomethacin, suggesting that the action of pranidipine does not involve prostanoid metabolism. NG-nitro-L-arginine completely prevented the action of Pranidipine. The drug affected neither nitric oxide (NO) synthase activity nor the level of cyclic GMP in the vessel. Pranidipine did not affect the sensitivity of the contractile proteins to calcium. Pranidipine also did not alter cyclic GMP-induced relaxation in a-toxinskinned vascular preparations. Pranidipine also prolonged glyceryl trinitrate-induced relaxation in the endothelium denuded rat aorta. Furthermore, pranidipine enhanced relaxation of the aorta induced by glyceryl trinitrate even in the presence of methylene blue, a guanylyl cyclase inhibitor. This action was not modified by iberiotoxin or by charybdotoxin, two inhibitors of the calciumactivated potassium channel. The results strongly suggest that pranidipine enhances cyclic GMPindependent NO-induced relaxation of smooth muscle by a mechanism other than through NOinduced hyperpolarization. These effects were in direct contrast to amlodipine, another new 1,4dihydropyridine calcium antagonist.

Effect of cilostazol on delayed cerebral vasospasm after subarachnoid hemorrhage in rats: evaluation using black blood magnetic resonance imaging.

The purpose of this study was to use black blood magnetic resonance imaging (BB-MRI) to assess delayed cerebral vasospasm (DCV) after subarachnoid hemorrhage (SAH) in rats, and evaluate whether delayed treatment with the anti-platelet agent cilostazol was effective on DCV. BA vasospasm was sequentially assessed at 1, 2, and 3 h, and 1-6 days after SAH by BB-MRI. BB-MRI clearly visualized biphasic vasospasm; early vasospasm at 1 h later and the maximal DCV at day 2. Cilostazol was perorally administered twice at day 1 after having confirmed significant DCV using BB-MRI. The effect of cilostazol on DCV was evaluated at day 2. Cilostazol significantly attenuated DCV and suppressed the levels of malondialdehide and 8-isoprostane in CSF after SAH. This study shows that BB-MRI is a useful and less invasive method for the evaluation of DCV, and cilostazol may be effective on DCV.

Venodilator effects of pranidipine, a 1,4-dihydropyridine Ca2+ channel antagonist, in rats: comparison with nifedipine and amlodipine.

The effects of pranidipine, a dihydropyridine Ca2+ channel antagonist, on mean circulatory filling pressure, an index of body venous tone, were compared with those of other dihydropyridines (nifedipine and amlodipine) and nitroglycerin in anaesthetized hexamethonium- and norepinephrine-treated rats. In this study, the compounds were used at doses having a equi-hypotensive effect. Intravenous bolus injection of pranidipine (10 and 30 microg/kg) significantly decreased mean circulatory filling pressure in a dose-dependent manner, as did nitroglycerin (30 and 100 microg/kg). Nifedipine (30 and 100 microg/kg), however, did not affect mean circulatory filling pressure. Amlodipine (1000 and 3000 microg/kg) decreased mean circulatory filling pressure only at the higher dose. These results suggest that pranidipine has a greater venodilator effect than nifedipine and amlodipine.

Cilostazol Inhibits Accumulation of Triglyceride in Aorta and Platelet Aggregation in Cholesterol-Fed Rabbits

Cilostazol is clinically used for the treatment of ischemic symptoms in patients with chronic peripheral arterial obstruction and for the secondary prevention of brain infarction. Recently, it has been reported that cilostazol has preventive effects on atherogenesis and decreased serum triglyceride in rodent models. There are, however, few reports on the evaluation of cilostazol using atherosclerotic rabbits, which have similar lipid metabolism to humans, and are used for investigating the lipid content in aorta and platelet aggregation under conditions of hyperlipidemia. Therefore, we evaluated the effect of cilostazol on the atherosclerosis and platelet aggregation in rabbits fed a normal diet or a cholesterol-containing diet supplemented with or without cilostazol. We evaluated the effects of cilostazol on the atherogenesis by measuring serum and aortic lipid content, and the lesion area after a 10-week treatment and the effect on platelet aggregation after 1- and 10-week treatment. From the lipid analyses, cilostazol significantly reduced the total cholesterol, triglyceride and phospholipids in serum, and moreover, the triglyceride content in the atherosclerotic aorta. Cilostazol significantly reduced the intimal atherosclerotic area. Platelet aggregation was enhanced in cholesterol-fed rabbits. Cilostazol significantly inhibited the platelet aggregation in rabbits fed both a normal diet and a high cholesterol diet. Cilostazol showed anti-atherosclerotic and anti-platelet effects in cholesterol-fed rabbits possibly due to the improvement of lipid metabolism and the attenuation of platelet activation. The results suggest that cilostazol is useful for prevention and treatment of atherothrombotic diseases with the lipid abnormalities.

OPC-28326, a selective peripheral vasodilator with angiogenic activity, mitigates postinfarction cardiac remodeling

Although OPC-28326, 4-(N-methyl-2-phenylethylamino)-1-(3,5-dimethyl-4-propionyl-aminobenzoyl) piperidine hydrochloride monohydrate, was developed as a selective peripheral vasodilator with α2-adrenergic antagonist properties, it also reportedly exhibits angiogenic activity in an ischemic leg model. The purpose of this study was to examine the effect of OPC-28326 on the architectural dynamics and function of the infarcted left ventricle during the chronic stage of myocardial infarction. Myocardial infarction was induced in male C3H/He mice, after which the mice were randomly assigned into two groups: a control group receiving a normal diet and an OPC group whose diet contained 0.05% OPC-28326. The survival rate among the mice (n = 18 in each group) 4 wk postinfarction was significantly greater in the OPC than control group (83 vs. 44%; P < 0.05), and left ventricular remodeling and dysfunction were significantly mitigated. Histologically, infarct wall thickness was significantly greater in the OPC group, due in part to an abundance of nonmyocyte components, including blood vessels and myofibroblasts. Five days postinfarction, Ki-67-positive proliferating cells were more abundant in the granulation tissue in the OPC group, and there were fewer apoptotic cells. These effects were accompanied by activation of myocardial Akt and endothelial nitric oxide synthase. Hypoxia within the infarct issue, assessed using pimonidazole staining, was markedly attenuated in the OPC group. In summary, OPC-28326 increased the nonmyocyte population in infarct tissue by increasing proliferation and reducing apoptosis, thereby altering the tissue dynamics such that wall stress was reduced, which might have contributed to a mitigation of postinfarction cardiac remodeling and dysfunction.