Michiko Muramatsu

Cyclo-oxygenase-2 enhances basic fibroblast growth factor-induced angiogenesis through induction of vascular endothelial growth factor in rat sponge implants

Angiogenesis is reportedly enhanced by prostaglandins (PGs). In the present study, we investigated whether or not cyclo‐oxygenase (COX)‐2 mediated angiogenesis in chronic and proliferate granuloma. In rat sponge implants, angiogenesis was gradually developed over a 14‐day experimental period as granuloma formed. This angiogenesis was enhanced by topical injections of human recombinant basic fibroblast growth factor (bFGF). In sponge granuloma, mRNA of COX‐1 was constitutively expressed, whereas that of COX‐2 was increased with neovascularization in parallel with that of vascular endothelial growth factor (VEGF). Topical injections of bFGF increased the expression of COX‐2 mRNA. bFGF‐stimulated angiogenesis was inhibited by indomethacin or selective COX‐2 inhibitors, NS‐398, nimesulide, and JTE‐522. The levels of PGE2 and 6‐keto‐PGF1α in the sponge granuloma were increased with bFGF 13 fold and 9 fold, respectively, and these levels were markedly reduced by NS‐398. The expression of VEGF mRNA in the granuloma was also enhanced by bFGF, and was reduced by NS‐398. Topical injections of PGE2 and beraprost sodium, a PGI2 analogue, increased the expression of VEGF mRNA, with angiogenesis enhancement. The enhanced angiogenesis by bFGF was significantly inhibited by topical injections of VEGF anti‐sense oligonucleotide. These results suggested that COX‐2 may enhance bFGF‐induced neovascularization in sponge granuloma by PG‐mediated expression of VEGF, and that a COX‐2 inhibitor would facilitate the management of conditions involving angiogenesis.

(−)-Phenylahistin: A new mammalian cell cycle inhibitor produced by aspergillus ustus

Abstract (−)-Phenylahistin is a fungal diketopiperazine metabolite consisting of L -phenylalanine and isoprenylated dehydrohistidine, and it showed an inhibitory activity on the cell cycle progression of P388 cells in the G2/M phase.

Eplerenone Inhibits Atherosclerosis in Nonhuman Primates

Aldosterone may be involved in the pathogenesis of atherosclerosis. We investigated the effect of eplerenone, a selective mineralocorticoid receptor blocker, on atherosclerosis in monkeys fed a high-cholesterol diet. Monkeys fed a high-cholesterol diet for 9 months were divided into 3 groups: those treated with a low dose of eplerenone (30 mg/kg per day); those treated with a high dose of eplerenone (60 mg/kg per day); and the placebo-treated group. The normal group consisted of monkeys fed a normal diet. There were no significant differences in blood pressure and cholesterol levels between the placebo- and eplerenone-treated groups. On the other hand, monocyte chemoattractant protein-1 and malondialdehyde-modified LDL were significantly higher in the placebo-treated group than in the normal group, whereas they were suppressed in the eplerenone-treated groups. The ratio of intimal volume to total volume by intravascular ultrasound analysis imaging of the aortas was dose-dependently lower in the eplerenone-treated groups than in the placebo-treated group. Acetylcholine-induced vasorelaxation was significantly weaker in the placebo-treated group than in the normal group, but the vasorelaxation was strengthened in the eplerenone-treated groups. A significant upregulation of angiotensin-converting enzyme activity was observed in the placebo-treated group, but the activity was suppressed in the eplerenone-treated groups. In conclusion, eplerenone may strengthen the endothelium-dependent relaxation and suppress angiotensin-converting enzyme activity in the vasculature, thus preventing the development of atherosclerosis in nonhuman primates.

Significance of Angiotensin II Receptor Blocker Lipophilicities and Their Protective Effect against Vascular Remodeling

Although the lipophilicities of the various angiotensin II receptor blockers (ARBs) are very different, the relationship between lipophilicity and the protective effect against vascular remodeling is unclear. In this study, we compared the protective effects of a highly lipophilic ARB, telmisartan, and an ARB with low lipophilicity, losartan, on vascular function and oxidative stress in stroke-prone spontaneously hypertensive rats (SHR-SP). SHR-SP received oral placebo, 1 mg/kg telmisartan, or 10 mg/kg losartan for 2 weeks. The blood pressure (BP) in SHR-SP was significantly higher than that in Wistar-Kyoto (WKY) rats before treatment, and the BP was reduced equally in telmisartan- and losartan-treated SHR-SP compared to placebo-treated SHR-SP. Acetylcholine-induced vasorelaxation in isolated carotid arteries was significantly weaker in SHR-SP than in WKY rats, but in both telmisartan- and losartan-treated SHR-SP, acetylcholine-induced vasorelaxation was significantly higher than in placebo-treated SHR-SP. Moreover, acetylcholine-induced vasorelaxation in telmisartan-treated rats was significantly stronger than in losartan-treated SHR-SP. The expression of the endothelial nitric oxide synthase gene was significantly higher in telmisartan- and losartan-treated rats than in placebo-treated SHR-SP, and was significantly higher in telmisartan-treated rats than in losartan-treated rats. In contrast, the expression of the NAD(P)H oxidase subunit p22phox gene in telmisartan-treated SHR-SP was significantly lower than that in losartan-treated SHR-SP. Immunohistochemistry showed that angiotensin II expression in the aorta was significantly lower in telmisartan-treated SHR-SP than in losartan-treated SHR-SP. In conclusion, a highly lipophilic ARB, telmisartan, may be useful for preventing NAD(P)H oxidase activity, and thereby for conferring vascular protection.

Chymase mediates mast cell-induced angiogenesis in hamster sponge granulomas

We investigated the contribution of mast cell chymase in mast cell-dependent angiogenesis using the hamster sponge-implant model, where angiogenesis in the granulation tissue surrounding the subcutaneously implanted sponge was evaluated by measuring the hemoglobin content. Daily local injection of compound 48/80 (3-100 microg/site/day), a potent mast cell activator, induced formation of granulomas and angiogenesis in time- and dose-dependent manners. This angiogenic response was inhibited by chymase inhibitors including chymostatin (> or = 1 nmol/site/day), soybean trypsin inhibitor (SBTI; > or = 1.4 nmol/site/day) and lima bean trypsin inhibitor (LBTI; > or = 3.3 nmol/site/day), but not by a tryptase inhibitor like leupeptin (> or = 700 nmol/site/day). Although pyrilamine (> or = 2,580 nmol/site/day), a histamine H1 receptor antagonist, and protamine (300 microg/site/day) also inhibited angiogenesis, these effects were much less pronounced than those by chymase inhibitors. Furthermore, antigen-induced angiogenesis in hamsters pre-sensitized with ovalbumin was also inhibited by the chymase inhibitors by 60-70%. Our results suggest that chymase is a major mediator in mast cell-mediated angiogenesis.

Effects of chymase inhibitor on angiotensin II-induced abdominal aortic aneurysm development in apolipoprotein E-deficient mice

OBJECTIVE Chymase may play an important role in abdominal aortic aneurysm (AAA) development through matrix metalloproteinase (MMP)-9 activation. The purpose of this study was to determine whether chymase is involved in angiotensin (Ang) II-induced AAA development in apolipoprotein E (apoE)-deficient mice. METHODS AND RESULTS In this study, Ang II (1000 ng/kg/min; vehicle group) or saline (saline group) was administered to 16-week-old, male, apoE-deficient mice for 4 weeks. To examine the effects of chymase inhibition on AAA development, oral NK3201 (30 mg/kg/day) was given for the same period as the Ang II infusion. AAAs developed at the suprarenal region of the abdominal aorta in the Ang II-treated vehicle group, but they were not observed in the saline group. On the other hand, the severity and luminal area of the AAAs in the Ang II-treated vehicle group were significantly suppressed by NK3201 treatment. MMP-9 activity was significantly lower in the Ang II-treated+NK3201-treated group than in the Ang II-treated vehicle group. Furthermore, there were significantly fewer monocyte/macrophage cells in the Ang II-treated+NK3201-treated group than in the Ang II-treated vehicle group. CONCLUSIONS Chymase is involved in Ang II-induced AAA development in apoE-deficient mice.

Cytotoxic effects of NSL-1406, a new thienopyrimidine derivative, on leukocytes and osteoclasts.

We synthesized a series of thienopyrimidine derivatives and examined their cytotoxic effects on several cell lines. One of the derivatives, NSL-1406, was shown to exert potent cytotoxic effects on leukemia cell line including P388 cells and J774 cells. It was also inhibitory on mouse osteoclasts and suppressed the in vitro bone resorption by osteoclasts at nanomolar concentrations.

Role of Chymase-Dependent Angiotensin II Formation in Regulating Blood Pressure in Spontaneously Hypertensive Rats

Vascular smooth muscle cells in spontaneously hypertensive rats (SHR) express angiotensin II-forming chymase (rat vascular chymase [RVCH]), which may contribute to blood pressure regulation. In this study, we studied whether chymase-dependent angiotensin II formation contributes to the regulation of blood pressure in SHR. The systolic blood pressure in 16-week-old Wistar-Kyoto (WKY) rats was 113±9 mmHg, compared to 172±3 mmHg in SHR. Using synthetic substrates for measuring angiotensin-converting enzyme (ACE) and chymase activities, it was found that both ACE and chymase activities in extracts from SHR aortas were significantly higher than in those from WKY rat aortas. Using angiotensin I as a substrate, angiotensin II formation in SHR was found to be significantly higher than that in WKY rats, and its formation was completely suppressed by an ACE inhibitor, but not by a chymase inhibitor. RVCH mRNA expression could not be detected in aorta extracts from either WKY rats or SHR. In carotid arteries isolated from WKY rats and SHR, angiotensin I-induced vasoconstriction was completely suppressed by an ACE inhibitor, but not by a chymase inhibitor. Angiotensin I-induced pressor responses in both WKY rats and SHR were also completely inhibited by an ACE inhibitor, but they were not affected by a chymase inhibitor. In SHR, an ACE inhibitor and an angiotensin II receptor blocker showed equipotent hypotensive effects, but a chymase inhibitor did not have a hypotensive effect. These results indicated that chymase-dependent angiotensin II did not regulate blood pressure in SHR in the present study.

The significance of chymase in the progression of abdominal aortic aneurysms in dogs.

In this study, we investigated the effect of a specific chymase inhibitor, NK3201, in the progression of abdominal aortic aneurysm in a dog experimental model. Abdominal aortic aneurysms were induced in dogs by injecting elastase into the abdominal aorta. NK3201 (1 mg/kg per day, p.o.) or a placebo was started 3 days before elastase injection and continued for 8 weeks after the injection. On abdominal ultrasound, the aortic diameter was seen to gradually expand in the placebo-treated group, but not in the NK3201-treated group. Eight weeks after elastase injection, the ratio of the medial area to the total area in the placebo-treated group was significantly smaller than that in the normal group, but it was significantly larger than that in the NK3201-treated group. In addition to chymase activity, angiotensin II–forming and matrix metalloproteinase-9 activities were significantly higher in the placebo-treated group than in the normal group; in the NK3201-treated group, all of these activities were significantly decreased. On immunohistochemical analyses, there was a significantly greater number of chymase-positive cells in the placebo-treated group than in the normal group, but the number was significantly smaller in the NK3201-treated group than in the placebo-treated group. Thus, chymase inhibition may become a useful strategy for preventing abdominal aortic aneurysms.

The regressive effect of an angiotensin II receptor blocker on formed fatty streaks in monkeys fed a high-cholesterol diet.

Objectives To clarify the regressive effect of an angiotensin II type 1 receptor blocker (ARB) on already formed fatty streaks, we investigated the effect of the administration of an ARB, olmesartan, on formed fatty streaks in monkeys fed a high-cholesterol diet. Methods After the monkeys were fed a high-cholesterol diet for 6 months, intimal hyperplasia was clearly observed on intravascular ultrasound. For the next 6 months, the high-cholesterol diet was continued, and olmesartan (3 mg/kg per day) or placebo was administered. A control group was fed a normal diet for 12 months. Results Olmesartan did not significantly affect blood pressure or plasma cholesterol levels throughout the experiment. After 6 months of treatment with olmesartan, intimal hyperplasia was significantly lower than before treatment. Acetylcholine-induced relaxation in isolated carotid arteries was significantly less in the high-cholesterol diet placebo-treated group compared to the normal diet group, whereas its response was improved by olmesartan. Serum levels of monocyte chemoattractant protein were significantly increased with a high cholesterol load, but they were significantly suppressed by olmesartan. Conclusions We have demonstrated for the first time that an ARB, olmesartan, was found to have a regressive effect on formed fatty streaks in monkeys.