Seitaro Mutoh

Smooth muscle cell migration induced by inflammatory cell products and its inhibition by a potent calcium antagonist, nilvadipine

The chemotactic activities of inflammatory cell products for rat aortic smooth muscle cells (SMC) were examined in modified Boyden chambers. A checker board analysis revealed that interleukin-1 (IL-1), leukotriene B4 (LTB4), platelet-derived growth factor (PDGF) and inflammatory exudate from zymosan-activated air pouches stimulated chemotaxis of SMC. The chemotaxis, irrespective of the attractants used, was strongly inhibited by nilvadipine, a potent calcium antagonist, and the IC50 values were around 1 x 10(-10) M. Removal of extracellular calcium abolished the chemotactic activities of the attractants. These results suggest that inflammatory cells such as macrophages and polymorphonuclear leukocytes (PMN) have an important role in the migration of SMC into the intima during atherogenesis, and that nilvadipine might be useful for preventing and treating atherosclerosis.

Inhibition of arterial thrombosis by a protease-activated receptor 1 antagonist, FR171113, in the guinea pig.

The antiplatelet and antithrombotic effects of FR171113, 3-(4-chlorophenyl)-2-(2,4-dichlorobenzoylimino)-5-(methoxycarbonyl methylene)-1,3-thiazolidin-4-one, a non-peptide protease-activated receptor 1 (PAR1) antagonist, were evaluated in guinea pigs. FR171113 inhibited Ser-Phe-Leu-Leu-Arg-Asn-NH2 (a synthetic PAR1 agonist peptide)-induced and thrombin-induced aggregation of guinea pig platelets in a concentration-dependent manner in vitro (IC50=1.5 and 0.35 microM, respectively). Subcutaneous administration of FR171113 (0.1-3.2 mg/kg) produced a dose-dependent inhibition of platelet aggregation ex vivo. The ED50 value of FR171113 for platelet aggregation was 0.49 mg/kg s.c. However, FR171113 did not have an inhibitory effect on ADP- or collagen-induced platelet aggregation in vitro and ex vivo. One hour after FR171113 treatment at 1.0 mg/kg s.c., significant inhibition of arterial thrombosis without a prolongation of thrombin time or coagulation time was seen in the FeCl3-induced carotid artery thrombosis model in guinea pigs. Furthermore, FR171113 did not prolong bleeding time even at 32 mg/kg s.c., which is a much higher dose than that required in the thrombosis model. These observations indicate that FR171113 has desirable antiplatelet effects both in vitro and in vivo and that its in vivo antithrombotic activity is efficacious without causing a prolongation of bleeding time.

Effects of FK228, a novel histone deacetylase inhibitor, on human lymphoma U-937 cells in vitro and in vivo.

FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide] is a novel histone deacetylase inhibitor that shows therapeutic efficacy in Phase I trials of patients with malignant lymphoma. However, its mechanism of action has not been characterized. In this study, we examined the in vitro and in vivo effects of FK228 on human lymphoma U-937 cells. FK228 very strongly inhibited the growth of U-937 cells with an IC(50) value of 5.92 nM. In a scid mouse lymphoma model, mice treated with FK228 once or twice a week survived longer than control mice, with median survival times of 30.5 (0.56 mg/kg) and 33 days (0.32 mg/kg), respectively (vs. 20 days in control mice). Remarkably, 2 out of 12 mice treated with FK228 (0.56 mg/kg once or twice a week) survived past the observation period of 60 days. The apoptotic population of U-937 cells time-dependently increased to 37.7% after 48 hr of treatment with FK228. In addition, FK228 induced G1 and G2/M arrest and the differentiation of U-937 cells to the CD11b(+)/CD14(+) phenotype. Expression of p21(WAF1/Cip1) and gelsolin mRNA increased up to 654- and 152-fold, respectively, after 24hr of treatment with FK228. FK228 caused histone acetylation in p21(WAF1/Cip1) promoter regions, including the Sp1-binding sites. In conclusion, (i) FK228 prolonged the survival time of scid mice in a lymphoma model, and (ii) the beneficial effects of FK228 on human lymphoma may be exerted through the induction of apoptosis, cell cycle arrest, and differentiation via the modulation of gene expression by histone acetylation.

Antitumor efficacy of FK228, a novel histone deacetylase inhibitor, depends on the effect on expression of angiogenesis factors

UNLABELLED It has been recently demonstrated that histone deacetylase inhibitors inhibit angiogenesis, but their mechanism of action has not been characterized well. In this study, we examined the in vitro and in vivo effects of FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide], an HDAC inhibitor, on the expression of angiogenesis factors in FK228-sensitive PC-3 prostate and FK228-resistant ACHN renal cancer cells. FK228 suppressed the expression of VEGF mRNA in PC-3 cells, but not in ACHN cells. FK228 also suppressed the expression of basic fibroblast growth factor (bFGF) mRNA in both PC-3 and ACHN cells. Under conditions of hypoxia, FK228 suppressed the expression of VEGF mRNA without modulating the expression of hypoxia-inducible factor-1 alpha mRNA in PC-3 cells. FK228 induced the highest acetylation of histone H3 and H4 in the P2 region of the VEGF promoter, which includes the hypoxia-inducible factor-1 alpha binding site that plays an important role in regulating the expression of VEGF gene. Moreover, FK228 reduced the amount of VEGF and bFGF protein, and their mRNA levels in PC-3 xenograft implanted in nude mice, but did not reduce them in ACHN xenograft. IN CONCLUSION (i) FK228 showed a suppressive effect on the expression of angiogenesis factors, such as VEGF and bFGF, in PC-3 xenograft but not in ACHN xenograft, which suggests that the effect on the expression of angiogenesis factors is important for the antitumor efficacy of FK228; (ii) FK228 caused histone acetylation of the VEGF promoter regions, which may contribute to the suppression of VEGF gene expression.

Inflammatory responses in cuff-induced atherosclerosis in rabbits

Cuff-treatment of the rabbit carotid artery produced a diffuse intimal thickening which resembled early lesions of atherosclerosis. A limited amount of focal endothelial damage occurred first (0.5 h), leukocytes infiltrated the subendothelium and extensive endothelial denudation occurred at 24 h. At 3 days, the regenerating endothelium covered the denuded area, and the media was edematous. At 7 days proliferation of intimal cells became visible. Maximum intimal thickening occurred at 3 weeks. Daily injection of dexamethasone (0.01-10 mg/kg i.m.) and ticlopidine (1-100 mg/kg i.m.) dose-dependently attenuated the intimal thickening. Indomethacin had little effect. Inflammatory exudate from zymosan-activated air pouch induced chemotaxis of rat smooth muscle cells (SMC) in vitro. Similar chemotactic activity was observed with leukotriene B4 (LTB4) but not with the other lipoxygenase products tested. The exudate contained reasonable amounts of LTB4, which would account for its chemotactic activity. Dexamethasone inhibited the chemotaxis by the exudate and proliferation of SMC. These results are discussed in relation to the mechanism of atherogenesis. It is concluded that leukocytes play a major role in cuff-induced intimal thickening, and that their products cause endothelial denudation and SMC chemotaxis. Involvement of platelet aggregation in atherogenesis is also suggested.

Discovery of quinazolinone and quinoxaline derivatives as potent and selective poly(ADP-ribose) polymerase-1/2 inhibitors.

Two classes of quinazolinone derivatives and quinoxaline derivatives were identified as potent and selective poly(ADP‐ribose) polymerase‐1 and 2 (PARP‐1) and (PARP‐2) inhibitors, respectively. In PARP enzyme assays using recombinant PARP‐1 and PARP‐2, quinazolinone derivatives displayed relatively high selectivity for PARP‐1 and quinoxaline derivatives showed superior selectivity for PARP‐2. SBDD analysis via a combination of X‐ray structural study and homology modeling suggested distinct interactions of inhibitors with PARP‐1 and PARP‐2. These findings provide a new structural framework for the design of selective inhibitors for PARP‐1 and PARP‐2.

Prevention of progressive joint destruction in collagen‐induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR255031

1 FR255031 (2‐[(7S)‐7‐[5‐(4‐ethylphenyl)‐2‐thienyl]‐1,1‐dioxido‐4‐(2‐pyridinylcarbonyl)hexahydro‐1,4‐thiazepin‐7‐yl]‐N‐hydroxyacetamide) is a novel synthetic matrix metalloproteinase (MMP) inhibitor that inhibits human collagenases (MMP‐1, MMP‐8 and MMP‐13), gelatinases (MMP‐2 and MMP‐9) and membrane type 1 MMP (MT1‐MMP/MMP‐14). FR255031 also inhibits rat collagenase and gelatinase. We studied the effect of FR255031 and Trocade, an inhibitor of collagenase and MMP‐14, on a rat collagen‐induced arthritis (CIA) model. 2 Rat CIA was induced by intradermal injection of type II collagen (IIC) and oral administration of FR255031 or Trocade was performed for 28 days. Body weight loss, hind paw swelling, elevation of serum anti‐IIC antibody, and histological and radiographic scores were evaluated. 3 FR255031 markedly inhibited cartilage degradation in a dose‐dependent manner in the CIA model, but Trocade failed to prevent the degradation. 4 FR255031 at a dose of 100 mg kg−1 also had statistically significant effects on bone destruction and pannus formation and on the recovery of body weight loss on day 28. 5 These results indicate that FR255031 is effective for rat CIA, especially on joint cartilage destruction. These data suggest that as well as collagenases or MT‐MMP, gelatinases are also involved in joint destruction in arthritis.

Multiple modes of action of tacrolimus (FK506) for neuroprotective action on ischemic damage after transient focal cerebral ischemia in rats.

While the immunosuppressant tacrolimus (FK506) is known to be neuroprotective following cerebral ischemia, the mechanisms underlying its neuroprotective properties are not fully understood. To determine the mode of action by which tacrolimus ameliorates neurodegeneration after transient focal ischemia, we therefore evaluated the effect of tacrolimus on DNA damage, release of cytochrome c, activation of microglia and infiltration of neutrophils following a 60-min occlusion of the middle cerebral artery (MCA) in rats. In this model, cortical brain damage gradually expanded until 24 h after reperfusion, whereas brain damage in the caudate putamen was fully developed within 5 h. Tacrolimus (1 mg/kg) administered immediately after MCA occlusion significantly reduced ischemic damage in the cerebral cortex, but not in the caudate putamen. Tacrolimus decreased both apoptotic and necrotic cell death at 24 h and reduced the number of cytochrome c immunoreactive cells at 8 h after reperfusion in the ischemic penumbra in the cerebral cortex. In contrast, tacrolimus did not show significant neuroprotection for necrotic cell death and reduction of cytochrome c immunoreactive cells in the caudate putamen. Tacrolimus also significantly decreased microglial activation at 8 h and inflammatory markers (cytokine-induced neutrophil chemoattractant and myeloperoxidase [MPO] activity) at 24 h after reperfusion in the ischemic cortex but not in the caudate putamen. These results collectively suggest that tacrolimus ameliorates the gradually expanded brain damage by inhibiting both apoptotic and necrotic cell death, as well as suppressing inflammatory reactions.

Role of inflammatory responses in initiation of atherosclerosis: effects of anti-inflammatory drugs on cuff-induced leukocyte accumulation and intimal thickening of rabbit carotid artery

Immediately after a cuff-sheathing of rabbit carotid artery, a large number of leukocytes adhered to injured endothelium then infiltrated into the media. These inflammatory responses were followed by an atherosclerotic change, intimal thickening, of the artery. A simultaneous injection of dexamethasone (10 mg/kg i.m.) inhibited the leukocyte accumulation by 74% when evaluated 18 h thereafter. Similarly, 39% inhibition was obtained with the same dose of FR110302, a potent 5-lipoxygenase inhibitor. On the other hand, the same dose of indomethacin, a cyclooxygenase inhibitor, had little effect on the leukocyte accumulation. The intimal thickening which was evaluated 3 weeks after the cuff-treatment was attenuated by a daily dose (10 mg/kg i.m.) of dexamethasone or FR110302 but not by one of indomethacin. The inhibition by the two former drugs were 91 and 58%, respectively. In vitro, the three drugs in concentrations up to 10 microM hardly affected endothelial adhesion of PMN which was induced by LPS or IL-1. Though 10 microM of FR110302 and indomethacin significantly decreased PMN chemotaxis induced by LTB4, the decreases were less than that at 10 microM dexamethasone. These results confirm a possible linkage between inflammation and atherosclerosis, and suggest that 5-lipoxygenase products contribute to the initiation and development of atherosclerosis.

Effects of FK228, a novel histone deacetylase inhibitor, on tumor growth and expression of p21 and c-myc genes in vivo

In this study, we examined the effects of FK228 (FR901228, depsipeptide) on tumor growth and expression of p21 and c-myc genes in vivo. FK228 induced the expression of p21 mRNA and decreased c-myc mRNA in tumor xenograft sensitive to FK228. However, FK228 did not sufficiently modulate the expression of p21 mRNA and increased the expression of c-myc in tumor xenograft less sensitive to FK228. The modulation of p21 and/or c-myc genes may be critical for the marked antitumor activity of FK228 in vivo.