Yukio Katsuki

Repeated administration of low-dose lipopolysaccharide induces preterm delivery in mice: a model for human preterm parturition and for assessment of the therapeutic ability of drugs against preterm delivery.

OBJECTIVE Our purpose was to establish a new animal model for human preterm delivery for assessment of the protective effect of drugs against preterm delivery. STUDY DESIGN C3H/HeN, C3H/HeN, and BALB/c female mice impregnated by C3H/HeN, B6D2F1, and B6D2F1 male mice, respectively, were treated intraperitoneally with Escherichia coli lipopolysaccharide (0 to 100 microgram/kg, single dose or repeated doses at 1- to 6-hour intervals) on days 12 through 17 of pregnancy. On day 15 of pregnancy, the C3H/HeN females that had been impregnated by B6D2F1 males and administered lipopolysaccharide were treated intraperitoneally with indomethacin (1000 microgram/kg), ritodrine hydrochloride (1000 microgram/kg), urinary trypsin inhibitor (25 x 10(4) units/kg), or gabexate mesylate (100 mg/kg); preterm or term delivery was recorded for these mice. RESULTS C3H/HeN females impregnated by B6D2F1 males revealed the highest (100%) incidence of preterm delivery when the females were treated with 50 microgram/kg lipopolysaccharide twice at a 3-hour interval on day 15 or 17 of pregnancy. Indomethacin and urinary trypsin inhibitor used separately significantly decreased the incidence of preterm delivery, but only urinary trypsin inhibitor, and not any of the other drugs, significantly increased the incidence of term delivery in the mice. CONCLUSION A new animal model for investigation of preterm delivery was established, and its usefulness for assessment of the protective effect of drugs against preterm delivery was demonstrated.

Trophoblastic apoptosis in mice with preterm delivery and its suppression by urinary trypsin inhibitor

Objective To investigate histopathologic changes of the placenta in mice with preterm delivery induced by lipopoly-saccharide and the effect of urinary trypsin inhibitor. Methods Female C3H/HeN mice impregnated by male B6D2F1 mice were treated with lipopolysaccharide (50 μg/kg, intraperitoneally) or lipopolysaccharide plus urinary trypsin inhibitor (25 × 104 U/kg, intraperitoneally). At 3, 6, 9, and 18 hours after the second dose of lipopolysaccharide, and at delivery in the control and urinary trypsin inhibitor-treated groups, the concentrations of interleukin-1α and tumor necrosis factor-α were determined in serum and amniotic fluid. Subsequently, the placentas were examined. In the same manner, we examined mice treated with interleukin-1α (250 μg/kg, subcutaneously) on day 15 of pregnancy and intact mice on days 15 and 18 of pregnancy as well as at delivery. To assess the direct action of cytokines, we cultured placental slices with tumor necrosis factor-α, interleukin-1a, or tumor necrosis factor-α plus urinary trypsin inhibitor and examined them morphologically. Results Control mice were characterized by trophoblastic apoptosis and increased serum levels of tumor necrosis factor-α and interleukin-1α. In contrast, urinary trypsin inhibitor-treated mice showed suppression of apoptosis and lower cytokine levels. Interleukin-1α induced trophoblastic apoptosis and increased the serum level of tumor necrosis factor-α. The in vitro study showed that tumor necrosis factor-α directly induced trophoblastic apoptosis in placental slices. Conclusion We demonstrated that trophoblastic apoptosis occurs in the placentas of a mouse model with preterm delivery induced by lipopolysaccharide. We postulated that apoptosis may lead to placental abruption, and its development may be prevented by treatment with urinary trypsin inhibitor.

Dienogest, a synthetic steroid, suppresses both embryonic and tumor-cell-induced angiogenesis

Orally administered dienogest (17alpha-cyanomethyl-17beta-hydroxy-estra-4,9-diene-3-one) is efficacious against human hormone-dependent cancer xenografts in severely immunodeficient mice and in rats with experimental endometriosis, but its mechanisms of action remain unclear. We assessed the effect of dienogest on angiogenesis, because these two diseases that are sensitive to dienogest are known to be angiogenesis-dependent. Topical dienogest treatment dose-dependently inhibited embryonic angiogenesis, the ID(50) value being 6.4 nmol/egg. Oral administration of dienogest (1 mg kg(-1) day(-1)) for 5 consecutive days significantly suppressed angiogenesis induced by S-180 mouse tumor cells in the mouse dorsal air sac assay. In vitro experiments showed that dienogest at concentrations up to 10 microM had little or no effect on the proliferation of plasminogen activator activity or formation of tube-like structures by microvascular endothelial cells. These results suggest that dienogest is a new, orally active antagonist of angiogenesis, and that its anti-angiogenic action may be involved in its therapeutic effects on cancer xenografts and endometriosis that we observed previously.

Effects of ethyl all-cis-5,8,11,14,17-icosapentaenoate on the physical properties of arterial walls in high cholesterol diet-fed rabbits

The effects of ethyl all-cis-5,8,11,14,17-icosapentaenoate (EPA-E) on in vivo physical properties of arteriosclerotic aorta and femoral artery in high cholesterol diet (HCD)-fed rabbits were studied. The aortic pulse wave velocity (PWV) of rabbits fed HCD for 12 weeks (control group) tended to be higher than that of rabbits fed a normal diet (normal group). Because the PWVs in HCD-fed rabbits administered orally with 30 and 300 mg/kg/day EPA-E were significantly lower than the PWV of the control group, the distensibility of arteriosclerotic aorta was improved with administration of EPA-E. The stiffness parameter (β) value as an in vivo indicator of arteriosclerosis was significantly higher in the control group than in the normal group and improved with administration of EPA-E to almost the same level as that of the normal group. The β-values were in significant negative correlation with medial elastin content and medial smooth muscle cell (SMC) density in thoracic aorta and in positive correlation with the free cholesterol content in abdominal aortic SMC. On the other hand, they were not correlated with either the cross-sectional area of intimal thickening lesions or the plasma lipid levels measured simultaneously. The femoral PWVs were, like those in the aorta, higher in the control group as compared with the normal group, and the changes were improved with administration of EPA-E. These results show that EPA-E improved the in vivo distensibility of arteriosclerotic arteries in HCD-fed rabbits. The mechanism of this improvement with EPA-E appeared to be related to histologic amelioration in the aortic medial elastic fibers and SMC and to protection from free cholesterol accumulation in SMC as one of the probable causes.

Role of urinary trypsin inhibitor in the maintenance of pregnancy in mice

Objective To investigate the mechanisms whereby urinary trypsin inhibitor prevents lipopolysaccharide-induced preterm delivery in mice. Methods On day 15 of pregnancy, C3H/HeNCrg female mice impregnated by Crg:B6D2F1 male mice were treated intraperitoneally with lipopolysaccharide (50 μg/kg, twice at a 3-hour interval) to induce preterm delivery. Urinary trypsin inhibitor (2.5 × 104, 7.5 × 104, or 25 × 104 units/kg, ten times at 1-hour intervals) or saline solution was administered intraperitoneally to the animals. Results The incidence of preterm delivery was significantly decreased on a dose-related basis by urinary trypsin inhibitor treatment. Urinary trypsin inhibitor prevented the morphologic and functional changes in fetal membranes and cervical ripening preceding the onset of preterm delivery. Urinary trypsin inhibitor also suppressed the increase in plasma and amniotic fluid concentrations of interleukin-1α, interleukin-6, and tumor necrosis factor-α after the lipopoly-saccharide dosing in this animal model for preterm delivery. Conclusion Urinary trypsin inhibitor prevents the pathogenicity of preterm delivery through the suppression of cytokine production.

Ability of intrauterine bacterial lipopolysaccharide to cause in situ uterine contractions in pregnant rabbits.

Background. To investigate the ability of bacterial lipopolysaccharide delivered by the intrauterine route to cause uterine contractions in rabbits, and to assess the suppressive effect of urinar trypsin inhibitor on them.

Preventive effect of ritodrine hydrochloride and/or urinary trypsin inhibitor against lipopolysaccharide-induced preterm delivery in mice

Background. The purpose of this study was to confirm the preventive effect of ritodrine hydrochloride (ritodrine) alone or ritodrine plus urinary trypsin inhibitor (UTI) in a mouse model of preterm delivery.

Effect of dienogest on bleeding time, coagulation, fibrinolysis, and platelet aggregation in female rats.

We investigated the effect of dienogest on bleeding time, coagulation, fibrinolysis, and platelet aggregation in female rats compared with that of medroxyprogesterone acetate (MPA) and danazol, in order to elucidate the reason for relatively high incidence of bleeding in dienogest-treated patients with endometriosis. Dienogest caused no change in the bleeding time at a single dose of 100 mg/kg or at a repeated dose of 10 mg/kg per day for 2 weeks. The drug increased the fibrinogen level, coagulation factor II and V activities, and antithrombin III activity, but had no effect on fibrinolysis or on platelet aggregation at repeated doses of 1 and 10 mg/kg per day for 4 weeks. MPA significantly shortened the bleeding time at the same doses as dienogest. MPA increased the fibrinogen level and plasminogen activity, potentiated the platelet aggregation, and increased the platelet cholesterol-to-phospholipid ratio at a repeated dose of 10 mg/kg per day for 4 weeks. Danazol significantly shortened the bleeding time like MPA. Danazol increased the fibrinogen level, coagulation factor II, V, VII, VIII, IX, X, XI, and XII activities, and antithrombin III activity, but had no influence on the platelet aggregation at repeated doses of 10 and 100 mg/kg per day for 4 weeks. In comparison with MPA and danazol, dienogest may induce a relatively high incidence of bleeding in patients with endometriosis partially because of its minimal effect on hemostasis.

Effects of dienogest (a synthetic steroid) on coagulation, fibrinolysis, and platelet aggregation in female monkeys.

We investigated the effects of dienogest (0.1-10 mg/kg per day, p.o.) on coagulation, fibrinolysis and platelet aggregation in female rhesus monkeys. Then, we also examined those of medroxyprogesterone acetate (MPA, 10 mg/kg per day, p.o.) or danazol (10-1000 mg/kg per day, p.o.) on these parameters in the same species. In addition, we assessed the effects of dienogest (1 and 3 mg/kg per day, p.o.) or MPA (10 mg/kg per day, p.o.) on platelet aggregation and platelet lipids in female cynomolgus monkeys. At doses of 0.3 mg/kg or greater, dienogest increased the levels of several coagulation and anticoagulation factors, but had no effect on the prothrombin time, activated partial thromboplastin time, fibrinolysis, or platelet aggregation. MPA (10 mg/kg) had no effect on coagulation or fibrinolysis, but significantly potentiated platelet aggregation in response to ADP and collagen and also increased the platelet cholesterol-to-phospholipid ratio. Danazol (10 mg/kg or more) increased the activities of coagulation factors V, VII, VIII, X, XI, and XII in comparison to dienogest and MPA. Consequently, dienogest caused less potentiation of platelet aggregation than MPA and less potentiation of coagulation than danazol.

高コレステロール食飼育ウサギにおける動脈壁弾性と血管内皮依存性弛緩反応およびethyl all-cis-5，8，11，14，17-icosapentaenoate(EPA-E)の影響

We studied the elasticity and endothelium-dependent relaxation (EDR) of the aorta in 1% cholesterol diet (HCD)-fed rabbits. Furthermore, the effects of ethyl all-cis-5,8,11,14, 17-icosapentaenoate (EPA-E) were examined in this model of atherosclerosis. After 12 weeks of feeding with HCD, the animals showed increase in plasma total cholesterol level, formation of atherosclerotic plaque, decrease in aortic elasticity and impairment of EDR to acetylcholine (ACh). The levels of aortic elasticity in HCD-fed rabbits administered orally with EPA-E (300 mg/kg for 12 weeks) were almost the same as those of rabbits fed a normal diet, although EPA-E showed no effects on the plasma total cholesterol level and formation of atherosclerotic plaque in HCD-fed rabbits. On EDR in response to ACh and cyclic GMP formation in the HCD-fed rabbit aorta, EPA-E improved the impairment of these parameters, but not significantly. Therefore, EPA-E had little effect on the endothelium in this model of atherosclerosis, although EPA-E improved the decrease in the aortic elasticity. Because the levels of aortic elasticity showed no significant correlation with the magnitude of EDR to ACh or the size of atherosclerotic plaque, the decrease of aortic elasticity in this model of atherosclerosis was thought to have little relation to the dysfunction of the endothelium.