Xanthoangelols isolated from Angelica keiskei inhibit inflammatory-induced plasminogen activator inhibitor 1 (PAI-1) production.

The folk medicine Angelica keiskei (Ashitaba) exhibits antitumor, antioxidant and antidiabetic activities and it has recently attracted attention as a health food. Ashitaba is thought to have antithrombotic properties, but this has not yet been scientifically proven. The elevation of plasma plasminogen activator inhibitor 1 (PAI-1), an inhibitor of fibrinolysis results in a predisposition to the risk of thrombosis. The present study showed that Ashitaba exudates injected intraperitoneally and orally administered over long-term suppressed the lipopolysaccharide (LPS) induced PAI-1 increase in mouse plasma. We also found that xanthoangelol, xanthoangelols B and D, the components of Ashitaba exudates, significantly inhibited TNFα-induced PAI-1 production from human umbilical vein endothelial cells (HUVECs). These findings suggest that Ashitaba can decrease elevated PAI-1 production, and that daily consumption of Ashitaba product might maintain anticoagulant status by inhibiting elevations in PAI-1 under inflammatory conditions.

Experimental study on the hemostatc activity of Pollen Typhae: a traditional folk medicine used by external and oral application.

Pollen Typhae is the traditional Chinese herbal medicine widely used to treat the hemorrhagic diseases both by external and oral application. The present study examines the hemostatic properties and its components of Pollen Typhae. Pollen extract significantly reduced prothrombin time (PT), activated partial prothrombin time (APTT) and recalcification time. Pollen extract directly activated factor XII in the coagulation cascade. Acidic polysaccharide in the pollen that adsorbed to the diethylaminoethyl (DEAE) column was the causative agent of factor XII activation. These results suggested that an electronegative charge attributed to an acidic polysaccharide in the pollen extract contributed to the hemostatic activity. We then examined the hemostatic activity of administered pollen extract in the mouse tail bleeding model. Tail bleeding was significantly decreased after oral administration of the pollen extract, whereas the acidic polysaccharide fraction did not affect the duration of tail bleeding. These results suggest that the oral anticoagulant effect of Pollen Typhae is attributed to compounds other than acidic polysaccharides. We concluded that the activation of the intrinsic coagulation pathway by the acidic polysaccharide contributes to the external hemostatic property of Pollen Typhae, and the action of components such as flavonoids that possess anticoagulant activity are causative agent when orally administered.

PERIOD2 is a circadian negative regulator of PAI-1 gene expression in mice.

An increased level of obesity-induced plasma plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular disease. To determine whether the circadian clock component PERIOD2 (PER2) is involved in the regulation of PAI-1 gene expression, we performed transient transfection assays in vitro, and generated transgenic (Tg) mice overexpressing PER2. We then compared PAI-1 expression in Tg and wild-type (WT) mice with or without obesity induced by a high-fat/high-sucrose diet. PER2 suppressed CLOCK:BMAL1- and CLOCK:BMAL2-dependent transactivation of the PAI-1 promoter in vitro. Furthermore, nuclear translocation is dispensable for PER2 to suppress CLOCK:BMAL1-dependent transactivation of the PAI-1 promoter, because functional loss of the nuclear localization domain did not affect either the interaction with BMAL1 or the suppressive role of PER2. The diurnal expression of clock and clock-controlled genes was disrupted in a gene-specific manner, whereas that of PAI-1 mRNA was significantly damped in the hearts of PER2 Tg mice fed with a normal diet. Obesity-induced plasma PAI-1 increase was significantly suppressed in Tg mice in accordance with cardiac PAI-1 mRNA levels, whereas body weight gain and changes in metabolic parameters were identical between WT and Tg mice. Endogenous PAI-1 gene expression induced by transforming growth factor-beta1 was significantly attenuated in embryonic fibroblasts derived from Tg mice compared with those from WT mice. Our results demonstrated that PER2 represses PAI-1 gene transcription in a BMAL1/2-dependent manner. The present findings also suggest that PER2 attenuates obesity-induced hypofibrinolysis by downregulating PAI-1 expression independently of metabolic disorders.

In-vitro and clinical evaluation of an oral mucosal adhesive film containing indomethacin.

To develop a new mucoadhesive film containing an analgesic combining clinical efficacy and patient comfort, we prepared and evaluated a two-layered film consisting of an adhesive layer containing indomethacin (IM) as the active ingredient and carboxyvinyl polymer (CP) as a bonding agent and a nonadhesive layer containing polyethylene glycol (PEG) to improve film texture. In in vitro and in vivo adhesive tests, the optimal concentration of CP that could be applied to the mucous membrane was 0.2% or 0.3%. Stability testing determined that the optimal storage conditions and expiration period were 4 degrees C without shade and 4 weeks, respectively. The film was clinically evaluated in patients with oral pain. IM at concentrations of 0.5% and 1% provided optimum analgesic effects, and the effects were the greatest in the 1% IM group. The addition of PEG to the nonadhesive layer reduced the number of patients experiencing discomfort at the site where the film was applied. Therefore this film formulation may be useful for local analgesic application due to its low dose requirement, moderate adhesion, and comfortable texture.

Controlled indomethacin release from mucoadhesive film: in vitro and clinical evaluations.

To develop a film formulation allowing controlled release for long-term analgesia, we selected ethyl cellulose (EC) as a novel additive, prepared a film formulation using indomethacin (IM film), and evaluated it in vitro and clinically. In the in vitro experiments, the effects of the EC concentration on the release rate of IM and on the adhesion force to the mucous membrane were investigated. The addition of 10% EC resulted in more sustained slow release compared with no EC, and the adhesion of the film with 10% EC added was similar to that of films containing carboxyvinyl polymer, which we reported previously showed significantly increased adhesion. A two-layered film consisting of an adhesive layer with 2% or 1% IM and 10% EC and a nonadhesive layer with 2% polyethylene glycol as a softening agent, was investigated for clinical use. Film consisting of an adhesive layer with 2% IM and 10% EC exhibited rapid onset of potent analgesia and was expected to prolong the duration of analgesia. These results suggest that IM film with EC added may be useful clinically, since it shows both immediate analgesic effects and prolonged duration of release.

Sole existence of antithrombin antibody in patients with systemic lupus erythematosus showing tendency of its antigenic determinants directing against exosite II (antithrombin/heparin binding site) of thrombin.

We conducted an investigation to determine the antigenic determinants of antithrombin antibody (aThr), which has recently been recognized as a new antiphospholipid antibody mostly co-existing with antiprothrombin antibody, employing patients with systemic lupus erythematosus and antiphospholipid syndrome. Using an enzyme-linked immunosorbent assay we found aThr in 34 of 83 patients (40.9%), and 27 of these 34 patients (79.4%) with aThr were all negative for other antiphospholipid antibodies. An optical density value of six of 30 patients (20.0%) with aThr showed more than a 40% reduction of reactivity to thrombin with the addition of antithrombin in a dose-dependent manner. The inhibition percentage of aThr to thrombin was prominently increased to 11 of 30 (37%) along with its inhibition rate (100% at the highest) by the co-existence of heparin. Seven out of 30 patients with aThr (23.3%) showed a reduction of the optical density value with the addition of hirudin. Our findings suggest that aThr exists solely in patients with systemic lupus erythematosus without other antiphospholipid antibodies, and the antigenic determinants of aThr are directed to exosite I (hirudin binding site) and exosite II (antithrombin/heparin binding site) on the thrombin surface, with exosite II predominance. Accordingly, aThr could be an isolated and additional new marker of thrombosis/hemostasis. Since our patients who were positive only for aThr do not have a past history of antiphospholipid-associated complications at this stage, however, further long-term follow-up and additional studies in these clinical settings are needed to verify our hypothesis in the future.

CLOCK regulates the circadian rhythm of kaolin-induced writhing behavior in mice.

Kaolin-induced writhing reaction is a simple and convenient model of bradykinin-induced pain for assessment of analgesic actions. In this study, we demonstrated that the number of kaolin-induced writhing reaction was fluctuated in a circadian manner that peaked at the end of the resting period (dusk) and reduced during the active (dark) period in mice. Circadian rhythm of the writhing intensity was completely phase-shifted by a time-imposed restricted feeding. On the other hand, 24 h of food deprivation did not affect the writhing intensity, suggesting that the endogenous clock that can be entrained to the scheduled feeding is responsible to the circadian intensity of the writhing reaction. Day/night fluctuation of the writhing intensity was completely abolished and the writhing reaction was significantly reduced in the circadian clock deficient Clock-mutant mice, although the kaolin-induced bradykinin production and blood pressure suppression were not affected in these mutant mice. Our present study suggested that the circadian variation of the pain sensitivity is governed by the food-entrainable endogenous clock and by the circadian clock molecules in mammals.

Food deprivation induces adipose plasminogen activator inhibitor-1 (PAI-1) expression without accumulation of plasma PAI-1 in genetically obese and diabetic db/db mice.

Relationships between energy intake and fibrinolytic functions have been documented in detail. We evaluated food deprivation (FD) as a means of modulating fibrinolytic activity in genetically obese and diabetic db/db mice and in their lean counterparts. Twelve hours of FD induced considerable gene expression of plasminogen activator inhibitor-1 (PAI-1) in both epididymal (3.8-fold, p < 0.05) and intestinal (2.4-fold, p < 0.05) adipose tissues without affecting plasma PAI-1 levels in db/db mice, whereas the FD did not affect these parameters in wild-type mice. Importantly, 24 hours of FD increased the plasma PAI-1 content in wild-type (1.9-fold, p < 0.01) but not in db/db mice, although adipose PAI-1 mRNA levels were significantly increased in db/db mice. The plasma PAI-1 content significantly correlated with hepatic PAI-1 mRNA levels in wild-type (r = 0.84, p < 0.01) and in db/db (r = 0.63, p < 0.01) mice. However, plasma PAI-1 did not correlate with adipose PAI-1 expression in db/db mice, although adipose tissue in general is thought to be the principal site of PAI-1 production in obesity. Hepatic PAI-1 expression was closely correlated with serum levels of free fatty acids in wild-type (r = 0.72, p < 0.01), but not in db/db mice. Adipose PAI-1 expression significantly correlated with serum corticosterone levels in both genotypes (wild-type, r = 0.52, p < 0.05; db/db, r = 0.51, p < 0.01), suggesting that adipose PAI-1 expression is up-regulated by fasting-induced glucocorticoids. The present findings suggested that fasting differentially affects fibrinolytic activity in obese and lean subjects and that PAI-1 expression in the liver as well as in adipose tissues comprises an important determinant of increased risk for cardiovascular disease in obesity.

Circadian variations in coagulation and fibrinolytic factors among four different strains of mice.

This study examined circadian variation in coagulation and fibrinolytic parameters among Jcl:ICR, C3H/HeN, BALB/cA, and C57BL/6J strains of mice. Plasma plasminogen activator inhibitor 1 (PAI-1) levels fluctuated in a circadian manner and peaked in accordance with the mRNA levels at the start of the active phase in all strains. Fibrinogen mRNA levels peaked at the start of rest periods in all strains, although plasma fibrinogen levels remained constant. Strain differences in plasma antithrombin (AT) activity and protein C (PC) levels were then identified. Plasma AT activity was circadian rhythmic only in Jcl:ICR, but not in other strains, although the mRNA levels remained constant in all strains. Levels of plasma PC and its mRNA fluctuated in a circadian manner only in Jcl:ICR mice, whereas those of plasma prothrombin, factor X, factor VII, prothrombin time (PT), and activated partial thrombin time (APTT) remained constant in all strains. These results suggest that genetic heterogeneity underlies phenotypic variations in the circadian rhythmicity of blood coagulation and fibrinolysis. The circadian onset of thrombotic events might be due in part to the rhythmic gene expression of coagulation and fibrinolytic factors. The present study provides fundamental information about mouse strains that will help to understand the circadian variation in blood coagulation and fibrinolysis.

Comparative study of circadian variation in numbers of peripheral blood cells among mouse strains: unique feature of C3H/HeN mice.

We examined strain differences in numbers of blood cells and their circadian rhythms in male Jcl:ICR, BALB/cA, C57BL/6J and C3H/HeN mice. The total numbers of circulating white blood cells (WBCs) were increased during subjective day and night, and the peaks in the active period were common to all strains. However, the number of WBCs in C3H/HeN mice remained lower and plasma levels of corticosterone (CS) were slightly higher throughout the day compared with the other strains. The numbers of circulating red blood cells (RBC) also differed according to strain. The numbers of RBCs, hematocrit (HCT) and hemoglobin (HGB) were considerably lower in C3H/HeN mice compared with the other strains, although mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) were highest among the tested strains. We found that serum erythropoietin (EPO) levels were considerably higher in C3H/HeN mice than in the other three strains. The high EPO level might be related to the unique features of RBCs in C3H/HeN mice. The present observations provide basic information about the numbers of peripheral blood cells and their circadian rhythm in mouse models and also demonstrate a unique feature of C3H/HeN mice.

Clock mutation affects circadian regulation of circulating blood cells.

BACKGROUND: Although the number of circulating immune cells is subject to high-amplitude circadian rhythms, the underlying mechanisms are not fully understood. METHODS: To determine whether intact CLOCK protein is required for the circadian changes in peripheral blood cells, we examined circulating white (WBC) and red (RBC) blood cells in homozygous Clock mutant mice. RESULTS: Daytime increases in total WBC and lymphocytes were suppressed and slightly phase-delayed along with plasma corticosterone levels in Clock mutant mice. The peak RBC rhythm was significantly reduced and phase-advanced in the Clock mutants. Anatomical examination revealed hemoglobin-rich, swollen red spleens in Clock mutant mice, suggesting RBC accumulation. CONCLUSION: Our results suggest that endogenous clock-regulated circadian corticosterone secretion from the adrenal gland is involved in the effect of a Clock mutation on daily profiles of circulating WBC. However, intact CLOCK seems unnecessary for generating the rhythm of corticosterone secretion in mice. Our results also suggest that CLOCK is involved in discharge of RBC from the spleen.

HLA-B polymorphism in Japanese HIV-1-infected long-term surviving hemophiliacs.

Approximately 30% of patients with hemophilia in Japan were infected with human immunodeficiency virus (HIV) in early 1980s through contaminated blood products. In 1995, a cohort of HIV-infected, asymptomatic patients with hemophilia was set up for follow-up study. Although the patients met the criteria for long-term non-progressor (LTNP) at the entry to the cohort, some of them later developed lymphopenia during five more years of observation. We collected blood samples from 80 long-term survivors; 42 of them did not require antiviral therapy, but the rest were under treatment. Analysis of HLA-B genotype revealed that carriers of known HIV-resistant alleles such as HLA-B*5701, B*5801, and alleles of B27 antigenic group were not increased in frequency, but that HLA-B*1507 was increased in the cohort (6.25% vs. 1.03%, OR = 6.40, p = 0.039). We also observed the decrease in carriers of HLA-B*5401 (3.75% vs. 14.95%, OR = 0.22, p = 0.016). HLAB* 5401 is a relatively common allele in East Asian populations and belongs to the same B22 antigenic group as B55 and B56 which were reported to associate with rapid progression. Our data indicated that HLA class I is one of the host factors involved in the retardation of HIV disease progression as also reported in the previous studies; however, the alleles associated with this resistance were not the same because of divergent host genetic background.

Tissue-specific augmentation of circadian PAI-1 expression in mice with streptozotocin-induced diabetes.

Diabetes is associated with an excess risk of cardiac events, and the risk for infarction is partly determined by plasminogen activator inhibitor-1 (PAI-1). We found that plasma total and active PAI-1 levels increased in a circadian manner in mice with streptozotocin (STZ)-induced diabetes. Circadian expression of PAI-1 mRNA in the lung, heart, liver, and kidney increased in a tissue-specific manner. Peak to peak comparisons revealed that the mRNA expression levels increased by 1.7, 1.7, 1.2, and 1.6-fold in the heart, lung, liver, and kidney, respectively. In contrast, the circadian expression of the clock gene, mPer2, was preserved in the diabetic mice, suggesting that the altered expression of PAI-1 mRNA did not arise due to impaired circadian clocks. Our results suggest that impairment of the coagulation and fibrinolytic systems induced by diabetes is partly due to impaired circadian PAI-1 fluctuation at the level of mRNA expression.

Oxidized phospholipids in oxidized low-density lipoprotein reduce the activity of tissue factor pathway inhibitor through association with its carboxy-terminal region.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that inhibits the initial reactions of blood coagulation. In this study, we explored the nature of active components that reduce the anticoagulant activity of TFPI in oxidized low-density lipoprotein (ox-LDL). The organic solvent-soluble fraction obtained from ox-LDL was fractionated by normal-phase HPLC. The binding profile of each fraction to TFPI showed a single peak eluting near purified oxidized phospholipid. To explore further the components in oxidized phospholipid that inhibit TFPI activity, we used oxidized phospholipids that mimic the biological activity of ox-LDL. The oxidation products of 1- and/or 2-oleoyl phosphatidylcholine or phosphatidylethanolamine were the most potent inhibitors of TFPI activity, whereas those of arachidonyl phosphatidylcholine possessed only a weak inhibitory effect on the TFPI activity. These oxidized phospholipids mainly associated with the C-terminal basic region of the TFPI molecule. The results indicate that oxidation products of delta-9 unsaturated phospholipids are candidate active components of ox-LDL that impair the function of TFPI through specific association with its C-terminal basic region.