Short- and long-term treatment with folic acid suppresses thrombus formation in atherogenic mice in vivo.

In the present study, we examined the effects of short- and long-term treatment with folic acid (FA) on thrombus formation in vivo in atherogenic mice to explore a novel agent for the prevention of atherothrombotic disease. Apolipoprotein E and low-density lipoprotein receptor double deficient (ApoE(-/-)LDLR(-/-)) mice were orally administrated a single bolus of FA (20mg/kg) or fed an atherogenic diet with or without FA (0.02, 0.5, and 1.5mg/kg) for 12 weeks. Thrombus formation and endothelial function were assessed in vivo using the He-Ne laser-induced carotid artery thrombus formation test and the flow-mediated vasodilation method. Platelet reactivity was assessed ex vivo using haemostatometry. Short-term treatment with FA markedly increased plasma folate levels and significantly suppressed laser-induced thrombus formation in apoE(-/-)LDLR(-/-) mice. Short-term treatment with FA suppressed platelet reactivity in apoE(-/-)LDLR(-/-) mice, but FA treatment did not affect endothelial function or plasma homocysteine levels. Long-term treatment with FA increased plasma folate levels dose-dependently. Thrombus formation and endothelial dysfunction were suppressed by treatment with 0.5 and 1.5mg/kg of FA, respectively, but not with 0.02mg/kg of FA, whereas platelet reactivity was not altered by treatment with any dose of FA. Long-term treatment with all doses of FA decreased the plasma homocysteine levels in apoE(-/-)LDLR(-/-) mice, although this result was not consistent with its anti-thrombotic action. In conclusion, our data showed that short- and long-term treatment with FA could suppress in vivo thrombus formation in an atherogenic setting, independent of its hypohomocysteinemic action.

Experimental antithrombotic effects of sesame seed whole grains and extracts.

Prevention of arterial thrombotic diseases has a high priority in developed countries. An inappropriate diet is known to enhance the risks for acute thrombotic events, and nutritional products experimentally shown to be antithrombotic, might contribute beneficial effects. The present study forms part of a series of investigations into the antithrombotic effect of various foods and vegetables. Roasted and crushed whole grains from six varieties of sesame seeds were added to the diet of mice. Antithrombotic activity was measured in the carotid artery in vivo, using a He-Ne laser-induced thrombosis technique after 12 weeks. Col/Chichibu/Maruteru-2/1995 and T016 varieties showed significant antithrombotic activity, whilst 00037803 was prothrombotic. The acute effects of purified ingredients, sesamin, sesamolin and sesamol, given orally or intra-arterially, were also examined after a single dose. The most effective ingredient was sesamol, followed by sesamolin and sesamin. Daily intake of specific antithrombotic sesame whole grains or purified active ingredients might help to prevent atherothrombotic diseases.

Impaired endothelial function may be due to decreased aortic tetrahydrobiopterin, assessed by a new flow-mediated vasodilation in vivo in hypercholesterolemic/atherogenic mice.

Tetrahydrobiopterin (BH4) is an important cofactor for endothelial nitric oxide synthase activity. The relationship between endothelial function in vivo and aortic BH4 level is not fully understood, however. In the present study, we aimed to clarify whether reduction of aortic BH4 levels contributes to endothelial dysfunction in vivo using spontaneously hyperlipidemic mice. To estimate endothelial function in vivo and in real-time state, we developed a flow-mediated vasodilation (FMV) method in mice, which measured changes in the diameter of the femoral artery in response to increased blood flow. C57BL/6 mice and apoE/low-density lipoprotein receptor double knock-out mice were fed a low-fat diet (LFD) or a high-fat diet (HFD) for 12 weeks from 6 weeks of age. HFD feeding impaired FMV in double knock-out mice, but not in C57BL/6 mice. Furthermore, HFD feeding reduced plasma NOx concentration and aortic BH4 level in double knock-out mice. Conversely, exogenous injection of BH4 (2 mg/kg) markedly increased aortic BH4 levels and restored endothelial function. In conclusion, we demonstrated that HFD feeding impaired nitric oxide-mediated endothelial function and reduced BH4 level in vivo, and that acute augmentation of aortic BH4 levels improved endothelial function. These findings indicate that BH4 is a critical determinant of nitric oxide-mediated endothelial function in hypercholesterolemia.

The antithrombotic effects of carrot filtrates in rats and mice.

The prevention of arterial thrombotic disease has a high priority in developed countries. An inappropriate diet is known to enhance the risk for acute thrombotic events and a regular diet with proven antithrombotic effects might be a beneficial way to prevent disease. The present study was undertaken as a part of a series of investigations to examine vegetables and fruits for antithrombotic activity. A shear-induced in vitro platelet reactivity/thrombolysis method [Global Thrombosis Test, formerly Gorog Thrombosis Test] was used to screen 15 different varieties of carrot for antiplatelet and thrombolytic activities. Effective varieties were further investigated using an in vivo, laser-induced thrombosis model in mice. The different carrot varieties demonstrated a variable effect on thrombosis in vitro and in vivo. In particular, a variety designated SAKATA-0421 exerted an antithrombotic effect in vivo independent from heat treatment of the filtrate at 100 degrees C for 10 min. There was no significant correlation between antithrombotic activity and the levels of polyphenolics and any other biochemical parameter, including antioxidant activity, alpha-carotene and beta-carotene, alpha-tocopherol and ascorbic acid. Different varieties of carrot demonstrated a range of antithrombotic and prothrombotic activities. After oral intake, the particular heat resistant variety (SAKATA-0421) showed antithrombotic effect in vivo possibly due to antiplatelet reactivity and/or spontaneous thrombolytic activity. The present study added a new variety to the list of antithrombotic fruits and vegetables.

Long-term intake of rosemary and common thyme herbs inhibits experimental thrombosis without prolongation of bleeding time.

OBJECTIVE: Our earlier study demonstrated an inhibition of experimental thrombogenesis after acute administration of rosemary and common thyme. The present study aimed to investigate the effect of long-term intake of these herbs on platelets, thrombus formation and bleeding time. METHODS: Western-style high-fat diet containing 5% or 0.5% dried rosemary or common thyme was given to male 6 week old C57BL/6 mice for 12 weeks. Shear-induced platelet reactivity was measured in vitro by haemostatometry. The antithrombotic effect was assessed by the laser-induced thrombosis technique in the carotid artery of mice. Bleeding time was tested by the tail cut method. Endothelial function was assessed by the flow-mediated vasodilation test. RESULTS: Rosemary (5% and 0.5%) significantly inhibited arterial thrombus formation. Only the higher concentration of common thyme (5%) had a significant antithrombotic effect. In 5% concentration, both rosemary and common thyme significantly inhibited platelet reactivity and enhanced the flow-mediated vasodilation. CONCLUSIONS: Long-term daily intake of rosemary and common thyme has an antithrombotic effect, which is probably due to inhibition of platelets and stimulation of endothelial cells. The antithrombotic effect was not accompanied by prolongation of bleeding time.

A rice variety (HYJA-Ri4) enhances arterial thrombus formation in an animal model of thrombosis.

Epidemiological evidence exists for a causative role of inappropriate diets in the development and clinical outcome of thrombotic diseases. Our previous studies have shown that some fruits and vegetables have antithrombotic activity. As rice is a principal ingredient of everyday food in Japan, the aim of the present study was to determine rice varieties with antithrombotic effect. Western-style high-fat diet containing flour prepared from whole-grain rice of different varieties was fed to atherosclerosis-prone (apolipoprotein E- and low density lipoprotein receptor-deficient) mice for 3 months. A laser-induced carotid-artery thrombosis model was used. None of the five tested rice varieties showed an antithrombotic effect. The effect of HYJA-Ri1, HYJA-Ri2, HYJA-Ri3 and HYJA-Ri5 rice on experimental thrombus formation did not differ significantly from the controls. However, HYJA-Ri4 rice significantly enhanced the thrombotic reaction (p = 0.0078), thus having a prothrombotic effect. Our findings suggest that HYJA-Ri4 rice should not be included in an antithrombotic diet.

Testing various fruits for anti-thrombotic effect: i. Mulberries.

Prevention of arterial thrombotic disease has high priority in developed countries. As inappropriate diet predispose to acute thrombotic events, regular intake of an antithrombotic diet may be a convenient and effective way of prevention. The present study was performed to examine antithrombotic effect of mulberry varieties. A shear-induced in vitro platelet reactivity/thrombolysis test (Gorog Thrombosis Test) was used to screen for antiplatelet and thrombolytic activities. In case of effectiveness, it was followed by an in vivo test of laser-induced thrombosis in mice. Antioxidant capacity was assessed by superoxide anion and radical scavenging activities. Total polyphenolics, anthocyanin and citrate contents were also measured. The tested varieties showed different effect in vitro on platelet reactivity and endogenous thrombolytic activity. Varieties inhibiting platelet reactivity were antithrombotic in vivo regardless inhibition or enhancement of thrombolysis. Those mulberry varieties, which enhanced platelet reactivity in vitro, were prothrombotic only if inhibitory activity on endogenous thrombolysis coexisted with the platelet effect. Antioxidant activities and polyphenolics content did not affect platelets and the overall thrombotic status. However, antioxidant activities and polyphenolics content significantly correlated with the endogenous thrombolytic activity. These data showed that mulberry varieties can be grouped into subclasses with either anti- or prothrombotic activities. Antioxidant activities and polyphenolic contents do not affect platelets but may enhance endogenous thrombolysis, thus causing an overall antithrombotic effect.

Effect of dietary vegetable and animal proteins on atherothrombosis in mice.

OBJECTIVE: It is believed that vegetable and fish, but not animal, proteins prevent thrombosis. The present study compared the effect of long-term intake of purified vegetable and animal proteins (casein, pork, egg white, chicken, white and red fish, soybean, and potato) and powders from whole vegetable and animal meats (soybean, pork, chicken, and horse mackerel) on thrombotic tendency. METHODS: Western-style high-fat diets with various proteins were given to mice deficient in apolipoprotein-E and low-density lipoprotein receptor for 8 or 12 wk. Thrombotic tendency was assessed by a laser-induced thrombosis test, severity of atherosclerosis was assessed by morphometric analysis of the aortic lesions, and reactivity of platelets was measured by a shear-induced platelet function test. RESULTS: There was no difference in thrombotic tendency between the vegetable and animal protein diets with the exception of pork protein, which showed a prothrombotic effect. The latter was not observed in animals kept on a high-protein diet. Thrombotic tendency was not affected by preparation of dietary powders from whole pork, chicken, or horse mackerel meats and casein (control), all of which contained the same percentages of protein and lipid. In contrast, soybean powder showed an antithrombotic effect, which was mainly due to an inhibition of atherogenesis rather than to platelet inhibition. CONCLUSION: Neither the purified animal nor the vegetable protein diet affected thrombotic tendency. Soybean powder inhibited atherothrombosis mainly by inhibiting the atherosclerotic process.

Endothelial dysfunction precedes atherosclerotic lesions and platelet activation in high fat diet-induced prothrombotic state.

INTRODUCTION: Earlier we have demonstrated a prothrombotic state in spontaneously atherogenic rodents kept on Western-style high fat diet. The aim of the present study was to investigate the cellular mechanism of such prothrombotic state. MATERIALS AND METHODS: Two kinds of diets, Western-style high fat diet containing 20% fat (w/w) and 0.05% cholesterol (w/w) and low fat diet containing 7% fat without cholesterol based on AIN93G, were added to diet-sensitive apolipoprotein E and low-density lipoprotein receptor double deficient male mice for 12 or 18 weeks from 6 weeks of age. Atherosclerosis was assessed by morphometry of the aortic wall or lipid-stained lesions. Endothelial function was measured by flow-mediated vasodilation (FMV) of the femoral artery. Platelet reactivity was measured ex vivo by a shear-induced platelet aggregation test. RESULTS AND CONCLUSIONS: 12 weeks feeding of mice with high fat diet significantly impaired FMV, as compared with mice fed with low fat diet (P<0.05). In contrast, there was no significant difference in the lipid-stained areas and in the reactivity of platelets between the two groups. 18 weeks feeding with high fat diet significantly impaired FMV (P<0.05) and enhanced both lipid-stained areas (P<0.05) and platelet reactivity (P<0.01). These findings show that in high fat diet-induced prothrombotic state, endothelial dysfunction precedes both the morphologically detectable lesions and the enhancement of platelet reactivity.

Dietary diacylglycerol extenuates arterial thrombosis in apoE and LDLR deficient mice.

INTRODUCTION: Prevention of arterial thrombotic diseases has high priority in developed countries. An appropriate diet is thought to be the best way to prevent or reduce the risk of mortality from such diseases. The aim of the present study was to evaluate the effect of diacylglycerol (DAG)-rich diets on arterial thrombosis. MATERIAL AND METHODS: Diet-sensitive congenital apolipoprotein E (ApoE) and LDL receptor (LDLR) double deficient mice were used. Thrombosis was assessed by the rate and extent of thrombus formation in the carotid artery of mice after laser irradiation. Plasma total cholesterol and triglyceride levels were measured by enzymatic assays. Four kinds of diets were used: high fat (Western-style) diet contained 20% fat (w/w) and 0.05% cholesterol (w/w); the TAG-rich and the DAG-rich high fat diet contained 20% TAG or DAG oil (w/w) with very similar fatty acid composition and 0.05% cholesterol ; Low fat (Japanese-style) diet contained 7% fat, without cholesterol. These diets were on the basis of AIN93G and were given to mice for 8 weeks from 6 weeks of age. RESULTS: Western-style high fat and TAG-rich high fat diets significantly increased thrombogenicity compared with low fat diet. DAG-rich high fat diet showed the lowest value, and the extent of thrombogenicity was equivalent to the low fat diet group. Fasting plasma total cholesterol level of DAG-rich high fat and low fat diet groups were significantly lower than that of TAG-rich high fat and high fat diet groups. Fasting plasma triglyceride levels in DAG-rich high fat diet group were significantly lower than in the TAG-rich high fat diet group. CONCLUSIONS: Dietary DAG but not TAG oil extenuates arterial thrombus formation. The mechanism of this effect is unclear and further investigated.

Mechanism of the antithrombotic effect of dietary diacylglycerol in atherogenic mice.

INTRODUCTION: We have shown earlier that diacylglycerol (DAG) but not triacylglycerol (TAG) inhibited thrombus formation. The aim of the present study was to investigate the mechanism of this antithrombotic effect of DAG. MATERIALS AND METHODS: Four different diets, the (1) Western-style high-fat diet (HFD) containing 20% lipid and 0.05% cholesterol (w/w), (2) TAG-rich and (3) DAG-rich HFDs containing 20% lipid and 0.05% cholesterol, but all lipid replaced by TAG or DAG oil with very similar fatty acid composition and the (4) Japanese-style low-fat diet (LFD) containing 7% oil but no cholesterol were given to apolipoprotein E and low-density lipoprotein (LDL) receptor double-deficient mice. Atherogenicity was assessed by morphology, mapping the whole aorta and measuring the total area of lipid-stained lesions. Endothelial function was measured by the flow-mediated vasodilation test. Platelet reactivity was assessed from native blood sample by a shear-induced platelet function test (hemostatometry). Serum lipoprotein profile was measured by HPLC. RESULTS: Both the Western-style and the TAG-rich HFDs have accelerated atherosclerosis. In contrast, DAG-rich HFD inhibited the atherosclerotic process to an extent comparable with the Japanese-style LFD. There was no significant difference in platelet and coagulant activity between the studied diet groups. DAG-rich but not the TAG-rich HFD significantly suppressed serum LDL cholesterol level. CONCLUSIONS: The present findings suggest that the mechanism of antithrombotic and anti-atherogenic effect of DAG may involve the protection of the vascular endothelium from injury and lowered serum LDL cholesterol.

An experimentally antithrombotic strawberry variety is also effective in humans.

Prevention of arterial thrombotic diseases is of high priority in developed countries. As inappropriate diet is regarded as an important risk factor of thrombotic events, daily intake of an antithrombotic diet may offer a convenient and effective way of prevention. Earlier we used animal models of thrombosis to find fruits and vegetables with potential antithrombotic activity. Among various strawberry varieties tested, a particular variety (KYSt-4, Nohime) showed a significant antithrombotic effect. The aim of the present investigation was to extend this study to humans, by testing the experimentally active KYSt-4 and inactive KYSt-10 variety for effectiveness in humans after oral intake. Filtrates of strawberries were prepared and administered orally. Thrombotic status was tested by a novel global test (Gorog Thrombosis Test). The strawberry variety (KYSt-4; Nohime) which earlier inhibited experimental thrombosis showed antithrombotic effects in humans, while the experimentally inactive variety (KYSt-10) as well as the relevant control (water) were ineffective.

Anti-thrombotic effect of strawberries.

Because of the high mortality, prevention of arterial thrombotic disease has top priority in developed countries. As inappropriate diet is known to predispose to acute thrombotic events, regular intake of an anti-thrombotic diet may offer a convenient and effective method of prevention. As part of a systematic investigation into the anti-thrombotic effect of fruits and vegetables, strawberry varieties were tested in this study. An in vitro platelet function test (haemostatometry) was used for screening strawberry filtrates. Those that showed significant antiplatelet effect were further assessed with a laser-induced thrombosis test in mice. Measurement of flow-mediated vasodilation in the femoral artery of mice reflected the effect on the vascular endothelium. Correlation between the effects on platelet reactivity in vitro and the antioxidant activity (hypoxanthine/xanthine oxidase test) or phenolic compound content was assessed. Strawberry varieties KYSt-4 (Nohime), KYSt-11 (Kurume IH-1) and KYSt-17 (Kurume 58) showed significant antiplatelet activity both in vitro and, after oral administration, in vivo. Both KYSt-11 and KYSt-17, but not KYSt-4, significantly reduced flow-mediated vasodilation; that is, caused endothelial dysfunction. Antiplatelet activities were heat stable. Significant correlation was found between antiplatelet and antioxidant activities (P=0.049, R=0.23) or total phenolic compounds (P=0.0096, R=0.36). Of the tested strawberry varieties, KYSt-4, KYSt-11 and KYSt-17 showed significant anti-thrombotic effect. The dual mechanism of the effect may involve a direct inhibition of both platelet function and antioxidant activities.

Testing various herbs for antithrombotic effect.

OBJECTIVE: Prevention of arterial thrombotic diseases has high priority in developed countries. Because people who eat inappropriate food are prone to thrombotic events, regular intake of an antithrombotic diet may offer a convenient and effective way of prevention. The aim of the present study was to find herbs that have antithrombotic effects. METHODS: Herbs were crushed and centrifuged and the juice obtained was filtered. The effect of filtrate on platelet-rich thrombus formation was assessed by a shear-induced in vitro platelet function test (hemostatometry). Filtrates showing significant antithrombotic activity were further assessed by using a laser-induced in vivo thrombosis test in mice. The effect of orally administered herb filtrates on flow-mediated vasodilation was also measured. RESULTS: Common thyme and rosemary showed significant antithrombotic activity in vitro and in vivo. Neither herb affected flow-mediated vasodilation. CONCLUSION: Some herbs possess antithrombotic effects in experimental models of thrombosis. The mechanism of the antithrombotic effect of common thyme and rosemary may involve a direct inhibitory effect on platelets.

Anti-thrombotic effect of proanthocyanidin, a purified ingredient of grape seed.

INTRODUCTION: Moderate and regular consumption of wine reduces the risk of acute coronary thrombotic events. The mechanism of the anti-thrombotic effect of wine is not clear. Extract or purified ingredients of grapes have not yet been studied for anti-thrombotic effect. MATERIALS AND METHODS: Anti-thrombotic effect of proanthocyanidin, a highly purified ingredient of grape seed, was assessed by a shear-induced thrombosis test in vitro and by a laser-induced thrombosis test in the mouse carotid artery, in vivo. RESULTS AND CONCLUSIONS: Intravenously (20 mg/kg body weight, BW) or orally (2 x 200 mg/kg BW) administered proanthocyanidin significantly inhibited the laser-irradiation induced thrombus formation in the carotid artery (both P=0.01). Subsequent to oral administration of proanthocyanidin, in vitro platelet reactivity to shear stress has been inhibited. The latter suggests that the in vivo anti-thrombotic effect of proanthocyanidin may be due to a direct inhibitory effect on platelets.

An onion variety has natural antithrombotic effect as assessed by thrombosis/thrombolysis models in rodents.

INTRODUCTION: Prevention of arterial thrombotic diseases has a high priority in developed countries. As inappropriate diet has been shown to be an important risk factor for thrombotic events, regular antithrombotic diet may offer a convenient and effective way of prevention. The aim of the present study was to test onion extracts for antithrombotic effect and to identify the effective varieties in Allium cepa. MATERIALS AND METHODS: A shear-induced platelet function test (haemostatometry) was used to screen for antithrombotic potential. Onion extracts showing significant antithrombotic activity in vitro were further assessed in vivo by using a laser-induced thrombosis test in mice. RESULTS AND CONCLUSIONS: An onion variety, Toyohira, showed significant antithrombotic activity both in vitro and in vivo. Toyohira showed thrombolytic activity in addition to the antiplatelet effect. Superkitamomiji, 2935A, and K83211 showed only thrombolytic activity. Quercetin, the richest flavonoid in onion, was measured, but no correlation was found between quercetin content and antithrombotic activity. It is concluded that onion A. cepa can be classified into varieties with or without antithrombotic and thrombolytic effects. This should be taken into account in future population studies on the antithrombotic effects of vegetables.