Takako Tomita

Green tea polyphenols (flavan 3-ols) prevent oxidative modification of low density lipoproteins: an ex vivo study in humans

Oxidation of low density lipoprotein (LDL) plays crucial roles in atherogenesis. We previously reported that green tea polyphenols (flavan 3-ols), especially epigallocatechingallate (EGCg) and epicatechingallate, exerted potent inhibitory effects on LDL oxidation in vitro. To examine whether intake of green tea polyphenols renders LDL resistant to ex vivo oxidation in humans, 22 male volunteers aged between 22 and 32 years were recruited and assigned the same dietary regimen for 2 weeks. After a 1-week baseline period, they were equally divided into two groups: control and tea. The tea group ingested 300 mg of green tea polyphenol extract twice daily for 1 week. Plasma EGCg concentration at the end of the experiment was 56 nmol/L on average (56% in free form) in the tea group; no EGCg was detected before the experiment. Plasma concentrations of lipids, ascorbate, alpha-tocopherol, and lipid peroxides did not change before and after the experiment in either group, but beta-carotene was higher in the tea group (P< 0.01 by paired Studentst-test). LDL (0.1 mg/mL) was incubated with 5 microM Cu(2+) and the oxidation was measured by absorbance at 234 nm. The lag time was significantly prolonged by 13.7 min in the tea group (P < 0.05 by paired Studentst-test, before versus after), whereas such a change was not observed in the control group. These results suggest that daily consumption of seven to eight cups (approximately 100 mL each cup) of green tea may increase resistance of LDL to in vivo oxidation, leading to reduction in the risk of cardiovascular diseases.

Antiatherosclerotic effect of the edible mushrooms Pleurotus eryngii (Eringi), Grifola frondosa (Maitake), and Hypsizygus marmoreus (Bunashimeji) in apolipoprotein E-deficient mice

In the present study, we examined the antiatherosclerotic effects of 3 edible mushrooms, Pleurotus eryngii (Eringi), Grifola frondosa (Maitake), and Hypsizygus marmoreus (Bunashimeji), in atherosclerosis-susceptible C57BL/6J, apolipoprotein E-deficient (apoE(-/-)) mice. Male apoE(-/-) mice (6 weeks of age) were fed a normal diet (cholesterol concentration <66 mg/100 g) or a normal diet containing 3% dried Eringi, Maitake, or Bunashimeji mushroom powder for 10 weeks. Food intake, body weight, serum total cholesterol (TC), and serum triacylglycerols (TG) were measured periodically during the experimental period. At the end of the experiment (at 16 weeks of age), the atherosclerotic lesion area was measured in cross-sections of the aortic root. Serum TC concentrations in the Bunashimeji group were significantly lower than that in the control group at 8, 10, 12, 14, and 16 weeks of age. Serum TC concentrations in the Eringi, and Maitake groups were significantly lower than that in the control group only at 12 weeks of age. There was no significant difference in the serum TG concentrations in all groups during the experimental period. The atherosclerotic lesions were significantly decreased in the Eringi, Maitake, and Bunashimeji groups than that in the control group at the end of the experiment. Dietary supplementation with the Bunashimeji mushroom powder had the strongest antiatherosclerotic effect among 3 mushrooms. In conclusion, supplementation of the 3 edible mushrooms prevents the development of atherosclerosis, even normal diet. Antiatherosclerotic effect is partly via lowering of serum TC concentrations; further mechanisms should be investigated.

Concurrent formation of peroxynitrite with the expression of inducible nitric oxide synthase in the brain during middle cerebral artery occlusion and reperfusion in rats.

Peroxynitrite is assumed to play a crucial role in brain damage associated with the overproduction of nitric oxide (NO). The purpose of this study is to examine time-dependent changes of nitrite and nitrate (NOx) concentration in the circulation, and peroxynitrite formation as well as the expression of inducible nitric oxide synthase (iNOS) in the penumbra of rat brains during transient middle cerebral artery occlusion (MCAO) of Wistar rat for 2 h and reperfusion for 4-70 h. NOx concentration in the circulation was continuously monitored at the right jugular vein by microdialysis. The expression of iNOS was detected at 22-70 h after reperfusion in vascular walls and the cortex. Nitrotyrosine, a marker of peroxynitrite, appeared 4 h after reperfusion in the cortex, increasing substantially at 22-46 h in vascular walls. NOx level in dialysate increased immediately after MCAO. After a gradual decrease, the level increased again 4 h after reperfusion, reaching a maximum at 46 h. Brain myeloperoxidase activity, a marker of neutrophil infiltration, was not detected 4 h after reperfusion, but greatly increased at 22 h and then decreased. These results suggest that a marked increase of NOx level in the circulation might reflect the expression of iNOS, while neuronal NOS may contribute to peroxynitrite formation in the cortex observed at an earlier phase of reperfusion. This study indicates that monitoring NOx level in the circulation serves to assess the progress of stroke, and to determine appropriate therapeutic measures.

Basic aggregation properties of washed rat platelets: Correlation between aggregation, phospholipid degradation, malondialdehyde, and thromboxane formation

The basic characteristics of washed rat platelets in aggregation and biochemical responses to thrombin and collagen were investigated. Maximum aggregation response to thrombin and collagen was observed in the presence of 1-3 mM extracellular CA2+. Breakdown of endogenous phospholipids was followed by HPTLC. Phosphatidylinositol degraded most rapidly and greatly, followed by phosphatidylethanolamine, and phosphatidylcholine. Sphingomyelin did not change on stimulation. Formation of malondialdehyde was closely associated with thrombin- and collagen-induced aggregation within the range of the concentration employed. In addition, it was correlated with TXB2 formation and phosphatidylinositol degradation. These results indicated that, despite known species variation, thrombin-induced aggregation is mediated by TXA2 and malondialdehyde formation reflects both cyclo-oxygenase and thromboxane synthase activities, and phospholipid degradation in rat platelets as well as in human platelets.

Rapid and reversible inhibition by low density lipoprotein of the endothelium-dependent relaxation to hemostatic substances in porcine coronary arteries. Heat and acid labile factors in low density lipoprotein mediate the inhibition.

The effects of hemostatic substances on the vascular tone in porcine coronary arteries and the influence of low density lipoprotein on tension were investigated. Thrombin induced a marked concentration-dependent relaxation in prostaglandin F2 alpha-precontracted strips with intact endothelium, whereas it produced a modest constriction in endothelium-denuded arteries. Methylene blue abolished the relaxation, but indomethacin did not affect it significantly. An exposure of the intact strips to low density lipoprotein resulted in a marked inhibition of the relaxation to thrombin but did not interfere with vasodilation by sodium nitroprusside. The inhibition by low density lipoprotein was reversed completely by washing. In contrast, high density lipoprotein lacked such inhibitory effects. Adenosine diphosphate, calcium ionophore A23187, and platelet-activating factor also produced relaxation in the intact strips. An exposure of the strips to low density lipoprotein almost abolished relaxation to these substances. The inhibition was also reversible. Heat treatment or acid treatment of low density lipoprotein resulted in a complete loss of the inhibitory effects, but diisopropyl fluorophosphate treatment did not alter the effect. It is concluded that low density lipoprotein may play a new pathological role in promotion of coronary vasospasm through rapid and reversible inhibition in endothelium-dependent relaxation to hemostatic substances.

Antiatherogenic Effects of a Novel Lipoprotein Lipase-Enhancing Agent in Cholesterol-Fed New Zealand White Rabbits

Following our report that administration of 4-diethoxyphosphorylmethyl-N-(4-bromo-2-cyanophenyl) benzamide (NO-1886) to rats elevated postheparin lipoprotein lipase (LPL) activity through an increase in the enzyme mass, we now investigate antiatherogenic effects of NO-1886 in cholesterol-fed New Zealand White rabbits. For 20 weeks, four groups of male rabbits received regular rabbit chow (the normal control), 0.25% cholesterol-containing chow (the control), and cholesterol chow supplemented with 0.5% and 1.0% NO-1886, respectively. Postheparin LPL activity at week 10 was raised by 30% in 0.5% of the NO-1886 group and 40% in 1.0% of the NO-1886 group compared with those in the control. The area under the curve of plasma cholesterol level was not different in three cholesterol-fed groups whereas the area under the curve of HDL cholesterol was approximately twofold greater in the two NO-1886 groups than in the control, and the area under the curve of plasma triglyceride was reduced to the level of the normal control. LPL activity was correlated with HDL cholesterol (r = .764, n = 18) and triglyceride (r = -.627, n = 18). Relative atheromatous area, aortic cholesterol, and triglyceride contents were reduced to approximately 25%, 60%, and 55%, respectively, of the control values by NO-1886 ingestion. Multiple regression analysis of LPL, HDL cholesterol, and triglyceride indicated that HDL cholesterol was the most powerful protector against aortic cholesterol accumulation, and triglyceride was the one to protect against the atheromatous area. We concluded that NO-1886 prevented the development of atherosclerosis through increasing LPL activity with a consequent increase in HDL cholesterol and a decrease in triglyceride without a significant influence of plasma cholesterol level.

Mechanism of pathophysiological effects of diesel exhaust particles on endothelial cells.

The suspension of diesel exhaust particles (DEP) inhibited endothelium-dependent relaxation (EDR). The mechanism of the impairment of EDR by DEP was investigated with cultured porcine endothelial cells (PEC) and NO synthase (NOS) cell free system. Incubation of PEC with DEP (50-150 μg/ml) for 10-30 min did not induce cell damage. Bradykinin-induced endothelium-dependent relaxing factor (EDRF) release from PEC was bioassayed by cyclic GMP formation in RFL-6 cells. A 10-min preincubation of PEC with DEP (0.1-100 μg/ml) inhibited EDRF release. NOS activity from rat cerebellum cytosol was measured either by the conversion of 3H-l-arginine to (3)H-l-citrulline or the NO(2)(-) formation. A 10-min preincubation of NOS with DEP (0.1-100 μg/ml) did not affect the formation of (3)H-l-citrulline. In contrast, it inhibited NO(2)(-) formation. These results suggest that DEP neither induced cell damage nor inhibited EDRF release from PEC, but DEP scavenged NO to block its physiological action.

Hypoaggregability of washed platelets from stroke-prone spontaneously hypertensive rats (SHRSP).

The aggregation properties of washed SHRSP platelets were investigated in comparison with normotensive WKY platelets at prehypertensive (4 weeks), early hypertensive (11 weeks) and late hyperten- sive (17 weeks) ages in the absence of plasma factors. The number of platelets in SHRSP was markedly lower with the development of hypertension than that in WKY. The thrombin- and collagen-induced aggregation was markedly reduced in the platelets from 11 and 17 week old SHRSP compared with that of age-matched WKY, whereas the degree of platelet aggregation in 4 week old SHRSP showed a tendency to be even greater than that in WKY. The changes in blood pressure and platelet aggregability were correlated inversely. ADP did not induce aggregation in the same system used for thrombin and collagen stimulation but in another system it aggregated washed rat platelets. Aggregation responses to ADP and ionophore A23187 were also significantly lower in 14 week old SHRSP platelets than age-matched WKY platelets. Together with other evidence, these results suggest that defective Ca2+ function, rather than the presence of exhausted platelets, is responsible for hypoaggregability in SHRSP platelets. Stroke Vol 15, No I, 1984

Inhibition of cholesterylester accumulation by 17β-estradiol in macrophages through activation of neutral cholesterol esterase

Premenopausal women are at a lower risk of coronary heart disease relative to age matched men. However, the underlying mechanisms are not clearly understood. This article studies the effects of 17 beta-estradiol (17 beta-E2) at physiological concentrations on the cholesterylester metabolism in macrophages (J774 A.1 cells) with a particular focus on neutral cholesterol esterase (N-CEase). Cells were incubated with beta-VLDL, [1-14C]oleic acid and 17 beta-E2 (0.25 and 2.5 nM). 17 beta-E2 dose-dependently reduced cholesteryl-[1-14C]oleate (14C-CO) at 36 h and 48 h relative to the control. It also stimulated hydrolysis of 14C-CO in foam cells on 36 h and 48 h incubation. In addition, 17 beta-E2 markedly increased N-CEase activity at 24 h and 36 h. This increase preceded the enhanced hydrolysis of cholesterylester, 17 alpha-E2 (inactive isomer), estrone and estriol had no stimulatory action on N-CEase, whereas progesterone and testosterone inhibited it. 17 beta-E2-treatment (24 h) increased the activity of cyclic AMP-dependent protein kinase (A-kinase). DEAE-cellulose column chromatography revealed that an isoform (type II) of A-kinase appeared in 17 beta-E2-treated cells in addition to type I of A-kinase found in the control cells. These results suggest that inhibition of cholesterylester accumulation in macrophages by 17 beta-E2 is mediated by an enhancement of N-CEase activity possibly through an increase in A-kinase.

ENHANCEMENT OF ENDOTHELIUM-DEPENDENT RELAXATION IN THE AORTA FROM STROKE-PRONE SPONTANEOUSLY HYPERTENSIVE RATS AT DEVELOPMENTAL STAGES OF HYPERTENSION

1. Endothelium‐dependent relaxation (EDR) in aortic rings from young (8 weeks) and adult (16 weeks and 20 weeks) stroke‐prone spontaneously hypertensive rats (SHRSP) was investigated in comparison with age‐matched Wistar‐Kyoto rats (WKY).