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We previously identified seven peptides in serum that are associated with hypertensive disorders of pregnancy (HDP). However, the significance of these peptides in the general population is unknown. The aim of this study was to clarify the relationships of HDP-associated peptides with hypertension and other cardiovascular risks in adult men. We investigated the relationships of peptide levels with cardiovascular risk factors, including adiposity, blood pressure, blood lipids and glycemic status, in men (mean age: 46.4 years) who were receiving annual health checkups at their workplace. The concentrations of the abovementioned seven peptides in serum were measured simultaneously using a mass spectrometer. Among the seven peptides, only a peptide with m/z 2091 (P-2091) derived from fibrinogen-α showed a significant correlation with diastolic blood pressure (Spearman's rank correlation coefficient [r], -0.446). Another peptide with m/z 2378 (P-2378) originating from complement component 4 showed a significant positive correlation with body mass index (r, 0.273) and a significant inverse correlation with HDL cholesterol (r, -0.336). In addition, a peptide with m/z 3156 (P-3156) derived from an inter-α-trypsin inhibitor showed significant inverse correlations with body mass index (r, -0.258) and triglycerides (r, -0.334). There was no significant correlation of the levels of any of the seven peptides with hemoglobin A1c. Among the seven peptides related to HDP, P-2091, P-2378 and P-3156 were inversely associated with diastolic blood pressure, HDL cholesterol and triglycerides, respectively. Therefore, these peptides are possible biomarkers for discriminating cardiovascular risk in a general population.

BackgroundResveratrol has been shown to inhibit platelet aggregation. However, the mechanism for this action of resveratrol remains to be clarified. The purpose of this study was to elucidate the Ca2+-related mechanism for the inhibitory action of resveratrol on platelet aggregation.MethodsCa2+entry and subsequent aggregation of human platelets induced by different stimulants including thrombin, thapsigargin, and 1-oleoyl-2-acetylglycerol (OAG) were measured by the fluorescence method and light transmittance method, respectively. Each stimulant was added to a nominally Ca2+-free medium containing platelets, and then CaCl2was added to the medium to induce Ca2+influx into platelets.ResultsThapsigargin-induced Ca2+entry into platelets and subsequent platelet aggregation were significantly inhibited in the presence of resveratrol at 6.25 μM or higher concentrations, while OAG-induced Ca2+entry and subsequent platelet aggregation were not affected by resveratrol at concentrations up to 50 μM. In the nominally Ca2+-free medium, thrombin induced a small transient increase in intracellular Ca2+concentrations, which was attenuated in the presence of resveratrol at 12.5 μM or higher concentrations. Thrombin-induced Ca2+entry into platelets and subsequent platelet aggregation were significantly inhibited in the presence of resveratrol at 12.5 μM or higher concentrations.ConclusionsThe results suggest that resveratrol inhibits thrombin-induced platelet aggregation through decreasing Ca2+release from its stores and inhibiting store-operated Ca2+influx into platelets.

Background Although ethanol is known to inhibit platelet aggregation, the effects of another variant of alcohol, methanol, have not been reported. The purpose of this study was to determine whether methanol and its metabolite, formic acid, affect Ca2+ entry into and subsequent aggregation of platelets in vitro. Methods Ca2+ entry into and aggregation of human platelets were measured by spectrofluorometry using Fura-2/AM as an indicator and the light transmission method, respectively. Results Thrombin-induced platelet aggregation was significantly augmented by methanol at pharmacological concentrations (0.5–2%) in a concentration-dependent manner. Methanol at 2% significantly attenuated thapsigargin-induced platelet aggregation, which was not significantly affected by lower concentrations (0.5 and 1%) of methanol. Methanol (0.5–2%) did not significantly affect platelet aggregation induced by 1-oleoyl-2-acetyl-sn-glycerol (OAG), or Ca2+ entry into platelets induced by thrombin, thapsigargin, or OAG. Platelet aggregation induced by thrombin, thapsigargin, or OAG was significantly inhibited by formic acid at toxic concentrations (0.01% or higher). Ca2+ entry into platelets induced by thrombin or thapsigargin was also significantly inhibited by formic acid at 0.01% or higher, while that induced by OAG was not affected by formic acid at 0.005 and 0.01% and augmented by that at 0.02%. Conclusions Methanol at pharmacological doses has diverse effects on platelet aggregation, depending on the aggregation stimuli, without affecting Ca2+ entry into platelets. Formic acid at toxic concentrations has an inhibitory action on platelets aggregation, which was partly explained by the reduction of Ca2+ entry into platelets.

The complement system has been proposed to play a significant role in the regulation of T-cell responses. However, the precise mechanism underlying C4-induced immune tolerance remains to be clarified. We recently reported that monomeric C4b inhibits CXCL10 production from blood cells. The purpose of this study was to verify the active site of monomeric C4b.We investigated the in vitro effects of a C4b-derived peptide (VPAGSARPVAFSVVPTAAA), named HP2 (highly homologous peptide 2), on the IFN-β-induced production of CXCL10 in human blood and the in vivo effects of the administration of HP2 on Th1/2 cytokine production in the spleen in mice. We also tested whether the administration of HP2 influences symptoms of experimentally induced ulcerative colitis in mice.HP2 inhibited CXCL10 production in human blood, and the administration of HP2 significantly suppressed the production of Th1 cytokines, such as IL-2, IFN-γ, and TNF-α, in spleen cells isolated from mice. The administration of HP2 in the mice significantly improved the symptoms of colitis, with down-regulation of colitogenic CD4(+)CD45RB(high) T cells and up-regulation of CD4(+)LAP/TGF-β1(+) T cells.The amino acid sequence described above is suggested to be the active site in C4b for the inhibition of Th1 cytokine production. These results should contribute to the development of new drugs suppressing autoimmune responses.

The aim of this study was to clarify the role of diacylglycerol kinase (DGK)γ in vascular endothelial cells. The mRNA and protein expression of DGKγ and inducible nitric oxide synthase (iNOS) in rat aortic endothelial cells (RAECs) were investigated using RT-PCR, immunocytochemical, and immunoblot analyses. In RAECs, immunoreactivity of DGKγ was detected in the cytoplasm as a tubular or reticular structure. DGKγ immunoreactivity colocalized with those for GM130 and Golgin 97 but not with that for protein disulfide isomerase (PDI). In the presence of brefeldin A, DGKγ immunoreactivity was markedly decreased and displayed an aggregation-like pattern. After treatment of RAECs with nocodazole, DGKγ immunoreactivity was detected in Golgi stacks, which were severely segmented and appeared in vesicular shape. Stimulation with IL-1β increased mRNA expression of DGKγ, which was strongly attenuated by SB203580, a p38 MAPK inhibitor. IL-1β also induced expression of iNOS, which was observed as a tubular structure, and this distribution coincided with DGKγ immunoreactivity. Brefeldin A reduced both iNOS immunoreactivity and DGKγ immunoreactivity. iNOS expression was impaired by DGK inhibitors, R59022 and R59949. These results suggest that DGKγ is upregulated by IL-1β through the p38 MAPK pathway and may be involved in protein trafficking of iNOS in vascular endothelial cells.

Alcohol and tobacco are important modifiable risk factors for prevention of atherosclerosis and cardiovascular disease. There is a strong association between habitual alcohol drinking and smoking. Alcohol shows diverse effects on progression of atherosclerosis mainly through its blood pressure–elevating and high-density lipoprotein (HDL) cholesterol–raising actions. In addition to atherogenic actions, smoking modifies associations between alcohol consumption and cardiovascular risk factors. A positive association of alcohol consumption with blood pressure and inverse associations of alcohol consumption with low-density lipoprotein (LDL) cholesterol and non-HDL cholesterol were more prominent in smokers than in nonsmokers. Both in smokers and nonsmokers, HDL cholesterol tended to be higher with an increase in alcohol consumption. Therefore, blood pressure elevation and LDL cholesterol reduction by drinking are suggested to be greater in smokers than in nonsmokers. Further prospective studies on whether and how smoking influences associations of alcohol consumption with atherosclerotic risk factors and cardiovascular events are needed.

The expression pattern of diacylglycerol kinase (DGK) and the biological significance of DGKepsilon in vascular smooth muscle cells were investigated. mRNA expression for DGKalpha, DGKepsilon, and DGKzeta was detected in isolated rat aortic smooth muscle cells (RASMCs) and A7r5 cells by reverse transcription with polymerase chain reaction analysis. An immunocytochemical study revealed intense DGKepsilon in a filamentous pattern, parallel to the long axis of cell, and on actin stress fibers as shown by double-staining with fluorescent phalloidin. DGKalpha was detected sparsely in the cytoplasm and nucleus, and DGKzeta was observed as a granular pattern in the nucleus. In order to elucidate the functional significance of DGKepsilon, its immunoreactivity was examined in RASMCs incubated with serotonin, a vasoconstrictive agonist. When RASMCs were stimulated with serotonin, the cells lost their polarization and shortened, i.e., contracted. In RASMCs contracted by serotonin, DGKepsilon was detected diffusely in the cytoplasm without a filamentous stress fiber pattern. Protein and mRNA expression of DGKepsilon in RASMCs was significantly increased by stimulation with serotonin. Inhibition of Rho-associated kinases by Y-27632 or inhibition of actin polymerization by cytochalasin B resulted in a decrease in the intensity of DGKepsilon immunoreactivity on stress fibers. The results suggest that DGKepsilon interacts with actin stress fibers and is involved in their stability in vascular smooth muscle cells.

Background and aims: Only limited information has been available on the effects of age on the relationship between obesity and other atherosclerotic risk factors, such as blood pressure and serum lipids. The purpose of this study was to investigate the effects of age on the relationships of obesity with blood pressure and serum cholesterol concentrations. Methods: A community-based cross-sectional study was performed on 157,902 workers in Yamagata, Japan. Blood pressure and serum total and HDL cholesterol concentrations were measured, and body mass index (BMI) and atherogenic index were calculated. The correlations of BMI with blood pressure, serum cholesterol concentrations and atherogenic index in different age groups were compared. Results: BMI showed significant positive correlations with systolic and diastolic blood pressures, serum total cholesterol level and atherogenic index, and showed a significant negative correlation with serum HDL cholesterol level. The relationships of BMI with systolic and diastolic blood pressures became weaker with advancing age in both men and women after 30 and 40 years of age, respectively. The relationships of BMI with serum total cholesterol level and atherogenic index also became weaker with advancing age after 30 years of age in men and after 40 years of age in women. There was no age-dependent tendency in the relationship between BMI and HDL cholesterol, however. The above age-dependent changes were more prominent in men than in women. Conclusion: The relationships of obesity with blood pressure, serum total cholesterol level and atherogenic index in the elderly are much weaker than in the young.

Twenty male volunteers 20-25 years old were examined to determine their serum concentrations of interleukin-6 (IL-6), acute phase proteins and lipids before, immediately after and one hour after a load of 90 watts for 20 minutes using a Monark ergometer, and the same parameters were reexamined after eicosapentaenoic acid (EPA) intake of 1.125 g/day for 2 weeks. By EPA intake, EPA level of the membranes of red blood cells increased significantly by 75.6% and docosahexanoic acid (DHA) by 53.8%. The IL-6 level increased significantly by 17% and C-reactive protein (CRP) by 11.7%, but fibrinogen (Fbg) decreased by 9.6%. After EPA intake, at one hour after the load, the change rate of IL-6 decreased to that of before EPA. The change rate of α(1)-acid glycoprotein ( α(1)-AGP) increased in the group in which IL-6 was unchanged and did so significantly in the group in which it increased, but tended to increase in the group in which it decreased. Thus the change rate of sialic acids (SA) increased significantly in both the IL-6 unchanged and increased groups. It is suggested that EPA activated IL-6, which was related to the increase of α(1)-AGP as an activator of immunity. The change rate of sialic acid (SA) as an index of acute phase protiens was correlated significantly and positively with that of total cholesterol and HDL-cholesterol.