Atherosclerosis is believed to be an inflammatory condition of the arterial wall. It has become apparent that various types of cells of innate and adaptive immunity participate in atherogenesis. T cells are of particular interest because they mediate pathogenic immune responses involved in the acceleration of atherosclerosis. Recent studies from several independent groups indicated that subsets of regulatory T cells (Tregs) actively mediate immunologic tolerance and inhibit atherosclerosis development or progression through the down-regulation of effector T-cell responses. It is likely that there is an imbalance between pathogenic effector T cells and Tregs under atherosclerotic conditions. Recent evidence suggests that in addition to the thymus, gut-associated lymphoid tissues are the main sites for the generation of several subsets of peripherally inducible Tregs. This indicates that intervention in the gut environment to promote an endogenous regulatory immune response may serve as a possible therapeutic approach to suppress atherosclerotic diseases. In this review, we discuss not only the possible role of Tregs in the prevention of atherosclerosis, but also promising strategies to prevent or cure atherosclerotic diseases by promoting an endogenous regulatory immune response, particularly by oral immune modulation.

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Morus alba (white mulberry) has been used in traditional Chinese medicine as an anti-headache, diuretic, expectorant, and anti-diabetic agent. In previous studies, extracts of Morus alba demonstrated favorable biological properties, such as antioxidant activity, suppression of lipoxygenase (LOX)-1, cytotoxicity against cancer cells, and inhibition of the invasion and migration of cancer cells.
Aim: This study further evaluated the effects of morusinol, a flavonoid derived from Morus alba root bark, on platelet aggregation and thromboxane B₂ (TXB₂) formation in vitro and thrombus formation in vivo.
Methods: The antiplatelet potential of morusinol was measured using in vitro rabbit platelet aggregation and TXB₂ formation assays. Arterial thrombus formation was investigated using an in vivo ferric chloride (FeCl₃)-induced thrombosis model.
Results: Morusinol significantly inhibited collagen- and arachidonic acid-induced platelet aggregation and TXB₂ formation in cultured platelets in a concentration-dependent manner. Thrombus formation was reduced by 32.1, 42.0, and 99.0% for collagen-induced TXB₂ formation, and 8.0, 24.1, and 29.2% for arachadonic acid-induced TXB₂ formation, with 5, 10, and 30 µg/mL morusinol, respectively. Moreover, oral morusinol (20 mg/kg) or aspirin (20 mg/kg) for three days significantly increased the time to occlusion in vivo by 20.3±5.0 or 6.8±2.9 min, respectively, compared with the control (1% CMC, carboxymethyl cellulose).
Conclusion: Taken together, these results indicate that morusinol may significantly inhibit arterial thrombosis in vivo due to antiplatelet activity. Thus, morusinol may exert beneficial effects on transient ischemic attacks or stroke via the modulation of platelet activation.

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Aim: To evaluate the potential of the lipoprotein-associated phospholipase A₂ (Lp-PLA₂) level as a biomarker in the prediction of prognostic outcome in patients with acute ischemic stroke (IS).
Methods: From October 2008 to March 2010, 130 patients with acute IS were prospectively enrolled in the study and their medical records were reviewed. A blood sample was collected from each patient 48 hours after acute IS, as well as from 20 healthy volunteers as controls. Messenger-RNA (mRNA) expression of Lp-PLA₂ of peripheral-blood mononuclear cells (PBMNCs) relative to that of β actin was measured using quantitative reverse transcription polymerase chain reaction (RT-PCR).
Results: Patients with acute IS exhibited significantly higher Lp-PLA₂ mRNA expression of PBMNCs than the control group (p <0.0001). Lp-PLA₂ mRNA expression of PBMNCs in patients with a major adverse clinical outcome (MACO) (defined as recurrent stroke or death) within 90 days was significantly higher than in patients without MACO (p=0.006). Furthermore, elevated Lp-PLA₂ mRNA expression was strongly associated with old age, diabetes mellitus, a positive history of significant coronary arterial disease and significant stenosis of the extra-cranial carotid arteries (all p <0.04), and positively correlated with the body mass index, leukocyte count, and serum levels of total cholesterol and low-density lipoprotein cholesterol. Multivariate analysis revealed that Lp-PLA₂ mRNA expression of PBMNCs was a significant independent predictor of MACO within 90 days (p= 0.011).
Conclusion: Elevated Lp-PLA₂ mRNA expression of PBMNCs seems to be a potential biomarker for predicting an unfavorable outcome in patients with acute IS.

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Aim: Ezetimibe reduces serum low-density lipoprotein cholesterol (LDL-C) levels by inhibiting intestinal cholesterol absorption; however, the effects of ezetimibe, including its pleiotropic effects, have not been fully clarified. The aims of our study were to examine the efficacy of ezetimibe for hypercholesterolemia and its pleiotropic effects.
Methods: Outpatients with hyper LDL-C levels were treated with 10 mg ezetimibe once a day for 12 weeks. Serum lipid profiles, atherosclerotic and inflammatory markers, glucose, insulin, liver function, and cholesterol absorption and synthesis markers were measured before and after treatment.
Results: Seventy-five patients treated with ezetimibe monotherapy and 16 patients treated with combined therapy of ezetimibe with different statins completed this study. Following 12 weeks of ezetimibe monotherapy, serum LDL-C, triglyceride, remnant-like particles cholesterol, matrix metalloproteinase-9, insulin, homeostasis model assessment of insulin resistance, high-sensitivity C-reactive protein, the relative mobility of the peak of the LDL fraction, and cholesterol absorption markers were significantly decreased, and high-density lipoprotein cholesterol and lathosterol were significantly increased. In the combined therapy group, LDL-C and cholesterol absorption markers were significantly decreased after treatment. In patients with a high basal ALT level that were treated with monotherapy, ALT and the AST/ALT ratio were significantly decreased and increased, respectively, after treatment.
Conclusion: Ezetimibe ameliorated not only atherogenic lipid profiles but also atherosclerotic and inflammatory markers, insulin sensitivity, and liver dysfunction in Japanese hypercholesterolemia patients, suggesting that ezetimibe treatment is a beneficial and effective strategy for the treatment of hypercholesterolemia, especially patients with obesity, metabolic syndrome, and/or fatty liver.

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Aim: In subjects with metabolic syndrome (MetS) endothelial dysfunction is a very consistent finding. Processes leading to endothelial dysfunction and atherosclerosis involve the altered control of subclinical inflammation by innate immune defenses that possibly include mannose-binding lectin (MBL). We investigated the associations of MBL with traits of MetS and early atherosclerosis in obese subjects before and after marked weight reduction.
Methods: In a prospective longitudinal study, MBL concentrations of 96 severely obese subjects with and without MetS (Ø BMI with MetS 41.0±7.9kg/m², Ø BMI without MetS 39.4±7.7kg/m²) were examined in association with markers of insulin resistance, dyslipidemia, adipokines, and subclinical atherosclerosis before and after marked weight loss (Ø weight loss 20±8 kg after 3 months of participation in a standardized weight reduction program), in addition to the study of 25 seemingly healthy lean subjects (BMI 20-25kg/m²).
Results: MBL concentrations did not differ between healthy lean and severely obese subjects independently of the presence of metabolic abnormalities. In severely obese subjects there was no significant difference concerning the cardiovascular risk profile, apolipoproteins, inflammatory and metabolic parameters, and markers of endothelial dysfunction and atherosclerosis between subjects with functional MBL deficienct (MBL<778ng/mL) and MBL sufficient (MBL≥778ng/mL) obesity. Marked weight loss did not influence MBL levels.
Conclusions: Our findings suggest that plasma levels of MBL did not differ between healthy lean and severely obese subjects. MBL did not affect cardiovascular risk factors, or markers of endothelial dysfunction and early atherosclerosis in severely obese patients before and after marked weight loss.

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Aim: Atherosclerotic disease is a major health problem among the elderly, which arises from a complex interaction between genetic and environmental factors. The catechol-O-methyltransferase (COMT) gene encodes an enzyme that degrades catecholamines and estrogens to less active metabolites. The objective of this study was to examine whether polymorphisms of the COMT gene affected the severity of atherosclerotic disease in a Japanese elderly population.
Method: A total of 1536 autopsy cases of hospital deaths were assessed for the degree of pathological atherosclerotic index (PAI), coronary stenotic index (CSI) and intracranial stenotic index (ICAI), which were obtained by macroscopic examination of the luminal surface of formalin-fixed arteries. Two single nucleotide polymorphisms (SNPs) in the COMT gene, rs4633 (C/T) and rs4680 (G/A) were genotyped. The rs4680 (G/A) corresponds to a functional SNP with the substitution of valine to methionine.
Result: The CC genotype of rs4633 (C/T) and the GG genotype of rs4680 (G/A) showed a significantly higher degree of PAI and the association remained positive after adjustment for age, hypertension, diabetes, smoking and drinking (p=0.035 and p=0.031, respectively). There were no significant associations between COMT genotypes and CSI or ICAI. When male and female subjects were analyzed separately, the association was observed only in female subjects (p=0.012 and p=0.027) after adjustment for age, hypertension, diabetes, smoking and drinking.
Conclusion: The functional SNP in the COMT gene associated with the severity of atherosclerosis in a Japanese elderly population, whereby the influence of the genotype appears to be stronger in females than in males.

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Aim: Dual antiplatelet therapy (DAPT) with aspirin and clopidogrel is essential after percutaneous coronary intervention (PCI). Clopidogrel is a prodrug and changed into active metabolite by cytochrome p450 enzymes (CYPs), especially CYP2C19. Proton pump inhibitors (PPIs) are used for the prevention of aspirin-induced gastrointestinal bleeding. PPIs are also metabolized by CYP2C19, although the degree of its contribution is dependent on the kind of PPI. Omeprazole, a PPI, has been reported to weaken the antiplatelet effects of clopidogrel. Famotidine, a histamine receptor type 2 (H2) blocker, could also be an alternative to PPIs. The aim of this study was to evaluate the effects of PPIs and an H2 blocker on the antiplatelet function of clopidogrel.
Methods: Patients receiving DAPT due to prior PCI, who took either omeprazole or rabeprazole, were enrolled (n=25). The initial PPI was changed to the other PPI as a crossover study. In another study, patients undergoing DAPT without taking PPIs or H2 blockers were enrolled (n=30) and famotidine was added.
Results: Platelet aggregability when taking omeprazole was higher than when taking rabeprazole, evaluated by an optical aggregometer using collagen as a stimulus (p=0.0051) and by the VerifyNow P2Y12 assay (p=0.0060). Platelet aggregability when taking rabeprazole was comparable to that in control patients (n=15). Concomitant use of famotidine had no effect.
Conclusion: Omeprazole significantly reduced the antiplatelet effect of clopidogrel and this effect on clopidogrel was stronger than that of rabeprazole. Concomitant use of famotidine had no effect on the antiplatelet effect of clopidogrel.

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Aim: The cardio-ankle vascular index (CAVI) reflects overall arterial stiffness from the aorta to the ankle, independent of blood pressure. We aimed to investigate the association of fat burden assessed by visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT) and epicardial adipose tissue (EAT) with CAVI in an asymptomatic population.
Methods: A total of 260 asymptomatic Korean individuals who had CAVI, abdominal computed tomography (CT) and coronary CT were evaluated retrospectively. The VAT, SAT, EAT and SAT to VAT ratio (SVR) were measured and assessed for correlation with CAVI.
Results: Different fat compartments showed different correlations with arterial stiffness as assessed by CAVI. The amount of fat measured by VAT (r= 0.129, p= 0.037), EAT (r=0.193, p= 0.002) and SVR (r=−0.168, p= 0.007) showed a significant correlation with CAVI, whereas the amount of total abdominal fat and SAT did not (p= 0.261 and p= 0.434 respectively). From step-wise multivariate regression analysis including age, pulse pressure, fasting blood sugar level, VAT, SVR and EAT, EAT (p= 0.036) and age (p<0.001) showed significant associations with CAVI. When quartiles of CAVI were assessed, EAT showed serial increment, whereas SVR showed a stepwise decrease from the first quartile to fourth quartile of CAVI (p=0.041).
Conclusion: VAT, EAT and SVR, which reflect metabolic risk, have shown significant correlations with arterial stiffness measured by CAVI. EAT showed an independent association with arterial stiffness after adjusting for covariables by multivariable correlation analysis. Among the different parameters reflecting fat burden, EAT showed the strongest correlation with CAVI.

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Aim: Alanine aminotransferase (ALT) and γ-glutamyltransferase (GGT) are associated with insulin resistance and arteriosclerotic disease. Since adiposity raises liver enzyme levels and causes insulin resistance, adipocytokines are thought to underlie the relationship between liver enzymes and insulin resistance. To clarify this hypothesis, we conducted a cross-sectional epidemiological study in a Japanese general population.
Methods: The study subjects were 903 middle-aged to elderly persons. Plasma levels of adiponectin and leptin were measured, while other clinical parameters were obtained from personal health records of medical check-ups. Insulin resistance was assessed by a homeostasis model assessment index (HOMA-IR).
Results: Plasma levels of ALT (r=0.379, p<0.001), GGT (r=0.225, p<0.001), adiponectin (r= −0.346, p<0.001) and leptin (r=0.369, p<0.001) were significantly correlated with insulin resistance even on subgroup analysis by sex. Further, any combination of liver enzymes and adipocytokines was synergistically associated with insulin resistance (p<0.001) after adjustment for possible covariates (ALT*adiponectin: β=−0.098, p<0.001, ALT*leptin: β=0.129, p<0.001, GGT*adiponectin: β=−0.054, p=0.054, GGT*leptin: β=0.126, p<0.001); however, in simple obese subjects with normal adipocytokine levels, liver enzymes were not associated with insulin resistance (mean HOMA-IR: worsened adipocytokine +/visceral obesity +, 2.01±1.14; +/−, 1.39±0.84; −/+, 1.23± 0.55; −/−, 1.03±0.57; p<0.001).
Conclusion: Plasma levels of ALT and GGT were independent determinants of insulin resistance only in subjects with a worsened adipocytokine profile. Use of liver enzyme levels as a marker of insulin resistance requires stratification by adipocytokine profile.

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