Recent epidemiologic studies have revealed that hypertriglyceridemia is associated with atherosclerosis independent of other coronary risk factors. However, it is difficult to select patients at high risk for coronary artery disease using only serum triglyceride levels compared with low-density lipoprotein cholesterol levels since multiple factors are associated with elevating triglycerides. Atherosclerotic diseases with high triglyceride levels can be found in patients with familial combined hyperlipidemia, diabetes mellitus, and metabolic syndrome, in which remnant lipoproteins accumulate in the circulating blood. Recent researches have paid attention to remnant lipoproteins as atherogenic particles with the development of methods for measuring remnant cholesterol levels and apolipoprotein B-48 levels directly from human serum. Measurement of these parameters in addition to serum triglycerides may help to distinguish high-risk patients and enable us to prevent or suppress the progression of atherosclerotic diseases in those patients. However, questions remain to be answerd to evaluate the significance of remnant lipoproteins. Here, we focus on three issues: the underlying problems in measuring remnant lipoprotein cholesterol, the assessment of postprandial hyperlipidemia as an atherogenic condition, and finally a review of our experimental and clinical findings about the mechanisms by which remnant lipoproteins induce atherosclerosis.

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Aim: In this study, transnasal endoscopy was used to observe the time-course changes of gastrointestinal mucosa with low-dose aspirin (ASA), and the preventive effect of rebamipide was compared with placebo.
Methods: Twenty healthy H.pylori-negative subjects were divided between those receiving 100 mg aspirin with placebo, and those receiving 100 mg aspirin+300 mg rebamipide for 7 days daily. Transnasal endoscopy was performed at 0, 2, 6 and 24 hrs on the first day, and then on the third and seventh days.
Results: Ulcers, in the duodenum at 24 hrs and in the antrum at 72 hrs, improved with continuous ASA. Erosions were mainly observed in the duodenum; erosions amounted to 14 at 3 days and 19 at 7 days in the placebo group. No ulcers and some erosions were mainly observed in the duodenum; erosions amounted to 5 at 3 days and 3 at 7 days in the rebamipide group.
Conclusion: In short-term gastrointestinal damage induced by ASA, damage was observed in the duodenum most frequentiy, and peak damage was at 24hrs and 72 hrs. Almost all damage improved gradually in spite of continuous ASA. Rebamipide reduced the damage of low-dose aspirin-induced GI complications.

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Aim: Angiotensin (Ang) II-induced abdominal aortic aneurysm (AAA) in apoE-deficient mice has been used as a model of human AAA, but it has been unclear why the progression of AAA continues after stopping the Ang II infusion. The involvement of vascular Ang II-forming enzymes in the progression of AAA was studied.
Methods: ApoE-deficient mice were infused with Ang II (1,000 ng/kg/min) for 4 weeks and evaluated until 20 weeks after the Ang II infusion. Just after and 20 weeks after stopping the Ang II infusion, the degree of AAA, as well as the ACE and chymase activities, was evaluated. An Ang II receptor blocker (candesartan, 30 mg/kg/day) and an angiotensin-converting enzyme (ACE) inhibitor (lisinopril, 60 mg/kg/day) were given for 20 weeks after stopping the Ang II infusion.
Results: The aortic diameter expanded just after stopping the Ang II infusion and progressed for a further 20 weeks after the infusion was stopped. Just after stopping the infusion, aortic ACE and chymase activities were significantly increased, but only the increase in chymase activity continued until 20 weeks after the infusion was stopped. Candesartan and lisinopril significantly attenuated aortic diameter expansion.
Conclusion: The increases in vascular Ang II-forming activities were involved in the progression of AAA after stopping the Ang II infusion.

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Aim: The effect of statins in preventing cardiac events in Japanese coronary artery disease (CAD) patients was studied in a retrospective investigation of 148 patients diagnosed with CAD by coronary angiography (CAG).
Methods: Sixty-five patients received statins within 2 weeks after CAG, and 83 patients did not receive statins after CAG.
Results: In the statin group, total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) were reduced significantly with statin administration (p<0.01). In the non-statin group, baseline levels of TC, LDL-C and high-density lipoprotein cholesterol were not changed significantly at the end of the follow-up period. As for the effect of statin in preventing cardiac events, the incidence of cardiac events was significantly lower (p<0.0003) in the statin group (n=5: 8%) than in the non-statin group (n=28: 34%). In subanalysis of 37 patients whose TC at the time of initial CAG was less than 200 mg/dL, none of the statin group (n=17) suffered a cardiac event. This was significantly lower than the incidence of cardiac events in the non-statin group (n=5: 25%; p<0.05).
Conclusion: The present study demonstrated that lowering LDL-C of Japanese CAD patients by statin administration is effective to prevent cardiac events, particularly, a second percutaneous coronary intervention (PCI) for restenosis of a coronary artery following the initial PCI whether or not these patients had hypercholesterolemia.

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Aim: Human urotensin-II (UII) is a cyclic neuropeptide with potent vasoconstrictive activity in the vasculature. The expression of UII and its receptor (UT) mRNA is detected at high levels in the brain. We evaluated the relationship between plasma UII levels and vascular dementia (VaD) caused by stroke or atherosclerotic small vessel disease.
Methods: Carotid artery intima-media thickness (IMT), plaques, plasma levels of immunoreactive UII (IR-UII), and atherosclerotic biomarkers were determined in 42 patients with VaD, 197 with Alzheimer's disease (AD), and 47 non-demented elderly controls.
Results: Age, gender, body mass index, systolic blood pressure (SBP), fasting plasma glucose, insulin, triglycerides, high-density lipoprotein cholesterol, leptin, and plasminogen activator inhibitor-1 levels were not significantly different among these groups. IR-UII, low-density lipoprotein (LDL) cholesterol, lipoprotein(a), lipid peroxides, interleukin-6, and high-sensitive C-reactive protein (hs-CRP) levels, and maximum IMT were significantly higher in VaD than in AD patients or controls. IR-UII level showed a significantly positive correlation with SBP or maximum IMT. Multivariate logistic regression analysis revealed a significantly independent association between IR-UII levels or increased maximum IMT (≥1.1 mm) and VaD as compared with SBP, LDL cholesterol, and interleukin-6 levels.
Conclusion: Increased plasma IR-UII levels and carotid atherosclerosis may be involved in the pathogenesis and progression of VaD.

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Aim: Lipoprotein lipase (LPL) plays an important role in lipid metabolism. There is an association between the common S447X polymorphism in the LPL gene and high-density lipoprotein cholesterol (HDL-C) levels, and some association between circulating HDL-C and adiponectin levels has been suggested; however, it is not known whether there is any association between the S447X polymorphism and adiponectin levels. The aim of this study was to investigate whether the LPL S447X polymorphism was associated with adiponectin in the general population.
Methods: LPL S447X was analyzed in 277 community-dwelling subjects (123 men and 154 women, mean age; 65±13 years) in the Mima study. Whole samples were genotyped using a fluorescent allele-specific DNA primer assay system. The allele frequencies and any associations with serum lipid and adiponectin levels were investigated.
Results: The allele frequencies were S=0.875 and X=0.125 for the LPL S447X polymorphism. The carriers of the X allele had significantly higher levels of adiponectin and HDL-C than non-carriers. The presence of the X allele was significantly associated with higher adiponectin levels, independent of age, sex, body mass index, smoking and HDL-C in multiple regression analyses.
Conclusion: The LPL S447X polymorphism might therefore be significantly associated with higher adiponectin levels, independent of HDL-C.

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Aim: ATP-binding cassette transporter G1 (ABCG1) is a cholesterol transporter that plays a role in cholesterol efflux in the presence of high-density lipoprotein (HDL). Moreover, HDL-mediated cholesterol efflux is increased in cultured ABCG1 overexpressed cells; however, the physiological roles of ABCG1 and its contribution to atherosclerosis in humans remain unclear.
Methods: The effect of ABCG1 -257T>G mutation on transcription activity was determined by a reporter assay. One hundred nine Japanese men with coronary artery disease (CAD) were analyzed. ABCG1 -257T>G polymorphism was assessed by mutation-selective PCR methods to identify T/T, T/G, and G/G genotypes.
Results: The reporter assay showed that ABCG1 transcription activity was significantly lower (p<0.01) in the G allele of -257T>G polymorphism compared with that in the T allele. Clinically, there were no significant differences in serum triglyceride and total, LDL, and HDL cholesterol levels, or other risk factors. Logistic regression analysis revealed significant additive and dominant effects on the frequency of patients with multi-vessel disease compared with single-vessel disease (T/T vs. T/G vs. G/G, odds ratio:2.1, p=0.027; T/T vs. T/G and. G/G, odds ratio:3.5, p=0.005).
Conclusion: This is the first report to show that a novel ABCG1 -257T>G promoter polymorphism influences CAD severity in Japanese men.

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Aim: We previously identified a quantitative trait locus (QTL) on rat chromosome 5 that appeared to be primarily controlled by the sympathetic nervous system. Because sympathetic overactivity is related to hypertension, solute carrier family 6, member 9 (SLC6A9) is a candidate gene for the connection of this QTL with blood pressure regulation. In the present study, we therefore explored the role of SLC6A9 genetic variations in human essential hypertension (EH).
Methods: We evaluated three single nucleotide polymorphisms (SNPs) (rs2286245, rs3791124 and rs2486001) in 758 essential hypertension patients and 726 controls. Polymorphism-related genotypes were determined with TaqMan assays.
Results: The allelic frequency of rs2286245 (C versus T, p=0.032) showed significant differences between EH and normotensive controls (NT) groups. The genotypic distribution of rs3791124 in its dominant model (AA+GA versus GG, p=0.027) also showed significant differences between EH and NT groups. The genotype and allele distributions of rs2486001 did not exhibit any significant differences.
Conclusion: We found an association between SLC6A9 gene polymorphisms and essential hypertension in a Japanese population, suggesting that SLC6A9 is a susceptibility locus for essential hypertension.

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Aim: The objective of this study was to investigate the relation between serum high molecular weight (HMW)-adiponectin concentration and fasting and postprandial blood glycemic and lipid parameters in type 1 diabetic patients.
Methods: Type 1 diabetic patients treated with short-acting insulin analogs and healthy volunteers were recruited. The subjects were divided into two groups based on the mean HMW-adiponectin value of 12.2 mg/L: low HMW-adiponectin (men/women=7/2) and high HMW-adiponectin (men/women=3/8). Blood samples were collected after an overnight fast, and 30180 min after consuming white bread (B) or white bread with butter (BB).
Results: Type 1 diabetic patients with high HMW-adiponectin had lower triglyceride and remnant like particle (RLP)-triglyceride concentrations than those with low HMW-adiponectin (p<0.01), and had a higher lipoprotein lipase mass (p<0.01) than healthy subjects (men/women=8/6). After B and BB meals, type 1 diabetic patients with high HMW-adiponectin consistently had lower triglyceride and RLP-triglyceride concentrations for up to 180 min than those with low HMW-adiponectin (p<0.01); however, apoB48 did not differ between these two groups. Fasting and postprandial plasma glucose were higher in both diabetic groups than in healthy subjects (p<0.001), with no significant difference between patient groups.
Conclusion: HMW-adiponectin is more strongly associated with very low density lipoprotein remnant metabolism than glucose utilization in type 1 diabetic patients receiving short-acting insulin analogs.

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Aim: A point mutation of apolipoprotein A-I, V156E, is naturally occurring and is found in Oita, Japan. V156E of apoA-I (apoA-Ioita) shows unique structural and functional properties and more potent antioxidant activity against copper-mediated low-density lipoprotein (LDL) oxidation in vitro.
Methods: A reconstituted high-density lipoprotein (rHDL)-containing V156E (V156E-rHDL) suppressed the accumulation of [3H]-cholesteryl ester (CE)-LDL in THP-1 cells. To compare the short-term anti-atherosclerotic effects of V156E and wild-type (WT) apo A-I, palmitoyloleoyl phosphatidylcholine (POPC)-rHDL-containing V156E or WT was infused into atherosclerotic apo-E-deficient mice. Each POPC-rHDL was injected via the tail vein at a dosage of 150 mg/kg body weight in 0.4 mL Tris-buffered saline (TBS), and blood was collected 24 and 48 hours post-injection.
Results: Serum interleukin (IL)-6 levels were decreased 28% in the V156E-rHDL group 48 hours post-injection. Injection with V156E-rHDL induced a reduction of the lipid-stained area in lesions by 26% and the number of macrophages detected in the lesions was decreased by 40% compared to with WT-rHDL injection. Serum lecithin:cholesterol acyltransferase and paraoxonase activity was enhanced in V156E-rHDL-injected mice. The antioxidant ability in the ferric reduced ability serum assay was also increased in the V156E group at both 24 and 48 hours post-injection, with a decrease of serum lipid hydroperoxide concentration.
Conclusion: Blood infusion of V156E-rHDL resulted in potent lesion regression activity and enhanced anti-inflammatory and antioxidant activities in apo-E deficient mice compared to the WT-rHDL group. These results provide evidence suggesting that V156E-apoA-I may be a good candidate for HDL therapy.

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Aim: Calcium channel blockers (CCBs) inhibit the migration of vascular smooth muscle cells (VSMC) by mechanisms that remain poorly understood. The purpose of the present study was to characterize the signaling mechanisms by which CCBs inhibit VSMC migration.
Methods and Results: Nifedipine potently inhibited platelet-derived growth factor (PDGF)-induced chemotaxis, collagen I-induced haptotaxis, and wound-induced migration of human aortic VSMC. In addition, nifedipine inhibited PDGF-induced membrane ruffling and lamellipodium formation. PDGF-induced VSMC migration was significantly inhibited by PP2, a selective inhibitor of the Src kinase family, and was also significantly inhibited by the expression of kinase-inactive Src, suggesting that Src is required for VSMC migration. Nifedipine also inhibited PDGF-induced Src activation (by 60±4% with 30 µM) and tyrosinephosphorylation of Cas, paxillin, and cortactin, which are actin-associated substrates of Src. RNA interference-induced knockdown of the Ca²⁺-dependent tyrosine kinase, Pyk2, resulted in inhibition of PDGF-induced Src activation and migration. Finally, nifedipine inhibited PDGF-induced Pyk2 activation in a dose-dependent manner.
Conclusion: These data suggest that nifedipine interferes with VSMC migration via inhibition of the Pyk2-Src axis and inhibition of actin remodeling processes, including membrane ruffling and lamellipodium formation.

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Aim: Equol is the main active product of daidzein metabolism, produced via specific microflora in the gut. This study aimed to clarify the effects of equol on oxidized low-density lipoprotein (OX-LDL)-stimulated apoptosis in human umbilical vein endothelial cells (HUVECs).
Methods: HUVECs were cultured in the presence of OX-LDL, and cell apoptosis was monitored by evaluating of DNA fragmentation and the production of cytoplasmic histone-associated DNA fragments. We simultaneously evaluated the level of cellular superoxide and nitric oxide (NO) and the effects of the anti-oxidant activity of equol on apoptosis.
Results: We found that equol inhibited the induction of apoptosis in response to exposure of HUVECs to OX-LDL. Treatment of cells with equol led to a significant reduction in superoxide production by NAD(P)H oxidase and also to a significant increase in NO production. We further observed an effect of equol on the suppression of OX-LDL uptake.
Conclusions: These results suggested that equol might contribute to a reduced level of OX-LDL-stimulated apoptosis linked to the reduced generation of intracellular reactive oxygen species (ROS).

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Aim: Menopause is a risk factor for cardiovascular disease (CVD) in women because of the reduction in endogenous estrogen. Recently, single nucleotide polymorphisms (SNPs) of the estrogen receptor α (ESR-1) gene (c.454-397T>C) associated with the prognosis of myocardial infarction in postmenopausal women were identified; however, the mechanism by which genetic variation of ESR-1 contributes to the pathogenesis of CVD is unknown. Circulating levels of adipokines and inflammatory cytokines predict CVD risk; hence, this study aimed to investigate whether ESR-1 genotypes (c.454-397T>C) might influence circulating levels of adipokines and inflammatory cytokines in postmenopausal women with type 2 diabetes.
Methods: Sixty-three postmenopausal women with type 2 diabetes were recruited. Serum levels of adiponectin, resistin, interleukin-6 (IL-6), and high-sensitive C-reactive protein (hs-CRP) were determined.
Results: The genotype of ESR-1 was closely associated with serum adiponectin, which was decreased in subjects with the T allele and was lowest in those with the T/T genotype. Multiple logistic regression analysis revealed independent contribution of the homozygote for the T allele to low serum levels of adiponectin.
Conclusion: The T allele of the c.454-397T>C SNP of ESR-1 is associated with low serum levels of adiponectin, which may lead to a high risk of CVD in postmenopausal women.

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Background: Arterial calcification is associated with cardiovascular (CV) disease, to be leading to vessel wall stiffness and causing the management of hemodynamics in the elderly more difficult. Here, we compared the extent of calcification in the aortic arch by reviewing chest X-rays to that in the abdominal aorta as assessed by more detailed examinations. In addition, the validity of the grading and the relationship of this useful grading to clinical risk factors were evaluated.
Methods and Results: The extent of aortic arch calcification (AAC) on a postero-anterior plain chest X-ray was divided into four grades (0 to 3). First, AAC grade was assessed in patients who underwent two quantitative examinations for abdominal aortic calcification; lateral radiograph of lumbar spine and/or computer tomography, and was positively correlated with the abdominal aortic calcification level. Subsequently, AAC grade in 239 out-patients (115 men; mean age, 61.9 years) was also evaluated, and was 0, 1, 2, and 3 in 46%, 22%, 29%, and 4% of the population, respectively, was significantly associated with pulse pressure and intima-media thickness. AAC grade in patients with diabetes or renal dysfunction was significantly higher than in those without each risk, but there was no association with other risk factors. In addition, AAC grade was positively correlated with risk factor clustering.Conclusion: Assessment of AAC detectable on a chest X-ray is very useful and its grade reflects the magnitude of calcified change in the whole aorta. In addition, AAC evaluation may provide supportive information for atherosclerotic risk stratification.

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Aim: Endoglin is a homodimeric transmembrane glycoprotein that has been demonstrated to affect transforming growth factor β (TGF-β) signaling and endothelial nitric oxide synthase (eNOS) expression by affecting SMAD proteins in vitro. Thus, in this study we stepped forward to elucidate whether endoglin is co-expressed with SMAD2, phosphorylated SMAD2/3 proteins and eNOS in vivo in atherosclerotic lesions in ApoE/LDLR double knockout mice. In addition, we sought whether endoglin expression as well as the expression of SMAD2, phosphorylated SMAD2/3 and eNOS is affected by atorvastatin treatment.
Methods: Two-month-old female ApoE/LDLR double knockout mice were divided into two groups. The control group was fed with the western type diet whereas in the atorvastatin group, atorvastatin at dose 100 mg/kg per day was added to the same diet. Immunohistochemical and western blot analysis of endoglin, SMAD2, phosphorylated SMAD2/3 and eNOS expressions in aorta were performed.
Results: The biochemical analysis showed that administration of atorvastatin significantly decreased level of total cholesterol, VLDL, LDL, TAG, and significantly increased level of HDL cholesterol. Fluorescence immunohistochemistry showed endoglin co-expression with SMAD2, phosphorylated SMAD2/3 and eNOS in aortic endothelium covering atherosclerotic lesions in both control and atorvastatin treated mice. Western blot analysis demonstrated that atorvastatin significantly increased expression of endoglin, SMAD2, phosphorylated SMAD2/3, and eNOS in mice aorta.
Conclusion: These findings suggest, that endoglin might be interesting marker of endothelial dysfunction and/or atherogenesis which is upregulated by statins implicating potential beneficial role of endoglin and its pathway in atherosclerosis.

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Aim: Obesity is a well known strong risk factor for coronary artery disease (CAD). We prospectively investigated the influence of body mass index (BMI) on the inhibitory effects of pravastatin against the development of coronary atherosclerosis.
Methods: In 56 patients with stable CAD, 3-dimensional intravascular ultrasound was performed in matched coronary segments at the baseline and after 6-month treatment with pravastatin.
Results: The plaque volume was significantly reduced by 11% after treatment (p<0.001 vs. baseline). The percent plaque volume was positively correlated with the baseline BMI (r=0.37, p<0.001), and negatively correlated with the serum total cholesterol / high-density lipoprotein cholesterol ratio (r=0.27, p<0.05) and total leukocyte count (r=0.27, p<0.05). Multivariate regression analysis showed that BMI was an independent predictor of the change in plaque volume (beta coefficient: 0.326; 95% CI: 0.003 to 0.037; p<0.05). No correlations were found between BMI and changes in the serum levels of any other lipids, apolipoproteins, or hs-CRP.
Conclusion: The present study demonstrated that an increase in BMI attenuated pravastatin-induced coronary atherosclerosis regression. The results may provide new insight into the framework for the treatment of obese patients with CAD.

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Aim: The aim of our study is to elucidate the interactive effects on lipid metabolism of fenofibrate and two fish oils with EPA and DHA contents in mice.
Methods: Female C57BL/6J mice were fed purified experimental diets containing safflower oil (SO), EPA-rich menhaden oil (MO) or DHA-rich tuna oil (TO) with or without 0.1% fenofibrate for 8 weeks. At the end of the experiments, we measured plasma lipids and hepatic triglycerides and cholesterol, and the hepatic mRNA expression of lipogenic and lipidolytic genes.
Results: Plasma TG levels fell in the group fed MO or TO alone and fell significantly in all fenofibrate-treated groups. Although plasma total cholesterol levels fell significantly in fish oil-fed groups, fenofibrate treatments increased significantly plasma total cholesterol levels in these fish oil groups, but not in the group fed SO alone; however, hepatic triglyceride and total cholesterol levels markedly decreased in MO-or TO-fed mice. In lipid synthesis, the hepatic mRNA level of SREBP-1c was not reduced in either fish oil group; however, Insig-1 mRNA decreased in MO and TO feeding groups by about half and FAS or SCD-1 mRNA decreased significantly in MO and TO feeding groups, compared with the SO feeding group. In both fish oil groups, SREBP-2 mRNA decreased significantly and HMG-CoA reductase mRNA also decreased with/without fenofibrate. On the other hand, fenofibrate supplementation significantly induced the mRNA expression of AOX and UCP-2, which play a role in lipid catabolism, in all diets. CYP7A1 mRNA increased markedly in mice fed MO diet with fenofibrate, compared with TO diet with fenofibrate.
Conclusion: These data suggest that differences in dietary contents of EPA and DHA do not influence the inhibition of lipogenesis, and that fenofibrate supplementation stimulates fatty acid oxidation, regardless of the oil type; however, cholesterol catabolism was induced by a combination of EPA-rich fish oil and fenofibrate, which suggests that EPA has a greater synergistic ability for cholesterol catabolism induction by fenofibrate than DHA.

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Aim: Tangier disease (TD), caused by deficiency of ATP-binding cassette transporter A1, is characterized by the absence of high density lipoprotein and the accumulation of cholesteryl esters in many tissues. Recently, it has been reported that ABCA1 is expressed in pancreatic β cells and mice with specific inactivation of ABCA1 in β cells showed markedly impaired insulin secretion, suggesting that ABCA1 deficiency may be involved in diabetes. The aim of the current study was to confirm these findings by the oral glucose tolerance test (OGTT) in human subjects with ABCA1 deficiency.
Methods and Results: Four Japanese patients with TD were investigated by OGTT with 75 g glucose. In all TD patients, the plasma glucose concentration after 30 min progressively increased, indicating a type 2 diabetic pattern; however the plasma insulin concentration did not respond well to glucose increase. The calculated insulinogenic index was significantly lower in TD patients than in non-diabetic controls (0.055±0.034 vs 0.775±0.538, mean±SD, p<0.05, respectively).
Conclusions: Although the number of TD patients was very small in the current study, these observations indicated a possible mechanism that glucose-stimulated insulin secretion might be impaired in human TD patients with ABCA1 mutations. Taken together, ABCA1 may be involved in insulin secretion from pancreatic β-cells.

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