Aim: Severe gastrointestinal bleeding sometimes occurs in patients with aortic stenosis (AS), known as Heyde's syndrome. This syndrome is thought to be caused by acquired von Willebrand syndrome and is characterized by reduced large von Willebrand factor (vWF) multimers. However, the relationship between the severity of AS and loss of large vWF multimers is unclear.
Methods: We examined 31 consecutive patients with severe AS. Quantitative evaluation for loss of large vWF multimers was performed using the conventional large vWF ratio and novel large vWF multimer index. This novel index was defined as the ratio of large multimers of patients to those of controls.
Results: Loss of large vWF multimers, defined as the large vWF multimer index ＜80%, was detected in 21 patients (67.7%). The large vWF multimer ratio and the large vWF multimer index were inversely correlated with the peak aortic gradient (R=-0.58, p=0.0007, and R=-0.64, p＜0.0001, respectively). Anemia defined as hemoglobin ＜9.0 g/dl was observed in 12 patients (38.7%), who were regarded as Heyde's syndrome. Aortic valve replacement was performed in 7 of these patients, resulting in the improvement of anemia in all patients from a hemoglobin concentration of 7.5±1.0g/dl preoperatively to 12.4±1.3 g/dl postoperatively (p＜0.0001).
Conclusions: Acquired von Willebrand syndrome may be a differential diagnosis in patients with AS with anemia. The prevalence of AS-associated acquired von Willebrand syndrome is higher than anticipated.

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Aim: Macrophage foam cell formation is the most prominent characteristic of the early stages of atherosclerosis. Ubiquitin Fold Modifier 1 (UFM1) is a new member of the ubiquitin-like protein family, and its underlying mechanism of action in macrophage foam cell formation is poorly understood. Our current study focuses on UFM1 and investigates its role in macrophage foam cell formation.
Methods: Using real-time quantitative PCR (qRT-PCR) and western blot analysis, we first analyzed the UFM1 expression in mouse peritoneal macrophages (MPMs) from ApoE－/－ mice in vivo and in human macrophages treated with oxLDL in vitro. Subsequently, the effects of UFM1 on macrophages foam cell formation were determined by Nile Red staining and direct lipid analysis. We then examined whether UFM1 affects the process of lipid metabolism in macrophages. Lastly, with the method of small interfering RNA (siRNA), we delineated the mechanism of UFM1 to attenuate lipid accumulation in THP-1 macrophages.
Results: UFM1 is dramatically upregulated under atherosclerosis conditions both in vivo and in vitro. Moreover, UFM1 markedly decreased macrophage foam cell formation. Mechanistic studies revealed that UFM1 increased the macrophage cholesterol efflux, which was due to the increased expression of ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1). Furthermore, the upregulation of ABCA1 and ABCG1 by UFM1 resulted from liver X receptor α (LXRα) activation, which was confirmed by the observation that LXRα siRNA prevented the expression of ABCA1 and ABCG1. Consistent with this, the UFM1-mediated attenuation of lipid accumulation was abolished by such inhibition.
Conclusions: Taken together, our results showed that UFM1 could suppress foam cell formation via the LXRα-dependent pathway.

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Aim: The purpose of this study was to assess the association with automated US measurements of carotid artery intima-media thickness (cIMT) with anthropometric, laboratory data and ultrasonographic measurements of fat, in order to identify potential markers that could be used to prevent the development and progression of cardiovascular pathology in adolescents.
Method: Forty-five patients aged 10 to 17 years were enrolled in this study. Blood samples and anthropometric measurements were obtained from all subjects. All patients underwent an ultrasonic assessment of subcutaneous tissue, pre-peritoneal fat, and intra-abdominal fat. All patients received an US assessment of the common carotid artery intima-media thickness.
Results: There was a positive association of minimum beds of pre-peritoneal fat, on both sides, with cIMT. Additionally, cIMT on the right side was positively associated with HOMA-IR. In our multivariate analysis, HOMA-IR remained independently associated with cIMT (left) and measurement of the minimum bed of pre-peritoneal fat was associated with right cIMT. There was no association of cIMT with sex, BMI z-score, demographic variables or laboratory findings.
Conclusion: Pre-peritoneal fat and HOMA-IR are associated with automated ultrasound measurements of both carotid intima-media thickness. Carotid intima-media thickness was not associated with any other demographic variables nor with other laboratory findings when assessed with our automated US method.

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Aim: Ischemic stroke (IS) is a multifactorial disease caused by environmental risk factors and genetic susceptibility. However, few studies have assessed whether gene–gene interactions among cytochrome P450 (CYP) pathway genes influence the risk of IS. The aim of the present study was to investigate the association of 10 variants of eight CYP pathway genes with IS and to determine whether these gene–gene interactions increase the risk of IS.
Methods: Ten variants of eight CYP pathway genes were examined using mass spectrometry methods in 396 patients with IS and 378 controls. Gene–gene interactions were analyzed using generalized multifactor dimensionality reduction (GMDR) methods.
Results: Apart fro m variant rs9333025, there were no significant differences in the genotype distributions of the other nine variants between the two groups using the single–locus analytical approach. However, the GMDR analysis showed a significant gene–gene interaction among rs17110453, rs751141, and rs9333025, which scored 10 for cross-validation consistency and nine for the sign test (p=0.011). Individual patients with the combination of 17110453CC, rs751141GG, and rs9333025GG had a significantly higher risk for IS than those with the combination of 17110453AA, rs751141AA, and rs9333025AA [odds ratio (OR)=2.86, 95% confidence interval (CI): 1.24–7.26, p=0.004]. Logistic regression analysis showed that certain gene–gene interactions among rs17110453, rs751141, and rs9333025 predict a higher risk for IS (OR=2.36, 95% CI: 1.228–5.297, p=0.005).
Conclusion: The three-loci interaction may confer a higher risk for IS. The combinatorial analysis used in this study may be helpful to elucidate complex genetic risk factors for IS.

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Aims: 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have multiple pleiotropic effects, such as anti-inflammatory and vascular endothelium protection, that are independent of their low-density-lipoprotein (LDL) cholesterol lowering effects. However, whether different statins exert diverse effects on inflammation, insulin resistance, and the progression of carotid atherosclerosis [as indicated by the intima-media thickness (CIMT)] in patients with dyslipidemia remains unclear.
Methods: A total of 146 patients with hypercholesterolemia without known cardiovascular disease were randomly assigned to receive 5 mg/day of atorvastatin (n=73) or 1 mg/day of pitavastatin (n=73).
Results: At baseline, age, gender, blood pressure, lipid profiles, and the serum monocyte chemoattractant protein (MCP)-1, homeostasis model assessment of insulin resistance (HOMA-IR) and CIMT values were comparable between the groups. After 12 months of treatment, atorvastatin and pitavastatin equally reduced the LDL cholesterol levels; however, atorvastatin increased the HOMA-IR by ＋26% and pitavastatin decreased this parameter by －13% (p＜0.001). The MCP-1 values were reduced by －28% in the patients treated with pitavastatin and only －11% in those treated with atorvastatin (p=0.016). A greater percent decrease in the mean CIMT from baseline was observed in the patients treated with pitavastatin than in those treated with atorvastatin (－4.9% vs. －0.5%, p=0.020).
Conclusions: These data indicate that, while these agents significantly and equally reduce the LDL cholesterol levels, atorvastatin and pitavastatin have different effects on inflammation, insulin resistance, and the progression of carotid atherosclerosis in patients with dyslipidemia.

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Aim: Vascular endothelial dysfunction is considered an early predictor of atherosclerosis. It has been proven that elevated blood levels of free fatty acids pose a substantial risk for the development of cardiovascular disease. In this study, we examined the effects of palmitic acid (PA), a saturated fatty acid, on endothelial function by using the expression of adhesion molecule, cytokines, and inflammatory protein as indicators, as well as investigated the effects of eicosapentaenoic acid, an n-3 polyunsaturated fatty acid.
Methods: Human umbilical vein endothelial cells (HUVEC) were exposed to PA and EPA.
Results: When HUVEC were exposed to PA, there was an increase in the expression of adhesion molecule, cytokines, and inflammatory protein (ICAM-1, MCP-1, interleukin-6, PTX3). PA augmented the expression of long-chain acyl-CoA synthetase (ACSL) and the cyclin-dependent kinase inhibitor p21, and enhanced the phosphorylation of p65, a component of NF-κB. ACSL inhibition and siRNA-mediated ACSL3 knockdown suppressed the PA-induced increase in the expression of adhesion molecule, cytokines, and inflammatory protein, and ACSL inhibition suppressed the enhancement of p65 phosphorylation. In addition, p21 knockdown suppressed the PA-induced increase in the expression of MCP-1 and ICAM-1. EPA suppressed the PA-induced increase in the expression of ACSL and p21, the enhancement of p65 phosphorylation, as well as the associated increase in the expression of ICAM-1, MCP-1, interleukin-6, and PTX3.
Conclusions: These results suggest that the ACSL, p21, and NF-κB-dependent pathway may possibly be involved in PA-induced vascular endothelial dysfunction, and that EPA ameliorates this at least in part through the regulation of ACSL3 expression.

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Aim: We investigated the safety of 600/150 mg regimen of clopidogrel and the pharmacodynamics and pharmacokinetics of both 300/75 mg regimen and 600/150 mg regimen of clopidogrel in 72 Japanese subjects.
Methods: A randomized study was conducted in healthy Japanese male subjects. Eligible subjects were stratified by dose regimen (300 mg loading dose of clopidogrel on day 1 followed by a 75 mg maintenance dose from days 2 to 7 or a 600 mg loading dose of clopidogrel on day 1 followed by a 150 mg maintenance dose from days 2 to 7) and CYP2C19 metabolizer group [extensive metabolizers (EMs), intermediate metabolizers (IMs), and poor metabolizers (PMs)]. Platelet aggregation and platelet reactivity were evaluated by measuring the maximum platelet aggregation intensity (MAI) induced by 5 and 20 μM ADP, phosphorylation of vasodilator-stimulated phosphoprotein (VASP), and P2Y₁₂ reaction units (PRU) using the VerifyNow system, respectively. We also measured the plasma concentrations of clopidogrel and its active metabolite H4.
Results: No treatment emergent adverse events in the 300/75 mg and 600/150 mg regimen were observed in EMs, IMs, and PMs. All CYP metabolizer groups exhibited a lower MAI (%) induced by ADP in the 300/75 mg and 600/150 mg clopidogrel regimens, and MAI (%) in IM group was equipotent to EM irrespective of the clopidogrel dosage. The double dose regimen decreased MAI in the PM group as equipotent to the IM group receiving the standard dose regimen without the extension of bleeding time. No clear relationship of exposure to clopidogrel and CYP2C19 function was observed, whereas active metabolite H4 exposure was likely to be related to CYP2C19 function.
Conclusion: Clopidogrel in the 600/150 mg regimen was well tolerated. All CYP metabolizer groups exhibited a lower MAI (%) induced by ADP and anti-platelet activities analyzed by VASP and VerifyNow test in the 300/75 mg and 600/150 mg regimens in healthy Japanese subjects.

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Aim: High phosphorus conditions promote vascular calcification (VC) in both chronic kidney disease (CKD) patients and experimental models. However, the composition of medial calcification has not been accurately determined, so the objective of this study was to evaluate the mineral composition of calcification in a tissue culture model, not a cell culture system.
Methods: Aortic rings obtained from male Sprague-Dawley rats were incubated in serum-supplemented medium for 10 days. The inorganic phosphate (Pi) concentration of the medium was increased to induce VC, which was assessed by histology, imaging, and spectroscopy. The mineral composition of the calcification was analyzed using Fourier transform infrared (FTIR) spectroscopic imaging, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) mapping.
Results: The calcium content significantly increased only in aortic rings cultured for 10 days in the high-Pi medium (HiP: 3.8 mmol/L). The concentration of the phosphate transporter Pit-1 in the aortic tissue exposed to HiP was higher than that in the control incubated sections. The FTIR images and spectra indicated that PO₄^3－ was mostly distributed as hydroxyapatite in the medial calcification of aortic rings cultured in HiP. A small quantity of carbonate was identified. The SEM-EDX overlay map demonstrated that phosphorus and calcium simultaneously accumulated and localized in the area of medial calcification induced by exposure to HiP.
Conclusion: This is the first report of accurate determination of the chemical composition of aortic medial calcification. Exposure to high Pi concentration augments aortic calcification via an increase in Pit-1, which mainly contains calcium phosphate.

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Aim: An increasing number of studies have linked Helicobacter pylori (H. pylori) infection to extragastric diseases; however, the role of H. pylori in the pathogenesis of cardiovascular disease (CVD) remains controversial. We examined the association between H. pylori infection and risk of death from coronary heart disease (CHD) and stroke in a nested case-control study within a large prospective cohort study of Japanese subjects.
Methods: The cases were 627 subjects who died from CHD and stroke during the follow-up period until December 31, 2003, and 627 control subjects were selected and matched to cases on sex, age, and area. Commercial immunoassay IgG enzyme-linked immunosorbent assay kits were used for the determination of the seropositivity for H. pylori. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using a conditional logistic regression model.
Results: Overall, H. pylori infection was not associated with CVD (CHD and stroke) mortality risk. The multivariable OR was 0.96 (0.76–1.21) for the H. pylori positive subjects in comparison with H. pylori negative subjects. As for the subtype of CVD, H. pylori appears to be inversely associated with the risk of death from CHD, with an OR of 0.79 (0.50–1.25), but this was not statistically significant. No significant association was observed between H. pylori infection and stroke, with an OR of 1.02 (0.78–1.33).
Conclusion: The results of this nested case-control study suggest that there is no association between H. pylori infection and CHD and stroke mortality risk in otherwise healthy, elderly Japanese individuals.

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Aim: Efficiently inhibiting the formation of macrophage foam cells is indispensable for mitigating and treating atherosclerosis. Tenascin-C (TN-C) plays an important role in promoting atherosclerosis; therefore, it is essential to inhibit foam cell formation associated with TN-C for controlling atherosclerosis. Activating transcription factor 3 (ATF3) is one of the factors involved in regulating the complex process of foam cell formation. This study aimed to explore the role of TN-C and ATF3 in LPS-stimulated THP-1-derived macrophages.
Methods: RT-PCR was used for evaluating the expression of TN-C in LPS-stimulated THP-1 macrophages. Further, exogenous TN-C was introduced and incubated with cultured THP-1 macrophages to confirm the effect of TN-C on LPS-stimulated THP-1 macrophages. ATF3-modified THP-1 macrophages were constructed and verified by western blot. High performance liquid chromatography (HPLC) assay and Oil red O staining were applied for detecting cholesteryl ester/total cholesterol (CE/TC) and lipid formation in THP-1 macrophages.
Results: The expression of TN-C was determined to be upregulated in LPS-stimulated THP-1 macrophages in a dose- and time-dependent manner. HPLC assay and Oil red O staining confirmed that TN-C can enhance LPS-induced THP-1 macrophage foam cell formation. Moreover, ATF3 can act as a negative regulatory factor for inhibiting TN-C-induced foam cell formation by suppressing TLR-4 in LPS-stimulated THP-1 macrophages.
Conclusion: ATF3 can inhibit TN-C-induced foam cell formation in LPS-stimulated THP-1 macrophages by suppressing TLR-4. It may be a useful molecular target to control TN-C-induced foam cell formation in atherosclerosis.

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