Aim: Patients with severe aortic stenosis (AS) may have bleeding episodes due to the loss of high-molecular-weight (HMW) von Willebrand factor multimers (VWFMs). The absence of HMW-VWFMs and bleeding tendency are usually corrected after aortic valve replacement (AVR). To investigate the process of VWFM recovery and symptoms in patients with severe AS, we analyzed changes in VWF antigen (VWF:Ag), ADAMTS13 activity (ADAMTS13:AC), and platelet thrombus formation under high shear stress conditions.

Methods: Nine patients with severe AS undergoing AVR were analyzed.

Results: Evident deficiency of HMW-VWFMs was observed in six patients before surgery, which was rapidly restored within 8 days after AVR. Median levels of VWF:Ag before surgery, on postoperative days (PODs) 1, 8, 15, and 22, and one year after AVR were 78.1%, 130%, 224%, 155%, 134%, and 142%, respectively. In contrast, ADAMTS13:AC was 50.5%, 35.5%, 25.5%, 25.1%, 30.3%, and 84.6%, respectively. Preoperative thrombus formation but not surface coverage was significantly lower than that on POD 22, which was considered as normal level in each patient. Compared with preoperative levels, thrombus volume was significantly lower on POD 1, but rapidly increased by POD 8.

Conclusion: Bleeding tendency and loss of HMW-VWFMs observed in patients with severe AS before surgery was rapidly corrected after AVR. Instead, patients were in a VWF-predominant state between POD 8 and 22.

Aim: Cholesteryl ester transfer protein (CETP) is an important lipid transfer factor in plasma that enhances prothrombinase activity in purified systems. This study was conducted to test the association of plasma CETP activity with venous thrombosis (VTE) and to address the procoagulant mechanism of CETP activity in prothrombinase assays.

Methods: We measured CETP lipid transfer activity in plasmas of 49 male VTE patients and in plasmas of matched controls. CETP procoagulant activity was tested in purified prothrombinase systems.

Results: CETP lipid transfer activity levels were significantly higher in VTE patients than in controls (p=0.0008). A subset of patients carrying the CETP mutations Ala373Pro and Arg451Gln, which were also linked to the VTE risk, showed significantly higher plasma CETP activity than the noncarriers. The plasma CETP activity negatively correlated with APTT, suggesting that the CETP activity is associated with plasma coagulability. Recombinant (r) CETP bound to both factor Xa (Kd=15 nM) and Gla-domainless factor Xa (Kd=59 nM), whereas rCETP enhanced prothrombin activation by factor Xa, but not by Gla-domainless factor Xa. rCETP also required factor Va for enhancement of prothrombinase activity. When we addressed the effects of mutations in CETP on prothrombinase activity, Gln451-rCETP was found to have five-fold higher thrombin generation activity than wt-rCETP or Pro373-rCETP.

Conclusions: Elevated CETP lipid transfer activity in plasma was associated with the risk of VTE. Gln451-CETP, which is linked to VTE, has much higher procoagulant activity than wt-CETP. CETP might act as a physiologic procoagulant by mechanisms that involve its direct binding to factor Xa.

Aim: The vascular endothelium plays a key role in the pathophysiology of atherosclerosis. Flow-mediated dilation (FMD) is a novel way of assessing endothelial function. Cilostazol is a unique antiplatelet drug that also has the potential to improve endothelial function. The objective of this present study was to investigate the effects of cilosatzol on endothelial function as assessed by FMD.

Methods: Fifty-one patients with coronary artery disease (CAD) were assigned to one of two groups: the Cilostazol(+) group (with cilostazol) and Cilostazol(-) group (without cilostazol). In addition to conventional dual antiplatelet therapy with aspirin and clopidogrel/ticlopidine, the Cilostazol(+) group (n=27) was also given cilostazol (100 mg/day). The Cilostazol(-) group (n=24) did not receive cilostazol. FMD was assessed at enrollment and after 6–9 months.

Results: The FMD of both the Cilostazol(+) and Cilostazol(-) groups remained similar at 5.2 (interquartile range: 3.8–8.5) to 5.4 (interquartile range: 4.2–6.7) (P=0.29) and 5.0 (interquartile range: 3.6–6.4) to 4.9 (interquartile range: 4.0–7.0) (P=0.38), respectively. However, the diameters of the baseline and maximal brachial arteries tended to increase in the Cilostazol(+) group (baseline: 4.2±0.7 to 4.4±0.7, P=0.18; maximal: 4.5±0.7 to 4.6±0.7 P=0.22), whereas that of the Cilostazol(-) group tended to decrease (baseline: 4.1±0.6 to 3.9±0.5, P=0.10; maximal: 4.3±0.7 to 4.1±0.5, P=0.05). The rates of change in the baseline diameter (Cilostazol(+): 3.7±9.8% vs. Cilostazol(-): -3.8±12.2%, P=0.03) and maximal diameter (Cilostazol(+): +3.1±8.9% vs. Cilostazol(-): -4.4±12.0%, P=0.02) were significantly different.

Conclusion: Although cilostazol didn't affect the FMD, there was a significant difference in the rates of change in baseline and maximal brachial artery diameter. This may have a beneficial effect in patients with cardiovascular disease.

Aim: Advanced glycation end products (AGE) are considered to be among the critical pathogenic factors involved in the progression of diabetic complications. Skin autofluorescence (AF), a noninvasive measurement of AGE accumulation, has been recognized as a useful and convenient marker for diabetic vascular diseases in Caucasians. This study aimed to evaluate the association of tissue AGE, assessed using skin AF, with coronary artery calcification in Japanese subjects with type 2 diabetes.

Methods: In total, 122 Japanese subjects with type 2 diabetes enrolled in this cross-sectional study underwent multi-slice computed tomography for total coronary artery calcium scores (CACS) estimation and examination with a skin AF reader.

Results: Skin AF positively correlated with age, sex, diabetes duration, pulse wave velocity, systolic blood pressure, serum creatinine, and CACS. In addition, skin AF results negatively correlated with BMI, eGFR, and serum C-peptide concentration. According to multivariate analysis, age and systolic blood pressure showed strong positive correlation and eGFR showed negative correlation with skin AF values. Multiple linear regression analyses revealed a significant positive correlation between skin AF values and logCACS, independent of age, sex, diabetes duration, HbA1c, BMI, IMT, and blood pressure. However, skin AF showed no association with serum levels of AGE, such as Nε-(carboxymethyl) lysine and 3-deoxyglucosone.

Conclusion: Skin AF results positively correlated with CACS in Japanese subjects with type 2 diabetes. This result indicates that AGE plays a role in the pathogenesis of diabetic macrovascular disease. Measurement of skin AF values may be useful for assessing the severity of diabetic complications in Japanese subjects.

Aims: Clopidogrel is an antiplatelet drug primarily used to treat or prevent acute ischemic stroke (IS) or myocardial infarction (MI). This prodrug requires biotransformation to an active metabolite by cytochrome P450 (CYP) enzymes, and CYP single nucleotide polymorphisms (SNPs) could affect the efficiency of such biotransformation.

Methods: A total of 375 consecutive IS patients were genotyped for eight CYP SNPs using mass spectrometry. Platelet aggregation activity was measured before and after the 7–10 day treatment. Gene–gene interactions were analyzed using generalized multifactor dimensionality reduction (GMDR) analysis. All patients received clopidogrel therapy and were followed up for six months. Primary outcomes were evaluated as a composite of recurrent ischemic stroke (RIS), MI, and death. The secondary outcome was the modified Rankin Scale (mRS).

Results: Clopidogrel resistance occurred in 153 patients (40.8%). The frequency of CYP3A5 (rs776746) GG/AG and CYP2C19*2 (rs4244285) AA/AG genotypes was significantly higher in clopidogrel-resistant patients than in sensitive patients. There was a significant gene–gene interaction between CYP3A5 (rs776746) and CYP2C19*2 (rs4244285). CYP2C19*2 AA and its interaction with CYP3A5 GG were independent predictors of clopidogrel resistance and affected the activity of platelet aggregation. Diabetes mellitus, CYP2C19*2 (rs4244285), clopidogrel resistance, and the interaction of CYP2C19*2 with CYP3A5 were all independent risk factors for the primary outcomes of clopidogrel treatment. Clopidogrel-resistant patients were more likely to have poor outcomes (mRS ＞2 points) compared with clopidogrel-sensitive patients.

Conclusion: CYP SNPs and their interactions are associated with drug resistance and outcomes in acute IS patients.

Aim: Iron accumulation in foam cells was previously shown to be involved in atherogenesis. However, the mechanism for iron accumulation was not clarified. Ceruloplasmin (Cp) is an important factor in cellular iron efflux and was found to be downregulated in atherosclerotic plaques in our previous study. The current study is to investigate the role of Cp in atherosclerosis.

Methods: We used RAW264.7 cells, a well-accepted cell model of atherosclerosis, which were treated with lipopolysaccharides (LPS), ferric ammonium citrate (FAC) or deferoxamine, and oxidized low density lipoprotein (ox-LDL) to detect the regulation of Cp and its influence in iron efflux and lipid accumulation using biochemical and histological assays.

Results: Our results showed that the Cp protein level increased after 200-μM FAC treatment in LPS-activated RAW264.7 cells. Ox-LDL treatment (50 μg/ml) moderately reduced both mRNA and protein levels and ferroxidase activity of Cp (p＜0.05). No significant difference was observed in the expression of ferritin and ferroportin, two important iron-related proteins for iron storage and efflux, respectively, after ox-LDL treatment. However, co-treatment with ox-LDL and FAC drastically reduced the expression of Cp. Accordingly, the ferroxidase activities simultaneously decreased, whereas the protein levels of Ft and Fpn1 significantly increased, indicating further iron accumulation. Moreover, co-treatment with FAC and ox-LDL enhanced the accumulation of cholesterol compared with ox-LDL-only treatment to trigger apoptosis.

Conclusion: Our findings suggest that physiological interaction of iron and lipid obstructs iron efflux and accelerates the lipid accumulation in macrophages during foam cell formation, which implicates the role of iron in the pathology of atherosclerosis.

Aim: This study aims to determine the association between glucose metabolism and proinflammatory/anti-inflammatory properties of circulating monocytes or those of carotid plaques in patients who underwent carotid endarterectomy.

Methods: Clinical characteristics and expression levels of proinflammatory/anti-inflammatory markers in circulating monocytes/carotid plaques were examined in 12 patients with diabetes and 12 patients without diabetes.

Results: Circulating monocytes from patients with diabetes revealed higher tumor necrosis factor (TNF)-α and lower interleukin (IL)-10 expression levels compared with those from patients without diabetes, which was also observed in carotid plaques from patients with diabetes. Hyperglycemia revealed positive and negative correlations with the ratios of IL-6^＋ and IL-10^＋ cells in carotid plaques, respectively. Moreover, we determined a positive correlation between circulating monocytes and carotid plaques with respect to TNF-α and IL-6 expressions.

Conclusions: The inflammatory property of circulating monocytes was associated with that of carotid plaques. Hyperglycemia increased inflammatory properties and decreased anti-inflammatory properties of carotid plaques.

Aim: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is known to be a key molecule in the pathogenesis of atherosclerosis. Although high levels of serum soluble LOX-1 (sLOX-1) were demonstrated in patients with acute coronary syndrome, there are no reports about acute stroke patients. The aim of the present study was to evaluate the levels of sLOX-1 in acute stroke patients according to different stroke subtypes.

Methods: We enrolled a total of 377 patients with a stroke (men/women: 251/126; age: 40–79 years), 250 with ischemic stroke and 127 with intracerebral hemorrhage (ICH). Patients were admitted to our hospital within 3 days after the onset of stroke. As controls, we randomly selected age- and sex-matched subjects without a past history of cardiovascular disease according to stroke subtype from the community-based cohort of the Suita study. Serum LOX-1 levels were compared between stroke patients and healthy controls according to stroke subtype.

Results: Median values of serum sLOX-1 in stroke patients were significantly higher than those in controls (526 vs. 486 ng/L in ischemic stroke and 720 vs. 513 ng/L in ICH, respectively). Among subtypes of ischemic stroke, median sLOX-1 levels in atherothrombotic brain infarction (641 ng/L) only were significantly higher than those in controls (496 ng/L). Ischemic stroke [odds ratio (OR), 3.80; 95% confidence interval (CI), 1.86–7.74] and ICH (OR, 5.97; 95% CI, 2.13–16.77) were independently associated with high levels of sLOX-1 by multivariate logistic regression analysis.

Conclusions: Higher levels of sLOX-1 were observed in patients with acute stoke than in controls. High levels of sLOX-1 can be useful as biomarker for acute stroke.

Aims: Phospholipase A₂ receptor 1 (PLA₂R) has multiple biological functions other than functioning as a receptor for secretory PLA₂s. Two nonsynonymous polymorphisms in the C-type lectin-like domains (CTLD) 1 of PLA₂R gene have been associated with idiopathic membranous nephropathy. This study examined whether the same PLA₂R polymorphisms may alter functions of PLA₂R in cells expressing the variant PLA₂R. In addition, the clinical relevance of the experiment was examined.

Methods: Two nonsynonymous polymorphisms (T/C at rs3749117 and G/C at rs35771982) in CTLD1 of PLA₂R gene were completely linked. HEK293 cells expressing human wild-type PLA₂R (T at rs3749117 and G at rs35771982) or human mutant PLA₂R that had double mutations (C at rs3749117 and C at rs35771982) were constructed.

Results: HEK293 cells expressing mutant PLA₂R had lower migratory and proliferative responses to collagen I compared with cells expressing wild-type PLA₂R. In 580 male patients, PLA₂R gene polymorphisms were associated with an increase in maximum intima-media thickness (maxIMT) of the carotid artery. The multivariate regression model showed that PLA₂R gene polymorphisms were a risk factor of an increased maxIMT that was independent of conventional risk factors (OR=1.93, 95% CI: 1.17–3.19, p＜0.01).

Conclusions: The nonsynonymous common variants of PLA₂R gene altered biological functions in cells expressing variant PLA₂R. PLA₂R gene polymorphisms present a genetic risk of an increased IMT of the carotid artery in male. The functional changes in the variant PLA₂R may potentially be responsible for its association with an increased IMT of the carotid artery.

Aim: Antiphospholipid syndrome (APS) is characterized by recurrent thrombosis and/or gestational morbidity in patients with antiphospholipid autoantibodies (aPL). Over recent years, IgA anti-beta2-glycoprotein I (B2GPI) antibodies (IgA aB2GPI) have reached similar clinical relevance as IgG or IgM isotypes. We recently described the presence of immune complexes of IgA bounded to B2GPI (B2A-CIC) in the blood of patients with antecedents of APS symptomalology. However, B2A-CIC's clinical associations with thrombotic events (TEV) have not been described yet.

Methods: A total of 145 individuals who were isolate positive for IgA aB2GPI were studied: 50 controls without any APS antecedent, 22 patients with recent TEV (Group-1), and 73 patients with antecedents of old TEV (Group-2).

Results: Mean B2A-CIC levels and prevalence in Group-1 were 29.6±4.1 AU and 81.8%, respectively, and were significantly higher than those of Group-2 and controls (p＜0.001). In a multivariable analysis, positivity of B2A-CIC was an independent variable for acute thrombosis with a 22.7 odd ratio (confidence interval 5.1–101.6, 95%, p＜0.001). Levels of B2A-CIC dropped significantly two months after the TEV. B2A-CIC positive patients had lower platelet levels than B2A-CIC-negative patients (p＜0.001) and more prevalence of thrombocytopenia (p＜0.019). Group-1 had no significant differences in C3 and C4 levels compared with other groups.

Conclusion: B2A-CIC is strongly associated with acute TEV. Patients who did not develop thrombosis and were B2A-CIC positive had lower platelet levels, which suggest a hypercoagulable state. This mechanism is unrelated to complement-fixing aPL. B2A-CIC could potentially select IgA aB2GPI-positive patients at risk of developing a thrombotic event.