Recent technical innovation has enabled chemical modifications of small materials and various kinds of nanoparticles have been created. In clinical settings, nanoparticle-mediated drug delivery systems have been used in the field of cancer care to deliver therapeutic agents specifically to cancer tissues and to enhance the efficacy of drugs by gradually releasing their contents. In addition, nanotechnology has enabled the visualization of various molecular processes by targeting proteinases or inflammation. Nanoparticles that consist of poly (lactic-co-glycolic) acid (PLGA) deliver therapeutic agents to monocytes/macrophages and function as anti-inflammatory nanoparticles in combination with statins, angiotensin receptor antagonists, or agonists of peroxisome proliferator-activated receptor-γ (PPARγ). PLGA nanoparticle-mediated delivery of pitavastatin has been shown to prevent inflammation and ameliorated features associated with plaque ruptures in hyperlipidemic mice. PLGA nanoparticles were also delivered to tissues with increased vascular permeability and nanoparticles incorporating pitavastatin, injected intramuscularly, were retained in ischemic tissues and induced therapeutic arteriogenesis. This resulted in attenuation of hind limb ischemia. Ex vivo treatment of vein grafts with imatinib nanoparticles before graft implantation has been demonstrated to inhibit lesion development. These results suggest that nanoparticle-mediated drug delivery system can be a promising strategy as a next generation therapy for atherosclerotic vascular diseases.

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Aim: Matrix metalloproteinases (MMPs), angiotensin II (AII) and its receptors are implicated in atherosclerotic plaque instability, however the roles of the two receptor subtypes, ATR1 and ATR2, in MMP regulation remain uncertain. In this study, we investigated the effect of ATR1 and ATR2 blockade on the expression and activity of MMP-2, MMP-3 and MMP-9, in human carotid atheroma.
Methods: Atheroma samples (n=36) were obtained from patients undergoing carotid endarterectomy. The effects of ATR1 (irbesartan), ATR2 (PD123319) and combined ATR1 and ATR2 blockade on the expression and activity of the MMPs and the expression of tissue inhibitors of metalloproteinases (TIMPs) were investigated in explant culture experiments. Paired atheroma samples were incubated with the intervention or media control for 4 days. Protein levels (MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, TIMP-4, ATR1 and ATR2) were determined by ELISA. Overall gelatinase activity and specific activation were measured by chromogenic activity assays and zymography, respectively.
Results: ATR1 blockade, but not ATR2 blockade significantly reduced TIMP-1, TIMP-2 and TIMP-4 expression in atheroma supernatant. Combined ATR1 and ATR2 blockade significantly reduced MMP-2, MMP-3 and MMP-9 expression. MMP-2 and MMP-9 relative activation, and overall MMP-9 catalytic capacity were significantly increased by ATR1 blockade.
Conclusions: Our findings suggest that ATR1 blockade reduces TIMP expression and increases gelatinase activity in human carotid atheroma.

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Aim: Whether the association between serum γ-glutamyltransferase (γ-GTP) levels and total cardiovascular disease (CVD) mortality is independent of alcohol drinking in East Asian populations is not well known. We conducted a pooled analysis of Japanese men and women that enabled an analysis restricted to never-drinkers.
Methods: A total of 15,987 men and 25,053 women aged 40–79 years, pooled from seven cohort studies throughout Japan, were followed-up to examine sex-specific relationship between serum γ-GTP levels and total CVD mortality. Cox regression model was used that was adjusted for age, smoking status, body mass index, and systolic blood pressure and serum triglyceride, total cholesterol, aspartate aminotransferase, and alanine aminotransferase levels.
Results: During an average follow-up of 8.7 years, we documented 361 and 340 deaths from total CVD, 146 and 168 from stroke, and 101 and 53 from coronary heart disease (CHD) for men and women, respectively. Among the never-drinkers, hazard ratios (HRs) for mortality for one standard deviation of log-γ-GTP for men were 1.89 (1.00–3.58) for stroke, 1.04 (0.57–1.90) for CHD, and 1.43 (1.04–1.96) for total CVD. For women, HRs were 1.28 (1.06–1.54), 1.81 (1.34–2.44), and 1.30 (1.14–1.49), respectively.
Conclusion: γ-GTP may be a risk factor for total CVD mortality independent of alcohol drinking status in Japanese men and women.

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Aims: There is no community-based cohort study to examine the effect of very high level of high-density lipoprotein cholesterol (HDL-C) on coronary heart disease (CHD) and other cause-specific mortality. Therefore, we investigated the relationship between HDL-C including very high level and cause-specific mortality in a 20-year cohort study of the representative sample of Japanese.
Methods: We followed 7,019 individuals from the Japanese general population (2,946 men and 4,073 women). We defined HDL-C levels as follow: low (HDL-C ＜1.04 mmol/L), reference (1.04–1.55 mmol/L), high (1.56–2.06 mmol/L), very high (≥2.07 mmol/L). The multivariate adjusted hazard ratio (HR) for all-cause or cause-specific mortality was calculated using a Cox proportional hazards model adjusted for other traditional risk factors.
Results: During follow-up, we observed 1,598 deaths. No significant association was observed between HDL-C and all-cause mortality. Serum HDL-C also showed no association with stroke. In contrast, the risk for CHD among high HDL-C was lower than reference, HRs were 0.51 [95% confidence interval (CI): 0.21–1.23] in men, 0.33 (95% CI: 0.11–0.95) in women, and 0.41 (95% CI: 0.21–0.81) when men and women were combined. However, very high HDL-C did not show significant association with CHD and other cause-specific mortality.
Conclusions: HDL-C was not associated with all-cause and stroke mortality. In contrast, high serum HDL-C levels, at least up to 2.06 mmol/L, were protective against CHD, although further high levels were not. However, sample size of cause-specific death in very high HDL-C group was not enough even in this 20-year follow-up of 7,019 Japanese; larger cohort studies should be warranted.

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Aim: Hepatic effects of estrogen therapy on low-density lipoprotein (LDL) subfraction or oxidative stress have not been previously evaluated. The purpose of the present study was to investigate whether the differential hepatic effects of estrogen affect plasma distribution of small dense LDL and free radical production in postmenopausal women.
Methods: In all, 45 postmenopausal women were given 0.625 mg/day of oral conjugated equine estrogen (CEE) (n=15), 1.0 mg/day of oral 17β estradiol (E₂) (n=15), or 50 μg/day of transdermal 17βE₂ (n=15) for 3 months. Subjects received either estrogen alone or with dydrogesterone at 5 mg/day. Plasma concentrations of sex hormone-binding globulin (SHBG), lipids, metallic ions, and derivatives of reactive oxygen metabolites (d-ROMs) were measured.
Results: CEE, but not oral 17βE₂, increased the plasma concentrations of triglyceride, copper (Cu), and d-ROMs and the ratio of small dense LDL/total LDL cholesterol, a marker for plasma distribution of small dense LDL. Transdermal 17βE₂ decreased d-ROMs concentrations but did not significantly change other parameters. Plasma concentrations of SHBG increased in the 3 groups. Estrogen-induced changes in triglyceride correlated positively either with changes in SHBG (R=0.52, P=0.0002) or the ratio of small dense LDL/total LDL cholesterol (R=0.65, P＜0.0001). Changes in Cu also correlated positively either with changes in SHBG (R=0.85, P＜0.0001) or d-ROMs (R=0.86, P＜0.0001).
Conclusion: The hepatic effects of different routes or types of estrogen therapy may be associated with plasma distribution of small dense LDL and free radical production in postmenopausal women.

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Aim: To analyse the relationship between two potentially functional single-nucleotide polymorphisms (SNPs) of the miR-146a gene (rs2910164 and rs57095329) and the risk of atherosclerotic cerebral infarction (ACI).
Methods: A total of 297 patients with ACI and 300 matched healthy individuals were enrolled in the study. The miR-146a polymorphism was detected using the polymerase chain reaction–restriction fragment length polymorphism method.
Results: A significant difference in the C allele frequency at rs2910164 (p=0.028) was noted between patients with ACI and control subjects. In contrast, the genotype and allele frequencies of rs57095329 were not statistically associated with ACI. In addition, the decreased expression of miR-146a was significantly more frequent in ACI patients who were ApoEε4 (＋) carriers (p=0.0233), and rs2910164 G＞C was intimately associated with the ApoEε4-containing genotype in patients compared with the ApoEε4 (－) carriers (p=0.0323).
Conclusions: Our findings indicated that the C allele of rs2910164 miR-146a is an important risk factor for ACI, and ApoEε4 may function through attenuating miR-146a expression to enhance ACI susceptibility. This study provides new information about the possible relationship between miR-146a and ApoEε4 in the development of ACI, with potentially important therapeutic implications.

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Aims: We aimed to assess the relationship between lipid accumulation product (LAP) and stroke incidence and whether increased LAP would further enlarge the risk of stroke in participants with hypertension in an Inner Mongolian population of China.
Methods: Based on the baseline survey conducted in 2002–2003, a prospective cohort study was conducted among 2547 Mongolian people from Inner Mongolia, China. LAP was calculated by waist circumference (WC) and triglyceride (TG) concentration for men and women, respectively. Cox proportional hazards models and receiver operating characteristic (ROC) curves were used to evaluate the associations between LAP, hypertension, and stroke incidence.
Results: During the follow-up period, a total of 121 stroke events were observed. Participants with higher LAP were associated with higher risk of stroke [adjusted hazard ratio (HR), 1.67; 95% confidence interval (CI), 1.08–2.58] than the participants in the lower LAP group. However, no significant dose–response relationship was detected between LAP levels and risk of stroke (P-trend=0.103). The Kaplan–Meier curves showed that hypertensives with high LAP had highest cumulative stroke incidence rate (log-rank P ＜0.001). The multivariate-adjusted HRs (95% CIs) of stroke for normotensives with high LAP, hypertensives with low LAP, and hypertensives with high LAP were 1.80 (0.84–3.89), 2.94 (1.51–5.74), and 4.23 (2.15–8.34), respectively, compared with normotensives with low LAP.
Conclusions: The present study showed that high LAP was associated with an increased risk of future stroke, and hypertensives with high LAP had the highest risk of stroke among Inner Mongolians. These findings indicate that LAP and hypertension may be valuable to predict and prevent stroke incidence.

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Aim: New ischemic lesions in the brain can be detected in approximately 50% of patients undergoing carotid artery stenting (CAS). We wished to discover the laboratory-based predictors of new infarctions in the brain after CAS.
Methods: All consecutive patients with internal carotid artery stenosis of ≥70% with indication for CAS were enrolled in a prospective study for 16 months. All patients used dual antiplatelet therapy for ≥7 days before CAS. Neurologic examination and magnetic resonance imaging (MRI) of the brain were undertaken before and at 24 h after CAS. Samples of venous blood were collected at ＜24 h before CAS for the evaluation of hematology, reticulocytes, coagulation markers (PT, APTT, Fbg, Clauss), vWF antigen, PAI-1 activity, PAI-1 polymorphism 4G/5G, and the multiplate (aspirin and clopidogrel) resistance test. Blood samples for the assessment of anti-Xa activity were collected during CAS. Differences in the values of laboratory markers between patients with and without new ischemic lesions of the brain on control MRI were evaluated.
Results: The cohort comprised 81 patients (53 males; mean age, 67.3±7.2 years). New ischemic infarctions in the brain on control MRI were found in 46 (56.8%) patients. Three of seven patients with resistance to aspirin or clopidogrel had a new ischemic infarction in the brain. No significant differences for particular markers were found between patients with and without an ischemic lesion in the brain.
Conclusion: A high risk of a new ischemic infarction in the brain was detected in patients undergoing CAS, but a laboratory-based predictor of such an infarction could not be identified.

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Aim: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of serum low-density lipoprotein (LDL) cholesterol levels, and sortilin is linked to lipoprotein metabolism. Although statin therapy increases PCSK9 levels, effects of this therapy on plasma sortilin levels have not been evaluated. The purpose of the present study was to examine the effects of statins on plasma PCSK9 and sortilin levels, and association of statin-induced increase in PCSK9 levels with sortilin.
Methods: Serum lipid levels and plasma PCSK9 and sortilin levels were measured at baseline and 8 months after statin therapy in 90 statin-naive patients with coronary artery disease (CAD). Pitavastatin 4 mg/day was used to treat 44 patients and pravastatin 20 mg/day to treat the remaining 46 patients.
Results: For both statin groups, significant increases in hetero-dimer PCSK9 levels (pitavastatin: 31%, p＜0.0001; pravastatin: 34%, p=0.03) and decreases in sortilin levels (pitavastatin: －8%, p=0.02; pravastatin: －16%, p=0.002) were observed. Although a reduction in LDL cholesterol was greater in the pitavastatin group than in the pravastatin group, no significant differences were observed in percentage changes in hetero-dimer PCSK9 and sortilin levels. A significant positive correlation was observed between percentage changes in hetero-dimer PCSK9 levels and those in sortilin levels (pitavastatin: r=0.359, p=0.02; pravastatin: r=0.276, p=0.06).
Conclusions: Use of pitavastatin and pravastatin increased plasma PCSK9 and decreased sortilin levels. Statin-induced increases in PCSK9 were associated with changes in sortilin in statin-naive patients with CAD.

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Aim: The aim of this study was to investigate the cross-sectional association between arterial stiffness (AS) measured with the cardio-ankle vascular index (CAVI) and executive function in community-dwelling elderly people.
Methods: Subjects were 140 community-dwelling elderly people who participated in the study at Kobe, Japan during the period of August–September 2014, of which 126 (mean age±SD: 73.2±6.1, female: 67.5%) met the inclusion criteria and completed the study. Age, sex, body mass index, global cognition, existence of chronic disease, medication, smoking history, and years of education were assessed. The degree of AS was assessed using CAVI. Executive function was assessed using the Category Word Fluency Test (CWFT), Letter Word Fluency Test (LWFT), and Digit Symbol Substitution Test (DSST). We used a correlation analysis and multiple linear regression analysis to investigate whether higher CAVI was independently associated with lower executive function.
Results: In the univariate analysis, higher mean CAVI correlated with lower CWFT (rho=－0.21, p=0.020), LWFT (rho=－0.32, p＜0.001), and DSST (rho=－0.31, p＜0.001). In the multivariate analysis, higher mean CAVI was associated with lower LWFT (β=－0.21, p=0.046) after adjusting for confounding factors, although there was no association with CWFT (β=－0.05, p=0.61) and DSST (β=－0.06, p=0.51).
Conclusions: We found that high CAVI was associated with lower LWFT. These results suggest that arterial stiffness is associated with lower performance in phonemic fluency.

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Aim: Recent studies reported that low high-density lipoprotein (HDL)-mediated cholesterol efflux capacity rather than low HDL cholesterol (HDL-C) is strongly associated with the increased risk for coronary artery disease. It remains unclear whether exercised-based cardiac rehabilitation (CR) can increase HDL cholesterol efflux capacity.
Method: This study is a retrospective analysis of stored serum from patients with acute coronary syndrome (ACS) who participated in outpatient CR program following successful percutaneous coronary intervention. We employed a cell-based cholesterol efflux system including the incubation of ³H-cholesterol labeled macrophages with apolipoprotein B-depleted serum at the onset or early phase of ACS and at 6-month follow-up periods in 57 male and 11 female patients with ACS. Cardiopulmonary exercise tests were performed at the beginning and end of CR program.
Result: Fifty-seven patients completed the CR program. Compared with patients who dropped out from CR program (non-CR group), CR participants showed marked amelioration in serum lipid levels, increased efflux capacity, and improved exercise capacity. Spearman's rank correlation coefficient analysis revealed that the percent increases of efflux capacity were significantly associated with the percent increases in HDL-C (ρ=0.598, p＜0.0001) and apolipoprotein A1 (ρ=0.508, p＜0.0001), whereas no association between increases in efflux capacity and increases in cardiopulmonary fitness was observed. Increases in cholesterol efflux capacity were not seen in patients who continued smoking and those who did not achieve all risk factor targets and higher exercise tolerance.
Conclusion: CR can markedly increase both HDL-C and HDL cholesterol efflux capacity. These results suggest that CR is a very useful therapy for reverse cholesterol transport and secondary prevention.

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Homozygous familial hypercholesterolemia (HoFH) is a rare genetic disorder, which leads to premature cardiovascular diseases. Microsomal triglyceride transport protein (MTP) inhibitors, such as lomitapide, offer a new therapeutic approach for treating these patients. We evaluated the lipid lowering (LL) efficacy of lomitapide according to several gene variants in MTP. Four clinically and/or molecularly defined HoFH patients were treated with lomitapide in addition to conventional high intensity LL therapy and regular lipoprotein apheresis. Two patients responded to the therapy, with a significant reduction of LDL cholesterol (LDL-C＞50%, hyper-responders). Sequencing of all exonic and intronic flanking regions of the MTP gene in all patients revealed 36 different variants. The hyper-responders to lomitapide shared six common variants: rs17533489, rs79194015, rs745075, rs41275715, rs1491246, and rs17533517, which were not seen in hypo-responders (reduction in LDL-C＜50%). We suggest that in HoFH variants in the MTP gene may impact on the therapeutic response to lomitapide, but this requires further investigation.

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Whole exome sequencing (WES) technologies have accelerated genetic studies of Mendelian disorders, yielding approximately 30% diagnostic success. We encountered a 13-year-old Japanese female initially diagnosed with familial hypercholesterolemia on the basis of clinical manifestations of severe hypercholesterolemia (initial LDL cholesterol=609 mg/dl at the age of one) and systemic intertriginous xanthomas with histories of recurrent self-limiting episodes of fever and arthritis. Both her phenotypes seemed to co-segregate in a recessive manner. We performed WES on this patient, who was considered a proband. Among 206,430 variants found in this individual, we found 18,220 nonsense, missense, or splice site variants, of which 3,087 were rare (minor allele frequency ≤ 0.01 or not reported) in 1000 Genome (Asian population). Filtering by assuming a recessive pattern of inheritance with the use of an in silico annotation prediction tool, we successfully narrowed down the candidates to the compound heterozygous mutations in the ABCG5 gene (c.1256G＞A or p.Arg419His/c.1763-1G＞A [splice acceptor site]) and to the double-compound heterozygous mutations in the MEFV gene (c.329T＞C/C or p.Leu110Pro/c.442G＞C/C or p.Glu148Val). The patient was genetically diagnosed with sitosterolemia and familial Mediterranean fever using WES for the first time. Such a comprehensive approach is useful for identifying causative mutations for multiple unrelated inheritable diseases.

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