Oxidized low-density lipoprotein (oxLDL) is known to be a major risk factor for the initiation and development of atherosclerosis. It can elicit an array of atherogenic responses in multiple types of cells residing in the arterial wall, such as endothelial cells (ECs), macrophages, dendritic cells (DCs), and vascular smooth muscle cells (VSMCs). Although they have been studied for many years, the detailed mechanisms modulating oxLDL-induced inflammation have not been fully elucidated. Epigenetic mechanisms consist of DNA methylation, histone post-translational modifications (PTMs), and microRNA (miRNA) alterations. Recently, epigenetic factors, especially miRNAs, have emerged as novel components of the gene expression regulating oxLDL-triggered signal transduction. In addition to their regulatory roles in signaling molecules, increasing evidence suggests that the different genetic stability and cross-talk regulation among these epigenetic factors may be particularly important to the sustained inflammation initiated by temporal oxLDL stimulation. Therefore, in this review, we primarily focused on the functional role of miRNAs, as well as other epigenetic factors, on modulating oxLDL-induced signal transduction in different vascular cells, with a special emphasis on the crosstalk interactions between miRNAs and other epigenetic players that help translate transient environment insults into chronic inflammation. Moreover, we extensively discussed the potential applicability of miRNAs as disease biomarkers and therapeutic targets in diagnosing and treating atherosclerosis.

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This review highlights the pro-atherogenic roles of Ca²⁺-sensitive intracellular protease calpains. Among more than ten species of calpain isozymes, µ- and m-calpains have been characterized most extensively. These two isozymes are ubiquitously expressed in mammalian tissues, including blood vessels, and tightly regulate functional molecules in the vascular component cells through limited proteolytic cleavage. Indeed, previous cell-based experiments showed that calpains play significant roles in nitric oxide production in vascular endothelial cells (ECs), maintenance of EC barrier function and angiogenesis for maintaining vascular homeostasis. Recently, we demonstrated that modified-low density lipoprotein (LDL)-induced m-calpain causes hyperpermeability in ECs, leading to the infiltration of monocytes/macrophages and plasma lipids into the intimal spaces (Miyazaki T. et al., Circulation. 2011; 124: 2522-2532). Calpains also mediate oxidized LDL-induced apoptotic death in ECs. In monocytes/macrophages, calpains induce proteolytic degradation of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1), which results in impaired cholesterol efflux and subsequent macrophage foam cell formation. In vascular smooth muscle cells, calpains may be involved in the conversion from contractile phenotype to proliferative phenotype. In hepatocytes, calpains disrupt the biogenesis of high-density lipoprotein via proteolytic degradation of ABCA1. Thus, calpains may serve as novel candidate molecular targets for control of atherosclerosis.

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Aim: The concomitant use of angiotensin II receptor blocker (ARB) with low doses of hydrochlorothiazide (HCTZ) may provide additional antihypertensive activity. HCTZ induces hypokalemia and hyperglycemia, while ARB slightly induces hyperkalemia. Recently, it has been reported that ARB/ HCTZ did not worsen fasting blood sugar levels; however, the detailed glucose tolerance change effect with combination therapy of ARB/HCTZ compared to ARB alone therapy remains to be investigated.
Methods: Treated non-diabetes mellitus (DM) hypertensive patients taking a common dose of ARB regimens, not achieving blood pressure (BP) goals, were switched to 50 mg Losartan/12.5 mg HCTZ combinations, and the 75 g oral glucose tolerance test (75 g OGTT) was performed before switching and after switching at 3 months.
Results: This study included 30 patients aged 66.5±8.7 years, 67% women. Pre-switching BP 146.6±17.0/ 88.4±10.4 mmHg decreased and was maintained at a steady state, reaching 131.4±1.0/73.8±8.8 mmHg (p<0.001) 3 months later. After switching, blood glucose levels on the 75 g OGTT at fasting, 30, 60 and 120 minutes were significantly decreased. Homeostasis model assessment as an index of insulin resistance and the whole body insulin sensitivity index were significantly ameliorated.
Conclusions: On the 75 g OGTT, 50 mg Losartan with 12.5 mg HCTZ combinations did not worsen glucose tolerance; moreover, they improved BP, insulin resistance and sensitivity in non-DM Japanese patients with essential hypertension uncontrolled with ARBs alone.

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Aims: Statins not only reduce low-density lipoprotein (LDL) cholesterol, but also prevent the progression of kidney dysfunction. Ezetimibe, a cholesterol-absorption inhibitor, also lowers LDL cholesterol levels when added to statins; however, the effect of add-on ezetimibe on kidney function has had conflicting results.
Methods: We conducted an open-labeled, randomized, 12-month trial, comparing the effects of daily therapy with 20 mg fluvastatin either with or without 10 mg ezetimibe in 54 patients with dyslipidemia. The prespecified primary outcome was the percent change from baseline in kidney function, which was defined by the estimated glomerular filtration rate. The secondary outcomes were the changes in surrogate atherosclerotic markers. All analyses were by intention to treat.
Results: The primary outcome, the percent change from baseline (±SE) of the estimated glomerular filtration rate, was −5.5±1.9% in the fluvastatin-only group and 6.6±1.9% in the fluvastatin-plus-ezetimibe (combined-therapy) group (p=0.0002). Secondary outcomes, consisting of the cardioankle vascular index, augmentation index, ankle-brachial index, and maximum intima-media thickness of the carotid arteries, did not differ significantly between the two groups. At the end of the study, the mean (±SD) LDL cholesterol was 122±23 mg per deciliter in the fluvastatin group and 111±29 mg per deciliter in the combined-therapy group (a between-group difference of 9.2%, p= 0.036). Side-effect and safety profiles were similar in the two groups.
Conclusion: Combined therapy with fluvastatin 20 mg plus ezetimibe 10 mg daily resulted in a significant improvement in changes in the estimated glomerular filtration rate.

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Aim: Previous studies have revealed that blockade of the renin angiotensin system attenuates plaque vulnerability and reduces cardiovascular events; however, few studies have compared the effects of an angiotensin-converting enzyme inhibitor (ACEI) with an angiotensin receptor blocker (ARB) and evaluated combination therapy. The objective of this study was to compare the efficacy and mechanisms of plaque stabilization by ACEI or ARB and to determine the effects of combination therapy.
Methods: Twenty-eight male Japanese white rabbits were fed a high-cholesterol diet after balloon injury of the carotid arteries, then separated into ACEI (n= 7; imidapril 0.5 mg/kg/day), ARB (n= 7; TA606 4.5 mg/kg/day), combination (n= 7; imidapril 0.5 mg/kg/day+TA606 4.5 mg/kg/day), and vehicle (n= 7) groups.
Results: No difference in plaque volume was identified among the 4 groups. ACEI or ARB increased the thickness of the fibrous cap, collagen content and the number of smooth muscle cells in the intima (% smooth muscle cell in intima: ACEI, 36.3%; ARB, 36.4%; vehicle, 14.9%), and reduced the accumulation of macrophages (% macrophages in intima: ACEI, 20.1%; ARB, 24.0%; vehicle, 37.9%), suggesting the plaque-stabilizing effects of each drug. ACEI reduced matrix metalloproteinase (MMP)-9 expression and gelatinolytic activity in the intima. While ARB did not change gelatinolytic activity, accumulation ot T cell in the intima was suppressed. Combination therapy did not show additive effects.
Conclusion: These results suggest that ACEIs and ARBs have similar, but not additive, plaque-stabilizing effects. Each agent showed specific effects, with ACEIs decreasing gelatinolytic activity and ARBs suppressing T cell accumulation.

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Aim: Rev-Erb β gene plays crucial roles in circadian rhythm, lipid and glucose metabolism, and several diseases. The molecular mechanisms of the transcriptional regulation of Rev-Erb β that generate and determine the phase of the circadian oscillation remain unclear.
Methods: We analyzed the Rev-Erb β promoter by luciferase reporter assays, real-time bioluminescence monitoring assays and electrophoretic mobility shift assays.
Results: Luciferase reporter assays indicated that only the 5’ region and exon 1 have obvious promoter activity. Real-time bioluminescence monitoring assays revealed that E1, E2, E3, D boxes are important for maintenance of the amplitude of Rev-Erb β oscillation. Based on EMSA results, REV-ERBβ binds ROREs in the Bmal1 promoter region and inhibits Bmal1 promoter activity.
Conclusion: We provide direct evidence that three E-boxes and one D-box located in the first intron are crucial for the phase of circadian oscillation in Rev-Erb β expression and that the sequences upstream from its transcription start site function as a promoter with no circadian regulation. We also found that the E1 box affects the Rev-Erb β oscillation phase. Our results offer new insight into the role of Rev-Erb β in the circadian rhythm system.

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Aim: The inflammatory response following tissue injury after major surgery is known to affect endothelial function and vascular reactivity. In this study we evaluated the utility of bedside Digital Thermal Monitoring (DTM) as a surrogate for evaluating vascular function in the postoperative period.
Methods: Ischemia-induced reactive hyperemia variables were measured in sixty patients scheduled for major thoracic surgery using DTM (VENDYS 5000BC; Endothelix, Inc., Houston, TX, USA) at baseline and at 24, 48, 72 hours, and day 5 postoperatively. Furthermore, baseline DTM variables (TR, aTR and AUC_TR) and postoperative kinetics of these variables were compared among patients with and without preoperative chemo-radiation and cardiovascular risk factors.
Results: There were no significant differences in the DTM parameters measured at baseline and on each of the studied postoperative days. Compared to the baseline, the lowest measures of all variables were observed 24 hrs postoperatively and the highest measures of all variables were observed at 72 hrs. Patients with abdominal obesity and smoking had lower DTM values than the rest of the study group.
Conclusions: In our study, DTM as measured by the VENDYS 5000BC DTM system (Endothelix, Inc.) did not reveal significant changes in ischemia-induced reactive hyperemia (vascular reactivity) between the baseline and after surgery in the postoperative period. Patients with certain cardiovascular risk factors (abdominal obesity, smoking) had a significant lower DTM signal. Whether this novel non-invasive technique is able to serve as a perioperative diagnostic tool for patients in a clinical setting warrants further study.

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Aim: In the search for new antithrombotic drug candidates, the synthesis and anti-platelet activity of a new series of N-acylhydrazones that were designed as thrombin inhibitors has been previously described. The aim of this work was to further characterize the effects of these compounds on thrombin-induced platelet aggregation and induced thrombosis in vivo.
Methods: In this work, four compounds were tested, LASSBio-693, 694, 743 and 752, on platelet aggregation induced by thrombin, ADP and TRAP-4A. These compounds were further tested using a mouse pulmonary thromboembolism model induced by collagen (500 µg/kg) and norepinephrine (80 µg/kg) or thrombin (2,000 UI), and a deep venous thrombosis model.
Results: At 200 µM, the compounds showed between 36% and 82% inhibition (for L-743 and L-752, respectively) of thrombin-induced platelet aggregation. The receptor agonist of PAR-4, TRAP-4A (250 µM), was used and inhibition between 43% and 77% was observed for each compound (200 µM).
Compounds LASSBio-752 and 743 were the most effective in the venous thrombosis model, increasing the survival of the treated animals to 63% and 46%, respectively, in the model of collagen-induced thromboembolism and increasing to 80% (both) in the thrombin-induced model. LASSBio 743 was more effective for deep vein thrombosis, reducing the weight of the thrombus by approximately 70%. Conclusion: All compounds were administered orally and have shown effective antithrombotic action independently of the thrombotic stimulus. These results indicate that compounds LASSBio-743 and 752 are potential candidates for the treatment of cardiovascular diseases.

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Aim: Associations between long-term exposure to particulate matter (PM) and increased cardiovascular mortality have been reported, but few studies have investigated the associated incidence of cardiovascular disease, and none involved men.
Methods: We used data on 78,057 participants (37,121 men, 40,936 women) to examine the associations of long-term exposure to PM with mortality and the incidence of cardiovascular diseases. Average PM levels in the relevant area during the study period were used as an index of individual exposure to each subject. Cox models adjusted for age, sex, smoking, environmental smoking, body mass index, and alcohol consumption were used, with further adjustments for nitrogen dioxide and sulfur dioxide exposure, systolic blood pressure, total cholesterol, and diabetes mellitus.
Results: Increased PM levels were associated with an increased risk of incident coronary heart disease (CHD) and myocardial infarction (MI), particularly among smokers (adjusted hazard ratio (HR) per 10 µg/m³ increase in PM for CHD: 1.39, 95% confidence interval (CI) 1.01-1.93; for MI: 1.52, 1.08-2.13) and among women (for CHD: 1.63, 0.91-2.92; for MI: 1.99, 1.07-3.70); however, the observed positive association of PM exposure with the incidence of CHD and MI was not robust, and the association disappeared when one of the areas (Akita) was excluded. The association with CHD mortality was not statistically significant (adjusted HR per 10 µg/m³ increase in PM: 1.11, 0.92-1.33). There was an inverse association of long-term PM exposure with stroke mortality and incidence. There was no clear association between PM levels and lung cancer mortality and incidence.
Conclusions: Long-term exposure to PM might increase the risk of incident CHD in middle-aged Japanese men and women. Our findings also suggest that women and smokers are especially vulnerable to PM exposure.

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