Vascular endothelial cells play key roles in maintaining vascular and organ homeostasis. Adrenomedullin (AM), originally identified as a vasodilating peptide, is now recognized to be a pleiotropic molecule involved in both circulatory homeostasis and the pathogenesis of cardiovascular diseases. We have reported that knockout mice deficient in AM or receptor activity-modifying protein 2 (RAMP2), an AM-receptor accessory protein, show vascular endothelial cell deformities that are embryonically lethal. To directly clarify the pathophysiological functions of the vascular AM-RAMP2 system, we generated vascular endothelial cell-specific RAMP2 knockout mice. Using these mice, we found that the AM-RAMP2 system is a key determinant of vascular integrity and homeostasis from prenatal stages through adulthood. This review highlights the functions of AM-RAMP2 in vascular endothelial cells.

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Aim: 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are cholesterol-lowering drugs with a variety of pleiotropic effects including antithrombotic properties. Tissue factor pathway inhibitor (TFPI), which is produced predominantly in endothelial cells and platelets, inhibits the initiating phase of clot formation. We investigated the effect of fluvastatin on TFPI expression in cultured endothelial cells.
Methods: Human umbilical vein endothelial cells (HUVECs) were treated with fluvastatin (0–10μM). The expression of TFPI mRNA and antigen were detected by RT-PCR and western blotting, respectively. The effects of mevalonate intermediates, small GTP-binding inhibitors, and signal transduction inhibitors were also evaluated to identify which pathway was involved. A luciferase reporter assay was performed to evaluate the effect of fluvastatin on TFPI transcription. The stability of TFPI mRNA was estimated by quantitating its levels after actinomycin D treatment.
Results: Fluvastatin increased TFPI mRNA expression and antigen in HUVECs. Fluvastatin-induced TFPI expression was reversed by co-treatment with mevalonate or geranylgeranylpyrophosphate (GGPP). NSC23766 and Y-27632 had no effect on TFPI expression. SB203580, GF109203, and LY294002 reduced fluvastatin-induced TFPI upregulation. Moreover, fluvastatin did not significantly affect TFPI promoter activity. TFPI mRNA degradation in the presence of actinomycin D was delayed by fluvastatin treatment.
Conclusions: Fluvastatin increases endothelial TFPI expression through inhibition of mevalonate-, GGPP-, and Cdc42-dependent signaling pathways, and activation of the p38 MAPK, PI3K, and PKC pathways. This study revealed unknown mechanisms of the anticoagulant effect of statins and gave a new insight to its therapeutic potential for the prevention of thrombotic diseases.

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Aim: The aim of this study was to identify the age and sex-specific reference ranges for the non-high-density lipoprotein cholesterol (non-HDLC) levels in Japanese children.
Methods: The subjects included 441,431 schoolchildren (207,015 boys, 234,416 girls) 9-16 years of age who participated in a screening and care program for lifestyle-related diseases from 2006 to 2011. The serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) levels were measured, and the non-HDL-C levels were calculated. The serum lipid levels were analyzed according to age and sex.
Results: The overall mean non-HDL-C level was 105.7±24.0 mg/dL, with a sex difference: boys= 103.0±24.0 mg/dL and girls=108.2±23.8 mg/dL. In boys, the median non-HDL-C level decreased gradually from 104 mg/dL in the 9-year-old age group to 96 mg/dL in the 15-year-old age group. The 75th percentile level was approximately 120 mg/dL in the 9- to 11-year-old groups and decreased at approximately 113 mg/dL in the 12- to 15-year-old groups, whereas the 95th percentile level was approximately 150 mg/dL in the 9- to 11-year-old groups and decreased at approximately 140 mg/dL in the 13- to 15-year-old groups. In girls, the median non-HDL-C level remained unchanged at approximately 105 mg/dL, with 75th and 95th percentile levels of approximately 122 and 150 mg/dL, respectively.
Conclusions: The non-HDL-C levels vary by age and sex. The age- and sex-specific reference ranges for the non-HDL-C levels may be a valuable tool for management with respect to preventing the development of atherosclerosis in childhood.

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Aim: Our previous “J-BENEFIT (Japan BEzafibrate cliNical EFfectIveness and Tolerability)” study demonstrated that bezafibrate improves blood lipid profiles and glucose control in dyslipidemic patients with diabetes. However, bezafibrate did not significantly improve low-density lipoprotein cholesterol (LDL-C), although some patients showed decreases while others showed increases in the LDL-C levels. Therefore, a subgroup analysis of the J-BENEFIT study was conducted to identify factors influencing the bezafibrate-induced changes in the LDL-C levels.
Methods: Of the 3,316 patients in the J-BENEFIT study, 2,116 not treated with other lipid-lowering drugs were enrolled in the current study, and the effects of 24-week treatment with bezafibrate on the LDL-C levels were analyzed. A reduction in the LDL-C level of ≥ 25% occurred in 253 patients, and a logistic-regression analysis was used to identify factors associated with this improvement.
Results: Among the 2,116 overall patients, bezafibrate treatment significantly increased the LDL-C levels from 123.9±36.7 to 125.7±31.3 mg/dL. The subanalysis showed that the treatment responses varied according to the baseline LDL-C level, with significant decreases in the ≥ 160 and ≥ 140-＜160 mg/dL groups, no significant decrease in the ≥ 120-＜140 mg/dL group and a significant increase in the ＜120 mg/dL group. A multivariate logistic-regression analysis of the data for the patients with an LDL-C of ≥ 25% identified a female sex, the use of anti-hypertensive and hypoglycemic agents and a high baseline LDL-C level to be significant determinants of the LDL-C response to bezafibrate.
Conclusions: Our results showed that treatment with bezafibrate improves the LDL-C levels and lipid profiles in dyslipidemic diabetic patients, especially women, subjects co-treated with anti-hypertensive or hypoglycemic agents and those with high baseline LDL-C levels.

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Aim: Functional and structural abnormalities of the peripheral arteries are associated with renal dysfunction, independent of the presence of renal artery stenosis. This study investigated whether echolucent carotid plaque is associated with a future decline in the renal function in patients with coronary artery disease (CAD).
Methods: Ultrasound assessments of carotid plaque echolucency with integrated backscatter (IBS) analyses were performed in 327 patients with stable CAD and carotid plaque who exhibited a normal renal function (estimated glomerular filtration rate [eGFR] ≥60 mL/min/1.73 m²) at baseline. A lower IBS value reflects the presence of echolucent and lipid-rich unstable plaque. All patients were followed up for 36 months or until the occurrence of renal dysfunction, defined as an eGFR of ＜45 mL/min/1.73 m².
Results: During the follow-up period, 39 patients developed renal dysfunction. A multivariate logistic regression analysis showed that the presence of carotid plaque with a low IBS value was significantly associated with progression to renal dysfunction (odds ratios 0.48; 95% CI 0.30-0.78, p= 0.003). In addition, carotid plaque with a low IBS value had a significant incremental effect on the predictive value of known risk factors for renal dysfunction in analyses using c-statistics (AUC of the baseline risk model with and without IBS: 0.83 vs. 0.79, respectively, p=0.04), net reclassification improvement (index=0.549, p=0.001) and integrated discrimination improvement (index=0.068, p=0.002).
Conclusions: Echolucency of the carotid arteries is associated with future renal dysfunction in patients with stable CAD, indicating that the mechanisms related to plaque instability may be involved in the onset of renal dysfunction.

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Aim: Diabetes is accompanied by abdominal obesity, which produces various metabolic abnormalities. While metabolic factors have been considered to promote the development of coronary atherosclerosis in the early-stage of diabetes, it remains unknown whether the presence of obesity in early-stage diabetics affects the natural history of coronary atherosclerosis. We herein investigated the characteristics of the disease substrate in obese early-stage diabetics.
Methods: The DIANA (DIAbetes and diffuse coronary NArrowing) study was a serial evaluation of angiographic disease progression in early-stage diabetics with coronary artery disease. A total of 252 study subjects were stratified into non-obese (n=168) and obese groups (n=84). Obesity in Japanese subjects was defined as a body mass index ≥25 kg/m² according to the statement about Japanese obesity from the Japan Society for the Study of Obesity. Coronary atherosclerotic changes were evaluated by a quantitative computed analysis. The total lesion length (TLL=total length of all atherosclerotic lesions) was compared between the groups.
Results: The obese patients were younger (p=0.0002) and had higher levels of fasting (p=0.002) and postprandial insulin (p=0.01), and higher triglyceride levels (p=0.02). On serial angiographic evaluations, obese patients had greater disease progression, reflected by a larger percent change in the TLL (24.7±13.7 vs. 7.4±10.0%, p=0.04). However, the improvement of abnormal glucose tolerance was associated with a slowing of disease progression in both non-obese (−0.9±10.7 vs. ＋15.0±11.2%, p=0.04) and obese (＋4.2±22.8 vs. ＋55.5±26.5%, p=0.005) patients.
Conclusions: Obese patients with early-stage diabetes exhibit profound disease progression. Glycemic control attenuated the progression of their coronary atherosclerosis. Our findings indicate progressive but modifiable disease in obese early-stage diabetics under optimal glycemic management.

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Aim: To investigate the relationships between arterial stiffness and classic cardiovascular risk factors with respect to gender differences in addition to the prevalence of high arterial stiffness in Chongqing, China based on an examination of 18,336 subjects.
Methods: The cardio-ankle vascular index was used as a marker of arterial stiffness. The relationships between arterial stiffness and body mass index (BMI) as well as metabolic syndrome (MetS) were estimated using logistic regression models.
Results: The prevalence of high arterial stiffness was 12.74% in men and 9.91% in women. For age and BMI, compared with the reference group, men had higher adjusted odds ratios (ORs) in each group versus their female counterparts. For each individual index of MetS, the effects of waist circumference and systolic blood pressure (SBP) on high arterial stiffness exhibited remarkable gender differences, with women having higher ORs and adjusted ORs than men. As the sum of MetS traits increased, the ORs and adjusted ORs in the subjects also increased, with women having higher values than men in each group.
Conclusions: Gender-specific differences exist in the prevalence of high arterial stiffness among subjects compared by age, BMI and MetS, with varying effects of influence for these factors between genders.

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Aim: The current study investigated how prevalent the absence of a prior history of intermittent claudication would be in patients with critical limb ischemia (CLI) and examined the associated clinical features.
Methods: We used a database of 559 Japanese CLI patients participating in a multicenter prospective study. A history of intermittent claudication prior to CLI onset was surveyed at registration. The 95% confidence interval (CI) of its prevalence was calculated using the Clopper-Pearson method. Logistic regression analysis was performed to assess the association between the clinical features and the absence of preceding intermittent claudication.
Results: The study subjects were 73±10 years old and 67% were male. Tissue loss occurred in 82% of this population. The prevalence of the absence of prior intermittent claudication was 50% [95% CI: 46-55%]. In multivariate logistic regression analysis, a non-ambulatory status, diabetes mellitus, and regular dialysis were significantly and independently associated with the lack of a prior history of intermittent claudication (all p＜0.05). Indeed, the presence of these features was associated with a higher prevalence of the lack of the history. Regular dialysis, but not non-ambulatory status or diabetes mellitus, lost its statistical significance after further adjustment for the presence of isolated infrapopliteal lesions, whereas the presence of isolated infrapopliteal lesions itself was significantly associated with a lack of prior intermittent claudication.
Conclusions: The absence of a prior history of intermittent claudication was prevalent in CLI patients. Patients with a non-ambulatory status, diabetes mellitus, and regular dialysis were more likely to lack a prior history of intermittent claudication.

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Aim: This study was conducted to examine whether low serum levels of 25-hydroxyvitamin D (25OHD)are associated with a higher risk of incident peripheral artery disease (PAD) in a representative group of elderly people.
Methods: We followed 1568 community-dwelling elderly participants without PAD at the baseline (among a sample of 2097 initially eligible) over a mean of 4.4 years as part of the Progetto Veneto Anziani (Pro.V.A.) study. The baseline serum 25OHD levels were categorized as ＜24, 25-49, 50-74, ＞75 nmol/L, and incident PAD was defined as an ankle-brachial index below 0.9.
Results: At the baseline, there were no differences in known risk factors for PAD (BMI, waist circumference, diabetes, cardiovascular diseases, smoking habits, total cholesterol) or in the ankle-brachial index (ABI) between the groups with different serum 25OHD levels (＜24, 25-49, 50-74, ＞75 nmol/L). During a 4.4-year follow-up, 371 subjects developed PAD. The group with serum 25OHD levels ＞75 nmol/L was set as the reference group, and an adjusted Cox's regression analysis showed no association between low vitamin D levels and incident PAD during the follow-up: the hazard ratio ranged from 0.76 (95%CI: 0.41-1.42) for participants with serum 25OHD levels below 25 nmol/L to 1.32 (95%CI: 0.72-2.39) for those with serum 25OHD levels between 50-74 nmol/L (p for trend=0.08). These results did not change when participants were stratified by several risk factors for PAD.
Conclusions: Baseline hypovitaminosis D did not predict the onset of PAD over a 4.4-year follow-up in elderly people.

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