Critical limb ischemia (CLI) is commonly caused by atherosclerotic arterial obstruction or stenosis in the leg, as demonstrated by rest pain, skin ulcers and gangrene (Fontaine III or IV), often fails to respond to conservative treatments, and carries a high risk for limb amputation, with a particularly dismal prognosis. Although surgical revascularization techniques may be used for certain CLI patients, such techniques are not indicated for most CLI patients due to the diffuse nature of the responsible lesions, distal location of the obstruction, or coexisting systemic comorbidities. For such CLI patients with no alternative treatments, the potential utility of cell therapies has been investigated. Indeed many clinical trials are being carried out by academic sectors, and their achievements will facilitate clinical development by pharmaceutical companies.
In order to understand the situation regarding competitive international R&D of revascularization seeds for CLI, we surveyed the status of clinical trials. As a result, we identified 58 clinical trials on revascularization for CLI, with the majority in the early phase (<phase II: 82.7%). Revascularization seeds for CLI are in the development and competition phase, and promising seeds are expected to appear in the near future.
In this review, we discuss how to develop optimal regenerative medicine concerning the selection of cell origin, cell type, combination with growth factor, and the influence of concomitant disease.

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Aim: Apolipoprotein B-48 (apoB-48) is a major apolipoprotein of intestine-derived chylomicrons (CM) and CM remnants (CMR). Clinically overt hypothyroidism (OH) has been associated with premature and accelerated coronary atherosclerosis. To clarify the clinical significance of apoB-48 measurement in patients with thyroid disease, we investigated the correlations between the serum apoB-48 level and thyroid hormones.
Methods: From outpatients of Osaka University Hospital, patients with OH, subjects with subclinical hypothyroidism (SH) and subjects with normal thyroid function were collected and analyzed by measuring serum TSH, FT4 and FT3 levels. Serum apoB-48 levels were measured by a chemiluminescence enzyme immunoassay and the correlations with thyroid hormone levels or lipid profiles were assessed. These levels were compared among subjects with OH, SH and healthy controls.
Results: Serum apoB-48 level was correlated with TSH, total cholesterol (TC) and triglycerides (TG), but negatively with FT4 and FT3 level. LDL-C and HDL-C levels were not correlated with serum apoB-48 levels. Serum apoB-48 in patients with OH (7.4±5.9µg/mL) was significantly higher than in those with hyperthyroidism (5.1±3.5µg/mL; p<0.01) and normal subjects (4.7±3.7µg/mL; p<0.01), but decreased after levo-thyroxine replacement. ApoB-48, TG and TSH were significantly higher in SH subjects than normal subjects, suggesting that serum apoB-48 level depends on the thyroid function status, similar to TC, LDL-C and TG.
Conclusion: Increased serum apoB-48 concentrations and CMR may contribute to the increased risk of atherosclerosis and premature coronary artery disease in the hypothyroid state.

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Aim: Endothelial dysfunction is an initial step in the progression of atherosclerosis. Precise measurements of lipoprotein subclass distribution by high-performance liquid chromatography (HPLC) have been established. Here, we investigated the potential associations between lipoprotein subclass cholesterol concentrations and endothelial dysfunction evaluated by digital reactive hyperemia peripheral arterial tonometry (PAT).
Methods: We recruited 120 apparently healthy Japanese men. Endothelial function was assessed by digital reactive hyperemia PAT, expressed as the logarithmic-scaled reactive hyperemia index (RHI). Plasma cholesterol concentrations in lipoproteins and their subclasses were determined by HPLC with gel permeation columns.
Results: RHI was inversely correlated with age (r=−0.258, p=0.004), followed by LDL cholesterol (r=−0.236, p=0.010) and small LDL cholesterol (r=−0.223, p=0.014). In addition, RHI was significantly inversely associated with heart rate, hemoglobin A1c, total cholesterol, medium LDL cholesterol, apolipoprotein B100, and non-HDL cholesterol. In stepwise multiple regression analysis, age (β=−0.266, p=0.024), small LDL cholesterol (β=−0.213, p=0.015), and heart rate (β=−0.183, p=0.036) were found to be independent determinants of RHI (adjusted R² =0.132, p<0.001).
Conclusions: Small LDL cholesterol concentration was an important, independent determinant of endothelial dysfunction in men.

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Background: Cardiovascular diseases are the main causes of death in the Western world and are manifested by atherosclerosis. Depending on its intensity, regular aerobic exercise may be either beneficial or harmful to the atherosclerosis process.
Aim: The aim of this study was to verify the effects of aerobic exercise training of different intensities on the profile of atherosclerotic lesions and serum lipid, and in the hepatic oxidative balance of low-density lipoprotein receptor-deficient (LDLr^-/-) mice previously developed with atherosclerosis.
Methods: All animals were submitted to a three-month high-fat and high-cholesterol diet regime. The animals were then randomly divided into no exercise (G1, n=9), low-intensity aerobic exercise (G2, n=10, 8 weeks of treadmill running, 30 min/day⁻¹ at 8-10 m/min⁻¹) and moderate-intensity aerobic exercise (G3, n=10, 8 weeks of treadmill running, 30 min/day⁻¹ at 10-16m/min⁻¹) groups. Serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and triglycerides (TG), and oxidative damage (protein carbonyls and lipid hydroperoxides) were measured. The activity of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the liver tissue was assessed.
Results: G2 (0.015±0.005cm²) and G3 (0.014±0.001cm²) presented lower aortic fat deposition than G1 (0.039±0.005cm²). G2 and G3 exhibited higher HDL-C, TG and CAT activity, but lower lipid peroxidation and carbonyl protein than G1. SOD values were higher in G3 than G2 and G1, and GPx was higher in G2 than in G3 and G1.
Conclusions: Our protocols of low- and moderate-intensity aerobic exercise training (30 min daily for 8 weeks) induced similar benefits in LDLr^-/- mice with atherosclerosis.

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Aims: To investigate the prospective association between changes in the urinary albumin-creatinine ratio (ACR) and abnormal ankle-brachial index (ABI) in a community-based Chinese population.
Methods: This prospective cohort study included 799 residents aged 58.3±9.2 years and without a history of cardiovascular disease from an urban district of Beijing, China. Urinary ACR was measured at baseline, and at 4 and 6 years of follow-up. The 75th percentile of the baseline urinary ACR (5.82 mg/g) was used to define “high” ACR. The changes in urinary ACR were categorized as consistently low urinary ACR, intermittent high urinary ACR, and consistently high urinary ACR. ABI was measured at 6 years of follow-up. Multinomial logistic regression was used to evaluate the associations of changes in urinary ACR categories with the ABI categories.
Results: During 6 years of follow-up, 16.1% of participants (n= 128) had low ABI and 13.9% of participants (n= 111) had high ABI. After adjusting for potential confounders including baseline albuminuria, individuals who had consistently high urinary ACR or intermittent high urinary ACR had a significantly higher risk for low ABI than individuals who had consistently low urinary ACR, with odds ratios (OR) of 2.75 (95%CI, 1.37-5.52) and 2.06 (95%CI, 1.18-3.57), respectively. No independent association was observed between changes in urinary ACR and high ABI among participants.
Conclusion: Changes in urinary ACR below the definition for albuminuria predict low ABI among this community-based population without a history of cardiovascular disease.

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Aim: To identify predictors of coronary heart disease (CHD) in Japanese patients with type 2 diabetes (T2DM).
Methods: A matched case-control study was performed using 800 patients with T2DM admitted for treatment of hyperglycemia from January 2002 to June 2010. Cases comprised 16 patients who had developed acute myocardial infarction and/or received a coronary artery bypass by June 2010, and controls comprised 48 age- and sex-matched patients without CHD events. The mean age, glycated hemoglobin (HbA1c), and body mass index (BMI) were 61.5 yrs, 9.7% and 24.4 kg/m², respectively. The relationship of baseline variables, including lipid values, HbA1c, BMI, blood pressure, fasting blood sugar, 2h-post-breakfast blood sugar, delta blood sugar_0-2h, urinary albumin excretion, estimated glomerular filtration rate and treatment modalities (insulin/sulfonylurea/biguanide), to CHD development was analyzed by conditional logistic regression analysis.
Results: Total cholesterol (TC) (OR 2.35, 95%CI 1.11-4.98, p=0.03), non-HDL-cholesterol (OR 3.07, 95%CI 1.33-7.10, p=0.009), LDL-cholesterol (OR 2.84, 95%CI 1.24-6.51, p=0.01), non-HDL-cholesterol/HDL-cholesterol (OR 2.07, 95%CI 1.10-3.90, p=0.02) and LDL-cholesterol/ HDL-cholesterol (OR 2.74, 95%CI 1.22-6.15, p=0.01) were significantly related to CHD. Fold risk increment per 1-SD increase in basal TC, non-HDL-cholesterol, LDL-cholesterol, non-HDL-cholesterol/HDL-cholesterol and LDL-cholesterol/HDL-cholesterol was 2.33, 2.89, 2.52, 2.37 and 2.60, respectively. Only non-HDL-cholesterol was an independent risk factor. From the receiver operating characteristic curve, 3.89 mmol/L non-HDL-C was the best cutoff value. None of the non-lipid variables were significantly related to CHD.
Conclusion: Non-HDL-cholesterol was the most dominant predictor of the development of CHD in Japanese patients with T2DM.

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Aim: Vitamin D insufficiency and increased parathyroid hormone (PTH) levels have been suggested as prognostic indices for cardiovascular disease. Arterial stiffness, a surrogate marker for cardiovascular disease, is often increased in patients with primary hyperparathyroidism. PTH levels increase in patients with low 25-OH-vitamin D levels, but the influence of such an increase on arterial stiffness has not been investigated in postmenopausal women with reduced 25-OH-vitamin D levels. We therefore investigated the association between PTH and aortic stiffness in postmenopausal women with reduced 25-OH-vitamin D levels.
Methods: One hundred fifty postmenopausal women with 25-OH-vitamin D insufficiency (<30 ng/mL) were recruited. Aortic pulse wave velocity (aPWV), a measure of arterial stiffness, PTH and 25-OH-vitamin D levels were measured. Cardiovascular risk factors and markers of bone formation were evaluated.
Results: The 25-OH-vitamin D levels were associated with aPWV (rho=−0.23, p=0.006), but the association was not significant when controlling for PTH. Significant correlates of aPWV included age, body mass index, mean arterial pressure and PTH (rho=0.39, p<0.001). Arterial stiffness was predicted by logarithmically transformed PTH levels (β=0.23, p=0.007), independent of traditional cardiovascular risk factors and factors involved in bone formation. Increased PTH levels (>62 pg/mL) were associated with a 3.0-5.4-fold increased probability of having a mild-severe increase in aortic stiffness, irrespective of confounders.
Conclusion: Among postmenopausal women with reduced 25-OH-vitamin D levels, elevated PTH levels were a significant predictor of aortic stiffness, irrespective of cardiovascular risk factors and of factors involved in bone formation. PTH accounted for the association between 25-OH-vitamin D levels and aortic stiffness.

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Aim: The aim was to investigate the respective associations between lifestyle and proteinuria and the estimated glomerular filtration rate (eGFR).
Methods: The lifestyle habits of 25,493 middle-aged participants were investigated in a cross-sectional study to find habits that are associated with a low eGFR (<60 mL/min/1.73 m²) and/or the presence of proteinuria. The lifestyle habits of the participants were evaluated using a questionnaire. Unhealthy lifestyle habits were defined as follows: 1. obesity, 2. being a current/former smoker, 3. eating irregular meals, 4. having less than 5 hours sleep, 5. exercising less than once a week, and 6. drinking more than once a week. The associations among unhealthy habits, eGFR, and proteinuria were evaluated using multivariate analysis.
Results: The following lifestyle factors were significantly and independently associated with proteinuria: obesity (odds ratio (OR): 1.18, 95%C.I: 1.04-1.34), being a current/former smoker (OR: 1.26, 95%C.I: 1.11-1.42), eating irregular meals (OR: 1.40, 95%C.I: 1.22-1.61), sleeping less than 5 hours (OR: 1.38, 95%C.I: 1.15-1.65), and exercising less than once a week (OR: 1.18, 95%C.I: 1.05-1.33). In contrast, the following unhealthy lifestyle factors were not clearly associated with a low eGFR: obesity (OR: 1.05, 95%C.I: 0.95-1.17), being a current/former smoker (OR: 0.76, 95%C.I: 0.69-0.84), eating irregular meals (OR: 0.91, 95%C.I: 0.79-1.04), sleeping less than 5 hours (OR: 1.02, 95%C.I: 0.85-1.22), and exercising less than once a week (OR: 0.91, 95%C.I: 0.83-0.99).
Conclusion: Associations between proteinuria and unhealthy lifestyle habits were observed in our cross-sectional study. Unhealthy lifestyles should be monitored during the management of CKD patients with proteinuria.

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Aim: Low levels of soluble receptor for advanced glycation end-products (sRAGE) have been reported to be associated with coronary artery disease (CAD) and peripheral atherosclerosis. This study explored the relationship between circulating levels of sRAGE and the characteristics of coronary vessels detected by 64-slice computed tomography angiography (CTA).
Methods: In this cross-sectional study we included 127 consecutive patients with CAD but without acute coronary syndrome. Quantitative volumetric analysis of the lumen and plaque burden of the vessel wall (soft and calcific components) was performed for the three major coronary vessels. Each component was expressed as a percentage of vessel volume and utilized in per-patient analysis. The patients were classified into two groups according to the presence of calcium volume: non-calcified plaque (NCP) group (calcium volume %=0) and calcified plaque (CP) group (calcium volume % >0).
Results: In the NCP group, but not in the CP group, simple regression analysis revealed a negative association of total plaque burden % with sRAGE (β=−0.378, p=0.0019) and HDL cholesterol (β=−0.368, p=0.003) and a positive association with creatinine (β=0.258, p=0.041) and male gender (β=0.317, p=0.01). After adjusting for confounding factors, the total plaque burden % remained significantly associated only with sRAGE (β=−0.358, p=0.011).
Conclusions: Circulating sRAGE levels are associated in an inverse manner with non-calcified plaque burden, suggesting that it may be related with early atherosclerosis and plaque progression.

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