Shinji Koba

Clinical Significance of Small Dense Low-Density Lipoprotein Cholesterol Levels Determined by the Simple Precipitation Method

Objective—Recently, we established a simple method for the quantification of small dense LDL cholesterol (C) using heparin-magnesium precipitation. The small dense LDL-C level was identical to cholesterol in the denser LDL fraction with a density of 1.044 to 1.063 g/mL. The aim of this study was to examine clinical significance of this precipitation method for small dense LDL-C. Methods and Results—Small dense LDL-C was measured by a direct homogenous LDL-C assay in the supernatant that remained after heparin-magnesium precipitation with density <1.044 lipoproteins. In 313 normolipidemic subjects, the mean value of small dense LDL-C was 31±13 mg/dL. In 462 healthy subjects, small dense LDL-C levels were positively correlated with serum triglyceride and LDL-C and were inversely correlated with high-density lipoprotein cholesterol (HDL-C). Combined hyperlipidemia showed the highest small dense LDL-C level among the various types of hyperlipidemia. Patients with type 2 diabetes had an increased small dense LDL-C level (55±17). Patients with coronary heart disease also had increased small dense LDL-C levels (53±30) irrespective of the presence of diabetes, whereas their LDL-C levels were comparable to those of normolipidemic controls (111±31 versus 104±22). Conclusion—These results suggest that measurement of small dense LDL-C by the present precipitation method is useful to evaluate atherogenic risk and may be applicable to routine clinical examination.

Mechanisms of restenosis after coronary intervention: difference between plain old balloon angioplasty and stenting.

BACKGROUND Restenosis after coronary intervention remains an unsolved and important clinical problem. We histologically examined the mechanism of restenosis after both balloon injury and stenting. METHODS Coronary arteries of swine were subjected to balloon injury and stenting. Next, just after stenting or at 7, 14, or 28 days, the animals were sacrificed for the evaluation by morphometric analysis, histological observation, and immunostaining. RESULTS The neointimal area peaked at 14 days in the balloon injury group (BG) and increased linearly up to 28 days in the stent group (SG). At 28 days, the total vascular area in the BG was reduced to 78% of the control values. In the SG, the total vascular area remained enlarged. According to the phenotypic analysis, the vascular smooth muscle cells (VSMCs) in the neointimal area at 28 days were the contractile type in the BG and the synthetic type in the SG. Proliferating cell nuclear antigen (PCNA) and macrophage-positive cells were not observed in neointima in the BG at 28 days, whereas they were observed around the stent struts in the SG. In addition, numerous inflammatory cells, such as neutrophils and eosinophils, were also present in the SG. CONCLUSIONS Restenosis after balloon injury consisted of arterial remodeling and neointimal hyperplasia, whereas that after stenting consisted mostly of neointimal hyperplasia. The neointimal area in the SG lasted longer than that in the BG. Continuous inflammation may be an important factor in the restenosis of stenting.

Impact of Salusin-α and -β on Human Macrophage Foam Cell Formation and Coronary Atherosclerosis

Background— Human salusins, related bioactive polypeptides with mitogenic effects on vascular smooth muscle cells and fibroblasts and roles in hemodynamic homeostasis, may be involved in the origin of coronary atherosclerosis. Macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor (cholesterol influx), acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1; storage cholesterol ester converted from free cholesterol), and ATP-binding cassette transporter A1 (cholesterol efflux). Methods and Results— Serum salusin-α levels were decreased in 173 patients with angiographically proven coronary artery disease compared with 40 patients with mild hypertension and 55 healthy volunteers (4.9±0.6 versus 15.4±1.1 and 20.7±1.5 pmol/L, respectively; P<0.0001). Immunoreactive salusin-α and -β were detected in human coronary atherosclerotic plaques, with dominance of salusin-β in vascular smooth muscle cells and fibroblasts. After 7 days in primary culture, acetylated low-density lipoprotein–induced cholesterol ester accumulation in human monocyte-derived macrophages was significantly decreased by salusin-α and increased by salusin-β. Salusin-α significantly reduced ACAT-1 expression in a concentration-dependent manner. In contrast, salusin-β significantly increased ACAT-1 expression by 2.1-fold, with a maximal effect at 0.6 nmol/L. These effects of salusins were abolished by G-protein, c-Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase kinase inhibitors. ACAT activity and ACAT-1 mRNA levels were also significantly decreased by salusin-α and increased by salusin-β; however, neither salusin-α nor salusin-β affected scavenger receptor A function assessed by [125I]acetylated low-density lipoprotein endocytosis or scavenger receptor class A and ATP-binding cassette transporter A1 expression. Conclusions— Our results indicate that the 2 salusin isoforms have opposite effects on foam cell formation in human monocyte-derived macrophages. Development of atherosclerosis may be accelerated by salusin-β and suppressed by salusin-α via ACAT-1 regulation.

A Single Nucleotide Polymorphism in the Carboxylesterase Gene Is Associated with the Responsiveness to Imidapril Medication and the Promoter Activity

Imidapril is an angiotensin-converting enzyme inhibitor that is widely used in treating hypertension, although the responses vary among individuals. We investigated whether a single nucleotide polymorphism at position −816 of the carboxylesterase 1 (CES1) gene, which activates imidapril in the liver, is involved in the responsiveness to imidapril medication. A total of 105 Japanese hypertensives with systolic/diastolic blood pressures (SBP/DBP) of 140/90 mmHg or higher were prescribed 5–10 mg/day of imidapril. At baseline, blood pressure levels were not different between patients with and those without the −816C allele (AA vs. AC+CC groups). After 8 weeks of treatment, we classified the responders and non-responders based on the decline in their blood pressures, and found that the responder rate was significantly higher in the AC+CC group than in the AA group (p=0.0331). Also, the reduction in SBP was significantly greater in the AC+CC group than in the AA group (24.7±11.8 vs. 17.6±16.8 mmHg, p=0.0184). Furthermore, an in vitro reporter assay revealed that the −816C construct had significantly higher promoter activity (p<0.0001). These findings suggest that the A(−816)C polymorphism affects the transcriptional activity, and that this may account for the responsiveness to imidapril.

Remarkably high prevalence of small dense low-density lipoprotein in Japanese men with coronary artery disease, irrespective of the presence of diabetes

To examine how prevalence of the small dense LDL phenotype (LDL particle diameter < or =25.5 nm) is associated with coronary artery disease (CAD) in type 2 diabetic and non-diabetic Japanese men, an ethnic group with a low incidence of CAD, 85 non-diabetic men and 45 type 2 diabetic men with angiographically documented CAD, and 142 control men and 76 type 2 diabetic men without CAD were studied. Mean LDL particle diameter was determined using 2-16% polyacrylamide gel electrophoresis. LDL particle diameters in CAD patients were much smaller than those in controls (25.2+/-0.7 vs. 26.0+/-0.4 nm, mean+/-S.D., P<0.0001). LDL size was smaller in diabetic subjects (25.6+/-0.6 nm) and became even smaller in diabetics with CAD (25.0+/-1.0 nm). Prevalence of small dense LDL was markedly higher in both non-diabetic and diabetic CAD patients than that in non-diabetic and diabetic patients without CAD (71, 76, 23 and 42%, respectively). CAD patients had lower HDL-cholesterol and apo A1 levels, and higher triglyceride levels than those in diabetic and non-diabetic CAD-free patients, while total- and LDL-cholesterol levels were even lower in CAD group, and remnant-like particle-cholesterol, lipoprotein (a) and insulin levels were comparable among four groups. LDL size was significantly associated with triglyceride, HDL-cholesterol and glycemic control. Logistic regression analysis revealed that the small dense LDL phenotype was significantly associated with the incidence of CAD independent of low levels of HDL-cholesterol or high levels of triglyceride in both non-diabetic and diabetic cases. These results suggest that high prevalence of small dense LDL is a leading cause of CAD in both diabetic and non-diabetic Japanese men. Type 2 diabetes shows a greater capacity to reduce LDL size, which may contribute to the high incidence of CAD in the diabetic population.

Small vessel pathology and coronary hemodynamics in patients with microvascular angina

We studied the ultrastructure of cardiac myocytes and small blood vessels obtained by endomyocardial biopsy from 21 patients with microvascular angina. Ischemic ST segment depression during atrial pacing was recognised in all the patients who had normal coronary arteriograms and biopsy tissues were examined by light and electron microscopy. In patients with microvascular angina, insufficient increases in coronary sinus blood flow and in myocardial oxygen consumption measured with a Websters catheter were apparent during atrial pacing. Biopsy samples under the light microscope showed evidence of myocardial hypertrophy and sclerosis of small arteries and arterioles with perivascular fibrosis in 18 of 19 (95%) patients. Electron microscopy revealed that many endothelial nuclei in capillaries were swollen and that lumina of small arteries and arterioles were irregularly narrowed with proliferated and deformed medial smooth muscle cells. These findings suggest that disturbances in the coronary microcirculation in these patients is responsible for the ischemic changes in electrocardiograms.

Coronary vein infusion of multipotent stromal cells from bone marrow preserves cardiac function in swine ischemic cardiomyopathy via enhanced neovascularization

Few reports have examined the effects of adult bone marrow multipotent stromal cells (MSCs) on large animals, and no useful method has been established for MSC implantation. In this study, we investigate the effects of MSC infusion from the coronary vein in a swine model of chronic myocardial infarction (MI). MI was induced in domestic swine by placing beads in the left coronary artery. Bone marrow cells were aspirated and then cultured to isolate the MSCs. At 4 weeks after MI, MSCs labeled with dye (n=8) or vehicle (n=5) were infused retrogradely from the anterior interventricular vein without any complications. Left ventriculography (LVG) was performed just before and at 4 weeks after cell infusion. The ejection fraction (EF) assessed by LVG significantly decreased from baseline up to a follow-up at 4 weeks in the control group (P<0.05), whereas the cardiac function was preserved in the MSC group. The difference in the EF between baseline and follow-up was significantly greater in the MSC group than in the control group (P<0.05). The MSC administration significantly promoted neovascularization in the border areas compared with the controls (P<0.0005), though it had no affect on cardiac fibrosis. A few MSCs expressed von Willebrand factor in a differentiation assay, but none of them expressed troponin T. In quantitative gene expression analysis, basic fibroblast growth factor and vascular endothelial growth factor (VEGF) levels were significantly higher in the MSC-treated hearts than in the controls (P<0.05, respectively). Immunohistochemical staining revealed VEGF production in the engrafted MSCs. In vitro experiment demonstrated that MSCs significantly stimulated endothelial capillary network formation compared with the VEGF protein (P<0.0001). MSC infusion via the coronary vein prevented the progression of cardiac dysfunction in chronic MI. This favorable effect appeared to derive not from cell differentiation, but from enhanced neovascularization by angiogenic factors secreted from the MSCs.

Preventive Effects of Heregulin-β1 on Macrophage Foam Cell Formation and Atherosclerosis

Rationale: Human heregulins, neuregulin-1 type I polypeptides that activate proliferation, differentiation, and survival of glial cells, neurons, and myocytes, are expressed in macrophage foam cells within human coronary atherosclerotic lesions. Macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor class A (SR-A), acyl-coenzyme A:cholesterol acyltransferase (ACAT)1, and ATP-binding cassette transporter (ABC)A1. Objective: The present study clarified the roles of heregulins in macrophage foam cell formation and atherosclerosis. Methods and Results: Plasma heregulin-&bgr;1 levels were significantly decreased in 31 patients with acute coronary syndrome and 33 patients with effort angina pectoris compared with 34 patients with mild hypertension and 40 healthy volunteers (1.3±0.3, 2.0±0.4 versus 7.6±1.4, 8.2±1.2 ng/mL; P<0.01). Among all patients with acute coronary syndrome and effort angina pectoris, plasma heregulin-&bgr;1 levels were further decreased in accordance with the severity of coronary artery lesions. Expression of heregulin-&bgr;1 was observed at trace levels in intracoronary atherothrombosis obtained by aspiration thrombectomy from acute coronary syndrome patients. Heregulin-&bgr;1, but not heregulin-&agr;, significantly reduced acetylated low-density lipoprotein–induced cholesterol ester accumulation in primary cultured human monocyte-derived macrophages by reducing SR-A and ACAT1 expression and by increasing ABCA1 expression at both mRNA and protein levels. Heregulin-&bgr;1 significantly decreased endocytic uptake of [125I]acetylated low-density lipoprotein and ACAT activity, and increased cholesterol efflux to apolipoprotein (Apo)A-I from human macrophages. Chronic infusion of heregulin-&bgr;1 into ApoE−/− mice significantly suppressed the development of atherosclerotic lesions. Conclusions: This study provided the first evidence that heregulin-&bgr;1 inhibits atherogenesis and suppresses macrophage foam cell formation via SR-A and ACAT1 downregulation and ABCA1 upregulation.

Counteractive effects of omentin-1 against atherogenesis

AIMS Omentin-1, a novel adipocytokine expressed in visceral fat tissue, is negatively correlated with obesity, insulin resistance, and stable coronary artery disease (CAD). However, there have been no previous reports regarding the effects of omentin-1 on atherogenesis. METHODS AND RESULTS This study was performed to evaluate the atheroprotective effects of omentin-1 on human monocyte-derived macrophages, human aortic smooth muscle cells (HASMCs) in vitro, and aortic lesions in Apoe(-/-) mice in vivo. The histological expression of omentin-1 in coronary artery lesions and epicardial adipose tissues and its plasma levels were compared between CAD and non-CAD patients. Omentin-1 was abundantly expressed in human umbilical vein endothelial cells, macrophages, HASMCs, and human coronary artery SMCs in vitro. Omentin-1 promoted anti-inflammatory M2 phenotype during differentiation of human monocytes into macrophages. Omentin-1 suppressed oxidized low-density lipoprotein-induced foam cell formation associated with down-regulation of CD36, scavenger receptor class A, and acyl-CoA:cholesterol acyltransferase-1 and up-regulation of neutral cholesterol ester hydrolase in human macrophages. Omentin-1 suppressed angiotensin II-induced migration and platelet-derived growth factor-BB-induced proliferation, and collagen-1 and -3 expression in HASMCs. Four-week infusion of omentin-1 into Apoe(-/-) mice retarded the development of aortic atherosclerotic lesions with reduced contents of monocytes/macrophages, SMCs, and collagen fibres along with peritoneal M2-activated macrophages with inflammasome down-regulation and lowered plasma total cholesterol levels. Omentin-1 levels were markedly reduced in coronary endothelium and epicardial fat but increased in plasma and atheromatous plaques (macrophages/SMCs) in CAD patients compared with non-CAD patients. CONCLUSION This study provided the first evidence that omentin-1 may serve as a novel therapeutic target for atherosclerosis and CAD.

Japan Atherosclerosis Society (JAS) Guidelines for Prevention of Atherosclerotic Cardiovascular Diseases 2017

Toray Industries, Inc., Tokyo, Japan Department of Diabetes, Metabolism and Endocrinology, Chiba University Graduate School of Medicine, Chiba, Japan National Center for Geriatrics and Gerontology, Aichi, Japan Department of Internal Medicine and Cardiology, Gifu Prefectural General Medical Center, Gifu, Japan Division of Diabetes and Metabolism, Department of Internal Medicine, Iwate Medical University, Iwate, Japan Division of Endocrinology and Metabolism, Department of Medicine, Jichi Medical University, Tochigi, Japan Center for Integrated Medical Research, Hiroshima University Hospital, Hiroshima, Japan Egusa Genshi Clinic, Hiroshima, Japan Department of Cardiovascular Medicine, Juntendo University, Tokyo, Japan Preventive Medicine and Public Health, Keio University School of Medicine, Tokyo, Japan Biomedical Informatics, Osaka University, Osaka, Japan Division of Cardiology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan Department of Community Health Systems Nursing, Ehime University Graduate School of Medicine, Ehime, Japan Department of Vascular Medicine, Osaka City University Graduate School of Medicine, Osaka, Japan Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan Department of Vascular Surgery, Saitama Medical Center, Saitama, Japan Chief Health Management Department, Mitsui Chemicals Inc., Tokyo, Japan Department of Pediatrics, Showa University School of Medicine, Tokyo, Japan Department of Neurology, Kita-Harima Medical Center, Hyogo, Japan Department of Internal Medicine, Mizonokuchi Hospital, Teikyo University School of Medicine, Kanagawa, Japan Division of Cardiology, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan Tsukasa Health Care Hospital, Kagoshima, Japan Faculty of Nutrition, Division of Clinical Nutrition, Kobe Gakuin University, Hyogo, Japan Department of Food and Nutrition, Faculty of Human Sciences and Design, Japan Women’s University, Tokyo, Japan 25 Department of Preventive Cardiology, National Cerebral and Cardiovascular Center, Osaka, Japan Department of Medical Statistics, Toho University, Tokyo, Japan Department of Clinical Innovative Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan Department of Laboratory Medicine, Jikei University Kashiwa Hospital, Chiba, Japan Department of Geriatric and General Medicine, Osaka University Graduate School of Medicine, Osaka, Japan Department of Obstetrics and Gynecology, Aichi Medical University, Aichi, Japan 31 Department of Community Medicine, Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan Rinku General Medical Center, Osaka, Japan