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Aims: HDL particles have various anti-atherogenic functions, whereas HDL from atherosclerotic patients was demonstrated to be dysfunctional. One possible mechanism for the formation of dysfunctional HDL is the oxidation of its components. However, oxidized HDLs (Ox-HDLs) remain to be well investigated due to lack of reliable assay systems., Methods: We have developed a novel sandwich enzyme-linked immunosorbent assay (ELISA) for Ox-HDL by using the FOH1a/DLH3 antibody, which can specifically recognize oxidized phosphatidylcholine, a major component of HDL phospholipid (HDL-PL). We defined forced oxidation of 1 mg/L HDL-PL as 1 U/L Ox-HDL. We assessed serum Ox-HDL levels of normolipidemic healthy subjects ( n=94) and dyslipidemic patients (n=177)., Results: The coefficients of variation of within-run and between-run assays were 12.5% and 13.5%. In healthy subjects, serum Ox-HDL levels were 28.5±5.0 (mean±SD) U/L. As Ox-HDL levels were moderately correlated with HDL-PL (r=0.59), we also evaluated the Ox-HDL/HDL-PL ratio, which represents the proportion of oxidized phospholipids in HDL particles. In dyslipidemic patients, Ox-HDL levels were highly variable and ranged from 7.2 to 62.1U/L, and were extremely high (50.4±13.3U/L) especially in patients with hyperalphalipoproteinemia due to cholesteryl ester transfer protein deficiency. Regarding patients with familial hypercholesterolemia, those treated with probucol, which is a potent anti-oxidative and anti-hyperlipidemic drug, showed significantly lower Ox-HDL (16.2±5.8 vs. 30.2±5.4, p＜0.001) and Ox-HDL/HDL-PL ratios (0.200±0.035 vs. 0.229±0.031, p=0.015) than those without probucol., Conclusion: We have established a novel sandwich ELISA for Ox-HDL, which might be a useful and easy strategy to evaluate HDL functionality, although the comparison study between this Ox-HDL ELISA and the assay of HDL cholesterol efflux capacity remains to be done. Our results indicated that probucol treatment may be associated with lower Ox-HDL levels.

We report a case of Tangier disease with Leriche syndrome and bleeding tendency. In this male patient, nasal hemorrhage had been observed frequently throughout childhood. At 46 years old, he experienced effort angina, and coronary angiography demonstrated 75% stenosis in the right coronary artery. Orange-colored tonsils, mild hepatosplenomegaly and very low levels of serum high-density lipoprotein cholesterol (HDL-C) were observed, and the patient was diagnosed with Tangier disease. At 52 years old, effort angina recurred. Coronary angiography revealed 75% stenosis of the left main trunk, left anterior descending, and right coronary arteries. Stenosis of the brachiocephalic and right common iliac arteries was also recorded. Stents were implanted, and coronary artery bypass surgery was performed. At 53 years old, 15 months after surgery, the patient reported intermittent claudication, coldness of feet, and impotence. Aortic angiography showed progression of the stenosis at the bifurcation of the common iliac artery. The patient was diagnosed with Leriche syndrome, and aorta-left external iliac artery graft bypass surgery was performed. After surgery, oozing from subcutaneous tissue and leaking from the anastomotic region were observed. Additional analysis revealed two single-nucleotide polymorphisms (V825I and N935T) in the ATP-binding cassette transporter A1 (ABCA1) gene, and accumulation of small dense low-density lipoprotein together with low levels of HDL-C. In Tangier disease, HDL-C is markedly decreased because of ABCA1 deficiency. However, this is the first reported case to exhibit extensive atherosclerosis and bleeding tendency. This patient had atypical extensive and multiple atherosclerotic lesions, accompanied by Leriche syndrome and uncontrollable bleeding.

Aims: Progranulin (PGRN) is a multifunctional protein known to be involved in inflammation. However, the relation between PGRN and atherosclerosis remains elusive. The aim of this study was to define the role of PGRN in the development of atherosclerosis., Methods and Results: First, we checked the expression levels of PGRN in human atherosclerotic plaques. Immunohistochemical analysis showed that PGRN is strongly expressed in foam cells of atherosclerotic plaques. We also found that PGRN is expressed more abundantly in macrophages than in the smooth muscle cells of atherosclerotic lesions in ApoE(-/-) mice fed a high-fat diet for 12 weeks. Next, PGRN(-/-)ApoE(-/-) mice were generated to investigate the effect of PGRN on the development of atherosclerosis. PGRN(-/-)ApoE(-/-) mice exhibited severe atherosclerotic lesions compared with PGRN(+/+)ApoE(-/-) mice, despite their anti-atherogenic lipid profile. These results are partly due to enhanced expression of inflammatory cytokines, adhesion molecules, and decreased expression of endothelial nitric oxide synthase. In addition, lack of PGRN leads to accumulate excessive cholesterol in the macrophages and alter HDL-associated proteins., Conclusion: PGRN seems to be involved in the pathogenesis of atherosclerosis, possibly by various anti-atherogenic effects, including modulation of local and/or systemic inflammation.

In the present study, we measured carotid artery intima-media thickness (CIMT) and assessed several metabolic factors in middle-aged healthy Japanese men to clarify the relation between testosterone and atherosclerosis. The study comprised 176 male employees aged ≥40 years who visited Osaka University Healthcare Center for their annual health examinations. Serum total testosterone (TT) concentration was measured using radioimmunoassay (RIA) and serum free testosterone concentration was measured using analog ligand RIA (aFT). A multivariate model adjusted for age, body mass index, mean arterial pressure and treatment for hypertension demonstrated a significant association between aFT and CIMT. Even after adjustment for other clinically relevant factors, the significant association between aFT and CIMT was not attenuated. After adjustment for all other clinically relevant factors, both univariate and multivariate models ascertained the stepwise association that a level of aFT of ≤10.0 pg/mL was significantly associated with CIMT. However, the association between TT and CIMT was not significant in either univariate or multivariate models. We conclude that our finding showing that low serum aFT level is an influencing and independent risk factor for CIMT is of value in the clinical setting because no other studies, to our knowledge, have conducted multivariate analyses using the various metabolic factors included in the present analyses.

Aim: The clustering of dyslipidemia, impaired glucose tolerance and hypertension increases the morbidity and mortality from cardiovascular events. A class B scavenger receptor, CD36, is a receptor for oxidized LDL and a transporter of long-chain fatty acids. Because of the impaired uptake of oxidized LDL in CD36-deficient macrophages and from the results of CD36 knockout mice, CD36 deficiency (CD36-D) was supposed to be associated with reduced risks for coronary artery disease (CAD); however, CD36-D patients are often accompanied by a clustering of coronary risk factors. The current study aimed to investigate the morbidity and severity of cardiovascular diseases in CD36-D patients., Methods: By screening for CD36 antigen on platelets and monocytes using FACS or the absent myocardial accumulation of 123I-BMIPP by scintigraphy, 40 patients with type I CD36-D were collected, the morbidity of CAD and their features of atherosclerotic cardiovascular diseases were observed. Screening for CD36-D in both CAD patients (n = 319) and healthy subjects (n = 1,239) were underwent., Results: The morbidity of CAD was significantly higher in CD36-D patients than in the general population; 50% of patients (20 out of 40) had CAD identified by BMIPP scintigraphy and 37.5% (3 out of 8) by FACS screening, respectively. Three representative CD36-D cases demonstrated severe CAD and atherosclerosis. The frequency of CD36-D was three times higher in CAD patients than in healthy subjects (0.9% vs 0.3%, p < 0.0001)., Conclusion: The morbidity of CAD is significantly higher in CD36-D patients suffering from severe atherosclerosis, implying that the status of CD36-D might be atherogenic.

Background: Recently, remnant lipoprotein is expected to be a new therapeutic target in the age of 'beyond LDL-cholesterol'. The aim of this study was to clarify the clinical significance of remnant lipoprotein cholesterol (RemL-C) determination in annual health examinations with the focus on large artery atherosclerosis. Methods and results Subjects investigated were men (n = 528) and women (n = 318) who underwent annual health examinations at Osaka University. RemL-C was measured with a newly developed homogeneous assay. Carotid and aortic atherosclerosis was estimated by intima-media thickness (IMT) and cardio-ankle vascular index (CAVI), respectively. First, simple regression analysis revealed that the RemL-C levels positively correlated with maximum IMT, mean IMT and CAVI in the whole group (P < 0.05). Next, receiver operating characteristic curve analysis showed that the most effective levels of RemL-C for predicting carotid and aortic atherosclerosis were 0.21 mmol/L (P < 0.05) and 0.22 mmol/L (P < 0.01) or more, respectively. Odds ratios (ORs) of high RemL-C levels (0.21 mmol/L or more) for carotid and aortic atherosclerosis were significantly increased, especially in low-risk, apparently healthy women (OR: 4.20, P < 0.05 and 3.79, P < 0.01, respectively). Five out of 13 female low-risk cases (38%) with carotid atherosclerosis showed high serum RemL-C levels. It should be emphasized that conventional risk factors are still strong predictors for large artery atherosclerosis in the whole group., Conclusions: Our results indicate that high serum RemL-C level is a predictive hallmark for large artery atherosclerosis in apparently healthy women. Determination of RemL-C should be employed as one of the parameters in annual health examinations.

Plasma high density lipoprotein (HDL)-cholesterol levels are inversely correlated to the risk of atherosclerotic cardiovascular diseases. Reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which HDL particles play a crucial role to carry cholesterol derived from peripheral tissues to the liver. Recently, ATP-binding cassette transporters (ABCA1, ABCG1) and scavenger receptor (SR-BI) have been identified as important membrane receptors to generate HDL by removing cholesterol from foam cells. Adiponectin (APN) secreted from adipocytes is one of the important molecules to inhibit the development of atherosclerosis. Epidemiological studies have revealed a positive correlation between plasma HDL-cholesterol and APN concentrations in humans, although its mechanism has not been clarified. Therefore, in the present study, we investigated the role of APN on RCT, in particular, cellular cholesterol efflux from human monocyte-derived and APN-knockout (APN-KO) mice macrophages. APN up-regulated the expression of ABCA1 in human macrophages, respectively. ApoA-1-mediated cholesterol efflux from macrophages was also increased by APN treatment. Furthermore, the mRNA expression of LXRalpha and PPARgamma was increased by APN. In APN-KO mice, the expression of ABCA1, LXRalpha, PPARgamma, and apoA-I-mediated cholesterol efflux was decreased compared with wild-type mice. In summary, APN might protect against atherosclerosis by increasing apoA-I-mediated cholesterol efflux from macrophages through ABCA1-dependent pathway by the activation of LXRalpha and PPARgamma.

The reverse cholesterol transport (RCT) is one of the major protective systems against atherosclerosis, in which high-density lipoprotein (HDL) removes cholesterol from lipid-laden cells and delivers it to the liver. Scavenger receptor class B type I (SR-BI) is a HDL receptor in the liver and adrenal glands and is involved in the selective uptake of cholesteryl ester from HDL, which has been extensively, analyzed using rodent models. However, the expression and regulation of the human homologue of this receptor are not known yet. We previously reported that this receptor is expressed in in vitro differentiated macrophages and its expression is up-regulated by the addition of modified lipoproteins into the medium [Hirano K, Yamashita S, Nakagawa Y, et al. Expression of human scavenger receptor class B type I in cultured human monocyte-derived macrophages and atherosclerotic lesions. Circ Res 1999;85:108-16]. In order to further investigate the physiological significance of this receptor in humans, we have performed extensive immunohistochemical analyses with specimens of the liver and adrenal glands as well as arteries with different stages of atherosclerotic lesions. In human liver and adrenal glands, a positive SR-BI immunoreactivity was detected in both hepatic and adrenal parenchymal cells as well as Kupffer cells. These parenchymal cells had a strong signal on the cell surface, whereas Kupffer cells showed a heterogeneous and punctate pattern. In human aorta and coronary arteries, SR-BI was highly expressed in atherosclerotic plaques, but not in non-atherosclerotic lesions. Double immunostaining revealed that SR-BI was expressed in a subpopulation of macrophages, of which staining pattern was similar to that observed in Kupffer cells. These data clearly demonstrated that SR-BI was expressed with cell-specific fashions in both the initial and terminal step of RCT in humans. Thus, SR-BI might be physiologically relevant and have distinct tissue-specific functions.

Objectives: Atherosclerotic lesions of the coronary arteries are the pathological basis for myocardial infarction and ischemic cardiomyopathy. Progression of heart failure after myocardial infarction is associated with cardiac remodeling, which has been studied by means of coronary ligation in mice. However, this ligation model requires excellent techniques. Recently, a new murine model, HypoE mouse was reported to exhibit atherogenic Paigen diet-induced coronary atherosclerosis and myocardial infarction; however, the HypoE mice died too early to make possible investigation of cardiac remodeling. Therefore, we aimed to modify the HypoE mouse model to establish a novel model for ischemic cardiomyopathy caused by atherosclerotic lesions, which the ligation model does not exhibit. Methods and Results: In our study, the sustained Paigen diet for the HypoE mice was shortened to 7 or 10 days, allowing the mice to survive longer. The 7-day Paigen diet intervention starting when the mice were 8 weeks old was adequate to permit the mice to survive myocardial infarction. Our murine model, called the “modified HypoE mouse”, was maintained until 8 weeks, with a median survival period of 36 days, after the dietary intervention (male, n = 222). Echocardiography demonstrated that the fractional shortening 2 weeks after the Paigen diet (n = 14) significantly decreased compared with that just before the Paigen diet (n = 6) (31.4±11.9% vs. 54.4±2.6%, respectively, P<0.01). Coronary angiography revealed multiple diffuse lesions. Cardiac remodeling and fibrosis were identified by serial analyses of cardiac morphological features and mRNA expression levels in tissue factors such as MMP-2, MMP-9, TIMP-1, collagen-1, and TGF-β. Conclusion: Modified HypoE mice are a suitable model for ischemic cardiomyopathy with multiple diffuse lesions and may be considered as a novel and convenient model for investigations of cardiac remodeling on a highly atherogenic background. [ABSTRACT FROM AUTHOR]

Aging is one of the risk factors for atherosclerotic cardiovascular diseases, however, its molecular mechanism is currently unknown. Many types of cells in the atherosclerotic lesions are considered to have various biological abnormalities such as impaired lipid homeostasis and slow cell proliferation, which may be related to senescence at cellular levels. One of the common characteristics of senescent cells in vitro is the alteration of actin cytoskeletons, which were reported to be involved in the intracellular transport of lipids. Cholesterol efflux from the cells is the initial step of reverse cholesterol transport, a major protective system against atherosclerosis. Recently, we demonstrated that Cdc42, a member of the Rho-GTPase family, might be crucial for cellular lipid transport and cholesterol efflux based upon studies of Tangier cells that are deficient in ABCA1 gene. In the current review, we also indicate that the expression of Cdc42 is decreased in the cells from aged subjects in close association with the retarded intracellular lipid transport. Furthermore, the Cdc42 expression is reduced by culturing fibroblasts in vitro for a long duration. Werner syndrome (WS) is characterized by the early onset of senescent phenotypes including premature atherosclerotic cardiovascular diseases, although the underlying molecular mechanism for the enhanced atherosclerosis has not been fully understood yet. We examined the intracellular lipid transport and cholesterol efflux and the expression levels of cholesterol efflux-related molecules in skin fibroblasts obtained from patients with WS. Cholesterol efflux was markedly reduced in the WS fibroblasts in association with an increased cellular cholesterol content. Fluorescent recovery after photobleaching technique revealed that intracellular lipid transport around Golgi apparatus was markedly reduced when using a C6-NBD-ceramide as a tracer. Cdc42 protein and its guanosine 5′-triphosphate-bound active form were markedly reduced in the WS fibroblasts. The adenovirus-mediated complementation of wild-type Cdc42 corrected the impaired cholesterol efflux, intracellular lipid transport and cellular cholesterol levels in the WS fibroblasts. These data indicate that the reduced expression of Cdc42 might be responsible for the abnormal lipid transport, which in turn might be related to the accelerated cardiovascular manifestations in WS patients. The current review focuses on the impaired efflux of cholesterol from aged cells and its molecular mechanism as a basis for age-related enhancement of atherosclerosis. [ABSTRACT FROM AUTHOR]

Abstract: Background: Postprandial hyperlipidemia (PPHL) is an independent risk factor for coronary heart disease (CHD) which is based on the accumulation of chylomicrons (CM) and CM remnants containing apolipoprotein B-48 (apoB-48). Since atherosclerotic cardiovascular diseases are frequently observed even in subjects with normal serum triglyceride (TG) level, the correlation between fasting apoB-48 containing lipoproteins and carotid intima-media thickness (IMT) was analyzed in subjects with normal TG levels. Methods: From subjects who took their annual health check at the Osaka Police Hospital (n =245, male), one-hundred and sixty-four male subjects were selected to take part in this study; the excluding factors were: systolic blood pressure ≥140mmHg, intake of antihypertensive or antihyperlipidemic drugs, or age >65 years. The association between biochemical markers and IMT was analyzed and independent predictors of max-IMT were determined by multiple regression analysis in all subjects and in groups N-1 (TG<100mg/dl, n =58), N-2 (100≤TG<150mg/dl, n =53) and H (150≤TG mg/dl, n =53), respectively. Results: Fasting total cholesterol, LDL–cholesterol, HDL–cholesterol, apoB-100 and lnRemL–C (remnant lipoprotein–cholesterol) levels were not correlated with max-IMT, but lnTG and lnapoB-48 were significantly correlated with max-IMT in all subjects. LnapoB-48 and apoB-48/TG ratio were significantly correlated with max-IMT in group N-2. By multiple regression analysis, age and lnapoB-48 were independent variables associated with max-IMT in group N-2. Conclusion: Serum apoB-48 level might be a good marker for the detection of early atherosclerosis in middle-aged subjects with normal-range levels of blood pressure and TG. [Copyright &y& Elsevier]