Tomoyuki Yasuda

Tissue-Specific Liver X Receptor Activation Promotes Macrophage Reverse Cholesterol Transport In Vivo

Objective—We previously reported that a systemic liver X receptor (LXR) agonist promoted macrophage reverse-cholesterol transport (mRCT) in vivo. Because LXR are expressed in multiple tissues involved in RCT (macrophages, liver, intestine), we analyzed the effect of tissue-specific LXR agonism on mRCT. Methods and Results—In initial studies, the systemic LXR agonist GW3965 failed to promote mRCT in a setting in which LXR was expressed in macrophages but not in liver or intestine. To evaluate the effect of LXR activation specifically in small intestine on mRCT, wild-type mice were treated with either intestinal-specific LXR agonist (GW6340) or systemic LXR agonist (GW3965). Both GW3965 and GW6340 significantly promoted excretion of [3H]-sterol in feces by 162% and 52%, respectively. To evaluate the requirement for macrophage LXR activation, we assessed the ability of GW3965 to promote mRCT in wild-type mice using primary macrophages deficient in LXR&agr;/&bgr; vs wild-type macrophages. Whereas GW3965 treatment promoted fecal excretion compared with vehicle, its overall ability to promote mRCT was significantly attenuated using LXR&agr;/&bgr; knockout macrophages. Conclusion—We demonstrate that intestinal-specific LXR agonism promotes macrophage RCT in vivo and that macrophage LXR itself plays an important, but not predominant, role in promoting RCT in response to an LXR agonist.

RAGE mediates oxidized LDL-induced pro-inflammatory effects and atherosclerosis in non-diabetic LDL receptor-deficient mice

AIMS Receptor for advanced glycation end products (RAGE) plays a pivotal role in the genesis of diabetic vascular diseases. To further explore the mechanisms underlying atherosclerosis under non-diabetic conditions, we examined the effect of RAGE deficiency on atherosclerosis in hyperlipidaemic mice. METHODS AND RESULTS RAGE-/- mice were crossed with low-density lipoprotein receptor-deficient (LDLr-/-) mice to generate the double knockout (DKO) mice. After feeding with high-fat diet for 12 weeks, aortic atherosclerotic lesions were analysed histologically in these mice. Although there were no differences in serum levels of glucose and known RAGE ligands between DKO and LDLr-/- mice, DKO mice exhibited a significant decrease in the size and macrophage content in atherosclerotic lesions compared with LDLr-/- mice. Expression of intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the aorta was lower in DKO mice than in LDLr-/- mice. Fluorescence-based assays revealed that oxidative stress in the vessel wall was attenuated in DKO mice than in LDLr-/- mice. Cell culture experiments revealed that RAGE mediated oxidative LDL-induced activation of p42/44 mitogen-activated protein kinases and oxidative stress in macrophages. CONCLUSION Oxidative LDL may be a ligand of RAGE in the hyperlipidaemic state. RAGE inactivation inhibits the atherosclerosis through reducing oxLDL-induced pro-inflammatory responses and oxidative stress in hyperlipidaemia.

Impact of Combined Deficiency of Hepatic Lipase and Endothelial Lipase on the Metabolism of Both High-Density Lipoproteins and Apolipoprotein B–Containing Lipoproteins

Rationale: Hepatic lipase (HL) and endothelial lipase (EL) are extracellular lipases that both hydrolyze triglycerides and phospholipids and display potentially overlapping or complementary roles in lipoprotein metabolism. Objective: We sought to dissect the overlapping roles of HL and EL by generating mice deficient in both HL and EL (HL/EL-dko) for comparison with single HL-knockout (ko) and EL-ko mice, as well as wild-type mice. Methods and Results: Reproduction and viability of the HL/EL-dko mice were impaired compared with the single-knockout mice. The plasma levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, non–HDL cholesterol, and phospholipids in the HL/EL-dko mice were markedly higher than those in the single-knockout mice. Most notably, the HL/EL-dko mice exhibited an unexpected substantial increase in small low-density lipoproteins. Kinetic studies with [3H]cholesteryl ether–labeled very-low-density lipoproteins demonstrated that the HL/EL-dko mice accumulated counts in the smallest low-density lipoprotein–sized fractions, as assessed by size exclusion chromatography, suggesting that it arises from lipolysis of very-low-density lipoproteins. HDL from all 3 lipase knockout models had an increased cholesterol efflux capacity but reduced clearance of HDL cholesteryl esters versus control mice. Despite their higher HDL cholesterol levels, neither HL-ko, EL-ko, nor HL/EL-dko mice demonstrated an increased rate of macrophage reverse cholesterol transport in vivo. Conclusions: These studies reveal an additive effect of HL and EL on HDL metabolism but not macrophage reverse cholesterol transport in mice and an unexpected redundant role of HL and EL in apolipoprotein B lipoprotein metabolism.

Endothelial cell-selective adhesion molecule modulates atherosclerosis through plaque angiogenesis and monocyte-endothelial interaction

Endothelial cell-selective adhesion molecule (ESAM) is a new member of the immunoglobulin superfamily, which is expressed in vascular endothelial cells. Previous studies have demonstrated that ESAM regulates angiogenesis, endothelial permeability, and leukocyte transmigration. However, little is known concerning the role of ESAM in atherosclerosis. In this study, we assessed the effects of ESAM inactivation on atherosclerosis in mice. ESAM-/- mice were bred with apoE-/- mice to generate double knockout mice, and the aortic lesion size of apoE-/- and ESAM-/-apoE-/- mice was compared histologically. Although plasma cholesterol levels were higher in ESAM-/-apoE-/- mice, the lesion size was markedly smaller than in apoE-/- mice. ESAM-/-apoE-/- mice exhibited a decrease in the number of vasa vasorum and macrophages in the vessel wall. In vitro adhesion assays showed that THP-1 cells, which did not express ESAM, bound to the ESAM-coated culture plates, suggesting that ESAM may interact with heterophilic ligand(s) on monocytes. Moreover, downregulation of ESAM by siRNA in the endothelial monolayer diminished transendothelial migration of THP-1 cells. In conclusion, ESAM inactivation can reduce susceptibility to atherosclerosis by inhibiting plaque neovascularization and macrophage infiltration into the atheroma.

Targeted deletion of endothelial lipase increases HDL particles with anti-inflammatory properties both in vitro and in vivo

Previous studies have shown that targeted deletion of endothelial lipase (EL) markedly increases the plasma high density lipoprotein cholesterol (HDL-C) level in mice. However, little is known about the functional quality of HDL particles after EL inhibition. Therefore, the present study assessed the functional quality of HDL isolated from EL−/− and wild-type (WT) mice. Anti-inflammatory functions of HDL from EL−/− and WT mice were evaluated by in vitro assays. The HDL functions such as PON-1 or PAF-AH activities, inhibition of cytokine-induced vascular cell adhesion molecule-1 expression, inhibition of LDL oxidation, and the ability of cholesterol efflux were similar in HDL isolated from WT and EL−/− mice. In contrast, the lipopolysaccharide-neutralizing capacity of HDL was significantly higher in EL−/− mice than that in WT mice. To evaluate the anti-inflammatory actions of HDL in vivo, lipopolysaccharide-induced systemic inflammation was generated in these mice. EL−/− mice showed higher survival rate and lower expression of inflammatory markers than WT mice. Intravenous administration of HDL isolated from EL−/− mice significantly improved the mortality after lipopolysaccharide injection in WT mice. In conclusion, targeted disruption of EL increased HDL particles with preserved anti-inflammatory and anti-atherosclerotic functions. Thus, EL inhibition would be a useful strategy to raise ‘good’ cholesterol in the plasma.

Pitavastatin decreases the expression of endothelial lipase both in vitro and in vivo.

AIMS In addition to their cholesterol-lowering effect, statins increase high-density lipoprotein cholesterol (HDL-C) levels. Endothelial lipase (EL) is a regulator of plasma HDL-C levels. In the present study, the effects of statins on EL expression were investigated. METHODS AND RESULTS The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor pitavastatin suppressed basal and cytokine-treated EL expression in endothelial cells. Concomitant treatment with mevalonate or geranylgeranyl pyrophosphate completely reversed the inhibitory effect of pitavastatin, suggesting that geranylgeranylated proteins are involved in the inhibition of EL expression by statins. Inhibition of RhoA activity by overexpression of a dominant-negative mutant of RhoA or a Rho kinase inhibitor decreased EL levels. Pitavastatin reduced phospholipase activities of endothelial cells, and concomitant treatment with mevalonate reversed its inhibitory effect. Pitavastatin reduced RhoA activity and EL expression in mouse tissues. Furthermore, plasma EL concentrations in human subjects were measured by enzyme-linked immunosorbent assays. Plasma EL levels were negatively associated with plasma HDL levels in 237 patients with cardiovascular diseases, and pitavastatin treatment reduced plasma EL levels and increased HDL-C levels in 48 patients with hypercholesterolaemia. CONCLUSION These findings suggest that statins can reduce EL expression in vitro and in vivo via inhibition of RhoA activity. The inhibition of EL expression in the vessel wall may contribute to the anti-atherogenic effects of statins.

Serum Trans-Fatty Acid Concentration Is Elevated in Young Patients With Coronary Artery Disease in Japan

BACKGROUND Adverse effects of dietary intake of trans-fatty acids (TFA) on the incidence of coronary artery disease (CAD) are well recognized in Western countries. The risk of TFA, however, has not been well clarified in Japan. We investigated the association of serum TFA concentration with serum lipid profile, coronary risk factors, and prevalence of CAD. METHODSANDRESULTS A total of 902 patients, who were hospitalized at Kobe University Hospital from July 2008 to March 2012 and gave written informed consent, were enrolled in this study. Among them, 463 patients had CAD, and 318 patients had metabolic syndrome (MetS). Serum TFA, elaidic acid (trans-9-C18:1) and linolelaidic acid (trans-9, 12-C18:2), were measured on gas chromatography/mass spectrometry. Serum TFA level had a positive correlation with body mass index, waist circumference, low-density lipoprotein cholesterol, triglycerides, and apolipoprotein B48, and an inverse correlation with age and high-density lipoprotein cholesterol. Fasting serum TFA, by age quartile in the young generation with CAD and/or MetS, was higher than that in patients without CAD and/or MetS. On multivariate logistic regression, TFA was identified as a CAD risk after adjustment for classical risk factors. CONCLUSIONS Serum TFA concentration was elevated in young patients with CAD and/or MetS. Diet-derived TFA may cause a serious health problem, particularly in the young generation in Japan.

Fasting serum concentration of apolipoprotein B48 represents residual risks in patients with new-onset and chronic coronary artery disease.

BACKGROUND To identify new therapeutic targets for coronary artery disease (CAD), we investigated whether fasting serum concentration of apolipoprotein (apo) B48 could be a marker for CAD. METHODS Patients with CAD were divided into those with new-onset CAD [i.e., those receiving percutaneous coronary intervention (PCI) for the first time] and those with chronic CAD (i.e., those receiving follow-up coronary angiography). Fasting serum biochemical analyses were performed on admission and 6 months after the PCI. RESULTS On admission, serum LDL-C concentrations in patients with chronic CAD (n=138), presumably receiving statin treatment, were lower than in patients with new-onset CAD (n=50, p<0.02) or without CAD (n=71, p<0.001). Nevertheless, apoB48 was higher in CAD patients than in those without CAD (p<0.001). After adjusting for classic cardiovascular risk factors, multivariate logistic regression analyses showed apoB48 to be an independent predictor of coronary risk in new-onset or chronic CAD, irrespective of the LDL-C levels. Moreover, apoB48 was markedly increased during the follow-up period in CAD patients having new lesion progression after the prior PCI. CONCLUSION Fasting serum apoB48 concentration could be a marker of new onset as well as chronic CAD, and predict new lesion progression in secondary prevention.

Trans-fatty acid promotes thrombus formation in mice by aggravating antithrombogenic endothelial functions via Toll-like receptors

SCOPE Since excessive intake of trans-fatty acid (TFA) increases the risk of myocardial infarction, we investigated the effects of TFA on thrombus formation using animal and cell culture experiments. METHODS AND RESULTS C57BL/6 mice were fed a diet containing TFA or cis-fatty acid (5% each of total calories) or a chow diet for 4 weeks, and thrombus formation was induced in the carotid artery by He-Ne laser irradiation. The high-TFA diet significantly promoted thrombus formation in the carotid artery compared to the chow or cis-fatty acid diet. TFA activated the inflammatory signaling pathway in cultured endothelial cells and in mice; aortic gene expression levels of antithrombogenic molecules, including thrombomodulin and tissue factor pathway inhibitor, were decreased, and the expression levels of prothrombogenic molecules were increased in TFA-treated mice. TFA markedly upregulated the prothrombogenic molecules and downregulated the antithrombogenic molecules in endothelial cells. In addition, TFA induced phosphorylation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, and nuclear factor-κB. The TFA-activated signal pathways and prothrombogenic phenotypic changes of endothelial cells were inhibited by genetic or pharmacological inactivation of Toll-like receptors 2 and 4. CONCLUSION TFA aggravates the antithrombogenic phenotypes of vascular endothelial cells via Toll-like receptors and promotes thrombus formation in mice.

Endothelial Lipase Modulates Pressure Overload–Induced Heart Failure Through Alternative Pathway for Fatty Acid Uptake

Lipoprotein lipase has been considered as the only enzyme capable of generating lipid-derived fatty acids for cardiac energy. Endothelial lipase is another member of the triglyceride lipase family and hydrolyzes high-density lipoproteins. Although endothelial lipase is expressed in the heart, its function remains unclear. We assessed the role of endothelial lipase in the genesis of heart failure. Pressure overload–induced cardiac hypertrophy was generated in endothelial lipase−/− and wild-type mice by ascending aortic banding. Endothelial lipase expression in cardiac tissues was markedly elevated in the early phase of cardiac hypertrophy in wild-type mice, whereas lipoprotein lipase expression was significantly reduced. Endothelial lipase−/− mice showed more severe systolic dysfunction with left-ventricular dilatation compared with wild-type mice in response to pressure overload. The expression of mitochondrial fatty acid oxidation–related genes, such as carnitine palmitoyltransferase-1 and medium-chain acyl coenzyme A dehydrogenase, was significantly lower in the heart of endothelial lipase−/− mice than in wild-type mice. Also, endothelial lipase−/− mice had lower myocardial adenosine triphosphate levels than wild-type mice after aortic banding. In cultured cardiomyocytes, endothelial lipase was upregulated by inflammatory stimuli, whereas lipoprotein lipase was downregulated. Endothelial lipase–overexpression in cardiomyocytes resulted in an upregulation of fatty acid oxidation–related enzymes and intracellular adenosine triphosphate accumulation in the presence of high-density lipoprotein. Endothelial lipase may act as an alternative candidate to provide fatty acids to the heart and regulate cardiac function. This effect seemed relevant particularly in the diseased heart, where lipoprotein lipase action is downregulated.