Liver-specific Lxr inhibition represses reverse cholesterol transport in cholesterol-fed mice.

BACKGROUND AND AIMS: High density lipoprotein (HDL) exerts an anti-atherosclerotic effect via reverse cholesterol transport (RCT). Several phases of RCT are transcriptionally controlled by Liver X receptors (Lxrs). Although macrophage Lxrs reportedly promote RCT, it is still uncertain whether hepatic Lxrs affect RCT in vivo. METHODS: To inhibit Lxr-dependent pathways in mouse livers, we performed hepatic overexpression of sulfotransferase family cytosolic 2B member 1 (Sult2b1) using adenoviral vector (Ad-Sult2b1). Ad-Sult2b1 or the control virus was intravenously injected into wild type mice and Lxrα/ß double knockout mice, under a normal or high-cholesterol diet. A macrophage RCT assay and an HDL kinetic study were performed. RESULTS: Hepatic Sult2b1 overexpression resulted in reduced expression of Lxr-target genes - ATP-binding cassette transporter G5/G8, cholesterol 7α hydroxylase and Lxrα itself - respectively reducing or increasing cholesterol levels in HDL and apolipoprotein B-containing lipoproteins (apoB-L). A macrophage RCT assay revealed that Sult2b1 overexpression inhibited fecal excretion of macrophage-derived 3H-cholesterol only under a high-cholesterol diet. In an HDL kinetic study, Ad-Sult2b1 promoted catabolism/hepatic uptake of HDL-derived cholesterol, thereby reducing fecal excretion. Finally, in Lxrα/ß double knockout mice, hepatic Sult2b1 overexpression increased apoB-L levels, but there were no differences in HDL levels or RCT compared to the control, indicating that Sult2b1-mediated effects on HDL/RCT and apoB-L were distinct: the former was Lxr-dependent, but not the latter. CONCLUSIONS: Hepatic Lxr inhibition negatively regulates circulating HDL levels and RCT by reducing Lxr-target gene expression.

Atorvastatin Reduces Circulating S100A12 Levels in Patients with Carotid Atherosclerotic Plaques - A Link with Plaque Inflammation.

AIMS: Inflammation is involved in various processes of atherosclerosis development. Serum C-reactive protein (CRP) levels, a predictor for cardiovascular risk, are reportedly reduced by statins. However, several studies have demonstrated that CRP is a bystander during atherogenesis. While S100A12 has been focused on as an inflammatory molecule, it remains unclear whether statins affect circulating S100A12 levels. Here, we investigated whether atorvastatin treatment affected S100A12 and which biomarkers were correlated with changes in arterial inflammation. METHODS: We performed a prospective, randomized open-labeled trial on whether atorvastatin affected arterial (carotid and thoracic aorta) inflammation using 18fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG-PET/CT) and inflammatory markers. Thirty-one statin-naïve patients with carotid atherosclerotic plaques were randomized to either a group receiving dietary management (n=15) or one receiving atorvastatin (10mg/day, n=16) for 12weeks. 18F-FDG-PET/CT and flow-mediated vasodilation (FMD) were performed, the latter to evaluate endothelial function. RESULTS: Atorvastatin, but not the diet-only treatment, significantly reduced LDL-cholesterol (LDL-C, -43%), serum CRP (-37%) and S100A12 levels (-28%) and improved FMD (+38%). 18F-FDG-PET/CT demonstrated that atorvastatin, but not the diet-only treatment, significantly reduced accumulation of 18F-FDG in the carotid artery and thoracic aorta. A multivariate analysis revealed that reduction in CRP, S100A12, LDL-C, oxidized-LDL, and increase in FMD were significantly associated with reduced arterial inflammation in the thoracic aorta, but not in the carotid artery. CONCLUSIONS: Atorvastatin treatment reduced S100A12/CRP levels, and the changes in these circulating markers mirrored the improvement in arterial inflammation. Our observations suggest that S100A12 may be an emerging therapeutic target for atherosclerosis.

High-Density Lipoprotein Cholesterol Efflux Capacity as a Novel Prognostic Surrogate for Coronary Artery Disease.

AIM: We examined the impact of baseline high-density lipoprotein cholesterol efflux capacity (CEC) on major cardiac adverse events (MACE) in patients with coronary artery disease (CAD) during a long-term secondary prevention. METHOD: CEC was measured using a cell-based efflux system in (3)[H]-cholesterol-labeled J774 macrophages in apolipoprotein B-depleted plasma between January 2011 and January 2013. Patients with CAD were divided into 2 groups as a boundary CEC value of 1: 0.19 ≤ CEC ＜1 (impaired CEC group, mean CEC of 0.76±0.16, n=136), and 1 ≤ CEC ≤ 2.08 (enhanced CEC group, 1.20±0.19, n=44). MACE, comprised the incidence of cardiac death, non-fatal myocardial infarction, and any revascularizations (RV) without restenosis approximately 1 year after vascularization, was retrospectively investigated at September 2019. Impact of enhanced CEC on MACE among 22 variables was examined by applying a Cox proportional hazard model. RESULT: The frequency of MACE in impaired CEC group (16.9%, mean observational interval of 2111±888 days) was significantly higher than that in enhanced CEC group (2.3%, 2,252±685, p=0.013), largely driven by the significantly higher RV incidence (14.0 % versus 2.3 %, p=0.032). Enhancement of CEC was the significant predictor of MACE (hazard ratio: 0.11; 95% CI: 0.013-0.879; p=0.038). CONCLUSION: A baseline CEC level of more than 1 in patients with CAD brought favorable long-term clinical outcomes, suggesting that CEC is a useful prognostic and therapeutic surrogate for secondary prevention of CAD.

Suppressive Effect of Shiitake Extract on Plasma Ethanol Elevation.

Alcohol is usually consumed with meals, but chronic consumption is a leading cause of alcoholic liver diseases. We investigated if shiitake extracts with a high lentinic acid content (Shiitake-H) and without lentinic acid (Shiitake-N) could suppress the elevation in plasma ethanol concentrations by accelerating ethanol metabolism and preventing ethanol absorption from the gut. Shiitake-H and Shiitake-N suppressed the elevation in concentrations of ethanol and acetaldehyde in plasma, and promoted the activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the liver. However, these effects of Shiitake-H were more prominent than those of Shiitake-N. Furthermore, Shitake-H promoted ADH and ALDH activities in the stomach. We also examined the change in plasma ethanol concentration by injecting Shiitake-H or Shiitake-N into the ligated loop of the stomach or jejunum together with an ethanol solution. Shiitake-H suppressed the absorption of ethanol from the stomach and jejunum. In conclusion, Shiitake-H accelerates ethanol metabolism in the stomach and liver and inhibits ethanol absorption in the stomach and jejunum indicating that lentinic acid is a functional component in shiitake.

Allergenicity of Deamidated and/or Peptide-Bond-Hydrolyzed Wheat Gliadin by Transdermal Administration.

Hydrochloric acid (HCl)-treated wheat protein (HWP) is widely used in various products, including foods, cosmetics and shampoos. Recently, immediate hypersensitivity towards facial soap containing HWP has been reported. HCl treatment of protein causes hydrolysis not only of main-chain amide bonds (peptide-bond hydrolysis) but also of side-chain ones (deamidation). We have already reported that gliadin, the main allergen in wheat, reduces allergenicity and increases digestibility by deamidation, indicating that deamidation and peptide-bond hydrolysis are effective to reduce the allergenicity of wheat protein. However, transdermally administered HWP is assumed to induce sensitization to orally administered wheat protein even in those who have been taking wheat products daily before sensitization. The present study was conducted to examine which structural change is responsible for the induction of cutaneous sensitization by comparing the allergenicity of deamidated and/or peptide-bond-hydrolyzed wheat gliadin. Because we have developed a deamidation method without causing peptide-bond hydrolysis, only deamidated wheat gliadin is available. Therefore, after deamidated-only, hydrolyzed-only, and deamidated and hydrolyzed gliadins were transdermally administered to mice for several weeks, the corresponding gliadin was intraperitoneally administered and allergenicity was evaluated. Transdermal administration of deamidated and hydrolyzed gliadin induced severe allergic reaction, while that of deamidated-only and hydrolyzed-only gliadin showed almost no allergic response. This result indicates that both deamidation and peptide-bond hydrolysis are necessary to increase the allergenic potency of transdermally administered wheat gliadin.

Sulfuric Odor Precursor S-Allyl-l-Cysteine Sulfoxide in Garlic Induces Detoxifying Enzymes and Prevents Hepatic Injury.

S-Allyl-l-cysteine sulfoxide (ACSO) is a precursor of garlic-odor compounds like diallyl disulfide (DADS) and diallyl trisulfide (DATS) known as bioactive components. ACSO has suitable properties as a food material because it is water-soluble, odorless, tasteless and rich in bulbs of fresh garlic. The present study was conducted to examine the preventive effect of ACSO on hepatic injury induced by CCl4 in rats. ACSO, its analogs and garlic-odor compounds were each orally administered via gavage for five consecutive days before inducing hepatic injury. Then, biomarkers for hepatic injury and antioxidative state were measured. Furthermore, we evaluated the absorption and metabolism of ACSO in the small intestine of rats and NF-E2-related factor 2 (Nrf2) nuclear translocation by ACSO using HepG2 cells. As a result, ACSO, DADS and DATS significantly suppressed the increases in biomarkers for hepatic injury such as the activities of aspartate transaminase (AST), alanine transaminase (ALT) and lactate dehydrogenase (LDH), and decreases in antioxidative potency such as glutathione (GSH) level and the activities of glutathione S-transferase (GST) and glutathione peroxidase (GPx). We also found ACSO was absorbed into the portal vein from the small intestine but partially metabolized to DADS probably in the small intestine. In in vitro study, ACSO induced Nrf2 nuclear translocation in HepG2 cells, which is recognized as an initial trigger to induce antioxidative and detoxifying enzymes. Taken together, orally administered ACSO probably reached the liver and induced antioxidative and detoxifying enzymes by Nrf2 nuclear translocation, resulting in prevention of hepatic injury. DADS produced by the metabolism of ACSO in the small intestine might also have contributed to the prevention of hepatic injury. These results suggest potential use of ACSO in functional foods that prevent hepatic injury and other diseases caused by reactive oxygen species (ROS).

Hepatic Overexpression of Endothelial Lipase Lowers High-Density Lipoprotein but Maintains Reverse Cholesterol Transport in Mice: Role of Scavenger Receptor Class B Type I/ATP-Binding Cassette Transporter A1-Dependent Pathways.

OBJECTIVE: Reverse cholesterol transport (RCT) is a major mechanism by which HDL (high-density lipoprotein) protects against atherosclerosis. Endothelial lipase (EL) reportedly reduces HDL levels, which, in theory, would increase atherosclerosis. However, it remains unclear whether EL affects RCT in vivo. APPROACH AND RESULTS: Adenoviral vectors expressing EL or luciferase were intravenously injected into mice, and a macrophage RCT assay was performed. As expected, hepatic EL overexpression markedly reduced HDL levels. In parallel, plasma 3H-cholesterol counts from the EL-expressing mice decreased by 85% compared with control. Surprisingly, there was no difference in fecal 3H-cholesterol excretion between the groups. Kinetic studies revealed increased catabolism/hepatic uptake of 3HDL-cholesteryl ether, resulting in no change in fecal HDL-cholesteryl ester excretion in the mice. To explore underlying mechanisms for the preservation of RCT despite low HDL levels in the EL-expressing mice, we investigated the effects of hepatic SR-BI (scavenger receptor class B type I) knockdown. RCT assay revealed that knockdown of SR-BI alone reduced fecal excretion of macrophage-derived 3H-cholesterol. Interestingly, hepatic EL overexpression under SR-BI inhibition further attenuated fecal tracer counts as compared with control. Finally, we observed that EL overexpression enhanced in vivo RCT under pharmacological inhibition of hepatic ABCA1 (ATP-binding cassette transporter A1) by probucol. CONCLUSIONS: Hepatic EL expression compensates for reduced macrophage-derived cholesterol efflux to plasma because of low HDL levels by promoting cholesterol excretion to bile/feces via an SR-BI pathway, maintaining overall RCT in vivo. In contrast, EL-modified HDL might negatively regulate RCT via hepatic ABCA1. Despite extreme hypoalphalipoproteinemia, RCT is maintained in EL-expressing mice via SR-BI/ABCA1-dependent pathways.

S-Allyl-L-cysteine sulfoxide, a garlic odor precursor, suppresses elevation in blood ethanol concentration by accelerating ethanol metabolism and preventing ethanol absorption from gut.

Alcoholic beverages are enjoyed together with meals worldwide, but their excessive intake is associated with an increased risk of various diseases. We investigated whether S-allyl-L-cysteine sulfoxide (ACSO), a sulfuric odor precursor of garlic, suppresses elevation in plasma ethanol concentration by accelerating ethanol metabolism and preventing ethanol absorption from the gut in rats. ACSO and garlic extract with a high ACSO content (Garlic-H) suppressed elevation in concentrations of ethanol and acetaldehyde in plasma and promoted the activities of alcohol dehydrogenase and aldehyde dehydrogenase. However, ACSO and Garlic-H did not affect plasma acetate so much. Furthermore, we examined the change in plasma ethanol concentration by injecting ACSO or Garlic-H into the ligated stomach or jejunum together with ethanol solution. ACSO and Garlic-H suppressed the absorption of ethanol from the stomach and jejunum, but suppression in the jejunum was less than in the stomach. In conclusion, ACSO inhibits ethanol absorption and accelerates ethanol metabolism.

Vitamin E supplementation improves high-densitiy lipoprotein and endothelial functions in end-stage kidney disease patients undergoing hemodialysis
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BACKGROUND AND AIMS: Patients with end-stage kidney disease (ESKD) undergoing hemodialysis (HD) have been shown to be at increased risk for cardiovascular disease (CVD). Decreased high-density lipoprotein cholesterol (HDL-C) and impaired cholesterol efflux capacity (CEC) have been reported in such patients, and effects of vitamin E supplementation on HDL functions are poorly understood. Therefore, the present study aimed to investigate effects of vitamin E supplementation on HDL and endothelial functions in ESKD patients undergoing HD. We also assessed the influence of diabetes and haptoglobin (Hp) phenotype on the effects of vitamin E. MATERIALS AND METHODS: Vitamin E (300 mg daily) was supplemented for 12 weeks, followed by a 10-week washout phase in 40 ESKD patients undergoing HD (20 diabetic and 20 nondiabetic patients). HDL functions, including CEC, antioxidant capacity, and anti-inflammatory activity, were investigated. In diabetic patients, endothelial function, as represented by flow-mediated vasodilatation (FMD), was also assessed. The findings were compared according to diabetic condition or Hp phenotype. RESULTS: Vitamin E significantly increased CEC, whereas antioxidant capacity and anti-inflammatory activity remained unchanged. Further, the improvement in CEC was maintained after the 10-week washout phase. Endothelial function was significantly improved in diabetic patients. Subanalyses based on diabetes or Hp phenotype revealed that neither diabetes nor Hp phenotype influenced the effects of vitamin E. CONCLUSION: In ESKD patients undergoing hemodialysis, vitamin E supplementation significantly improved the HDL function of CEC and, in diabetic patients, endothelial function. These effects were independent of Hp phenotype.
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Xanthohumol, a hop-derived prenylated flavonoid, promotes macrophage reverse cholesterol transport.

Xanthohumol, a prominent prenyl flavonoid from the hop plant (Humulus lupulus L.), is suggested to be antiatherogenic since it reportedly increases high-density lipoprotein (HDL) cholesterol levels. It is not clear whether xanthohumol promotes reverse cholesterol transport (RCT), the most important antiatherogenic property of HDL; therefore, we investigated the effects of xanthohumol on macrophage-to-feces RCT using a hamster model as a CETP-expressing species. In vivo RCT experiments showed that xanthohumol significantly increased fecal appearance of the tracer derived from intraperitoneally injected [3H]-cholesterol-labeled macrophages. Ex vivo experiments were then employed to investigate the detailed mechanism by which xanthohumol enhanced RCT. Cholesterol efflux capacity from macrophages was 1.5-fold higher in xanthohumol-fed hamsters compared with the control group. In addition, protein expression and lecithin-cholesterol acyltransferase activity in the HDL fraction were significantly higher in xanthohumol-fed hamsters compared with the control, suggesting that xanthohumol promoted HDL maturation. Hepatic transcript analysis revealed that xanthohumol increased mRNA expression of abcg8 and cyp7a1. In addition, protein expressions of liver X receptor α and bile pump export protein were increased in the liver by xanthohumol administration when compared with the control, implying that it stimulated bile acid synthesis and cholesterol excretion to feces. In conclusion, our data demonstrate that xanthohumol improves RCT in vivo through cholesterol efflux from macrophages and excretion to feces, leading to antiatherosclerosis effects. It remains to be elucidated whether enhancement of RCT by xanthohumol could prove valuable in humans.

Beneficial Effects of Exercise-Based Cardiac Rehabilitation on High-Density Lipoprotein-Mediated Cholesterol Efflux Capacity in Patients with Acute Coronary Syndrome.

AIM: Recent studies reported that low high-density lipoprotein (HDL)-mediated cholesterol efflux capacity rather than low HDL cholesterol (HDL-C) is strongly associated with the increased risk for coronary artery disease. It remains unclear whether exercised-based cardiac rehabilitation (CR) can increase HDL cholesterol efflux capacity. METHOD: This study is a retrospective analysis of stored serum from patients with acute coronary syndrome (ACS) who participated in outpatient CR program following successful percutaneous coronary intervention. We employed a cell-based cholesterol efflux system including the incubation of (3)H-cholesterol labeled macrophages with apolipoprotein B-depleted serum at the onset or early phase of ACS and at 6-month follow-up periods in 57 male and 11 female patients with ACS. Cardiopulmonary exercise tests were performed at the beginning and end of CR program. RESULT: Fifty-seven patients completed the CR program. Compared with patients who dropped out from CR program (non-CR group), CR participants showed marked amelioration in serum lipid levels, increased efflux capacity, and improved exercise capacity. Spearman's rank correlation coefficient analysis revealed that the percent increases of efflux capacity were significantly associated with the percent increases in HDL-C (ρ=0.598, p＜0.0001) and apolipoprotein A1 (ρ=0.508, p＜0.0001), whereas no association between increases in efflux capacity and increases in cardiopulmonary fitness was observed. Increases in cholesterol efflux capacity were not seen in patients who continued smoking and those who did not achieve all risk factor targets and higher exercise tolerance. CONCLUSION: CR can markedly increase both HDL-C and HDL cholesterol efflux capacity. These results suggest that CR is a very useful therapy for reverse cholesterol transport and secondary prevention.

Probucol-Oxidized Products, Spiroquinone and Diphenoquinone, Promote Reverse Cholesterol Transport in Mice.

OBJECTIVE: Oxidized products of probucol, spiroquinone and diphenoquinone, were shown to increase cell cholesterol release and plasma high-density lipoprotein (HDL) by inhibiting degradation of ATP-binding cassette transporter A1. We investigated whether these compounds enhance reverse cholesterol transport in mice. APPROACH AND RESULTS: Spiroquinone and diphenoquinone increased ATP-binding cassette transporter A1 protein (2.8- and 2.6-fold, respectively, P<0.01) and apolipoprotein A-I-mediated cholesterol release (1.4- and 1.4-fold, P<0.01 and P<0.05, respectively) in RAW264.7 cells. However, diphenoquinone, but not spiroquinone, enhanced cholesterol efflux to HDL (+12%, P<0.05), whereas both increased ATP-binding cassette transporter G1 protein, by 1.8- and 1.6-fold, respectively. When given orally to mice, both compounds significantly increased plasma HDL-cholesterol, by 19% and 20%, respectively (P<0.05), accompanied by an increase in hepatic and macrophage ATP-binding cassette transporter A1 but not ATP-binding cassette transporter G1. We next evaluated in vivo reverse cholesterol transport by injecting RAW264.7 cells labeled with (3)H-cholesterol intraperitoneally into mice. Both spiroquinone and diphenoquinone increased fecal excretion of the macrophage-derived (3)H-tracer, by 25% and 28% (P<0.01 and P<0.05), respectively. spiroquinone/diphenoquinone did not affect fecal excretion of HDL-derived (3)H-cholesterol, implying that macrophage-to-plasma was the most important step in spiroquinone/diphenoquinone-mediated promotion of in vivo reverse cholesterol transport. Finally, spiroquinone significantly reduced aortic atherosclerosis in apolipoprotein E null mice when compared with the vehicle. CONCLUSIONS: Spiroquinone and diphenoquinone increase functional ATP-binding cassette transporter A1 in both the macrophages and the liver, elevate plasma HDL-cholesterol, and promote overall reverse cholesterol transport in vivo. These compounds are promising as therapeutic reagents against atherosclerosis.

High-density lipoprotein cholesterol efflux capacity as a relevant predictor of atherosclerotic coronary disease.

BACKGROUND: We examined the clinical relevance of high-density lipoprotein cholesterol (HDL-C) efflux capacity from macrophage (cholesterol efflux capacity) as a predictor of atherosclerotic coronary artery disease (CAD) in comparison with that of conventional coronary and lipid risk variables in Japanese daily practice. METHODS AND RESULTS: Fasting blood sampling, including 6 routinely measured dyslipidemia-related variables, was performed at the time of coronary angiography (CAG) or multi-slice coronary computed tomography (MSCT) between January 2011 and January 2013. CAD, defined as native coronary atherosclerosis stenosis >50% by CAG or MSCT, was identified in 182 patients (CAD group), but not in 72 patients (non-CAD group). Cholesterol efflux capacity, measured using a cell-based efflux system in (3)[H]-cholesterol-labeled J774 macrophages in apolipoprotein B-depleted plasma, was significantly impaired in the CAD group compared with the non-CAD group (0.86 ± 0.26 vs. 1.02 ± 0.38; p = 0.001). After adjusting 15 patient and dyslipidemia-related variables using a propensity score matching analysis produced 55 patients in each arm, cholesterol efflux capacity in the CAD group remained to be significant compared with the non-CAD group (0.83 ± 0.24 vs. 0.97 ± 0.36; p = 0.019). Stepwise logistic regression analysis using a backward method after the baseline adjustment showed that cholesterol efflux capacity (odds ratio [OR]: 0.23; 95% confidence interval [CI]: 0.056-0.91; p = 0.037) was the single predictor of CAD, while other variables including HDL-C (p = 0.088) and apolipoprotein (apo) A-I (p = 0.681) were removed owing to those insignificance. The area under the receiver operating characteristic curve after the baseline adjustment was 0.67 (95% CI: 0.51-0.73, p = 0.048 by Hosmer-Lemeshow goodness-of-fit statistics). CONCLUSIONS: The present observational study conducted under daily clinical practice confirmed that cholesterol efflux capacity is a clinically relevant predictor of CAD among the conventional coronary risk factors and dyslipidemia-related variables.

Citrulline increases cholesterol efflux from macrophages in vitro and ex vivo via ATP-binding cassette transporters.

Reverse cholesterol transport (RCT) is a mechanism critical to the anti-atherogenic property of HDL. Although citrulline contributes to the amelioration of atherosclerosis via endothelial nitric oxide production, it remains unclear whether it affects RCT. This study was undertaken to clarify the effects of citrulline on expressions of specific transporters such as ATP binding cassette transporters (ABC)A1 and ABCG1, and the cholesterol efflux from macrophages to apolipoprotein (apo) A-I or HDL in vitro and ex vivo. Citrulline increased ABCA1 and ABCG1 mRNA and protein levels in THP-1 macrophages, translating into enhanced apoA-I- and HDL-mediated cholesterol efflux. In the human crossover study, 8 healthy male volunteers (age 30-49 years) consumed either 3.2 g/day citrulline or placebo for 1 week. Citrulline consumption brought about significant increases in plasma levels of citrulline and arginine. Supporting the in vitro data, monocyte-derived macrophages (MDM) differentiated under autologous post-citrulline sera demonstrated enhancement of both apoA-I- and HDL-mediated cholesterol efflux through increased ABCA1 and ABCG1 expressions, compared to MDM differentiated under pre-citrulline sera. However, the placebo did not modulate these parameters. Therefore, in addition to improving endothelium function, citrulline might have an anti-atherogenic property by increasing RCT of HDL.

Ezetimibe enhances macrophage reverse cholesterol transport in hamsters: contribution of hepato-biliary pathway.

Reverse cholesterol transport (RCT) is pivotal in the return of excess cholesterol from peripheral tissues to the liver for excretion in bile and eventually feces. RCT from macrophages is a critical anti-atherogenicity mechanism of HDL. As the cholesterol absorption inhibitor ezetimibe promoted RCT in mice, which lack cholesterol ester transfer protein (CETP), we investigated its effects in hamsters, which have CETP. A high-cholesterol diet (HC) increased cholesterol levels throughout lipoprotein fractions and ezetimibe markedly reduced VLDL/LDL cholesterol levels under both normal chow (NC) and HC. However, ezetimibe did not affect and reduced HDL-cholesterol levels under NC and HC, respectively. Intraperitoneal injection of (3)H-cholesterol pre-labeled macrophages in an in vivo RCT assay increased tracer accumulation in the liver but reduced it in bile under HC, and these changes were completely cancelled by ezetimibe. Under both NC and HC, ezetimibe reduced tracer levels in the liver but increased them in feces, indicating promotion of RCT in vivo. We performed a RCT assay using hamsters subjected to bile duct ligation (BDL) to clarify whether a transintestinal cholesterol efflux (TICE) pathway contributes to ezetimibe's enhancement of RCT. BDL markedly inhibited macrophage-derived (3)H-cholesterol excretion to feces and cancelled ezetimibe's stimulatory effect on RCT, suggesting that biliary cholesterol excretion is a major contributor in RCT promotion by ezetimibe but the contribution of the TICE pathway is minimal. In conclusions, ezetimibe exerts an additive anti-atherogenic property by enhancing RCT in hamsters. Our findings suggest that this property is independent of the TICE pathway.

Hepatic overexpression of idol increases circulating protein convertase subtilisin/kexin type 9 in mice and hamsters via dual mechanisms: sterol regulatory element-binding protein 2 and low-density lipoprotein receptor-dependent pathways.

OBJECTIVE: Low-density lipoprotein receptor (LDLR) is degraded by inducible degrader of LDLR (Idol) and protein convertase subtilisin/kexin type 9 (PCSK9), thereby regulating circulating LDL levels. However, it remains unclear whether, and if so how, these LDLR degraders affect each other. We therefore investigated effects of liver-specific expression of Idol on LDL/PCSK9 metabolism in mice and hamsters. APPROACH AND RESULTS: Injection of adenoviral vector expressing Idol (Ad-Idol) induced a liver-specific reduction in LDLR expression which, in turn, increased very-low-density lipoprotein/LDL cholesterol levels in wild-type mice because of delayed LDL catabolism. Interestingly, hepatic Idol overexpression markedly increased plasma PCSK9 levels. In LDLR-deficient mice, plasma PCSK9 levels were already elevated at baseline and unchanged by Idol overexpression, which was comparable with the observation for Ad-Idol-injected wild-type mice, indicating that Idol-induced PCSK9 elevation depended on LDLR. In wild-type mice, but not in LDLR-deficient mice, Ad-Idol enhanced hepatic PCSK9 expression, with activation of sterol regulatory element-binding protein 2 and subsequently increased expression of its target genes. Supporting in vivo findings, Idol transactivated PCSK9/LDLR in sterol regulatory element-binding protein 2/LDLR-dependent manners in vitro. Furthermore, an in vivo kinetic study using (125)I-labeled PCSK9 revealed delayed clearance of circulating PCSK9, which could be another mechanism. Finally, to extend these findings into cholesteryl ester transfer protein-expressing animals, we repeated the above in vivo experiments in hamsters and obtained similar results. CONCLUSIONS: A vicious cycle in LDLR degradation might be generated by PCSK9 induced by hepatic Idol overexpression via dual mechanisms: sterol regulatory element-binding protein 2/LDLR. Furthermore, these effects would be independent of cholesteryl ester transfer protein expression.

Intensive lipid lowering therapy with titrated rosuvastatin yields greater atherosclerotic aortic plaque regression: Serial magnetic resonance imaging observations from RAPID study.

OBJECTIVE: Although previous randomized clinical trials established a basis for lipid guidelines worldwide, they employed fixed doses of statins throughout trials (fire-and-forget approach). In the real clinical setting, however, statin doses are titrated to achieve target low-density lipoprotein cholesterol (LDL-C) levels (treat-to-target approach). The major objective was to investigate whether intensive lipid-lowering therapy using the treat-to-target approach yielded greater regression of aortic plaques. METHODS: We therefore performed a prospective, randomized trial comparing the effects of standard (achieve LDL-C levels recommended by the Japanese guidelines) and intensive (achieve 30% lower LDL-C levels than standard) rosuvastatin therapy for 1 year in 60 hypercholesterolemic patients with a primary endpoint of aortic atherosclerotic plaques evaluated by non-invasive magnetic resonance imaging (MRI). RESULTS: Average doses were 2.9 ± 3.1 and 6.5 ± 5.1 mg/day for standard (n = 29) and intensive therapy group (n = 31), respectively. Although both therapies significantly reduced LDL-C and high-sensitivity C-reactive protein (hsCRP) levels, LDL-C reduction was significantly greater in the intensive group (-46 vs. -34%). MRI study showed that thoracic aortic plaques were significantly regressed in both groups, with greater regression of thoracic plaque in the intensive group (-9.1 vs. -3.2%, p = 0.01). Multivariate analyses revealed that thoracic plaque regression was significantly correlated with hsCRP reduction, but not with changes in serum lipids, endothelial function, or doses of rosuvastatin. CONCLUSION: Intensive statin therapy with titration targeting lower LDL-C levels resulted in greater thoracic aortic plaque regression compared to standard therapy, which was correlated with hsCRP reduction, suggesting that intensive statin therapy could provide better clinical outcomes.

Rapid development of atherosclerosis in the world's smallest Microminipig fed a high-fat/high-cholesterol diet.

AIM: Experimental studies of human atherogenesis require an appropriate animal model that mimics human physiology and pathology. Because swine physiology is similar to human physiology, we developed a hyperlipidemia-induced atherosclerosis model using the recently developed world's smallest Microminipig(TM). METHODS: These animals weigh only 5kg at 3months of age, much smaller than any other miniature pig. We found that the administration of a high-fat/high-cholesterol diet containing at least 0.2% cholesterol without cholic acid for as little as eight weeks induces hypercholesterolemia and subsequent atherosclerosis in these animals. RESULTS: The serum levels of low-density lipoprotein cholesterol(LDL-C) and the percent distribution of cholesterol in the LDL fractions were markedly increased. The hepatic expression of LDL receptor and hydroxymethylglutaryl-CoA reductase was coordinately decreased. The cholesteryl ester transfer protein activity, which plays a role in reverse cholesterol transport, was detected in the serum of the Microminipigs. Niemann-Pick C1-like 1 protein was expressed in both the liver and small intestine; however, hepatic apoB mRNA editing enzyme was not expressed. As in humans, and in contrast to that observed in mice, most of the hepatic lipase activity was localized in the liver. These results suggest that the hyperlipidemia-induced gene expression profile linked to cholesterol homeostasis and atherogenesis is similar in Microminipigs and humans. CONCLUSION: We conclude that the characteristics of the Microminipig, including its easy handling size, make it an appropriate model for studies of atherosclerosis and related conditions.

Overexpression of stearoyl-coenzyme A desaturase 1 in macrophages promotes reverse cholesterol transport.

Stearoyl-coenzyme A desaturase 1 (SCD1) is the rate-limiting enzyme in the synthesis of monounsaturated fatty acids. However, the impact of SCD1 on atherosclerosis remains unclear. The aim of this study was to determine whether SCD1 affects macrophage reverse cholesterol transport (RCT) in mice. Compared to the control, adenoviral-mediated SCD1 overexpression in RAW264.7 macrophages increased cholesterol efflux to HDL, but not to apoA-I, without clear changes in ABCA1, ABCG1 and SR-BI expressions. While knockdown of ABCG1 and SR-BI did not affect the SCD1-induced cholesterol efflux to HDL, SCD1-overexpressing macrophages promoted the formation of both normal- and large-sized HDL in media, accompanying increased apolipoprotein A-I levels in HDL fractions. Transformation to larger particles of HDL was independently confirmed by nuclear magnetic resonance-based lipoprotein analysis. Interestingly, media transfer assays revealed that HDL generated by SCD1 had enhanced cholesterol efflux potential, indicating that SCD1 transformed HDL to a more anti-atherogenic phenotype. To study macrophage RCT in vivo, (3)H-cholesterol-labeled RAW264.7 cells overexpressing SCD1 or the control were intraperitoneally injected into mice. Supporting the in vitro data, injection of SCD1-macrophages resulted in significant increases in (3)H-tracer in plasma, liver, and feces compared to the control. Moreover, there was a shift towards larger particles in the (3)H-tracer distribution of HDL fractions obtained from the mice. In conclusion, macrophage-specific SCD1 overexpression promotes overall RCT through increased cholesterol efflux to HDL, suggesting that macrophage SCD1 achieves an anti-atherogenic effect by enhancing RCT.

Dipeptidyl peptidase-4 inhibitors attenuate endothelial function as evaluated by flow-mediated vasodilatation in type 2 diabetic patients.

BACKGROUND: Endothelial dysfunction is an independent predictor for cardiovascular events in patients with type 2 diabetes (T2DM). Glucagon like peptide-1 (GLP-1) reportedly exerts vasodilatory actions, and inhibitors of dipeptidyl peptidase-4 (DPP-4), an enzyme-degrading GLP-1, are widely used to treat T2DM. We therefore hypothesized that DPP-4 inhibitors (DPP-4Is) improve endothelial function in T2DM patients and performed 2 prospective, randomized crossover trials to compare the DPP-4I sitagliptin and an α-glucosidase inhibitor, voglibose (in study 1) and the DPP-4Is sitagliptin and alogliptin (in study 2). METHODS AND RESULTS: In study 1, 24 men with T2DM (46±5 years) were randomized to sitagliptin or voglibose for 6 weeks without washout periods. Surprisingly, sitagliptin significantly reduced flow-mediated vasodilatation (FMD; -51% compared with baseline, P<0.05) of the brachial artery despite improved diabetic status. In contrast, voglibose did not affect FMD. To confirm this result and determine whether it is a class effect, we conducted another trial (study 2) to compare sitagliptin and alogliptin in 42 T2DM patients (66±8 years) for 6 weeks with 4-week washout periods. Both DPP-4Is improved glycemic control but significantly attenuated FMD (7.2/4.3%, P<0.001, before/after sitagliptin; 7.0/4.8%, P<0.001, before/after alogliptin, respectively). Interestingly, FMD reduction was less evident in subjects who were on statins or whose LDL cholesterol levels were reduced by them, but this was not correlated with parameters including DPP-4 activity and GLP-1 levels or diabetic parameters. CONCLUSIONS: Our 2 independent trials demonstrated that DPP-4 inhibition attenuated endothelial function as evaluated by FMD in T2DM patients. This unexpected unfavorable effect may be a class effect of DPP-4Is. CLINICAL TRIAL REGISTRATION: URL: http://center.umin.ac.jp, Unique Identifiers: UMIN000005682 (sitagliptin versus voglibose) and UMIN000005681 (sitagliptin versus alogliptin).