There are errors in the legend for Fig 8. The first sentence “CD68+ cells but not smooth muscle actin+ cells differ among strains comprising the HMDP.” is inaccurate and the sentence has been removed. Differences were found, but these were not significant. This is discussed in the main body of the text. The description for panel L incorrectly corresponds to panel K and vice versa. The correct legend is below.

We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions.

Objective: Inflammation of vascular smooth muscle cells (VSMC) is intimately linked to atherosclerosis and other vascular inflammatory disease. Thioredoxin interacting protein (Txnip) is a key regulator of cellular sulfhydryl redox and a mediator of inflammasome activation. The goals of the present study were to examine the impact of Txnip ablation on inflammatory response to oxidative stress in VSMC and to determine the effect of Txnip ablation on atherosclerosis in vivo.Methods and results: Using cultured VSMC, we showed that ablation of Txnip reduced cellular oxidative stress and increased protection from oxidative stress when challenged with oxidized phospholipids and hydrogen peroxide. Correspondingly, expression of inflammatory markers and adhesion molecules were diminished in both VSMC and macrophages from Txnip knockout mice. The blunted inflammatory response was associated with a decrease in NF-kappa B nuclear translocation. Loss of Txnip in VSMC also led to a dramatic reduction in macrophage adhesion to VSMC. In vivo data from Txnip-ApoE double knockout mice showed that Txnip ablation led to 49% reduction in atherosclerotic lesion in the aortic root and 71% reduction in the abdominal aorta, compared to control ApoE knockout mice.Conclusion: Our data show that Txnip plays an important role in oxidative inflammatory response and atherosclerotic lesion development in mice. The atheroprotective effect of Txnip ablation implicates that modulation of Txnip expression may serve as a potential target for intervention of atherosclerosis and inflammatory vascular disease. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

Common forms of atherosclerosis involve multiple genetic and environmental factors. While human genome-wide association studies have identified numerous loci contributing to coronary artery disease and its risk factors, these studies are unable to control environmental factors or examine detailed molecular traits in relevant tissues. We now report a study of natural variations contributing to atherosclerosis and related traits in over 100 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP). The mice were made hyperlipidemic by transgenic expression of human apolipoprotein E-Leiden (APOE-Leiden) and human cholesteryl ester transfer protein (CETP). The mice were examined for lesion size and morphology as well as plasma lipid, insulin and glucose levels, and blood cell profiles. A subset of mice was studied for plasma levels of metabolites and cytokines. We also measured global transcript levels in aorta and liver. Finally, the uptake of acetylated LDL by macrophages from HMDP mice was quantitatively examined. Loci contributing to the traits were mapped using association analysis, and relationships among traits were examined using correlation and statistical modeling. A number of conclusions emerged. First, relationships among atherosclerosis and the risk factors in mice resemble those found in humans. Second, a number of trait-loci were identified, including some overlapping with previous human and mouse studies. Third, gene expression data enabled enrichment analysis of pathways contributing to atherosclerosis and prioritization of candidate genes at associated loci in both mice and humans. Fourth, the data provided a number of mechanistic inferences; for example, we detected no association between macrophage uptake of acetylated LDL and atherosclerosis. Fifth, broad sense heritability for atherosclerosis was much larger than narrow sense heritability, indicating an important role for gene-by-gene interactions. Sixth, stepwise linear regression showed that the combined variations in plasma metabolites, including LDL/VLDL-cholesterol, trimethylamine N-oxide (TMAO), arginine, glucose and insulin, account for approximately 30 to 40% of the variation in atherosclerotic lesion area. Overall, our data provide a rich resource for studies of complex interactions underlying atherosclerosis.

Hepcidin, the iron-regulatory hormone and acute phase reactant, is proposed to contribute to the pathogenesis of atherosclerosis by promoting iron accumulation in plaque macrophages, leading to increased oxidative stress and inflammation in the plaque (the "iron hypothesis"). Hepcidin and iron may thus represent modifiable risk factors in atherosclerosis. We measured hepcidin expression in Apoe(-/-) mice with varying diets and ages. To assess the role of macrophage iron in atherosclerosis, we generated Apoe(-/-) mice with macrophage-specific iron accumulation by introducing the ferroportin ffe mutation. Macrophage iron loading was also enhanced by intravenous iron injection. Contrary to the iron hypothesis, we found that hepatic hepcidin expression was not increased at any stage of the atherosclerosis progression in Apoe(-/-) or Apoe/ffe mice and that the atherosclerotic plaque size was not increased in mice with elevated macrophage iron. Our results strongly argue against any significant role of macrophage iron in atherosclerosis progression in mice.

Objective— To determine the efficacy of long-term anti-miR-33 therapy on the progression of atherosclerosis in high-fat, high-cholesterol–fed Ldlr –/– mice. Methods and Results— Ldlr –/– mice received saline, or control or anti-miR-33 oligonucleotides once a week for 14 weeks. The treatment was effective, as measured by reduced levels of hepatic miR-33 and increased hepatic expression of miR-33 targets. Analysis of plasma samples revealed an initial elevation in high-density lipoprotein cholesterol after 2 weeks of treatment that was not sustained by the end of the experiment. Additionally, we found a significant increase in circulating triglycerides in anti-miR-33–treated mice, compared with controls. Finally, examination of atheromata revealed no significant changes in the size or composition of lesions between the 3 groups. Conclusion— Prolonged silencing of miR-33 fails to maintain elevated plasma high-density lipoprotein cholesterol and does not prevent the progression of atherosclerosis in Ldlr –/– mice.

Recent studies demonstrated a strong positive association between blood trimethylamine-N-oxide (TMAO) level and risk for cardiovascular disease. Dietary choline is converted to trimethylamine (TMA) by the gut bacteria, and TMA is then converted to TMAO in the liver, mainly by flavin containing monooxygenase 3 (Fmo3). To examine how FMO3 expression influences atherogenesis, we administered two different FMO3 or control antisense oligonucleotdies (ASOs) to apolipoprotein E (apoE) null mice for 15 weeks. During the last 12 weeks of the ASO treatment, the mice received a chow diet containing 1% choline to enhance TMA production. Hepatic FMO3 mRNA levels were decreased by 80% in mice that received either FMO3 ASO #1 or #2 versus those that received the control ASO. As expected, circulating TMA and TMAO levels were significantly increased and decreased, respectively, in the mice that received the two FMO3 ASOs versus the controls. Other observed changes in the FMO3 ASO-treated mice included: decreased plasma triglyceride, HDL, and glucose levels. Significantly increased VLDL/IDL/LDL cholesterol was observed only in FMO3 ASO #2-treated mice versus the controls. FMO3 ASO treatment increased circulating total bile acid levels by more than 2-fold. Compared to controls, serum markers of hepatic inflammation and necrosis (ALT, AST) were significantly increased in FMO3 ASO treated mice. Histological examination revealed obvious hepatic steatosis and increased inflammatory cell infiltration, suggesting that FMO3 abrogation may be a reasonable model for non-alcoholic steatohepatitis (NASH). We also found a significant increase in quantitative triglyceride and cholesterol content of these livers. Transcriptional profiling by microarrays showed increased hepatic expression of inflammatory genes in mice that received FMO3 ASOs versus the controls. In addition, a significant increase in spleen weight in these mice suggests increased systemic inflammation. Finally, FMO3 knockdown increased atherosclerosis by more than 50% when compared to controls (p < 0.01). This work demonstrates that blocking FMO3 activity has a major impact on liver physiology and atherosclerosis. Our study suggests a protective role for FMO3 in atherosclerosis and liver function.

We examined the effects of a natural secondary bile acid, hyodeoxycholic acid (HDCA), on lipid metabolism and atherosclerosis in LDL receptor-null (LDLRKO) mice. Female LDLRKO mice were maintained on a Western diet for 8 wk and then divided into 2 groups that received chow, or chow + 1.25% HDCA, diets for 15 wk. We observed that mice fed the HDCA diet were leaner and exhibited a 37% (P<0.05) decrease in fasting plasma glucose level. HDCA supplementation significantly decreased atherosclerotic lesion size at the aortic root region, the entire aorta, and the innominate artery by 44% (P<0.0001), 48% (P<0.01), and 94% (P<0.01), respectively, as compared with the chow group. Plasma VLDL/IDL/LDL cholesterol levels were significantly decreased, by 61% (P<0.05), in the HDCA group as compared with the chow diet group. HDCA supplementation decreased intestinal cholesterol absorption by 76% (P<0.0001) as compared with the chow group. Furthermore, HDL isolated from the HDCA group exhibited significantly increased ability to mediate cholesterol efflux ex vivo as compared with HDL of the chow diet group. In addition, HDCA significantly increased the expression of genes involved in cholesterol efflux, such as Abca1, Abcg1, and Apoe, in a macrophage cell line. Thus, HDCA is a candidate for antiatherosclerotic drug therapy.

BACKGROUND:The human 9p21.3 chromosome locus has been shown to be an independent risk factor for atherosclerosis in multiple large-scale genome-wide association studies, but the underlying mechanism remains unknown. We set out to investigate the potential role of the 9p21.3 locus neighboring genes, including Mtap, the 2 isoforms of Cdkn2a, p16Ink4a and p19Arf, and Cdkn2b, in atherosclerosis using knockout mice models.METHODS AND RESULTS:Gene-targeted mice for neighboring genes, including Mtap, Cdkn2a, p19Arf, and Cdkn2b, were each bred to mice carrying the human APO*E3 Leiden transgene that sensitizes the mice for atherosclerotic lesions through elevated plasma cholesterol. We found that the mice heterozygous for Mtap developed larger lesions compared with wild-type mice (49623±21650 versus 18899±9604 μm(2) per section [mean±SD]; P=0.01), with morphology similar to that of wild-type mice. The Mtap heterozygous mice demonstrated changes in metabolic and methylation profiles and CD4(+) cell counts. The Cdkn2a knockout mice had smaller lesions compared with wild-type and heterozygous mice, and there were no significant differences in lesion size in p19Arf and Cdkn2b mutants compared with wild type. We observed extensive, tissue-specific compensatory regulation of the Cdkn2a and Cdkn2b genes among the various knockout mice, making the effects on atherosclerosis difficult to interpret.CONCLUSIONS:Mtap plays a protective role against atherosclerosis, whereas Cdkn2a appears to be modestly proatherogenic. However, no relation was found between the 9p21 genotype and the transcription of 9p21 neighboring genes in primary human aortic vascular cells in vitro. There is extensive compensatory regulation in the highly conserved 9p21 orthologous region in mice.

In this study, we examined the effects of dietary supplementation of two natural emulsifiers, hyodeoxycholic acid (HDCA) and D-limonene (D-Lim), on atherosclerotic lesion formation in LDLRKO mouse. Female LDLRKO mice were maintained on a western diet for 8 weeks, then divided into 3 groups that received the following diets for 15 weeks before euthanization, chow diet, chow diet + 1.25% HDCA, or chow diet + 5% D-Lim. We found that 1.25% HDCA was able to significantly suppress the development of plaque area throughout the aorta (beyond the root of the aorta) by 47% (p<0.01) over 15 weeks of treatment. 5% D-lim treated mice showed a 25% (p<0.05) reduction in plaque formation. Atherosclerotic lesion size at the innominate artery was decreased by 94% (p<0.0001) and 53% (p<0.05) in the HDCA and D-Lim groups, respectively, as compared to the chow diet group. Plasma VLDL/IDL/LDL cholesterol levels were significantly decreased by 61% (p<0.0001) and 23% (p=0.01) in the HDCA and D-Lim groups, respectively, as compared to the chow diet group. We found that HDCA supplementation decreased cholesterol absorption from gut by 76% (p<0.0001), whereas D-Lim had no effect on cholesterol absorption. Interestingly, hepatic mRNA level of Cyp7a1, a key enzyme in bile acid synthesis, was significantly increased by more than 100% (p<0.05) in D-Lim group, suggesting D-Lim may act as a bile sequestrant in the gut. In addition, HDL isolated from the HDCA and D-Lim groups exhibited significantly increased ability (49% and 45% increase, respectively) to mediate cholesterol efflux from cholesterol loaded RAW 264.7 cells as compared to those of the chow diet group. In summary, our data demonstrated that HDCA supplementation decreased plasma VLDL/IDL/LDL cholesterol levels mainly through its ability to block intestinal cholesterol absorption. D-Lim decreased plasma VLDL/IDL/LDL cholesterol levels probably through decreased bile acid re-absorption in the intestine and increased conversion of cholesterol into bile acid in the liver. HDCA and D-Lim improve the cholesterol efflux ability of HDL as well. These anti-atherogenic effects resulted in significantly decreased atherosclerosis in LDLRKO mice receiving HDCA or D-Lim. HDCA and D-Lim may be new candidates for anti-atherosclerotic drugs.