Judy Wu

Flavin containing monooxygenase 3 exerts broad effects on glucose and lipid metabolism and atherosclerosis

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.

Anti-miR-33 Therapy Does Not Alter the Progression of Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient 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.

Genetic Architecture of Atherosclerosis in Mice: A Systems Genetics Analysis of Common Inbred Strains

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.

Effect of 9p21.3 Coronary Artery Disease Locus Neighboring Genes on Atherosclerosis in Mice

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 &mgr;m2 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.The 9p21.3 region of the genome has been identified as the locus with strongest association to coronary artery disease (CAD) and myocardial infarction (MI) in multiple independent large scale genome-wide association studies (GWAS).1-3 The locus is within a 58kb region that is devoid of protein coding genes, suggestive of a regulatory function (Figure 1). Interestingly, the neighboring genes in the region include well-known tumor suppressor genes, including CDKN2A and CDKN2B.4-6 The CDKN2A locus encodes a cyclin-dependent protein kinase (CDK) inhibitory protein (CKI), p16INK4A, and a p53-regulatory protein, p19ARF. The CDKN2B gene encodes another CKI, p15INK4B. Another gene in the region is methylthioadenosine phosphorylase (MTAP), which encodes a ubiquitously expressed metabolic enzyme S-methyl-5′-thioadenosine phosphorylase7 that processes the polyamine biosynthesis byproduct in the methionine salvage pathway. Loss or inactivation of MTAP has frequently been observed in a number of different human tumors, and it has been shown to have a tumor suppressive role in a mice model.8 Figure 1 The landscape of the 9p21.3 region Multiple studies demonstrated a potential role for cell cycle regulatory mechanisms in atherosclerosis progression. Previously, the master tumor suppressor gene p53 has been implicated in the development of atherosclerosis in apolipoprotein E (ApoE)-null mice9, 10, affecting both cell proliferation and apoptosis within the atheroma. Another tumor suppressor gene, p21Waf1, was also shown to increase the atheroma size in ApoE-null mice11, whereas the tumor suppressor p27Kip1 was shown to protect against atherosclerosis.12 Correlations of the 9p21 locus SNP genotype to differential expression of the neighboring genes have been observed in several studies with inconsistent findings.13-15 A knockout (KO) mouse model involving the entire region orthologous to the 9p21.3 CAD locus showed significant decreases in the expressed levels of Cdkn2a and Cdkn2b, and increased proliferation of primary smooth muscle cells (SMC) and mouse embryonic fibroblasts (MEF), although an effect on atherosclerosis in vivo was not demonstrated.16 Mice deficient in the p19Arf gene were found to have increased atherosclerotic lesions in an ApoE null background with significant attenuation of apoptosis in lesions as well as in cultured primary macrophages and vascular smooth muscle cells.17 However, to date no observation regarding atherosclerotic phenotype has been made involving the other neighboring genes. We set out to survey the 9p21.3 orthologous region using knockout mice models to systematically address the role of the neighboring protein-coding genes in atherosclerosis. We chose the APOE*3 Leiden sensitizing model because it is dominant, simplifying the construction of the models, and also because it exhibits relatively modest elevations of cholesterol, more realistically modeling the human disease than other widely used models.

Hyodeoxycholic acid improves HDL function and inhibits atherosclerotic lesion formation in LDLR-knockout mice

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.—Shih, D. M., Shaposhnik, Z., Meng, Y., Rosales, M., Wang, X., Wu, J., Ratiner, B., Zadini, F., Zadini, G., Lusis, A. J., Hyodeoxycholic acid improves HDL function and inhibits atherosclerotic lesion formation in LDLR‐knockout mice. FASEB J. 27, 3805–3817 (2013). www.fasebj.org

The effect of 9p21.3 coronary artery disease locus neighboring genes on atherosclerosis in mice: Kim, Effect of 9p21.3 genes on atherosclerosis

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 &mgr;m2 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.The 9p21.3 region of the genome has been identified as the locus with strongest association to coronary artery disease (CAD) and myocardial infarction (MI) in multiple independent large scale genome-wide association studies (GWAS).1-3 The locus is within a 58kb region that is devoid of protein coding genes, suggestive of a regulatory function (Figure 1). Interestingly, the neighboring genes in the region include well-known tumor suppressor genes, including CDKN2A and CDKN2B.4-6 The CDKN2A locus encodes a cyclin-dependent protein kinase (CDK) inhibitory protein (CKI), p16INK4A, and a p53-regulatory protein, p19ARF. The CDKN2B gene encodes another CKI, p15INK4B. Another gene in the region is methylthioadenosine phosphorylase (MTAP), which encodes a ubiquitously expressed metabolic enzyme S-methyl-5′-thioadenosine phosphorylase7 that processes the polyamine biosynthesis byproduct in the methionine salvage pathway. Loss or inactivation of MTAP has frequently been observed in a number of different human tumors, and it has been shown to have a tumor suppressive role in a mice model.8 Figure 1 The landscape of the 9p21.3 region Multiple studies demonstrated a potential role for cell cycle regulatory mechanisms in atherosclerosis progression. Previously, the master tumor suppressor gene p53 has been implicated in the development of atherosclerosis in apolipoprotein E (ApoE)-null mice9, 10, affecting both cell proliferation and apoptosis within the atheroma. Another tumor suppressor gene, p21Waf1, was also shown to increase the atheroma size in ApoE-null mice11, whereas the tumor suppressor p27Kip1 was shown to protect against atherosclerosis.12 Correlations of the 9p21 locus SNP genotype to differential expression of the neighboring genes have been observed in several studies with inconsistent findings.13-15 A knockout (KO) mouse model involving the entire region orthologous to the 9p21.3 CAD locus showed significant decreases in the expressed levels of Cdkn2a and Cdkn2b, and increased proliferation of primary smooth muscle cells (SMC) and mouse embryonic fibroblasts (MEF), although an effect on atherosclerosis in vivo was not demonstrated.16 Mice deficient in the p19Arf gene were found to have increased atherosclerotic lesions in an ApoE null background with significant attenuation of apoptosis in lesions as well as in cultured primary macrophages and vascular smooth muscle cells.17 However, to date no observation regarding atherosclerotic phenotype has been made involving the other neighboring genes. We set out to survey the 9p21.3 orthologous region using knockout mice models to systematically address the role of the neighboring protein-coding genes in atherosclerosis. We chose the APOE*3 Leiden sensitizing model because it is dominant, simplifying the construction of the models, and also because it exhibits relatively modest elevations of cholesterol, more realistically modeling the human disease than other widely used models.

Effect of 9p21.3 Coronary Artery Disease Locus Neighboring Genes on Atherosclerosis in MiceCLINICAL PERSPECTIVE

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 &mgr;m2 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.The 9p21.3 region of the genome has been identified as the locus with strongest association to coronary artery disease (CAD) and myocardial infarction (MI) in multiple independent large scale genome-wide association studies (GWAS).1-3 The locus is within a 58kb region that is devoid of protein coding genes, suggestive of a regulatory function (Figure 1). Interestingly, the neighboring genes in the region include well-known tumor suppressor genes, including CDKN2A and CDKN2B.4-6 The CDKN2A locus encodes a cyclin-dependent protein kinase (CDK) inhibitory protein (CKI), p16INK4A, and a p53-regulatory protein, p19ARF. The CDKN2B gene encodes another CKI, p15INK4B. Another gene in the region is methylthioadenosine phosphorylase (MTAP), which encodes a ubiquitously expressed metabolic enzyme S-methyl-5′-thioadenosine phosphorylase7 that processes the polyamine biosynthesis byproduct in the methionine salvage pathway. Loss or inactivation of MTAP has frequently been observed in a number of different human tumors, and it has been shown to have a tumor suppressive role in a mice model.8 Figure 1 The landscape of the 9p21.3 region Multiple studies demonstrated a potential role for cell cycle regulatory mechanisms in atherosclerosis progression. Previously, the master tumor suppressor gene p53 has been implicated in the development of atherosclerosis in apolipoprotein E (ApoE)-null mice9, 10, affecting both cell proliferation and apoptosis within the atheroma. Another tumor suppressor gene, p21Waf1, was also shown to increase the atheroma size in ApoE-null mice11, whereas the tumor suppressor p27Kip1 was shown to protect against atherosclerosis.12 Correlations of the 9p21 locus SNP genotype to differential expression of the neighboring genes have been observed in several studies with inconsistent findings.13-15 A knockout (KO) mouse model involving the entire region orthologous to the 9p21.3 CAD locus showed significant decreases in the expressed levels of Cdkn2a and Cdkn2b, and increased proliferation of primary smooth muscle cells (SMC) and mouse embryonic fibroblasts (MEF), although an effect on atherosclerosis in vivo was not demonstrated.16 Mice deficient in the p19Arf gene were found to have increased atherosclerotic lesions in an ApoE null background with significant attenuation of apoptosis in lesions as well as in cultured primary macrophages and vascular smooth muscle cells.17 However, to date no observation regarding atherosclerotic phenotype has been made involving the other neighboring genes. We set out to survey the 9p21.3 orthologous region using knockout mice models to systematically address the role of the neighboring protein-coding genes in atherosclerosis. We chose the APOE*3 Leiden sensitizing model because it is dominant, simplifying the construction of the models, and also because it exhibits relatively modest elevations of cholesterol, more realistically modeling the human disease than other widely used models.