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1.  Macrophage Sortilin Promotes LDL Uptake, Foam Cell Formation, and Atherosclerosis 
Circulation research  2015;116(5):789-796.
Rationale
Non-coding gene variants at the SORT1 locus are strongly associated with LDL-C levels as well as with coronary artery disease (CAD). SORT1 encodes a protein called sortilin, and hepatic sortilin modulates LDL metabolism by targeting apoB-containing lipoproteins to the lysosome. Sortilin is also expressed in macrophages, but its role in macrophage uptake of LDL and in atherosclerosis independent of plasma LDL-C levels is unknown.
Objective
To determine the effect of macrophage sortilin expression on LDL uptake, foam cell formation, and atherosclerosis.
Methods and Results
We crossed Sort1−/− mice onto a ‘humanized’ Apobec1−/−; hAPOB Tg background and determined that Sort1 deficiency on this background had no effect on plasma LDL-C levels but dramatically reduced atherosclerosis in the aorta and aortic root. In order to test whether this effect was a result of macrophage sortilin deficiency, we transplanted Sort1−/−;LDLR−/− or Sort1+/+;LDLR−/− bone marrow into Ldlr−/− mice and observed a similar reduction in atherosclerosis in mice lacking hematopoetic sortilin without an effect on plasma LDL-C levels. In an effort to determine the mechanism by which hematopoetic sortilin deficiency reduced atherosclerosis, we found no effect of sortilin deficiency on macrophage recruitment or LPS-induced cytokine release in vivo. In contrast, sortilin deficient macrophages had significantly reduced uptake of native LDL ex vivo and reduced foam cell formation in vivo, whereas sortilin overexpression in macrophages resulted in increased LDL uptake and foam cell formation.
Conclusions
Macrophage sortilin deficiency protects against atherosclerosis by reducing macrophage uptake of LDL. Sortilin-mediated uptake of native LDL into macrophages may be an important mechanism of foam cell formation and contributor to atherosclerosis development.
doi:10.1161/CIRCRESAHA.116.305811
PMCID: PMC4602371  PMID: 25593281
Atherosclerosis; low density lipoprotein cholesterol; macrophage; foam cell; receptor-mediated endocytosis
2.  Tribbles-1 regulates hepatic lipogenesis through posttranscriptional regulation of C/EBPα 
The Journal of Clinical Investigation  2015;125(10):3809-3818.
Variants near the gene TRIB1 are significantly associated with several plasma lipid traits, circulating liver enzymes, and the development of coronary artery disease in humans; however, it is not clear how its protein product tribbles-1 regulates lipid metabolism. Here, we evaluated mice harboring a liver-specific deletion of Trib1 (Trib1_LSKO) to elucidate the role of tribbles-1 in mammalian hepatic lipid metabolism. These mice exhibited increased hepatic triglyceride (TG) content, lipogenic gene transcription, and de novo lipogenesis. Microarray analysis revealed altered transcription of genes that are downstream of the transcription factor C/EBPα, and Trib1_LSKO mice had increased hepatic C/EBPα protein. Hepatic overexpression of C/EBPα in WT mice phenocopied Trib1_LSKO livers, and hepatic knockout of Cebpa in Trib1_LSKO mice revealed that C/EBPα is required for the increased lipogenesis. Using ChIP-Seq, we found that Trib1_LSKO mice had increased DNA-bound C/EBPα near lipogenic genes and the Trib1 gene, which itself was transcriptionally upregulated by C/EBPα overexpression. Together, our results reveal that tribbles-1 regulates hepatic lipogenesis through posttranscriptional regulation of C/EBPα, which in turn transcriptionally upregulates Trib1. These data suggest an important role for C/EBPα in mediating the lipogenic effects of hepatic Trib1 deletion and provide insight into the association between TRIB1 and plasma lipids, and liver traits in humans.
doi:10.1172/JCI77095
PMCID: PMC4607129  PMID: 26348894
3.  Knockout of Adamts7, A Novel CAD Locus in Humans, Reduces Atherosclerosis in Mice 
Circulation  2015;131(13):1202-1213.
Background
Genome-wide association studies (GWAS) have established ADAMTS7 as a locus for coronary artery disease (CAD) in humans. Yet, these studies fail to provide directionality for the association between ADAMTS7 and CAD. Previous reports have implicated ADAMTS7 in the regulation of vascular smooth muscle cell (VSMC) migration, but a role and direction of impact for this gene in atherogenesis has not been shown in relevant model systems.
Methods and Results
We bred an Adamts7 whole body knockout (KO) mouse onto both the Ldlr and Apoe KO hyperlipidemic mouse models. Adamts7−/−/Ldlr−/− and Adamts7−/−/Apoe−/− mice displayed significant reductions in lesion formation in aortas and aortic roots as compared to controls. Adamts7 KO mice also showed reduced neointimal formation after femoral wire injury. Adamts7 expression was induced in response to injury and hyperlipidemia but was absent at later timepoints, and primary Adamts7 KO VSMCs showed reduced migration in the setting of TNFα stimulation. ADAMTS7 localized to cells positive for SMC markers in human CAD lesions, and sub-cellular localization studies in cultured VSMCs placed ADAMTS7 at the cytoplasm and cell membrane, where it co-localized with markers of podosomes.
Conclusions
These data represent the first in vivo experimental validation of the association of Adamts7 with atherogenesis, likely through modulation of vascular cell migration and matrix in atherosclerotic lesions. These results demonstrate that Adamts7 is proatherogenic, lending directionality to the original genetic association and supporting the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans, making it an attractive target for novel therapeutic interventions.
doi:10.1161/CIRCULATIONAHA.114.012669
PMCID: PMC4382454  PMID: 25712206
ADAMTS7; metalloproteinases; atherosclerosis; coronary artery disease; genome wide association study; knockout mouse
4.  Sortilin And Lipoprotein Metabolism: Making Sense Out Of Complexity 
Current opinion in lipidology  2014;25(5):350-357.
Purpose of review
Genome wide associations studies (GWAS) have been used as an unbiased tool to identify novel genes that contribute to variations in LDL-C levels in the hopes of uncovering new biology and new therapeutic targets for the treatment of atherosclerotic cardiovascular disease (ASCVD). The locus identified by GWAS with the strongest association with LDL-C and ASCVD is the 1p13 SORT1 locus. Here we review the identification and characterization of this locus, the initial physiological studies describing the role of SORT1 in lipoprotein metabolism, and recent work that has begun to sort out the complexity of this role.
Recent findings
Studies by several groups support an important role for sortilin in lipoprotein metabolism; however, the directionality of the effect of sortilin on plasma cholesterol and its role in the secretion of hepatic lipoproteins remains controversial. Studies by several groups support a role for sortilin in inhibiting lipoprotein export whereas other studies suggest that sortilin facilitates lipoprotein export.
Summary
Understanding the mechanism by which sortilin affects LDL-C will increase our understanding of the regulation of lipoprotein metabolism and hepatic lipoprotein export and may also allow us to harness the power of the 1p13 SORT1 locus for the treatment of ASCVD.
doi:10.1097/MOL.0000000000000110
PMCID: PMC4565516  PMID: 25101658
Sort1; genome wide association study; atherosclerotic cardiovascular disease; low-density lipoprotein cholesterol; lipid metabolism
5.  Permanent Alteration of PCSK9 With In Vivo CRISPR-Cas9 Genome Editing 
Circulation research  2014;115(5):488-492.
Rationale
Individuals with naturally occurring loss-of-function PCSK9 mutations experience reduced blood low-density lipoprotein cholesterol (LDL-C) levels and protection against cardiovascular disease.
Objective
The goal of this study was to assess whether genome editing using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system can efficiently introduce loss-of-function mutations into the endogenous PCSK9 gene in vivo.
Methods and Results
We used adenovirus to express Cas9 and a CRISPR guide RNA targeting Pcsk9 in mouse liver, where the gene is specifically expressed. We found that within three to four days of administration of the virus, the mutagenesis rate of Pcsk9 in the liver was as high as >50%. This resulted in decreased plasma PCSK9 levels, increased hepatic LDL receptor levels, and decreased plasma cholesterol levels (by 35%–40%) in the blood. No off-target mutagenesis was detected in 10 selected sites.
Conclusions
Genome editing with the CRISPR-Cas9 system disrupts the Pcsk9 gene in vivo with high efficiency and reduces blood cholesterol levels in mice. This approach may have therapeutic potential for the prevention of cardiovascular disease in humans.
doi:10.1161/CIRCRESAHA.115.304351
PMCID: PMC4134749  PMID: 24916110
Coronary disease; gene therapy; lipoprotein; molecular biology; prevention
6.  A null mutation in ANGPTL8 does not associate with either plasma glucose or type 2 diabetes in humans 
Background
Experiments in mice initially suggested a role for the protein angiopoietin-like 8 (ANGPTL8) in glucose homeostasis. However, subsequent experiments in model systems have challenged this proposed role. We sought to better understand the importance of ANGPTL8 in human glucose homeostasis by examining the association of a null mutation in ANGPTL8 with fasting glucose levels and risk for type 2 diabetes.
Methods
A naturally-occurring null mutation in human ANGPTL8 (rs145464906; c.361C > T; p.Q121X) is carried by ~1 in 1000 individuals of European ancestry and is associated with higher levels of plasma high-density lipoprotein cholesterol, suggesting that this mutation has functional significance. We examined the association of p.Q121X with fasting glucose levels and risk for type 2 diabetes in up to 95,558 individuals (14,824 type 2 diabetics and 80,734 controls).
Results
We found no significant association of p.Q121X with either fasting glucose or type 2 diabetes (p-value = 0.90 and 0.65, respectively). Given our sample sizes, we had >98 % power to detect at least a 0.23 mmol/L effect on plasma glucose and >95 % power to detect a 70 % increase in risk for type 2 diabetes.
Conclusion
Disruption of ANGPTL8 function in humans does not seem to have a large effect on measures of glucose tolerance.
Electronic supplementary material
The online version of this article (doi:10.1186/s12902-016-0088-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12902-016-0088-8
PMCID: PMC4730725  PMID: 26822414
Betatrophin; Angiopoietin-like 8; rs145464906
7.  Association of HDL cholesterol efflux capacity with incident coronary heart disease events: a prospective case-control study 
Summary
Background
Although HDL cholesterol concentrations are strongly and inversely associated with risk of coronary heart disease, interventions that raise HDL cholesterol do not reduce risk of coronary heart disease. HDL cholesterol efflux capacity—a prototypical measure of HDL function—has been associated with coronary heart disease after adjusting for HDL cholesterol, but its effect on incident coronary heart disease risk is uncertain.
Methods
We measured cholesterol efflux capacity and assessed its relation with vascular risk factors and incident coronary heart disease events in a nested case-control sample from the prospective EPIC-Norfolk study of 25 639 individuals aged 40–79 years, assessed in 1993–97 and followed up to 2009. We quantified cholesterol efflux capacity in 1745 patients with incident coronary heart disease and 1749 control participants free of any cardiovascular disorders by use of a validated ex-vivo radiotracer assay that involved incubation of cholesterol-labelled J774 macrophages with apoB-depleted serum from study participants.
Findings
Cholesterol efflux capacity was positively correlated with HDL cholesterol concentration (r=0·40; p<0·0001) and apoA-I concentration (r=0·22; p<0·0001). It was also inversely correlated with type 2 diabetes (r=–0·18; p<0·0001) and positively correlated with alcohol consumption (r=0·12; p<0·0001). In analyses comparing the top and bottom tertiles, cholesterol efflux capacity was significantly and inversely associated with incident coronary heart disease events, independent of age, sex, diabetes, hypertension, smoking and alcohol use, waist:hip ratio, BMI, LDL cholesterol concentration, log-triglycerides, and HDL cholesterol or apoA-I concentrations (odds ratio 0·64, 95% CI 0·51–0·80). After a similar multivariable adjustment the risk of incident coronary heart disease was 0·80 (95% CI 0·70–0·90) for a per-SD change in cholesterol efflux capacity.
Interpretation
HDL cholesterol efflux capacity might provide an alternative mechanism for therapeutic modulation of the HDL pathway beyond HDL cholesterol concentration to help reduce risk of coronary heart disease.
Funding
US National Institutes of Health, UK Medical Research Council, Cancer Research UK.
doi:10.1016/S2213-8587(15)00126-6
PMCID: PMC4648056  PMID: 26025389
8.  Lipidomic analyses of female mice lacking hepatic lipase and endothelial lipase indicate selective modulation of plasma lipid species 
Lipids  2014;49(6):505-515.
Hepatic lipase (HL) and endothelial lipase (EL) share overlapping and complementary roles in lipoprotein metabolism. The deletion of HL and EL alleles in mice raises plasma total cholesterol and phospholipid concentrations. However, the influence of HL and EL in vivo on individual molecular species from each class of lipid is not known. We hypothesized that the loss of HL, EL or both in vivo may affect select molecular species from each class of lipids. To test this hypothesis, we performed lipidomic analyses on plasma and livers from fasted female wild-type, HL-knockout, EL-knockout, and HL/EL-double knockout mice. Overall, the loss of HL, EL, or both, resulted in minimal changes to hepatic lipids; however, select species of CE were surprisingly reduced in the livers of mice only lacking EL. The loss of HL, EL, or both, reduced the plasma concentrations for select molecular species of triacylglycerol, diacylglycerol, and free fatty acid. On the other hand, the loss of HL, EL, or both, raised the plasma concentrations for select molecular species of phosphatidylcholine, cholesteryl ester, diacylglycerol, sphingomyelin, ceramide, plasmanylcholine, and plasmenylcholine. The increased plasma concentration of select ether phospholipids was evident in the absence of EL, thus suggesting that EL might exhibit a phospholipase A2 activity. Using recombinant EL, we showed that it could hydrolyse the artificial phospholipase A2 substrate 4-nitro-3-(octanoyloxy)benzoic acid. In summary, our study shows for the first time the influence of HL and EL on individual molecular species of several classes of lipids in vivo using lipidomic methods.
doi:10.1007/s11745-014-3907-6
PMCID: PMC4724200  PMID: 24777581
lipoproteins; hepatic lipase; endothelial lipase; knockout mice; lipidomics; mass spectrometry; phospholipase A2
9.  Perhexiline activates KLF14 and reduces atherosclerosis by modulating ApoA-I production 
The Journal of Clinical Investigation  2015;125(10):3819-3830.
Recent genome-wide association studies have revealed that variations near the gene locus encoding the transcription factor Krüppel-like factor 14 (KLF14) are strongly associated with HDL cholesterol (HDL-C) levels, metabolic syndrome, and coronary heart disease. However, the precise mechanisms by which KLF14 regulates lipid metabolism and affects atherosclerosis remain largely unexplored. Here, we report that KLF14 is dysregulated in the liver of 2 dyslipidemia mouse models. We evaluated the effects of both KLF14 overexpression and genetic inactivation and determined that KLF14 regulates plasma HDL-C levels and cholesterol efflux capacity by modulating hepatic ApoA-I production. Hepatic-specific Klf14 deletion in mice resulted in decreased circulating HDL-C levels. In an attempt to pharmacologically target KLF14 as an experimental therapeutic approach, we identified perhexiline, an approved therapeutic small molecule presently in clinical use to treat angina and heart failure, as a KLF14 activator. Indeed, in WT mice, treatment with perhexiline increased HDL-C levels and cholesterol efflux capacity via KLF14-mediated upregulation of ApoA-I expression. Moreover, perhexiline administration reduced atherosclerotic lesion development in apolipoprotein E–deficient mice. Together, these results provide comprehensive insight into the KLF14-dependent regulation of HDL-C and subsequent atherosclerosis and indicate that interventions that target the KLF14 pathway should be further explored for the treatment of atherosclerosis.
doi:10.1172/JCI79048
PMCID: PMC4607137  PMID: 26368306
10.  Reducing the burden of disease and death from familial hypercholesterolemia: A call to action 
American heart journal  2014;168(6):807-811.
Familial hypercholesterolemia (FH) is a genetic disease characterized by substantial elevations of low-density lipoprotein cholesterol, unrelated to diet or lifestyle. Untreated FH patients have 20 times the risk of developing coronary artery disease, compared with the general population. Estimates indicate that as many as 1 in 500 people of all ethnicities and 1 in 250 people of Northern European descent may have FH; nevertheless, the condition remains largely undiagnosed. In the United States alone, perhaps as little as 1% of FH patients have been diagnosed. Consequently, there are potentially millions of children and adults worldwide who are unaware that they have a life-threatening condition. In countries like the Netherlands, the United Kingdom, and Spain, cascade screening programs have led to dramatic improvements in FH case identification. Given that there are currently no systematic approaches in the United States to identify FH patients or affected relatives, the patient-centric nonprofit FH Foundation convened a national FH Summit in 2013, where participants issued a “call to action” to health care providers, professional organizations, public health programs, patient advocacy groups, and FH experts, in order to bring greater attention to this potentially deadly, but (with proper diagnosis) eminently treatable, condition.
doi:10.1016/j.ahj.2014.09.001
PMCID: PMC4683103  PMID: 25458642
11.  Tribbles-1: a novel regulator of hepatic lipid metabolism in humans 
Biochemical Society Transactions  2015;43(5):1079-1084.
The protein tribbles-1, encoded by the gene TRIB1, is increasingly recognized as a major regulator of multiple cellular and physiological processes in humans. Recent human genetic studies, as well as molecular biological approaches, have implicated this intriguing protein in the aetiology of multiple human diseases, including myeloid leukaemia, Crohn's disease, non-alcoholic fatty liver disease (NAFLD), dyslipidaemia and coronary artery disease (CAD). Genome-wide association studies (GWAS) have repeatedly identified variants at the genomic TRIB1 locus as being significantly associated with multiple plasma lipid traits and cardiovascular disease (CVD) in humans. The involvement of TRIB1 in hepatic lipid metabolism has been validated through viral-mediated hepatic overexpression of the gene in mice; increasing levels of TRIB1 decreased plasma lipids in a dose-dependent manner. Additional studies have implicated TRIB1 in the regulation of hepatic lipogenesis and NAFLD. The exact mechanisms of TRIB1 regulation of both plasma lipids and hepatic lipogenesis remain undetermined, although multiple signalling pathways and transcription factors have been implicated in tribbles-1 function. Recent reports have been aimed at developing TRIB1-based lipid therapeutics. In summary, tribbles-1 is an important modulator of human energy metabolism and metabolic syndromes and worthy of future studies aimed at investigating its potential as a therapeutic target.
doi:10.1042/BST20150101
PMCID: PMC4613491  PMID: 26517927
cardiovascular disease; GWAS; lipid metabolism; lipoproteins; Trib1; Tribbles
12.  High-Density Lipoprotein 
Circulation research  2013;113(12):1275-1277.
doi:10.1161/CIRCRESAHA.113.302667
PMCID: PMC4111252  PMID: 24311613
13.  Inhibition of endothelial lipase activity by sphingomyelin in the lipoproteins 
Lipids  2014;49(10):987-996.
Endothelial lipase (EL) is a major determinant of plasma HDL concentration, its activity being inversely proportional to HDL levels. Although it is known that it preferentially acts on HDL, compared to LDL and VLDL, the basis for this specificity is not known. Here we tested the hypothesis that sphingomyelin, a major phospholipid in lipoproteins is a physiological inhibitor of EL, and that the preference of the enzyme for HDL may be due to low sphingomyelin/ phosphatidylcholine (PtdCho) ratio in HDL, compared to other lipoproteins. Using recombinant human EL, we showed that sphingomyelin inhibits the hydrolysis of PtdCho in the liposomes in a concentration-dependent manner. While the enzyme showed lower hydrolysis of LDL PtdCho, compared to HDL PtdCho, this difference disappeared after the degradation of lipoprotein sphingomyelin by bacterial sphingomyelinase. Analysis of molecular species of PtdCho hydrolyzed by EL in the lipoproteins showed that the enzyme preferentially hydrolyzed PtdCho containing polyunsaturated fatty acids (PUFA) such as 22:6, 20:5, 20:4 at sn-2 position, generating the corresponding PUFA-lyso PtdCho. This specificity for PUFA-PtdCho species was not observed after depletion of sphingomyelin by sphingomyelinase. These results show that sphingomyelin not only plays a role in regulating EL activity, but also influences its specificity towards PtdCho species.
doi:10.1007/s11745-014-3944-1
PMCID: PMC4175046  PMID: 25167836
sphingomyelin/phosphatidyl choline ratio; lysophsphatidylcholine; substrate specificity; molecular species of phosphatidylcholine; sphingomyelinase; LC/MS; enzyme regulation
14.  Anacetrapib lowers LDL by increasing ApoB clearance in mildly hypercholesterolemic subjects 
The Journal of Clinical Investigation  2015;125(6):2510-2522.
BACKGROUND. Individuals treated with the cholesteryl ester transfer protein (CETP) inhibitor anacetrapib exhibit a reduction in both LDL cholesterol and apolipoprotein B (ApoB) in response to monotherapy or combination therapy with a statin. It is not clear how anacetrapib exerts these effects; therefore, the goal of this study was to determine the kinetic mechanism responsible for the reduction in LDL and ApoB in response to anacetrapib.
METHODS. We performed a trial of the effects of anacetrapib on ApoB kinetics. Mildly hypercholesterolemic subjects were randomized to background treatment of either placebo (n = 10) or 20 mg atorvastatin (ATV) (n = 29) for 4 weeks. All subjects then added 100 mg anacetrapib to background treatment for 8 weeks. Following each study period, subjects underwent a metabolic study to determine the LDL-ApoB-100 and proprotein convertase subtilisin/kexin type 9 (PCSK9) production rate (PR) and fractional catabolic rate (FCR).
RESULTS. Anacetrapib markedly reduced the LDL-ApoB-100 pool size (PS) in both the placebo and ATV groups. These changes in PS resulted from substantial increases in LDL-ApoB-100 FCRs in both groups. Anacetrapib had no effect on LDL-ApoB-100 PRs in either treatment group. Moreover, there were no changes in the PCSK9 PS, FCR, or PR in either group. Anacetrapib treatment was associated with considerable increases in the LDL triglyceride/cholesterol ratio and LDL size by NMR.
CONCLUSION. These data indicate that anacetrapib, given alone or in combination with a statin, reduces LDL-ApoB-100 levels by increasing the rate of ApoB-100 fractional clearance.
TRIAL REGISTRATION. ClinicalTrials.gov NCT00990808.
FUNDING. Merck & Co. Inc., Kenilworth, New Jersey, USA. Additional support for instrumentation was obtained from the National Center for Advancing Translational Sciences (UL1TR000003 and UL1TR000040).
doi:10.1172/JCI80025
PMCID: PMC4497759  PMID: 25961461
Cardiology; Therapeutics; Vascular Biology
15.  AAV Vectors Expressing LDLR Gain-of-Function Variants Demonstrate Increased Efficacy in Mouse Models of Familial Hypercholesterolemia 
Circulation research  2014;115(6):591-599.
Rationale
Familial hypercholesterolemia (FH) is a genetic disorder that arises due to loss-of-function mutations in the low-density lipoprotein receptor (LDLR) and homozygous FH (hoFH) is a candidate for gene therapy using adeno-associated viral (AAV) vectors. Proprotein convertase subtilisin/kexin type 9 (PCSK9) and inducible degrader of LDLR (IDOL) negatively regulate LDLR protein and could dampen AAV encoded LDLR expression.
Objective
We sought to create vectors expressing gain-of-function human LDLR variants that are resistant to degradation by human PCSK9 and IDOL and thereby enhance hepatic LDLR protein abundance and plasma LDL cholesterol reduction.
Methods and Results
Amino acid substitutions were introduced into the coding sequence of human LDLR cDNA to reduce interaction with hPCSK9 and hIDOL. A panel of mutant hLDLRs was initially screened in vitro for escape from PCSK9. The variant hLDLR-L318D was further evaluated using a mouse model of hoFH lacking endogenous LDLR and apolipoprotein B mRNA editing enzyme, APOBEC-1 (DKO). Administration of wild type hLDLR to DKO mice, expressing hPCSK9, led to diminished LDLR activity. However, LDLR-L318D was resistant to hPCSK9 mediated degradation and effectively reduced cholesterol levels. Similarly, the LDLR-K809R\C818A construct avoided hIDOL regulation and achieved stable reductions in serum cholesterol. An AAV8.LDLR-L318D\K809R\C818A vector that carried all three amino acid substitutions conferred partial resistance to both hPCSK9 and hIDOL mediated degradation.
Conclusion
Amino acid substitutions in the human LDLR confer partial resistance to PCSK9 and IDOL regulatory pathways with improved reduction in cholesterol levels and improve upon a potential gene therapeutic approach to treat homozygous FH subjects.
doi:10.1161/CIRCRESAHA.115.304008
PMCID: PMC4165546  PMID: 25023731
AAV; gene therapy; LDLR; PCSK9; IDOL; familial hypercholesterolemia
16.  Generation of iPSCs as a Pooled Culture Using Magnetic Activated Cell Sorting of Newly Reprogrammed Cells 
PLoS ONE  2015;10(8):e0134995.
Although significant advancement has been made in the induced pluripotent stem cell (iPSC) field, current methods for iPSC derivation are labor intensive and costly. These methods involve manual selection, expansion, and characterization of multiple clones for each reprogrammed cell sample and therefore significantly hampers the feasibility of studies where a large number of iPSCs need to be derived. To develop higher throughput iPSC reprogramming methods, we generated iPSCs as a pooled culture using rigorous cell surface pluripotent marker selection with TRA-1-60 or SSEA4 antibodies followed by Magnetic Activated Cell Sorting (MACS). We observed that pool-selected cells are similar or identical to clonally derived iPSC lines from the same donor by all criteria examined, including stable expression of endogenous pluripotency genes, normal karyotype, loss of exogenous reprogramming factors, and in vitro spontaneous and lineage directed differentiation potential. This strategy can be generalized for iPSC generation using both integrating and non-integrating reprogramming methods. Our studies provide an attractive alternative to clonal derivation of iPSCs using rigorously selected cell pools and is amenable to automation.
doi:10.1371/journal.pone.0134995
PMCID: PMC4539221  PMID: 26281015
17.  Higher plasma CXCL12 levels predict incident myocardial infarction and death in chronic kidney disease: findings from the Chronic Renal Insufficiency Cohort study 
European Heart Journal  2013;35(31):2115-2122.
Aims
Genome-wide association studies revealed an association between a locus at 10q11, downstream from CXCL12, and myocardial infarction (MI). However, the relationship among plasma CXCL12, cardiovascular disease (CVD) risk factors, incident MI, and death is unknown.
Methods and results
We analysed study-entry plasma CXCL12 levels in 3687 participants of the Chronic Renal Insufficiency Cohort (CRIC) Study, a prospective study of cardiovascular and kidney outcomes in chronic kidney disease (CKD) patients. Mean follow-up was 6 years for incident MI or death. Plasma CXCL12 levels were positively associated with several cardiovascular risk factors (age, hypertension, diabetes, hypercholesterolaemia), lower estimated glomerular filtration rate (eGFR), and higher inflammatory cytokine levels (P < 0.05). In fully adjusted models, higher study-entry CXCL12 was associated with increased odds of prevalent CVD (OR 1.23; 95% confidence interval 1.14, 1.33, P < 0.001) for one standard deviation (SD) increase in CXCL12. Similarly, one SD higher CXCL12 increased the hazard of incident MI (1.26; 1.09,1.45, P < 0.001), death (1.20; 1.09,1.33, P < 0.001), and combined MI/death (1.23; 1.13–1.34, P < 0.001) adjusting for demographic factors, known CVD risk factors, and inflammatory markers and remained significant for MI (1.19; 1.03,1.39, P = 0.01) and the combined MI/death (1.13; 1.03,1.24, P = 0.01) after further controlling for eGFR and urinary albumin:creatinine ratio.
Conclusions
In CKD, higher plasma CXCL12 was associated with CVD risk factors and prevalent CVD as well as the hazard of incident MI and death. Further studies are required to establish if plasma CXCL12 reflect causal actions at the vessel wall and is a tool for genomic and therapeutic trials.
doi:10.1093/eurheartj/eht481
PMCID: PMC4132636  PMID: 24306482
Atherosclerosis; Chemokines; Myocardial infarction; CXCL12
18.  Multiple rare alleles at LDLR and APOA5 confer risk for early-onset myocardial infarction 
Do, Ron | Stitziel, Nathan O. | Won, Hong-Hee | Jørgensen, Anders Berg | Duga, Stefano | Merlini, Pier Angelica | Kiezun, Adam | Farrall, Martin | Goel, Anuj | Zuk, Or | Guella, Illaria | Asselta, Rosanna | Lange, Leslie A. | Peloso, Gina M. | Auer, Paul L. | Girelli, Domenico | Martinelli, Nicola | Farlow, Deborah N. | DePristo, Mark A. | Roberts, Robert | Stewart, Alexander F.R. | Saleheen, Danish | Danesh, John | Epstein, Stephen E. | Sivapalaratnam, Suthesh | Hovingh, G. Kees | Kastelein, John J. | Samani, Nilesh J. | Schunkert, Heribert | Erdmann, Jeanette | Shah, Svati H. | Kraus, William E. | Davies, Robert | Nikpay, Majid | Johansen, Christopher T. | Wang, Jian | Hegele, Robert A. | Hechter, Eliana | Marz, Winfried | Kleber, Marcus E. | Huang, Jie | Johnson, Andrew D. | Li, Mingyao | Burke, Greg L. | Gross, Myron | Liu, Yongmei | Assimes, Themistocles L. | Heiss, Gerardo | Lange, Ethan M. | Folsom, Aaron R. | Taylor, Herman A. | Olivieri, Oliviero | Hamsten, Anders | Clarke, Robert | Reilly, Dermot F. | Yin, Wu | Rivas, Manuel A. | Donnelly, Peter | Rossouw, Jacques E. | Psaty, Bruce M. | Herrington, David M. | Wilson, James G. | Rich, Stephen S. | Bamshad, Michael J. | Tracy, Russell P. | Cupples, L. Adrienne | Rader, Daniel J. | Reilly, Muredach P. | Spertus, John A. | Cresci, Sharon | Hartiala, Jaana | Tang, W.H. Wilson | Hazen, Stanley L. | Allayee, Hooman | Reiner, Alex P. | Carlson, Christopher S. | Kooperberg, Charles | Jackson, Rebecca D. | Boerwinkle, Eric | Lander, Eric S. | Schwartz, Stephen M. | Siscovick, David S. | McPherson, Ruth | Tybjaerg-Hansen, Anne | Abecasis, Goncalo R. | Watkins, Hugh | Nickerson, Deborah A. | Ardissino, Diego | Sunyaev, Shamil R. | O’Donnell, Christopher J. | Altshuler, David | Gabriel, Stacey | Kathiresan, Sekar
Nature  2014;518(7537):102-106.
Summary
Myocardial infarction (MI), a leading cause of death around the world, displays a complex pattern of inheritance1,2. When MI occurs early in life, the role of inheritance is substantially greater1. Previously, rare mutations in low-density lipoprotein (LDL) genes have been shown to contribute to MI risk in individual families3–8 whereas common variants at more than 45 loci have been associated with MI risk in the population9–15. Here, we evaluate the contribution of rare mutations to MI risk in the population. We sequenced the protein-coding regions of 9,793 genomes from patients with MI at an early age (≤50 years in males and ≤60 years in females) along with MI-free controls. We identified two genes where rare coding-sequence mutations were more frequent in cases versus controls at exome-wide significance. At low-density lipoprotein receptor (LDLR), carriers of rare, damaging mutations (3.1% of cases versus 1.3% of controls) were at 2.4-fold increased risk for MI; carriers of null alleles at LDLR were at even higher risk (13-fold difference). This sequence-based estimate of the proportion of early MI cases due to LDLR mutations is remarkably similar to an estimate made more than 40 years ago using total cholesterol16. At apolipoprotein A-V (APOA5), carriers of rare nonsynonymous mutations (1.4% of cases versus 0.6% of controls) were at 2.2-fold increased risk for MI. When compared with non-carriers, LDLR mutation carriers had higher plasma LDL cholesterol whereas APOA5 mutation carriers had higher plasma triglycerides. Recent evidence has connected MI risk with coding sequence mutations at two genes functionally related to APOA5, namely lipoprotein lipase15,17 and apolipoprotein C318,19. When combined, these observations suggest that, beyond LDL cholesterol, disordered metabolism of triglyceride-rich lipoproteins contributes to MI risk.
doi:10.1038/nature13917
PMCID: PMC4319990  PMID: 25487149
19.  IL-1 and atherosclerosis: a murine twist to an evolving human story 
Inflammation is a critical component of atherosclerosis. IL-1 is a classic proinflammatory cytokine that has been linked to atherosclerosis. A clinical trial has been launched in which an antibody specific for IL-1β is being studied for its effects on cardiovascular events in patients with atherosclerosis. In this issue of the JCI, Alexander et al. report that mice lacking the receptor for IL-1 unexpectedly have features of advanced atherosclerosis that suggest the atherosclerotic plaques may be less stable. These findings illustrate the complexity of inflammatory pathways in atherosclerosis and suggest the need for careful calibration of antiinflammatory approaches to atherosclerosis.
doi:10.1172/JCI61163
PMCID: PMC3248309  PMID: 22201674
20.  Nuclear Receptors and microRNA-144 Coordinately Regulate Cholesterol Efflux 
Circulation research  2013;112(12):1529-1531.
The ATP-binding cassette transporter A1 (ABCA1) is a key mediator of cellular cholesterol efflux and HDL maturation. ABCA1 mRNA has an unusually long 3’ untranslated region, which makes it highly susceptible to microRNA (miRNA) targeting and repression. As such, multiple miRNAs have been reported to directly target ABCA1, including miR-33a/b, miR-26, miR-106b, and miR-758. Many of these miRNAs participate in feed-forward or feedback networks in controlling cholesterol and lipid homeostasis. Antisense oligonucleotide-based inhibition of miR-33 was found to increase HDL-C levels and regress atherosclerosis in mice and non-human primates. In this edition of Circulation Research, two separate studies identified novel miRNA networks driven by nuclear receptor induced miR-144 targeting of ABCA1 and cholesterol efflux. The first study reports that miR-144 serves to buffer uncontrolled ABCA1 activation in response to high cholesterol states and liver X receptor (LXR) activation in macrophages and liver. The second study highlights the role of miR-144 and ABCA1 in promotion of bile acid secretion in response to farensoid X receptor (FXR) activation in the liver. These studies suggest that anti-miR-144, like anti-miR-33, could be a novel approach to targeting HDL and reverse cholesterol transport.
doi:10.1161/CIRCRESAHA.113.301422
PMCID: PMC4043301  PMID: 23743223
21.  Apolipoprotein A-I and cholesterol efflux: The good, the bad, and the modified 
Circulation research  2014;114(11):1681-1683.
doi:10.1161/CIRCRESAHA.114.303974
PMCID: PMC4104775  PMID: 24855198
22.  Adipose Modulation of High-Density Lipoprotein Cholesterol 
Circulation  2011;124(15):1602-1605.
doi:10.1161/CIRCULATIONAHA.111.058453
PMCID: PMC3928975  PMID: 21986773
Editorials; high-density lipoprotein cholesterol; lipoproteins; obesity; adipose tissue; Reverse Cholesterol Transport
23.  Differential Association of Plasma Angiopoietin-Like Proteins 3 and 4 with Lipid and Metabolic Traits 
Objective
ANGPTL3 and ANGPTL4 are secreted proteins that inhibit lipoprotein lipase (LPL) in vitro. Genetic variants at the ANGPTL3 and ANGPTL4 gene loci are significantly associated with plasma lipid traits. The aim of this study was to evaluate the association of plasma angiopoietin-like protein 3 (ANGPTL3) and 4 (ANGPTL4) concentrations with lipid and metabolic traits in a large community-based sample.
Approach and Results
Plasma ANGPTL3 and ANGPTL4 levels were measured in 1770 subjects using a validated ELISA assay. A Pearson unadjusted correlation analysis and a linear regression analysis adjusting for age, gender and race were performed. ANGPTL3 levels were significantly positively associated with low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels (both P < 2 ×10−5) but not triglycerides. In contrast, ANGPTL4 levels were significantly negatively associated with LDL-C and HDL-C (both P < 2 × 10−5) and positively associated with triglycerides (P=0.003). In addition, ANGPTL4, but not ANGPTL3, levels were significantly positively associated with fasting blood glucose and metabolic syndrome.
Conclusions
Despite having similar biochemical effects in vitro, plasma ANGPTL3 and ANGPTL4 concentrations have nearly opposite relationships with plasma lipids. ANGPTL4 is strongly negatively associated with LDL-C and HDL-C and positively with multiple features of the metabolic syndrome including triglycerides, whereas ANGPTL3 is positively associated with LDL-C and HDL-C and not with metabolic syndrome traits including triglycerides. While ANGPTL3 and ANGPTL4 both inhibit LPL in vitro and influence lipoprotein metabolism in vivo, the physiology of these related proteins and their effects on lipoproteins is clearly divergent and complex.
doi:10.1161/ATVBAHA.113.302802
PMCID: PMC4104779  PMID: 24626437
Angiopoietin-Like Protein 3; Angiopoietin-Like Protein 4; lipid metabolism; low-density lipoprotein cholesterol; high-density lipoprotein cholesterol; triglycerides; epidemiology; lipids; lipoproteins; metabolic syndrome; diabetes mellitus
24.  Robust passive and active efflux of cellular cholesterol to a designer functional mimic of high density lipoprotein 
Journal of Lipid Research  2015;56(5):972-985.
The ability of HDL to support macrophage cholesterol efflux is an integral part of its atheroprotective action. Augmenting this ability, especially when HDL cholesterol efflux capacity from macrophages is poor, represents a promising therapeutic strategy. One approach to enhancing macrophage cholesterol efflux is infusing blood with HDL mimics. Previously, we reported the synthesis of a functional mimic of HDL (fmHDL) that consists of a gold nanoparticle template, a phospholipid bilayer, and apo A-I. In this work, we characterize the ability of fmHDL to support the well-established pathways of cellular cholesterol efflux from model cell lines and primary macrophages. fmHDL received cell cholesterol by unmediated (aqueous) and ABCG1- and scavenger receptor class B type I (SR-BI)-mediated diffusion. Furthermore, the fmHDL holoparticle accepted cholesterol and phospholipid by the ABCA1 pathway. These results demonstrate that fmHDL supports all the cholesterol efflux pathways available to native HDL and thus, represents a promising infusible therapeutic for enhancing macrophage cholesterol efflux. fmHDL accepts cholesterol from cells by all known pathways of cholesterol efflux: unmediated, ABCG1- and SR-BI-mediated diffusion, and through ABCA1.
doi:10.1194/jlr.M054635
PMCID: PMC4409287  PMID: 25652088
ATP-binding cassette transporter A1; atherosclerosis; macrophages; cholesterol efflux; ATP-binding cassette transporter G1; scavenger receptor class B type I
25.  Exome Sequencing in Suspected Monogenic Dyslipidemias 
Background
Exome sequencing is a promising tool for gene mapping in Mendelian disorders. We utilized this technique in an attempt to identify novel genes underlying monogenic dyslipidemias.
Methods and Results
We performed exome sequencing on 213 selected family members from 41 kindreds with suspected Mendelian inheritance of extreme levels of low-density lipoprotein (LDL) cholesterol (after candidate gene sequencing excluded known genetic causes for high LDL cholesterol families) or high-density lipoprotein (HDL) cholesterol. We used standard analytic approaches to identify candidate variants and also assigned a polygenic score to each individual in order to account for their burden of common genetic variants known to influence lipid levels. In nine families, we identified likely pathogenic variants in known lipid genes (ABCA1, APOB, APOE, LDLR, LIPA, and PCSK9); however, we were unable to identify obvious genetic etiologies in the remaining 32 families despite follow-up analyses. We identified three factors that limited novel gene discovery: (1) imperfect sequencing coverage across the exome hid potentially causal variants; (2) large numbers of shared rare alleles within families obfuscated causal variant identification; and (3) individuals from 15% of families carried a significant burden of common lipid-related alleles, suggesting complex inheritance can masquerade as monogenic disease.
Conclusions
We identified the genetic basis of disease in nine of 41 families; however, none of these represented novel gene discoveries. Our results highlight the promise and limitations of exome sequencing as a discovery technique in suspected monogenic dyslipidemias. Considering the confounders identified may inform the design of future exome sequencing studies.
doi:10.1161/CIRCGENETICS.114.000776
PMCID: PMC4406825  PMID: 25632026
genetics; human; DNA sequencing; Exome sequencing; lipids; Mendelian Genetics

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