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1.  High-Density Lipoprotein 
Circulation research  2013;113(12):1275-1277.
PMCID: PMC4111252  PMID: 24311613
2.  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.
PMCID: PMC4043301  PMID: 23743223
3.  Adipose Modulation of High-Density Lipoprotein Cholesterol 
Circulation  2011;124(15):1602-1605.
PMCID: PMC3928975  PMID: 21986773
Editorials; high-density lipoprotein cholesterol; lipoproteins; obesity; adipose tissue; Reverse Cholesterol Transport
4.  Apolipoprotein A-I and cholesterol efflux: The good, the bad, and the modified 
Circulation research  2014;114(11):1681-1683.
PMCID: PMC4104775  PMID: 24855198
5.  Targeting High Density Lipoproteins in the Prevention of Cardiovascular Disease? 
Current cardiology reports  2012;14(6):684-691.
Recent studies involving HDL-raising therapeutics have greatly changed our understanding of this field. Despite effectively raising HDL-C levels, niacin remains of uncertain clinical benefit. Synthetic niacin receptor agonists are unlikely to raise HDL-C or have other beneficial effects on plasma lipids. Despite the failure in phase 3 of two CETP inhibitors, two potent CETP inhibitors that raise HDL-C levels by > 100% (and reduce LDL-C substantially) are in late stage clinical development. Infusions of recombinant HDL containing ‘wild-type’ apoA-I or apoA-I Milano, as well as autologous delipidated HDL, all demonstrated promising early results and remain in clinical development. A small molecule that causes upregulation of endogenous apoA-I production is also in clinical development. Finally, upregulation of macrophage cholesterol efflux pathways through agonism of liver X receptors or antagonism of miR-33 remains of substantial interest. The field of HDL therapeutics is poised to transition from the ‘HDL-cholesterol hypothesis’ to the ‘HDL flux hypothesis’ in which the impact on flux from macrophage to feces is deemed to be of greater therapeutic benefit than the increase in steady-state concentrations of HDL cholesterol.
PMCID: PMC3517174  PMID: 22991041
Lipids; HDL; reverse cholesterol transport; niacin; GPR109A; cholesteryl ester transfer protein; CETP; anacetrapib; torcetrapib; dalcetrapib; evacetrapib; apoA-I; recombinant HDL; RVX-208; Liver X receptor; miR-33; cardiovascular disease
6.  Differential Association of Plasma Angiopoietin-Like Proteins 3 and 4 with Lipid and Metabolic Traits 
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.
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.
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
7.  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.
PMCID: PMC4409287  PMID: 25652088
ATP-binding cassette transporter A1; atherosclerosis; macrophages; cholesterol efflux; ATP-binding cassette transporter G1; scavenger receptor class B type I
8.  Exome Sequencing in Suspected Monogenic Dyslipidemias 
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.
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.
PMCID: PMC4406825  PMID: 25632026
genetics; human; DNA sequencing; Exome sequencing; lipids; Mendelian Genetics
9.  Genetic manipulation of the ApoF/Stat2 locus supports an important role for Type I Interferon signaling in atherosclerosis 
Atherosclerosis  2014;233(1):234-241.
Apolipoprotein F (ApoF) is a sialoglycoprotein that is a component of the HDL and LDL fractions of human serum. We sought to test the hypothesis that ApoF plays an important role in atherosclerosis in mice by modulating lipoprotein function. Atherosclerosis was assessed in male low density lipoprotein receptor knockout (LDLR KO) and ApoF/LDLR double knockout (DKO) mice fed a Western diet for 16 weeks. ApoF/LDLR DKO mice showed a 39% reduction in lesional area by en face analysis of aortas (p<0.05), despite no significant differences in plasma lipid parameters. ApoF KO mice had reduced expression of Interferon alpha (IFNα) responsive genes in liver and spleen, as well as impaired macrophage activation. Interferon alpha induced gene 27 like 2a (Ifi27l2a), Oligoadenylate synthetases 2 and 3 (Oas2 and Oas3) were significantly reduced in the ApoF KO mice relative to wild type controls. These effects were attributable to hypomorphic expression of Stat2 in the ApoF KO mice, a critical gene in the Type I IFN pathway that is situated just 425 base pairs downstream of ApoF. These studies implicate STAT2 as a potentially important player in atherosclerosis, and support the growing evidence that the Type I IFN pathway may contribute to this complex disease.
PMCID: PMC3936605  PMID: 24529150
ApoF; STAT2; Atherosclerosis; Type I IFN
10.  Systematic Cell-Based Phenotyping of Missense Alleles Empowers Rare Variant Association Studies: A Case for LDLR and Myocardial Infarction 
PLoS Genetics  2015;11(2):e1004855.
A fundamental challenge to contemporary genetics is to distinguish rare missense alleles that disrupt protein functions from the majority of alleles neutral on protein activities. High-throughput experimental tools to securely discriminate between disruptive and non-disruptive missense alleles are currently missing. Here we establish a scalable cell-based strategy to profile the biological effects and likely disease relevance of rare missense variants in vitro. We apply this strategy to systematically characterize missense alleles in the low-density lipoprotein receptor (LDLR) gene identified through exome sequencing of 3,235 individuals and exome-chip profiling of 39,186 individuals. Our strategy reliably identifies disruptive missense alleles, and disruptive-allele carriers have higher plasma LDL-cholesterol (LDL-C). Importantly, considering experimental data refined the risk of rare LDLR allele carriers from 4.5- to 25.3-fold for high LDL-C, and from 2.1- to 20-fold for early-onset myocardial infarction. Our study generates proof-of-concept that systematic functional variant profiling may empower rare variant-association studies by orders of magnitude.
Author Summary
Exome sequencing has proven powerful to identify protein-coding variation across the human genome, unravel the basis of monogenic diseases and discover rare alleles that confer risk for complex disease. Nevertheless, two key challenges limit its application to complex phenotypes: first, most alleles identified in a population are extremely rare; and second, most alleles are neutral on protein activities. Consequently, association tests that rely on enumerating rare alleles in cases and controls (termed rare variant association studies, RVAS) are typically underpowered, as the many neutral alleles dampen signals that arise from the few alleles that disrupt protein functions. Strategies to securely discriminate disruptive from neutral variants are immature, in particular for missense variants. Here we show that the statistical power of RVAS improves dramatically if variants are stratified according to their in vitro ascertained functions. We establish scalable technology to objectively profile the biological effects of exome-identified missense variants in the low-density lipoprotein receptor (LDLR) through systematic overexpression and complementation experiments in cells. We demonstrate that carriers of LDLR alleles, which our experiments identify as “disruptive-missense”, have higher plasma LDL-C, and that considering in vitro data may make it possible to reduce RVAS sample sizes by more than 2-fold.
PMCID: PMC4409815  PMID: 25647241
11.  Genetic Basis of Atherosclerosis: Insights from Mice and Humans 
Circulation Research  2012;110(2):337-355.
Atherosclerosis is a complex and heritable disease involving multiple cell types and the interactions of many different molecular pathways. The genetic and molecular mechanisms of atherosclerosis have in part been elucidated by mouse models; at least 100 different genes have been shown to influence atherosclerosis in mice. Importantly, unbiased genome-wide association studies have recently identified a number of novel loci robustly associated with atherosclerotic coronary artery disease (CAD). Here we review the genetic data elucidated from mouse models of atherosclerosis, as well as significant associations for human CAD. Furthermore, we discuss in greater detail some of these novel human CAD loci. The combination of mouse and human genetics has the potential to identify and validate novel genes that influence atherosclerosis, some of which may be candidates for new therapeutic approaches.
PMCID: PMC3357004  PMID: 22267839
CAD; Lipids; Mice; GWAS; Genome-wide
12.  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.
PMCID: PMC3248309  PMID: 22201674
13.  CPAP, Weight Loss, or Both for Obstructive Sleep Apnea 
The New England journal of medicine  2014;370(24):2265-2275.
Obesity and obstructive sleep apnea tend to coexist and are associated with inflammation, insulin resistance, dyslipidemia, and high blood pressure, but their causal relation to these abnormalities is unclear.
We randomly assigned 181 patients with obesity, moderate-to-severe obstructive sleep apnea, and serum levels of C-reactive protein (CRP) greater than 1.0 mg per liter to receive treatment with continuous positive airway pressure (CPAP), a weight-loss intervention, or CPAP plus a weight-loss intervention for 24 weeks. We assessed the incremental effect of the combined interventions over each one alone on the CRP level (the primary end point), insulin sensitivity, lipid levels, and blood pressure.
Among the 146 participants for whom there were follow-up data, those assigned to weight loss only and those assigned to the combined interventions had reductions in CRP levels, insulin resistance, and serum triglyceride levels. None of these changes were observed in the group receiving CPAP alone. Blood pressure was reduced in all three groups. No significant incremental effect on CRP levels was found for the combined interventions as compared with either weight loss or CPAP alone. Reductions in insulin resistance and serum triglyceride levels were greater in the combined-intervention group than in the group receiving CPAP only, but there were no significant differences in these values between the combined-intervention group and the weight-loss group. In per-protocol analyses, which included 90 participants who met prespecified criteria for adherence, the combined interventions resulted in a larger reduction in systolic blood pressure and mean arterial pressure than did either CPAP or weight loss alone.
In adults with obesity and obstructive sleep apnea, CPAP combined with a weight-loss intervention did not reduce CRP levels more than either intervention alone. In secondary analyses, weight loss provided an incremental reduction in insulin resistance and serum triglyceride levels when combined with CPAP. In addition, adherence to a regimen of weight loss and CPAP may result in incremental reductions in blood pressure as compared with either intervention alone.
PMCID: PMC4138510  PMID: 24918371
14.  Biodistribution of AAV8 Vectors Expressing Human Low-Density Lipoprotein Receptor in a Mouse Model of Homozygous Familial Hypercholesterolemia 
Recombinant adeno-associated viral vectors based on serotype 8 (AAV8) transduce liver with superior tropism following intravenous (IV) administration. Previous studies conducted by our lab demonstrated that AAV8-mediated transfer of the human low-density lipoprotein receptor (LDLR) gene driven by a strong liver-specific promoter (thyroxin-binding globulin [TBG]) leads to high level and persistent gene expression in the liver. The approach proved efficacious in reducing plasma cholesterol levels and resulted in the regression of atherosclerotic lesions in a murine model of homozygous familial hypercholesterolemia (hoFH). Prior to advancing this vector, called AAV8.TBG.hLDLR, to the clinic, we set out to investigate vector biodistribution in an hoFH mouse model following IV vector administration to assess the safety profile of this investigational agent. Although AAV genomes were present in all organs at all time points tested (up to 180 days), vector genomes were sequestered mainly in the liver, which contained levels of vector 3 logs higher than that found in other organs. In both sexes, the level of AAV genomes gradually declined and appeared to stabilize 90 days post vector administration in most organs although vector genomes remained high in liver. Vector loads in the circulating blood were high and close to those in liver at the early time point (day 3) but rapidly decreased to a level close to the limit of quantification of the assay. The results of this vector biodistribution study further support a proposed clinical trial to evaluate AAV8 gene therapy for hoFH patients.
PMCID: PMC4003465  PMID: 24070336
15.  High-Density Lipoprotein Hydrolysis by Endothelial Lipase Activates PPARα 
Circulation research  2006;98(4):490-498.
Although high-density lipoprotein (HDL) is known to inhibit endothelial adhesion molecule expression, the mechanism for this anti-inflammatory effect remains obscure. Surprisingly, we observed that HDL no longer decreased adhesion of U937 monocytoid cells to tumor necrosis factor (TNF)α-stimulated human endothelial cells (EC) in the presence of the general lipase inhibitor tetrahydrolipstatin. In considering endothelial mechanisms responsible for this effect, we found that endothelial lipase (EL) overexpression in both EC and non-EL–expressing NIH/3T3 mouse embryonic fibroblasts cells significantly decreased TNFα-induced VCAM1 expression and promoter activity in a manner dependent on HDL concentration and intact EL activity. Given recent evidence for lipolytic activation of peroxisome proliferator-activated receptors (PPARs)—nuclear receptors implicated in metabolism, atherosclerosis, and inflammation—we hypothesized HDL hydrolysis by EL is an endogenous endothelial mechanism for PPAR activation. In both EL-transfected NIH cells and bovine EC, HDL significantly increased PPAR ligand binding domain activation in the order PPAR-α≫-γ>-δ. Moreover, HDL stimulation induced expression of the canonical PPARα-target gene acyl-CoA-oxidase (ACO) in a PPARα-dependent manner in ECs. Conditioned media from EL-adenovirus transfected cells but not control media exposed to HDL also activated PPARα. PPARα activation by EL was most potent with HDL as a substrate, with lesser effects on LDL and VLDL. Finally, HDL inhibited leukocyte adhesion to TNFα-stimulated ECs isolated from wild-type but not PPARα-deficient mice. This data establishes HDL hydrolysis by EL as a novel, distinct natural pathway for PPARα activation and identifies a potential mechanism for HDL-mediated repression of VCAM1 expression, with significant implications for both EL and PPARs in inflammation and vascular biology.
PMCID: PMC4231787  PMID: 16439686
adhesion molecules; endothelial cells; HDL cholesterol; high-density lipoproteins; lipase; PPARs; transcriptional regulation
16.  Hepatic sortilin regulates both apolipoprotein B secretion and LDL catabolism 
The Journal of Clinical Investigation  2012;122(8):2807-2816.
Genome-wide association studies (GWAS) have identified a genetic variant at a locus on chromosome 1p13 that is associated with reduced risk of myocardial infarction, reduced plasma levels of LDL cholesterol (LDL-C), and markedly increased expression of the gene sortilin-1 (SORT1) in liver. Sortilin is a lysosomal sorting protein that binds ligands both in the Golgi apparatus and at the plasma membrane and traffics them to the lysosome. We previously reported that increased hepatic sortilin expression in mice reduced plasma LDL-C levels. Here we show that increased hepatic sortilin not only reduced hepatic apolipoprotein B (APOB) secretion, but also increased LDL catabolism, and that both effects were dependent on intact lysosomal targeting. Loss-of-function studies demonstrated that sortilin serves as a bona fide receptor for LDL in vivo in mice. Our data are consistent with a model in which increased hepatic sortilin binds intracellular APOB-containing particles in the Golgi apparatus as well as extracellular LDL at the plasma membrane and traffics them to the lysosome for degradation. We thus provide functional evidence that genetically increased hepatic sortilin expression both reduces hepatic APOB secretion and increases LDL catabolism, providing dual mechanisms for the very strong association between increased hepatic sortilin expression and reduced plasma LDL-C levels in humans.
PMCID: PMC3408750  PMID: 22751103
17.  Diabetes Reduces Bone Marrow and Circulating Porcine Endothelial Progenitor Cells, an Effect Ameliorated by Atorvastatin and Independent of Cholesterol 
Bone marrow derived endothelial progenitor cells (EPCs) are early precursors of mature endothelial cells which replenish aging and damaged endothelial cells. The authors studied a diabetic swine model to determine if induction of DM adversely affects either bone marrow or circulating EPCs and whether a HMG-CoA reductase inhibitor (statin) improves development and recruitment of EPCs in the absence of cholesterol lowering. Streptozotocin was administered to Yorkshire pigs to induce DM. One month after induction, diabetic pigs were treated with atorvastatin (statin, n = 10), ezetimibe (n = 10) or untreated (n = 10) and evaluated for number of bone marrow and circulating EPCs and femoral artery endothelial function. There was no effect of either medication on cholesterol level. One month after induction of DM prior to administration of drugs, the number of bone marrow and circulating EPCs significantly decreased (P < 0.0001) compared to baseline. Three months after DM induction, the mean proportion of circulating EPCs significantly increased in the atorvastatin group, but not in the control or ezetimibe groups. The control group showed progressive reduction in percentage of flow mediated vasodilatation (no dilatation at 3 months) whereas the atorvastatin group and ezetimibe exhibited vasodilatation, 6% and 4% respectively. DM results in significant impairment of bone marrow and circulating EPCs as well as endothelial function. The effect is ameliorated, in part, by atorvastatin independent of its cholesterol lowering effect. These data suggest a model wherein accelerated atherosclerosis seen with DM may, in part, result from reduction in EPCs which may be ameliorated by treatment with a statin.
PMCID: PMC4221094  PMID: 19051240
diabetes mellitus; endothelium; endothelial progenitor cells; statin; inflammation; cytometry
18.  Monogenic causes of elevated HDL cholesterol and implications for development of new therapeutics 
Clinical lipidology  2013;8(6):635-648.
Identification of the CETP, LIPG (encoding endothelial lipase) and APOC3 genes, and ana lysis of rare genetic variants in them, have allowed researchers to increase understanding of HDL metabolism significantly. However, development of cardiovascular risk-reducing therapeutics targeting the proteins encoded by these genes has been less straightforward. The failure of two CETP inhibitors is complex but illustrates a possible over-reliance on HDL cholesterol as a marker of therapeutic efficacy. The case of endothelial lipase exemplifies the importance of utilizing population-wide genetic studies of rare variants in potential therapeutic targets to gain information on cardiovascular disease end points. Similar population-wide studies of cardiovascular end points make apoC-III a potentially attractive target for lipid-related drug discovery. These three cases illustrate the positives and negatives of single-gene studies relating to HDL-related cardiovascular drug discovery; such studies should focus not only on HDL cholesterol and other components of the lipid profile, but also on the effect genetic variants have on cardiovascular end points.
PMCID: PMC4217288  PMID: 25374625
anacetrapib; apoC-III; CETP; cholesterol; dalcetrapib; endothelial lipase; evacetrapib; genetics; HDL; ISI-APOCIIIRX; torcetrapib
19.  Anti-oxidative and cholesterol efflux capacities of high-density lipoprotein are reduced in ischaemic cardiomyopathy 
European Journal of Heart Failure  2013;15(11):1215-1219.
Various pathological changes lead to the development of heart failure (HF). HDL is dysfunctional in both acute coronary syndrome, as measured by the HDL inflammatory index (HII) assay, and stable coronary disease, as measured by cholesterol efflux capacity. We therefore hypothesized that these functions of HDL are also impaired in subjects with ischaemic cardiomyopathy.
Methods and results
A case–control study was performed on subjects in the University of Pennsylvania Catheterization Study (PennCath) cohort of patients with angina. Cases had EF <50% and angiographic CAD (≥70% stenosis of any vessel; n = 23); controls included those with EF ≥55% and no CAD (n = 46). Serum from subjects was apolipoprotein-B depleted to isolate an HDL fraction. To measure HDL anti-oxidative capacity, the HDL fraction was incubated with LDL and a reporter lipid that fluoresces when oxidized. To measure cholesterol efflux capacity, the HDL fraction was also incubated with macrophages and tritium-labelled cholesterol. Mean HII was higher and efflux capacity lower in subjects with ischaemic cardiomyopathy (HII 0.26 vs. –0.028; efflux 0.80 vs. 0.92; P < 0.05). In a multivariable logistic regression model, both high HII and low efflux capacity were significant risk factors for HF [HII odds ratio (OR) 2.8, 95% confidence interval (CI) 2.0–3.9, P = 0.002; efflux OR 2.1, 95% CI 1.5–3.0, P = 0.03]. These effects persisted after adjustment for covariates and traditional risk factors for HF.
Subjects with reduced EF from ischaemia have lower HDL concentration and also impaired HDL function. HDL is a versatile lipoprotein particle with various anti-inflammatory and vasoprotective functions, whose impairment may contribute to ischaemic heart failure.
PMCID: PMC3888304  PMID: 23709232
Heart failure; Coronary artery disease; HDL cholesterol
20.  PPARα activation promotes macrophage reverse cholesterol transport through an LXR-dependent pathway 
Peroxisome proliferator-activate receptorα (PPARα) activation has been shown in vitro to increase macrophage cholesterol efflux, the initial step in reverse cholesterol transport (RCT). However, it remains unclear whether PPARα activation promotes macrophage RCT in vivo.
Methods and Results
We demonstrated that a specific potent PPARα agonist GW7647 inhibited atherosclerosis and promoted macrophage RCT in hypercholesterolemic mice expressing the human apoA-I gene. We compared the effect of GW7647 on RCT in human apoA-I transgenic (hA-ITg) mice with wild-type (WT) mice and showed that the PPARα agonist promoted RCT in hA-ITg mice to a much greater extent than in WT mice, indicating that human apoA-I expression is important for PPARα-induced RCT. We further investigated the dependence of the macrophage PPARα-LXR pathway on the promotion of RCT by GW7647. Primary murine macrophages lacking PPARα or LXR abolished the ability of GW7647 to promote RCT in hA-ITg mice. In concert, the PPARα agonist promoted cholesterol efflux and ABCA1/ABCG1 expression in primary macrophages and this was also by the PPARα-LXR pathway.
Our observations demonstrate that a potent PPARα agonist promotes macrophage RCT in vivo in a manner that is enhanced by human apoA-I expression and dependent on both macrophage PPARα and LXR expression.
PMCID: PMC3202300  PMID: 21441141
PPARα; LXR; cholesterol efflux; reverse cholesterol transport; apolipoprotein A-I
21.  Modulation of HDL Metabolism by the Niacin Receptor GPR109A in Mouse Hepatocytes 
Biochemical Pharmacology  2010;80(9):1450-1457.
The niacin receptor GPR109A is a Gi-protein coupled receptor which mediates the effects of niacin on inhibiting intracellular triglyceride lipolysis in adipocytes. However, the role of GPR109A in mediating the effects of niacin on high density lipoprotein (HDL) metabolism is unclear. We found niacin has no effect on HDL-C in GPR109A knockout mice. Furthermore, niacin lowered intracellular cAMP in primary hepatocytes mediated by GPR109A. We used an adeno-associated viral (AAV) serotype 8 vector encoding GPR109A under the control of the hepatic-specific thyroxine-binding globulin promoter to specifically overexpress GPR109A in mouse liver. Plasma HDL-C, hepatic ABCA1 and the HDL cholesterol production rate were significantly reduced in mice overexpressing GPR109A. Overexpression of GPR109A reduced primary hepatocyte free cholesterol efflux to apoA-I; conversely, GPR109A deficient hepatocytes had increased ABCA1-mediated cholesterol efflux. These data support the concept that the HDL-C lowering effect of niacin in wild-type mice is mediated through stimulation of GPR109A in hepatocytes; such an effect then leads to reduced hepatocyte ABCA1 expression and activity, decreased cholesterol efflux to nascent apoA-I, and reduced HDL-C levels. These results indicate that niacin-mediated activation of GP109A in liver lowers ABCA1 expression leading to reduced hepatic cholesterol efflux to HDL.
PMCID: PMC3328801  PMID: 20655299
niacin; GPR109A; HDL; ABCA1; AAV
22.  Discovery and validation of new molecular targets in treating dyslipidemia: The role of human genetics 
Trends in Cardiovascular Medicine  2009;19(6):195-201.
Several high profile failures of lipid-related therapeutics in clinical trials have led to intense interest in improved discovery and preclinical prioritization of potential targets. The careful study of patients with rare monogenic disorders has played a key role in establishing the causal role of cholesterol in atherosclerosis and highlighting viable drug targets. Systematic efforts to extend the association of common variants linked with lipid levels to coronary disease allow assessment of the vascular consequences of lifelong differences in lipids due to variation in specific molecules. This application of genetic epidemiology, termed Mendelian randomization, may prove useful in informing ongoing drug development efforts.
PMCID: PMC3328807  PMID: 20211435
23.  Pharmacologic Suppression of Hepatic ATP-Binding Cassette Transporter 1 Activity in Mice Reduces High-Density Lipoprotein Cholesterol Levels but Promotes Reverse Cholesterol Transport 
Circulation  2011;124(12):1382-1390.
The role of hepatic ATP-binding cassette transporter 1 (ABCA1) in maintaining plasma high density lipoprotein cholesterol (HDL-C) levels is well established, but its role in reverse cholesterol transport (RCT) is unclear. Probucol is a compound that reduces HDL-C levels but also reduces atherosclerosis in animal models and xanthomas in humans. The aim of the present study was to test the hypothesis that probucol inhibits hepatic ABCA1 activity, thereby reducing HDL-C levels but promoting RCT from macrophages.
Methods and Results
Wild-type (WT) C57BL/6 mice and scavenger receptor class B type I (SR-BI) knockout mice were fed a chow diet containing 0.5% probucol or normal chow for 2 weeks. In WT mice, probucol, despite decreasing HDL-C by >80%, effectively maintained macrophage RCT. In SR-BI knockout mice, probucol also substantially reduced HDL-C but significantly increased macrophage RCT. Furthermore, probucol significantly enhanced the excretion of HDL-derived cholesterol into feces in both WT and SR-BI knockout mice. Probucol inhibited ABCA1-dependent cholesterol efflux from mouse primary hepatocytes, and this effect was shown to be responsible for the effect of probucol on increasing the fecal excretion of HDL-derived cholesterol in vivo.
We demonstrate that pharmacological inhibition of hepatic ABCA1 activity with probucol reduced HDL-C levels but promoted RCT through diversion of HDL-derived cholesterol from efflux back into plasma instead to excretion in the bile. These results explain the beneficial effects of probucol on atherosclerosis and xanthomas despite its HDL-lowering effects and suggest that inactivation of hepatic ABCA1 leads to increased RCT despite reducing plasma HDL-C levels.
PMCID: PMC3323112  PMID: 21859969
atherosclerosis; cholesterol; HDL; lipids; lipoproteins; pharmacology
24.  Dense Genotyping of Candidate Gene Loci Identifies Variants Associated with High-Density Lipoprotein Cholesterol 
Plasma levels of high density lipoprotein cholesterol (HDL-C) are known to be heritable, but only a fraction of the heritability is explained. We used a high density genotyping array containing SNPs from HDL-C candidate genes selected on known biology of HDL-C metabolism, mouse genetic studies, and human genetic association studies. SNP selection was based on tagging-SNPs but also included low-frequency nonsynonymous SNPs.
Methods and Results
Association analysis in a cohort containing extremes of HDL-C (case-control, n=1733) provided a discovery phase, with replication in three additional populations for a total meta-analysis in 7,857 individuals. We replicated the majority of loci identified through genome wide association studies and present on the array (including ABCA1, APOA1/C3/A4/A5, APOB, APOE/C1/C2, CETP, CTCF-PRMT8, FADS1/2/3, GALNT2, LCAT, LILRA3, LIPC, LIPG, LPL, LRP4, SCARB1, TRIB1, ZNF664), and provide evidence suggestive of association in several previously unreported candidate gene loci (including ABCG1, GPR109A/B/81, NFKB1, PON1/2/3/4). There was evidence for multiple, independent association signals in five loci, including association with low frequency nonsynonymous variants.
Genetic loci associated with HDL-C are likely to harbor multiple, independent causative variants, frequently with opposite effects on the HDL-C phenotype. Cohorts composed of extreme individuals may be efficiently used in a case-control discovery of quantitative traits.
PMCID: PMC3319351  PMID: 21303902
lipids; genetic association; HDL cholesterol; cardiovascular diseases
25.  Beyond High-Density Lipoprotein Cholesterol Levels 
A number of therapeutic strategies targeting high-density lipoprotein (HDL) cholesterol and reverse cholesterol transport are being developed to halt the progression of atherosclerosis or even induce regression. However, circulating HDL cholesterol levels alone represent an inadequate measure of therapeutic efficacy. Evaluation of the potential effects of HDL-targeted interventions on atherosclerosis requires reliable assays of HDL function and surrogate markers of efficacy. Promotion of macrophage cholesterol efflux and reverse cholesterol transport is thought to be one of the most important mechanisms by which HDL protects against atherosclerosis, and methods to assess this pathway in vivo are being developed. Indexes of monocyte chemotaxis, endothelial inflammation, oxidation, nitric oxide production, and thrombosis reveal other dimensions of HDL functionality. Robust, reproducible assays that can be performed widely are needed to move this field forward and permit effective assessment of the therapeutic potential of HDL-targeted therapies.
PMCID: PMC3315104  PMID: 18534265

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