Increased Plasma High-density Lipoprotein Cholesterol Research Articles

Top Advances in Functional Genomics and Translational Biology for 2010

In recent years, major advances have been made in transcriptomics, metabolomics, proteomics, and particularly population genetics. More than 850 genome-wide association studies (GWAS) were published by the end of 2010, now covering a vast number of diseases and phenotypes and often incorporating tens of thousands of subjects or, in some cases, more than 100 000 subjects, increasing statistical power to identify and replicate contributing loci. Sequencing throughput advances and decreasing costs per sample have allowed the application of genome-wide genotyping and sequencing technology to larger DNA samples as well as transcriptome and epigenome features. This year was marked not only by continuing progress with GWAS, sequencing, and copy number variation studies but also by follow-up of GWAS and other findings with functional studies that uncover new molecular pathways contributing to disease and pave the way for future clinical applications. The American Heart Association Functional Genomics and Translational Biology Interdisciplinary Council (http://www.americanheart.org/presenter.jhtml?identifier=3016539) provides a forum for a multidisciplinary group of volunteers committed to making a substantial contribution to reduce the burden of heart disease and stroke through genetics, genomics, proteomics, and metabolomics research and translational science. With input from the Early Career Committee of the Council on Functional Genomics and Translational Biology, we considered many papers and selected 10 outstanding manuscript groups (15 articles) published in 2010 that reflect these advances, and we briefly summarize each below in chronological order.

Knockdown of the gene encoding Drosophila tribbles homologue 3 (Trib3) improves insulin sensitivity through peroxisome proliferator-activated receptor-γ (PPAR-γ) activation in a rat model of insulin resistance

Insulin action is purportedly modulated by Drosophila tribbles homologue 3 (TRIB3), which in vitro prevents thymoma viral proto-oncogene (AKT) and peroxisome proliferator-activated receptor-γ (PPAR-γ) activation. However, the physiological impact of TRIB3 action in vivo remains controversial. We investigated the role of TRIB3 in rats treated with either a control or Trib3 antisense oligonucleotide (ASO). Tissue-specific insulin sensitivity was assessed in vivo using a euglycaemic-hyperinsulinaemic clamp. A separate group was treated with the PPAR-γ antagonist bisphenol-A-diglycidyl ether (BADGE) to assess the role of PPAR-γ in mediating the response to Trib3 ASO. Trib3 ASO treatment specifically reduced Trib3 expression by 70% to 80% in liver and white adipose tissue. Fasting plasma glucose, insulin concentrations and basal rate of endogenous glucose production were unchanged. However, Trib3 ASO increased insulin-stimulated whole-body glucose uptake by ~50% during the euglycaemic-hyperinsulinaemic clamp. This was attributable to improved skeletal muscle glucose uptake. Despite the reduction of Trib3 expression, AKT2 activity was not increased. Trib3 ASO increased white adipose tissue mass by 70% and expression of Ppar-γ and its key target genes, raising the possibility that Trib3 ASO improves insulin sensitivity primarily in a PPAR-γ-dependent manner. Co-treatment with BADGE blunted the expansion of white adipose tissue and abrogated the insulin-sensitising effects of Trib3 ASO. Finally, Trib3 ASO also increased plasma HDL-cholesterol, a change that persisted with BADGE co-treatment. These data suggest that TRIB3 inhibition improves insulin sensitivity in vivo primarily in a PPAR-γ-dependent manner and without any change in AKT2 activity.

Differential effects of gemfibrozil and fenofibrate on reverse cholesterol transport from macrophages to feces in vivo

Gemfibrozil and fenofibrate, two of the fibrates most used in clinical practice, raise HDL cholesterol (HDLc) and are thought to reduce the risk of atherosclerotic cardiovascular disease. These drugs act as PPARα agonists and upregulate the expression of genes crucial in reverse cholesterol transport (RCT). In the present study, we determined the effects of these two fibrates on RCT from macrophages to feces in vivo in human apoA-I transgenic (hApoA-ITg) mice. [3H]cholesterol-labeled mouse macrophages were injected intraperitoneally into hApoA-ITg mice treated with intragastric doses of fenofibrate, gemfibrozil or a vehicle solution for 17days, and radioactivity was determined in plasma, liver and feces. Fenofibrate, but not gemfibrozil, enhanced [3H]cholesterol flux to plasma and feces of female hApoA-ITg mice. Fenofibrate significantly increased plasma HDLc, HDL phospholipids, hApoA-I levels and phospholipid transfer protein activity, whereas these parameters were not altered by gemfibrozil treatment. Unlike gemfibrozil, fenofibrate also induced the generation of larger HDL particles, which were more enriched in cholesteryl esters, together with higher potential to generate preβ-HDL formation and caused a significant increase in [3H]cholesterol efflux to plasma. Our findings demonstrate that fenofibrate promotes RCT from macrophages to feces in vivo and, thus, highlight a differential action of this fibrate on HDL.

Liver X Receptor Agonist Methyl-3β-Hydroxy-5α,6α-Epoxycholanate Attenuates Atherosclerosis in Apolipoprotein E Knockout Mice without Increasing Plasma Triglyceride

Background: Liver X receptors (LXRs) promote macrophage reverse cholesterol transport and cholesterol excretion from the body. The synthetic LXR ligands T0901317 and GW3965 were shown to significantly inhibit atherosclerosis in mice and to increase the expression of ATP-binding cassette transporter A1 (ABCA1) in the atherosclerotic lesions. However, these compounds increase plasma and hepatic triglyceride (TG) levels in mice. Methyl-3β-hydroxy-5α,6α-epoxycholanate (MHEC), synthesized from hyodeoxycholic acid, functions as an LXR agonist, but its role in atherogenesis and lipid metabolism remained to be elucidated. Methods: THP-1-derived macrophages were cultured in the medium con- taining various concentrations of MHEC or T0901317 (0–10 µmol/l) for 24 h. Reverse transcription polymerase chain reaction was used to quantify LXRα, LXRβ and ABCA1 mRNA levels in macrophages. Additionally, MHEC or T0901317 was orally administered at 10 mg/kg daily for 6 weeks in apolipoprotein E knockout (apoE^–/–) mice fed a high-cholesterol diet. Plasma lipids were determined enzymatically. The area of and ABCA1 expression in the aortic atherosclerotic lesions were measured by oil red O staining and immunohistochemistry, respectively. Results: Both MHEC and T0901317 equally stimulated LXRα and ABCA1 mRNA expression in a dose-dependent manner in THP-1-derived macrophages, but they did not induce LXRβ mRNA expression significantly. The plasma levels of total cholesterol, TG and high-density lipoprotein cholesterol were significantly higher in T0901317-treated mice than in the vehicle-treated control group. Interestingly, MHEC treatment dramatically increased plasma high-density lipoprotein cholesterol without altering plasma levels of total cholesterol and TG. Both MHEC and T0901317 equally inhibited the development of atherosclerotic lesions in apoE^–/– mice. The expression of ABCA1, a cholesterol efflux transporter, was greatly induced by the two LXR agonists in the artery wall. Conclusions: MHEC is a novel LXR agonist and it inhibits atherosclerosis in apoE^–/– mice without raising blood TG. Thus, MHEC relative to T0901317 may be a better therapeutic LXR agonist for the treatment of atherosclerosis.

Deletion of the High-Density Lipoprotein Receptor Scavenger Receptor BI in Mice Modulates Thrombosis Susceptibility and Indirectly Affects Platelet Function by Elevation of Plasma Free Cholesterol

Scavenger receptor BI (SR-BI) is a cell surface receptor that promotes the selective uptake of cholesteryl esters from high-density lipoprotein (HDL) by the liver. In mice, SR-BI deficiency results in increased plasma HDL cholesterol levels and enhanced susceptibility to atherosclerosis. The aim of this study was to investigate the role of SR-BI deficiency on platelet function. SR-BI-deficient mice were thrombocytopenic, and their platelets were abnormally large, probably because of an increased cholesterol content. The FeCl(3) acute injury model to study arterial thrombosis susceptibility showed that SR-BI wild-type mice developed total arterial occlusion after 24±2 minutes. In SR-BI-deficient mice, however, the time to occlusion was reduced to 13±1 minutes (P=0.02). Correspondingly, in SR-BI-deficient mice, platelets circulated in an activated state and showed increased adherence to immobilized fibrinogen. In contrast, platelet-specific disruption of SR-BI by bone marrow transplantation in wild-type mice did not alter plasma cholesterol levels or affect platelet count, size, cholesterol content, or reactivity, suggesting that changes in plasma cholesterol levels were responsible for the altered responsiveness of platelets in SR-BI-deficient mice. The function of SR-BI in HDL cholesterol homeostasis and prevention of atherosclerosis is indirectly also essential for maintaining normal platelet function and prevention of thrombosis.

Pioglitazone increases apolipoprotein A-I production by directly enhancing PPRE-dependent transcription in HepG2 cells

Pioglitazone, a hypoglycemic agent, has been shown to increase plasma HDL cholesterol, but the mechanism is incompletely understood. We further investigated effects of pioglitazone on transcriptional regulation of apolipoprotein (apo)A-I gene and functional properties of pioglitazone-induced apoA-I-containing particles. Pioglitazone dose-dependently stimulated apoA-I promoter activities in HepG2 cells. A peroxisome proliferator-activated receptor (PPAR)-response element located in site A (-214 to -192 bp, upstream of the transcription start site) of the promoter is required for pioglitazone-induced apoA-I gene transcription. Deletion of site A (-214 to -192 bp), B (-169 to -146 bp), or C (-134 to -119 bp), which clusters a number of cis-acting elements for binding of different transcription factors, reduced the basal apoA-I promoter activities, and no additional pioglitazone-sensitive elements were found within this region. Overexpression or knock-down of liver receptor homolog-1, a newly identified nuclear factor with strong stimulatory effect on apoA-I transcription, did not alter pioglitazone-induced apoA-I transcription. Pioglitazone-induced apoA-I transcription is mainly mediated through PPARalpha but not PPARgamma in hepatocytes. Pioglitazone induced production of HDL enriched in its subfraction containing apoA-I without apoA-II, which inhibited monocyte adhesion to endothelial cells in vitro. In conclusion, pioglitazone increases apoA-I production by directly enhancing PPAR-response element-dependent transcription, resulting in generation of apoA-I-containing HDL particles with increased anti-inflammatory property.

CAMP Responsive Element Binding Protein 1

Life scientists, especially those of us from a previous generation (or two), were trained to focus our efforts on generating large mean differences between groups or large changes with interventions while minimizing variability. The goal was to maximize effect size, thereby optimizing the chance of generating a statistically significant difference or change that, in turn, increased the odds that the data would be publishable. And, yes, even “way” back then, publications were still the “coin of the realm.” Article see p 294 However, investigators from more recent generations, and even some from previous generations, have finally begun to realize that the variability among individuals or their responses to interventions are also important. This realization was quite pointedly brought to my attention in an initial discussion with a geneticist about a possible collaboration. He asked, “How does blood pressure change with exercise training?” I responded, “Our previous reviews indicate that the average change in systolic and diastolic blood pressure with exercise training is …,” at which point he stopped me and asked, “No, what was the variability in the responses?” Not surprisingly, given my training in the previous generations as a life scientist, I did not have an answer. Thus began my path to recognizing the importance of the interindividual variability in responses to exercise training. This variability took on even more importance at that time in light of the relatively recently initiated Human Genome Project and its potential to produce a new era of personalized medicine. That thought-provoking question spurred me to revisit some of my previous data but now with a focus on the interindividual variability for the exercise training-induced responses of the cardiovascular (CV) disease risk factors that I studied throughout my career. Sure enough, interindividual variability in these responses is substantial! Most of us missed this potentially …

Pioglitazone Decreases Plasma Cholesteryl Ester Transfer Protein Mass, Associated With a Decrease in Hepatic Triglyceride Content, in Patients With Type 2 Diabetes

OBJECTIVEThiazolidinediones reduce hepatic steatosis and increase HDL cholesterol levels. In mice with human-like lipoprotein metabolism (APOE*3-Leiden.CETP transgenic mice), a decrease in hepatic triglyceride content is associated with a decrease in plasma cholesteryl ester transfer protein (CETP) mass and an increase in HDL levels. Therefore, the aim of the present study was to assess the effects of pioglitazone on CETP mass in patients with type 2 diabetes.RESEARCH DESIGN AND METHODSWe included 78 men with type 2 diabetes (aged 56.5 ± 0.6 years; HbA1c 7.1 ± 0.1%) who were randomly assigned to treatment with pioglitazone (30 mg/day) or metformin (2000 mg/day) and matching placebo, in addition to glimepiride. At baseline and after 24 weeks of treatment plasma HDL cholesterol levels and CETP mass were measured, and hepatic triglyceride content was assessed by proton magnetic resonance spectroscopy.RESULTSPioglitazone decreased hepatic triglyceride content (5.9 [interquartile range 2.6–17.4] versus 4.1 [1.9–12.3]%, P < 0.05), decreased plasma CETP mass (2.33 ± 0.10 vs. 2.06 ± 0.10 μg/ml, P < 0.05), and increased plasma HDL cholesterol level (1.22 ± 0.05 vs. 1.34 ± 0.05 mmol/l, P < 0.05). Metformin did not significantly change any of these parameters.CONCLUSIONSA decrease in hepatic triglyceride content by pioglitazone is accompanied by a decrease in plasma CETP mass and associated with an increase in HDL cholesterol levels. These results in patients with type 2 diabetes fully confirm recent findings in mice.

Genetic Association of the CCR5 Region With Lipid Levels in At-Risk Cardiovascular Patients

There is mounting evidence to suggest that chemokine receptor 5 (CCR5) plays an important role in the development and progression of atherosclerosis. A naturally occurring variant of the CCR5 gene CCR532, exists at allele frequencies of typically 10% in European populations and results in a nonfunctional CCR5 receptor. The CCR5Delta32 deletion and 26 other variants within the chemokine receptor 2-CCR5-chemokine receptor-like protein 2 (CCRL2) gene cluster spanning 59 kilobases of chromosome 3 were genotyped in 5748 subjects from the Treating to New Targets atorvastatin trial to determine whether genetic associations could be identified with circulating lipid values and cardiovascular disease. Our results demonstrate an association between the CCR5Delta32 deletion and increased plasma high-density lipoprotein cholesterol and decreased plasma triglycerides, both of which are beneficial from a cardiovascular perspective. Three single-nucleotide polymorphisms (rs1154428, rs6808835, and rs6791599) in CCRL2 in linkage disequilibrium (r(2)> or =0.65) with CCR5Delta32 and located up to 45 kilobases distal to it were associated with high-density lipoprotein cholesterol. The high-density lipoprotein cholesterol and triglycerides findings were replicated in an additional set of >6000 individuals from the Incremental Decrease in Endpoints through Aggressive Lipid Lowering atorvastatin trial. Our study provides evidence that a locus within the region of the genome encompassing the CCR5-CCRL2 region is associated with lipid levels and suggests that chemokine activity influences lipid levels in populations with preexisting cardiovascular disease. CLINICAL TRIAL REGISTRATION- clinicaltrials.gov. Identifier: TNT, NCT00327691; IDEAL, NCT00159835.

The Pharmacology and Off-Target Effects of Some Cholesterol Ester Transfer Protein Inhibitors

Inhibitors of cholesterol ester transfer protein (CETP) have the capacity to increase plasma high-density lipoprotein cholesterol to unprecedented levels. Still, hopes that CETP inhibition could reduce atherosclerosis were dented when the clinical development of one such inhibitor, torcetrapib, was halted because of an unexpected finding of increased cardiovascular and noncardiovascular mortality against a background of elevated blood pressure and plasma aldosterone levels. Recently, evidence has accumulated to show that these untoward effects may have been largely attributable to off-target toxicity of the compound, unrelated to the mechanism of CETP inhibition and not shared by other CETP inhibitors. In this review, we explore the rationale for CETP inhibition, compare the pharmacology of the small molecule CETP inhibitors that reached clinical development, and address the evidence relating to off-target adverse effects.

Changes in the atherogenic profile of patients with type 1 Gaucher disease after miglustat therapy

Objective Type 1 Gaucher disease (GD1) is an autosomal recessive lysosomal storage disorder associated with abnormal accumulation of glucocerebrosides. Plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), and high-density lipoprotein cholesterol (HDL-c) are decreased in GD1 patients. The effects of substrate reduction therapy (SRT) with miglustat on plasma lipids and atherogenic factors have not yet been examined. Here, we report plasma atherogenic profile data from GD1 patients undergoing long-term SRT. Methods Plasma was analysed in 26 GD1 patients treated with miglustat for up to 36 months. Ten patients were therapy-naïve and 16 had switched from enzyme replacement therapy (ERT); the interval between stopping ERT and starting SRT was 2–6 weeks. Plasma TC, triglycerides (TG), LDL-c, HDL-c, apolipoproteins (apoA-I, apoB, and Lp[a]), C-reactive protein (CRP) concentrations, and chitotriosidase activity were measured before SRT (baseline) and at 12, 24, and 36 months follow up. Results In therapy-naïve patients, miglustat significantly increased plasma HDL-c and apoA-I, and slightly increased TC; while TG, CRP concentrations, and TC/HDL-c ratios decreased significantly after 24 months. In contrast, there were no changes in HDL-c and apoA-I, or in the TC/HDL-c ratio in switch patients. However, a decrease in CRP was observed after 12 months. LDL-c and apoB were not significantly altered in either patient group. Conclusions Miglustat appears to have beneficial effects on plasma lipid, lipoprotein, and CRP concentrations in therapy-naïve GD1 patients, resulting in an improved atherogenic lipid profile. Further studies are required to determine the effect of miglustat on coronary heart disease risk.

Niacin Promotes Cholesterol Efflux through Stimulation of the PPARγ-LXRα-ABCA1 Pathway in 3T3-L1 Adipocytes

Background: The mechanism by which niacin increases plasma levels of high-density lipoprotein cholesterol (HDL-C) is not clearly understood yet. Adipocytes contain the largest pool of free cholesterol in the body and might play a significant role in cholesterol metabolism. Despite preferential accumulation in adipose tissue, it is not clear whether the actions of niacin on cholesterol efflux from adipocytes contribute to its HDL-raising effect. Methods: Fully differentiated 3T3-L1 adipocytes were incubated in the medium containing various concentrations of niacin (0–1.0 mmol/l) for 24 h. Reverse transcription polymerase chain reaction was used to evaluate peroxisome proliferator-activated receptor-γ (PPARγ), LXRα and ABCA1 mRNA expression in adipocytes. Cholesterol efflux rate was determined by measuring the release of radioactivity from ³H-cholesterolprelabeled cells into medium containing apolipoprotein A-I (ApoA-I). Results: Niacin dose-dependently stimulated PPARγ, LXRα and ABCA1 mRNA expression and promoted ApoA-I-induced cholesterol efflux in adipocytes. Treatment of PPARγ-selective antagonist GW9662 significantly abolished the niacin-induced increase in LXRα and ABCA1 mRNA expression and cholesterol efflux to ApoA-I. Conclusions: Niacin may promote cholesterol efflux from adipocytes to ApoA-I via activation of the PPARγ-LXRα-ABCA1 pathway. To some extent, this effect might help to explain the possible mechanism by which niacin increases plasma HDL-C levels.

Dietary capsanthin, the main carotenoid in paprika (Capsicum annuum), alters plasma high-density lipoprotein-cholesterol levels and hepatic gene expression in rats

The effects of dietary capsanthin, the main carotenoid in paprika (Capsicum annuum), on lipid metabolism were examined. Young male Wistar rats were fed diets containing paprika powder, paprika organic solvent extract, residue of paprika extract, and purified capsanthin. Administration of purified capsanthin for 2 weeks resulted in a significant increase in plasma HDL-cholesterol (P < 0.05) without detectable differences in plasma total cholesterol and TAG concentrations. A statistically significant correlation (r 0.567; P < 0.001) was found between dietary capsanthin concentrations and plasma HDL-cholesterol concentrations. Animals receiving diets containing two different capsanthin concentrations exhibited dose-dependent increases in plasma HDL-cholesterol (r 0.597; P < 0.005). While capsanthin was absent in the liver of animals fed the basal diet, it increased markedly in capsanthin-fed animals (P < 0.001). Quantitative analyses of hepatic mRNA levels revealed that capsanthin administration resulted in up-regulation of mRNA for apoA5 and lecithin cholesterol acyltransferase (LCAT), without significant differences in other mRNA levels related to HDL-cholesterol metabolism. These results suggest that capsanthin had an HDL-cholesterol-raising effect on plasma, and the potential to increase cholesterol efflux to HDL particles by increasing apoA5 levels and/or enhancement of LCAT activity.

Lecithin: Cholesterol Acyltransferase Expression Has Minimal Effects on Macrophage Reverse Cholesterol Transport In Vivo

Lecithin:cholesterol acyltransferase (LCAT) catalyzes the formation of plasma cholesteryl ester, plays a key role in high-density lipoprotein metabolism, and has been believed to be critical in the process of reverse cholesterol transport (RCT). The role of LCAT in RCT from macrophages was quantified with a validated assay involving intraperitoneal injection in mice of (3)H-cholesterol-labeled J774 macrophages and monitoring the appearance of tracer in plasma, liver, bile, and feces. Human LCAT overexpression in human apolipoprotein A-I transgenic mice substantially increased plasma high-density lipoprotein cholesterol levels but surprisingly did not increase macrophage RCT. Even in the setting of coexpression of scavenger receptor BI or cholesteryl ester transfer protein, both of which promoted the transfer of LCAT-derived high-density lipoprotein cholesterol ester to the liver, LCAT overexpression still had no effect on RCT. Serum from LCAT-overexpressing mice had reduced ability to promote cholesterol efflux from macrophages ex vivo via ABCA1. To determine the effect of LCAT deficiency on macrophage RCT, LCAT(-/-) and LCAT(+/-) mice were compared with wild-type mice. Despite extremely low plasma levels of high-density lipoprotein cholesterol, LCAT-deficient mice had only a 50% reduction in RCT. LCAT(+/-) mice had normal RCT despite a significant reduction in high-density lipoprotein cholesterol. Serum from LCAT-deficient mice had increased ability to promote ABCA1-mediated cholesterol efflux from macrophages ex vivo. These results demonstrate that LCAT overexpression does not promote an increased rate of macrophage RCT. Although LCAT activity does become rate limiting in the context of complete LCAT deficiency, RCT is reduced by only 50% even in the absence of LCAT. These data suggest that macrophage RCT may not be as dependent on LCAT activity as has previously been believed.

Moderate alcohol consumption increases plasma high-density lipoprotein cholesterol.

Moderate alcohol consumption increases plasma high-density lipoprotein cholesterol.

The role of reverse cholesterol transport in animals and humans and relationship to atherosclerosis

Reverse cholesterol transport (RCT) is a term used to describe the efflux of excess cellular cholesterol from peripheral tissues and its return to the liver for excretion in the bile and ultimately the feces. It is believed to be a critical mechanism by which HDL exert a protective effect on the development of atherosclerosis. In this paradigm, cholesterol is effluxed from arterial macrophages to extracellular HDL-based acceptors through the action of transporters such as ABCA1 and ABCG1. After efflux to HDL, cholesterol may be esterified in the plasma by the enzyme lecithin:cholesterol acyltransferase and is ultimately transported from HDL to the liver, either directly via the scavenger receptor BI or after transfer to apolipoprotein B-containing lipoproteins by the cholesteryl ester transfer protein. Methods for assessing the integrated rate of macrophage RCT in animals have provided insights into the molecular regulation of the process and suggest that the dynamic rate of macrophage RCT is more strongly associated with atherosclerosis than the steady-state plasma concentration of HDL cholesterol. Promotion of macrophage RCT is a potential therapeutic approach to preventing or regressing atherosclerotic vascular disease, but robust measures of RCT in humans will be needed in order to confidently advance RCT-promoting therapies in clinical development.

Scavenger Receptor BI-mediated Selective Uptake Is Required for the Remodeling of High Density Lipoprotein by Endothelial Lipase

Endothelial lipase (EL) is a negative regulator of high density lipoprotein (HDL) cholesterol plasma levels, and scavenger receptor BI (SR-BI) is involved in remodeling of HDL. The present study investigates the requirement of SR-BI for the effects of EL-mediated phospholipid hydrolysis on HDL metabolism in vivo. In vitro, selective uptake from EL-modified HDL was 129% higher than selective uptake from control HDL in SR-BI-overexpressing cells (p=0.01). In vivo overexpression of human EL by means of recombinant adenovirus decreased HDL plasma levels significantly (p<0.01). Fast protein liquid chromatography analysis and agarose gel electrophoresis revealed that EL expression resulted in the generation of small pre-beta HDL particles in wild-type mice, whereas in SR-BI-/- mice small HDL were preferentially removed. In kinetic experiments the fractional catabolic rate (FCR) of HDL cholesteryl ester increased by 110% (p<0.001), and the FCR of HDL apolipoproteins increased by 64% (p<0.001) in response to EL overexpression in wild-type mice. In SR-BI-/- mice a similar increase in the HDL apolipoprotein FCR occurred (p<0.001); however, there was no further increase in HDL cholesteryl ester catabolism. The apparent whole body selective uptake was increased 3-fold by EL in wild-type mice (p<0.001), whereas there was no selective uptake in SR-BI knock-out mice. EL overexpression increased hepatic selective uptake as well as holoparticle uptake (each p<0.01) in wild-type mice, whereas in SR-BI knock-out mice only holoparticle uptake increased (p<0.01). Our results indicate that SR-BI-mediated selective uptake of HDL cholesteryl ester is essential for the remodeling of large alpha-migrating HDL particles by EL.

Discovery of tetrahydro-cyclopenta[ b]indole as selective LXRs modulator

A series of tetrahydro-cyclopenta[ b]indoles modulating the activity of the liver-X-receptor (LXR) were derived from a high throughput screening hit. The potency and selectivity for LXRβ versus LXRα was improved. One compound, administered to wild-type mice modestly increased plasma HDL-cholesterol with no change in plasma triglycerides (TG) and reduced effects on liver TG content compared to T0901317. This novel series of LXR agonists shows promise to improve therapeutic efficacy with reduced potential to increase TG.

Beneficial effect of 3,4,5,6-tetrahydroxyxanthone on dyslipidemia in apolipoprotein E-deficient mice

Previous investigations have shown that decreased expression of angiopoietin-like protein 3 (Angptl3) is protective against dyslipidemia in atherosclerosis. The present study was conducted to test the effect of 3,4,5,6-tetrahydroxyxanthone, a xanthone compound, on dyslipidemia in apolipoprotein E-deficient (ApoE-/-) mice. Forty mice were randomly divided into 4 groups (n = 10): control group (C57BL/6J mice), ApoE-/-mice group, and two groups of ApoE-/- mice treated with 3,4,5,6-tetrahydroxyxanthone (10 or 30 mg/kg per day). Eight weeks after treatment, lipid levels in the blood and liver, expression of hepatic Angptl3, and adipose tissue lipoprotein lipase (LPL) were determined. Treatment with 3,4,5,6-tetrahydroxyxanthone (10 or 30 mg/kg) significantly decreased plasma and hepatic total cholesterol and triglyceride concentrations, increased plasma high-density lipoprotein cholesterol, and significantly downregulated expression of Angptl3 mRNA and protein concomitantly with upregulated expression of LPL mRNA. In addition, T0901317 (a liver X receptor ligand) caused elevated expression of hepatic Angptl3 mRNA and protein, and the effect of T0901317 was also abrogated by 3,4,5,6-tetrahydroxyxanthone in vivo and in vitro. The present results suggest that the beneficial effect of 3,4,5,6-tetrahydroxyxanthone on dyslipidemia may be related to reduced expression of Angptl3.

Abstract 3736: The HDL Receptor SR-BI Modulates Platelet Function and Susceptibility to Thrombosis in vivo

Objective: Scavenger receptor BI (SR-BI) is a cell surface receptor that promotes the selective uptake of cholesteryl esters from HDL by the liver. In mice, SR-BI deficiency leads to increased plasma HDL cholesterol levels and enhanced susceptibility to atherosclerosis. Recently, a family was identified in which heterozygous carriers of a mutation (P297S) in SR-BI showed elevated HDL cholesterol levels, suggesting that SR-BI is equally important in controlling HDL cholesterol levels in humans. Interestingly, SR-BI is also expressed on platelets, which play a crucial role in atherothrombosis. The aim of this study was to investigate the role of SR-BI in platelet function in both SR-BI knockout (KO) mice and human P297S carriers. Methods &amp; Results: Hematological analysis showed that SR-BI deficiency in mice caused thrombocytopenia (381±69x10 9 SR-BI KO platelets/L vs 878±162x10 9 wildtype (WT) platelets/L, p=0.001). Correspondingly, SR-BI deficiency increased tail bleeding times from 85±8 to 134±16 sec (p=0.02). SR-BI KO platelets were abnormally large (7.1±0.3 fl vs 5.8±0.0 fl for WT platelets, p=0.0004), probably caused by an increased cholesterol content. Platelet count and size of the human P297S carriers were, however, not significantly different from matched controls. The FeCl 3 acute injury model was used to study the effect of SR-BI deficiency in mice on the susceptibility to arterial thrombosis. WT mice developed total arterial occlusion after 24±2 min. In SR-BI KO mice, however, the time to total arterial occlusion was reduced 2-fold to 13±1 min (p=0.02). In line with this finding, SR-BI KO and P297S platelets circulated in an activated state and showed increased adherence to immobilized fibrinogen. The activated state of the platelets in vivo led to desensitization of the receptors for ADP and thromboxane that are essential for positive feedback mechanisms to enhance the platelet response. As a result, ex vivo aggregation of platelets from SR-BI KO mice and P297S carriers was inhibited in response to agonist stimulation as compared to control platelets. Conclusion: SR-BI is not only essential for HDL cholesterol homeostasis and atherosclerosis susceptibility, but also for maintaining normal platelet aggregation and prevention of thrombosis.