An effective way to reduce LDL cholesterol, the primary risk factor of atherosclerotic cardiovascular disease, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both hepatobiliary and transintestinal pathways. However, the lipoprotein that moves cholesterol through the plasma to the small intestine for transintestinal cholesterol efflux (TICE) is unknown. To test the hypothesis that hepatic very lowdensity lipoproteins (VLDL) support TICE, antisense oligonucleotides (ASO) were used to knockdown hepatic expression of microsomal triglyceride transfer protein (MTP), which is necessary for VLDL assembly. While maintained on a high cholesterol diet, Niemann-Pick C1-like 1 hepatic transgenic (L1Tg) mice, which predominantly excrete cholesterol via TICE, and wild type (WT) littermates were treated with control ASO or MTP ASO. In both WT and L1Tg mice, MTP ASO decreased VLDL triglyceride (TG) and cholesterol secretion. Regardless of treatment, L1Tg mice had reduced biliary cholesterol compared to WT mice. However, only L1Tg mice treated with MTP ASO had reduced fecal cholesterol excretion. Based upon these findings, we conclude that VLDL or a byproduct such as LDL can move cholesterol from the liver to the small intestine for TICE. Citation: Marshall SM, Kelley KL, Davis MA, Wilson MD, McDaniel AL, et al. (2014) Reduction of VLDL Secretion Decreases Cholesterol Excretion in Niemann-Pick C1-Like 1 Hepatic Transgenic Mice. PLoS ONE 9(1): e84418. doi:10.1371/journal.pone.0084418 Editor: Andrej A. Romanovsky, St. Joseph’s Hospital and Medical Center, United States of America Received October 14, 2013; Accepted November 7, 2013; Published January 3, 2014 Copyright: 2014 Marshall et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: National Heart, Lung, and Blood Institute of the NIH (http://www.nhlbi.nih.gov/) funded this work through the following awards: P01-HL049373 to L.L.R., K99/R00-HL096166 to J.M.B., and K99/R00-HL088528 to R.E.T. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have read the journal’s policy and have the following conflicts. R.M.C., R.G.L., and M.J.D. are employees of and have ownership interest in Isis Pharmaceuticals. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: brownm5@ccf.org (JMB); ryan.temel@uky.edu (RT) . These authors contributed equally to this work.

Elevated plasma low-density lipoprotein (LDL) and decreased high-density lipoprotein (HDL) cholesterol levels increase the risk of cardiovascular disease (CVD). While strategies to lower LDL cholesterol have been successful in reducing CVD-related mortality, there is still an unmet need for developing therapies to reduce the residual risk of atherosclerotic CVD. Plasma HDL cholesterol levels are inversely correlated with CVD risk and thus novel therapies to increase plasma HDL cholesterol levels have garnered much attention in recent years. MicroRNA-33a and b (miR-33a/b) are intronically-encoded microRNAs residing in the sterol response element binding protein genes SREBF2 and SREBF1 and suppress the expression of the genes involved in cholesterol efflux and fatty acid oxidation. Recent studies show that antagonism of miR-33 results in increased ABCA1expression and elevated plasma HDL levels in both mice and nonhuman primates. However, findings in mice suggest that long term miR-33 antagonism may lead to hepatic steatosis and increased production of very low-density lipoprotein (VLDL), the precursor to proatherogenic LDL. In the current study, cynomolgus monkeys were fed a high carbohydrate diet plus Kool Aid prior to administration of antisense oligonucleotides (ASOs) specific for miR-33a/b. As expected, miR-33 antagonism resulted in elevated plasma total cholesterol levels, predominantly due to increased HDL. No differences in plasma LDL or triglycerides were observed between treatment groups. MiR-33a/b antagonism had no effect on VLDL triglyceride secretion rates. Anti-miR-33 treatment did not promote hepatic lipid accumulation or adversely affect liver transaminase levels. Glucose tolerance tests revealed no differences in either plasma glucose or insulin levels in response to miR-33a/b antagonism. These findings suggest that anti-miR-33 therapy raises HDL cholesterol without negatively impacting either triglyceride or glucose homeostasis in nonhuman primates.

Circulating levels of the gut microbe-derived metabolite trimethylamine-N-oxide (TMAO) have recently been linked to cardiovascular disease (CVD) risk. Here, we performed transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT) and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 knockdown stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic endoplasmic reticulum (ER) stress and inflammation in part by decreasing hepatic oxysterol levels and subsequent LXR activation. FMO3 is thus identified as a central integrator of hepatic cholesterol and triacylglycerol metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMA/FMO3/TMAO pathway is a key regulator of lipid metabolism and inflammation.

Hyperlipidemia affects millions of people worldwide and is a major risk factor for cardiovascular disease. People with hyperlipidemia have elevated levels of serum cholesterol and an increased risk of thrombosis. Studies have suggested that oxidized lipoproteins, such as oxidized low-density lipoprotein (oxLDL), contribute to the development of a pro-thrombotic state. In this review, we discuss our recent studies demonstrating a role for hematopoietic cell-derived tissue factor (TF) expression in the activation of coagulation and increased thrombosis associated with hyperlipidemia. In addition, we investigated the effect of simvastatin on TF expression and coagulation. We found that simvastatin reduced leukocyte TF expression, TF⁺ microparticles, and coagulation. These results and earlier studies suggest that the anti-coagulant activity of statins is due, in part, to their ability to reduce monocyte TF expression in patients with cardiovascular disease.

An effective way to reduce LDL cholesterol, the primary risk factor of atherosclerotic cardiovascular disease, is to increase cholesterol excretion from the body. Our group and others have recently found that cholesterol excretion can be facilitated by both hepatobiliary and transintestinal pathways. However, the lipoprotein that moves cholesterol through the plasma to the small intestine for transintestinal cholesterol efflux (TICE) is unknown. To test the hypothesis that hepatic very low-density lipoproteins (VLDL) support TICE, antisense oligonucleotides (ASO) were used to knockdown hepatic expression of microsomal triglyceride transfer protein (MTP), which is necessary for VLDL assembly. While maintained on a high cholesterol diet, Niemann-Pick C1-like 1 hepatic transgenic (L1Tg) mice, which predominantly excrete cholesterol via TICE, and wild type (WT) littermates were treated with control ASO or MTP ASO. In both WT and L1Tg mice, MTP ASO decreased VLDL triglyceride (TG) and cholesterol secretion. Regardless of treatment, L1Tg mice had reduced biliary cholesterol compared to WT mice. However, only L1Tg mice treated with MTP ASO had reduced fecal cholesterol excretion. Based upon these findings, we conclude that VLDL or a byproduct such as LDL can move cholesterol from the liver to the small intestine for TICE.

Objective— Excessive caloric intake is associated with obesity and adipose tissue dysfunction. However, the role of dietary cholesterol in this process is unknown. The aim of this study was to determine whether increasing dietary cholesterol intake alters adipose tissue cholesterol content, adipocyte size, and endocrine function in nonhuman primates. Approach and Results— Age-matched, male African Green monkeys (n=5 per group) were assigned to 1 of 3 diets containing 0.002 (low [Lo]), 0.2 (medium [Med]), or 0.4 (high [Hi]) mg cholesterol/kcal. After 10 weeks of diet feeding, animals were euthanized for adipose tissue, liver, and plasma collection. With increasing dietary cholesterol, free cholesterol (FC) content and adipocyte size increased in a stepwise manner in visceral, but not in subcutaneous fat, with a significant association between visceral adipocyte size and FC content ( r 2 =0.298; n=15; P =0.035). In visceral fat, dietary cholesterol intake was associated with (1) increased proinflammatory gene expression and macrophage recruitment, (2) decreased expression of genes involved in cholesterol biosynthesis and lipoprotein uptake, and (3) increased expression of proteins involved in FC efflux. Conclusions— Increasing dietary cholesterol selectively increases visceral fat adipocyte size, FC and macrophage content, and proinflammatory gene expression in nonhuman primates. Visceral fat cells seem to compensate for increased dietary cholesterol by limiting cholesterol uptake/synthesis and increasing FC efflux pathways.

Circulating trimethylamine-N-oxide (TMAO) levels have recently been linked to cardiovascular disease (CVD) risk, but the mechanism underlying this association is unclear. Here we performed unbiased transcriptional profiling in mouse models of altered reverse cholesterol transport (RCT), and serendipitously identified the TMAO-generating enzyme flavin monooxygenase 3 (FMO3) as a powerful modifier of cholesterol metabolism and RCT. Knockdown of FMO3 in cholesterol-fed mice alters biliary lipid secretion, blunts intestinal cholesterol absorption, and limits the production of hepatic oxysterols and cholesteryl esters. Furthermore, FMO3 inhibition stimulates basal and liver X receptor (LXR)-stimulated macrophage RCT, thereby improving cholesterol balance. Conversely, FMO3 knockdown exacerbates hepatic ER stress and inflammation by altering the LXR-Lpcat3-driven phospholipid remodeling pathway. FMO3 is thus identified as a novel integrator of hepatic lipid metabolism, inflammation, and ER stress. These studies suggest that the gut microbiota-driven TMAO pathway may promote CVD pathogenesis in part by reciprocally regulating lipid metabolism and inflammation.

The LXR-regulated E3 ubiquitin ligase IDOL controls LDLR receptor stability independent of SREBP and PCSK9, but its relevance to plasma lipid levels is unknown. Here we demonstrate that the effects of the LXR-IDOL axis are both tissue and species specific. In mice, LXR agonist induces Idol transcript levels in peripheral tissues but not in liver, and does not change plasma LDL levels. Accordingly, Idol-deficient mice exhibit elevated LDLR protein levels in peripheral tissues, but not in the liver. By contrast, LXR activation in cynomolgus monkeys induces hepatic IDOL expression, reduces LDLR protein levels, and raises plasma LDL levels. Knockdown of IDOL in monkeys with an antisense oligonucleotide blunts the effect of LXR agonist on LDL levels. These results implicate IDOL as a modulator of plasma lipid levels in primates and support further investigation into IDOL inhibition as a potential strategy for LDL lowering in humans.

BackgroundMore than one-quarter of Americans have hypercholesterolemia and/or are being treated with cholesterol-lowering medications. Given the systemic nature of hypercholesterolemia and remaining questions regarding its effect on tendons at a local level, we sought to assess the utility of small versus large animal model systems for translational studies by exploring the effect of hypercholesterolemia on supraspinatus tendon elastic mechanical properties in mice, rats, and monkeys. We hypothesized that stiffness and elastic modulus would be increased in tendons across species due to hypercholesterolemia.Materials and methodsSupraspinatus tendons from normal (control) and high-cholesterol (HC) mice, rats, and monkeys were used in this study. After dissection, tendons were geometrically measured and tensile tested with tissue strain measured optically.ResultsOverall, HC animals had significantly altered plasma lipid profiles. Biomechanical testing showed a significant increase in stiffness compared with control in HC mice and rats, as well as a nonsignificant trend for HC monkeys. Elastic modulus was also significantly increased in HC mice and monkeys, with HC rats showing a trend.ConclusionsThe consistency of our findings across species and between small and large animals, combined with the fact that the aged mice were exposed to lifelong hypercholesterolemia (compared with rats and nonhuman primates, which were fed HC diets), suggests that these increased properties may be inherent to the effect of hypercholesterolemia on supraspinatus tendon rather than due to an effect of cumulative exposure time to the effects of HC. Further investigation is needed to confirm this concept.

Reverse cholesterol transport (RCT) can proceed through the classic hepatobiliary route or through the nonbiliary transintestinal cholesterol efflux (TICE) pathway. Scavenger receptor class B type I (SR-BI) plays a critical role in the classic hepatobiliary route of RCT. However, the role of SR-BI in TICE has not been studied. To examine the role of intestinal SR-BI in TICE, sterol balance was measured in control mice and mice transgenically overexpressing SR-BI in the proximal small intestine (SR-BIhApoCIII-ApoAIV-Tg). SR-BIhApoCIII-ApoAIV-Tg mice had significantly lower plasma cholesterol levels compared with wild-type controls, yet SR-BIhApoCIII-ApoAIV-Tg mice had normal fractional cholesterol absorption and fecal neutral sterol excretion. Both in the absence or presence of ezetimibe, intestinal SR-BI overexpression had no impact on the amount of cholesterol excreted in the feces. To specifically study effects of intestinal SR-BI on TICE we crossed SR-BIhApoCIII-ApoAIV-Tg mice into a mouse model that preferentially utilized the TICE pathway for RCT (Niemann-Pick C1-like 1 liver transgenic), and likewise found no alterations in cholesterol absorption or fecal sterol excretion. Finally, mice lacking SR-BI in all tissues also exhibited normal cholesterol absorption and fecal cholesterol disposal. Collectively, these results suggest that SR-BI is not rate limiting for intestinal cholesterol absorption or for fecal neutral sterol loss through the TICE pathway.

Transintestinal cholesterol excretion (TICE) is a recently discovered pathway by which cholesterol travels from plasma to the small intestine for direct excretion into the feces. Hallmarks of animal models with TICE include severely diminished biliary cholesterol secretion but near normal levels of hepatic cholesterol and fecal neutral sterol excretion. Using an ATP binding cassette transporter G8 (ABCG8) antisense oligonucleotide (ASO) to knock down ABCG8 specifically in liver (G8 HKD ), we created a novel mouse model with significantly decreased biliary cholesterol excretion but a 658% increase in hepatic cholesterol accumulation and a 78% reduction in fecal neutral sterol excretion, indicating a dysfunction in the TICE pathway. LXR agonists have previously been shown to stimulate the TICE pathway. In order to more definitively prove the TICE pathway was disfunctional in G8 HKD mice, we treated wild type (WT) and G8 HKD mice with the LXR agonist T0901317 and measured markers of TICE stimulation. As expected, in WT mice, T0901317 doubled biliary cholesterol concentrations. A similar effect was seen in G8 HKD mice treated with T0901317, but biliary cholesterol concentrations remained significantly less than their WT counterparts. These levels of biliary cholesterol closely mirrored hepatic ABCG8 mRNA expression. T0901317 stimulated fecal neutral sterol excretion by >1000% in wild type mice but only by 190% in G8 HKD mice. These data indicate that TICE is disfunctional in G8 HDK mice since the pathway was not stimulated to the same extent in WT and G8 HKD mice by an LXR agonist. Some controversy remains over whether the TICE pathway transports macrophage derived cholesterol. In order to address this issue, we performed a macrophage RCT assay on WT and TICE disfunctional G8 HKD mice. T0901317 stimulated macrophage RCT (fecal neutral sterol 3H dpm) by >2300% in wild type mice but only by 370% in G8 HKD mice. T0901317 increased fecal acidic sterol 3H count by 65-75% in both wild type and G8 HKD mice. These results indicate that macrophage RCT is impaired when the TICE pathway is decreased. In sum, our data shows that hepatic ABCG8 plays a key role in the TICE pathway and that impairing the TICE pathway through hepatic ABCG8 knockdown causes decreased macrophage RCT.

Recent studies have revealed a novel route for cholesterol disposal through intestine known as transintestinal cholesterol efflux (TICE) that significantly contributes to fecal neutral sterol loss. This pathway is an integral part of reverse cholesterol transport (RCT), yet major mechanisms regulating TICE are not well understood. Using an unbiased transcriptional profiling approach in mouse models of augmented TICE, we found that hepatic expression of the enzyme Flavin monoxygenase 3 (FMO3) was dramatically repressed. At the same time we identified this enzyme through transcriptional profiling, it was reported that plasma levels of its product trimethylamineoxide (TMAO) are highly predictive of atheroslcerosis in humans, and TMAO is proatherogenic in mice. To further understand FMO3’s role as a regulator of cholesterol metabolism we used antisense oligonucleotides (ASO) to knockdown FMO3 expression in mouse liver in C57BL/6 mice fed either low (0.02%) or high (0.2%) levels of dietary cholesterol. As expected, FMO3 knockdown (>90% knockdown in the liver) increased the TMA/TMAO ratio in plasma more than 3-fold. Interestingly, knockdown of FMO biliary cholesterol levels were reduced by 60%, whereas fecal cholesterol loss was quite normal in FMO3 ASO treated mice fed a high cholesterol diet, which phenocopies a previously described mouse model where TICE predominates (NPC1L1-liver transgenic mice). ASO-mediated knockdown of FMO3 also unexpectedly reduced hepatic cholesteryl ester (CE) storage by 70% in mice fed 0.2% cholesterol. In parallel, knockdown of FMO3 reduces plasma VLDL cholesterol levels and the secretion rate of VLDL cholesteryl ester, but not triacylglycerol in cholesterol fed mice. FMO3 knockdown also reduced the hepatic expression of several liver X receptor (LXR) target genes, while increasing expression of genes involved in cholesterol synthesis. Collectively, these studies have identified FMO3 as a novel regulator of hepatic cholesterol metabolism and TICE. Given that plasma levels of FMO3’s product (TMAO) are strongly associated with atherosclerosis development in humans, and production of TMAO promotes atherosclerosis in mice, these studies have important implications for future cardiovascular drug discovery.

Reverse cholesterol transport (RCT) is a process whereby cholesterol from foam cells in atherosclerotic plaques is removed by HDL, transported to the liver, secreted into bile, and excreted in the feces. Treatment of mice with LXR agonist can significantly increase RCT and inhibit atherosclerosis. Therefore, it would be anticipated that LXR agonists would also increase RCT and be anti-atherogenic in humans. However, cynomolgus monkeys treated with the LXR agonist GW3965 had significant increases in LDLc, which could be due to elevated hepatic expression of the inducible degrader of the LDL receptor (IDOL). IDOL is an E3 ubiquitin ligase that promotes the ubiquitination of the LDL receptor (LDLR) on its cytoplasmic domain, thereby targeting it for degradation. We recently discovered that GW3965 treatment of mice caused an ~3-fold increase in IDOL expression in the small intestine and consequently increased the turnover of the LDLR. In livers of GW3965 treated mice, LDLR protein was unchanged and IDOL expression was only increased by ~20%. However, treatment of human hepatoma cells with GW3965 increased IDOL 7-fold resulting in very low levels of LDLR protein expression. Therefore, we hypothesized that in humans and nonhuman primates, LXR agonist treatment would increase LDLc by inducing IDOL-dependent degradation of hepatic LDLR. After treating cynomolgus monkeys with GW3965 for 7 days, we found that HDLc was increased by ~20% while LDLc was increased by ~100%. The increase in LDLc was accompanied by a dramatic increase in plasma apoB100 and apoE concentration and a significant decrease in hepatic LDLR protein. We are currently determining the potential role of hepatic IDOL expression on these effects. We also found that unlike mice, monkeys treated with GW3965 did not display an increase in hepatic lipids, biliary cholesterol, and fecal neutral sterol excretion. These findings show that LXR activation has very different effects on lipoprotein metabolism and RCT in monkeys compared to mice. Our data suggest that LXR agonists are likely to exert both pro- and anti-atherosclerotic effects in primates and that these complex effects must be considered in the development of LXR agonists as drugs.

Hypercholesterolemia is a major risk factor for atherosclerosis. It also is associated with platelet hyperactivity, which increases morbidity and mortality from cardiovascular disease. However, the mechanisms by which hypercholesterolemia produces a procoagulant state remain undefined. Atherosclerosis is associated with accumulation of oxidized lipoproteins within atherosclerotic lesions. Small quantities of oxidized lipoproteins are also present in the circulation of patients with coronary artery disease. We therefore hypothesized that hypercholesterolemia leads to elevated levels of oxidized LDL (oxLDL) in plasma and that this induces expression of the procoagulant protein tissue factor (TF) in monocytes. In support of this hypothesis, we report here that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma microparticle (MP) TF activity. Furthermore, a high-fat diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte-derived TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity. Simvastatin treatment of hypercholesterolemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation, without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hypercholesterolemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6 complex.

Hyperlipidemia is associated with a prothrombotic state that includes activation of platelets. However, the molecular pathway that leads to the activation of the coagulation cascade has not been defined. Hyperlipidemia leads to the presence of low levels of oxidized lipoproteins, such as oxidized LDL (oxLDL), in the circulation and the accumulation of oxLDL in atherosclerotic lesions.. Monocytes and macrophages are activated by oxLDL binding to a TLR4/TLR6/CD36 receptor complex. We hypothesized that oxLDL induction of monocyte tissue factor (TF) expression and the release of TF-positive microparticles (MPs) produces a prothrombotic state. In support of this hypothesis, we found that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma MP TF activity. Furthermore, a western diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Inhibition of TF or a genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity consistent with a role of the TLR4/TLR6/CD36 complex in oxLDL induction of monocyte TF expression. Importantly, simvastatin treatment of hyperlipidemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hyperlipidemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6/CD36 receptor complex. Furthermore, simvastatin appears to act at multiple levels to reduce the prothrombotic state.

MicroRNAs have emerged as important post-transcriptional regulators of lipid metabolism, and are thus a new class of targets for therapeutic intervention. MicroRNA-33a and b (miR-33a/b) are recentl...

Genes involved in cholesterol biosynthesis and uptake are transcriptionally regulated in response to cellular sterol content in a coordinated manner. A number of these genes, including 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and LDL receptor (LDLR), undergo alternative splicing, resulting in reductions of enzyme or protein activity. Here we demonstrate that cellular sterol depletion suppresses, and sterol loading induces, alternative splicing of multiple genes involved in the maintenance of cholesterol homeostasis including HMGCR and LDLR, the key regulators of cellular cholesterol biosynthesis and uptake, respectively. These changes were observed in both in vitro studies of the HepG2 human hepatoma derived cell line, as well as in vivo studies of St. Kitts vervets, also known as African green monkeys, a commonly used primate model for investigating cholesterol metabolism. These effects are mediated in part by sterol regulation of polypyrimidine tract binding protein 1 (PTBP1), since knock-down of PTBP1 eliminates sterol induced changes in alternative splicing of several of these genes. Single nucleotide polymorphisms (SNPs) that influence HMGCR and LDLR alternative splicing (rs3846662 and rs688, respectively), have been associated with variation in plasma LDL-cholesterol levels. Sterol-induced changes in alternative splicing are blunted in carriers of the minor alleles for each of these SNPs, indicating an interaction between genetic and non-genetic regulation of this process. Our results implicate alternative splicing as a novel mechanism of enhancing the robust transcriptional response to conditions of cellular cholesterol depletion or accumulation. Thus coordinated regulation of alternative splicing may contribute to cellular cholesterol homeostasis as well as plasma LDL levels.

Cardiovascular disease remains the leading cause of mortality in westernized countries, despite optimum medical therapy to reduce the levels of low-density lipoprotein (LDL)-associated cholesterol. The pursuit of novel therapies to target the residual risk has focused on raising the levels of high-density lipoprotein (HDL)-associated cholesterol in order to exploit its atheroprotective effects. MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of lipid metabolism and are thus a new class of target for therapeutic intervention. MicroRNA-33a and microRNA-33b (miR-33a/b) are intronic miRNAs whose encoding regions are embedded in the sterol-response-element-binding protein genes SREBF2 and SREBF1 (refs 3-5), respectively. These miRNAs repress expression of the cholesterol transporter ABCA1, which is a key regulator of HDL biogenesis. Recent studies in mice suggest that antagonizing miR-33a may be an effective strategy for raising plasma HDL levels and providing protection against atherosclerosis; however, extrapolating these findings to humans is complicated by the fact that mice lack miR-33b, which is present only in the SREBF1 gene of medium and large mammals. Here we show in African green monkeys that systemic delivery of an anti-miRNA oligonucleotide that targets both miR-33a and miR-33b increased hepatic expression of ABCA1 and induced a sustained increase in plasma HDL levels over 12 weeks. Notably, miR-33 antagonism in this non-human primate model also increased the expression of miR-33 target genes involved in fatty acid oxidation (CROT, CPT1A, HADHB and PRKAA1) and reduced the expression of genes involved in fatty acid synthesis (SREBF1, FASN, ACLY and ACACA), resulting in a marked suppression of the plasma levels of very-low-density lipoprotein (VLDL)-associated triglycerides, a finding that has not previously been observed in mice. These data establish, in a model that is highly relevant to humans, that pharmacological inhibition of miR-33a and miR-33b is a promising therapeutic strategy to raise plasma HDL and lower VLDL triglyceride levels for the treatment of dyslipidaemias that increase cardiovascular disease risk.