Cardiovascular disease (CVD) remains the leading cause of mortality in westernized countries, despite optimum medical therapy to lower LDL cholesterol. The pursuit of novel therapies to target this residual risk has focused on raising levels of HDL cholesterol in order to exploit its atheroprotective effects1. MicroRNAs have emerged as important post-transcriptional regulators of lipid metabolism, and are thus a new class of targets for therapeutic intervention2. MicroRNA-33a and b (miR-33a/b) are intronic microRNAs embedded in the sterol response element binding protein genes SREBF2 and SREBF13–5, respectively, that repress expression of the cholesterol transporter ABCA1, a key regulator of HDL biogenesis. Recent studies in mice suggest that antagonizing miR-33a may be an effective strategy for raising plasma HDL3–5 and protecting from atherosclerosis6, however extrapolation of these findings to humans is complicated by the fact that mice lack miR-33b which is present only in the SREBF1 gene of higher mammals. Here we show in African green monkeys that systemic delivery of an anti-miR oligonucleotide that targets both miR-33a and miR-33b increases hepatic expression of ABCA1 and induces a sustained increase in plasma HDL over 12 weeks. Notably, miR-33 antagonism in this non-human primate model also increased the expression of miR-33 target genes involved in the oxidation of fatty acids (CROT, CPT1A, HADHB, PRKAA1) and reduced genes involved in fatty acid synthesis (SREBF1, FASN, ACLY, ACACA), resulting in a marked suppression of plasma VLDL triglyceride levels, a finding not previously observed in mice. These data establish, in a model highly relevant to humans, that pharmacological inhibition of miR-33a and b is a promising therapeutic strategy to raise plasma HDL and lower VLDL triglycerides for the treatment of dyslipidemias that increase cardiovascular disease risk.

SUMMARY 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 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.

Summary Recent evidence suggests that the intestine may play a direct facilitative role in reverse cholesterol transport (RCT), independent of hepatobiliary secretion. In order to understand the non-biliary pathway for RCT we created both genetic and surgical models of biliary cholesterol insufficiency. To genetically inhibit biliary cholesterol secretion we generated mice in which Niemann-Pick C1-Like 1 (NPC1L1) was overexpressed in the liver. Compared to controls, NPC1L1Liver-Tg mice exhibit a > 90% decrease in biliary cholesterol secretion, yet mass fecal sterol loss and macrophage RCT is normal. To surgically inhibit biliary emptying into the intestine, we have established an acute biliary diversion model. Strikingly, macrophage RCT persists in mice surgically lacking the ability to secrete bile into the intestine. Collectively, these studies demonstrate that mass fecal sterol loss and macrophage RCT can proceed in the absence of biliary sterol secretion, challenging the obligate role of bile in RCT.

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.

Summary 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.