Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL levels have remained elusive. Recent advances in the understanding of lipid metabolism have revealed that miR-33, an intronic microRNA located within the SREBF2 gene, suppresses expression of the cholesterol transporter ABC transporter A1 (ABCA1) and lowers HDL levels. Conversely, mechanisms that inhibit miR-33 increase ABCA1 and circulating HDL levels, suggesting that antagonism of miR-33 may be atheroprotective. As the regression of atherosclerosis is clinically desirable, we assessed the impact of miR-33 inhibition in mice deficient for the LDL receptor (Ldlr -/-mice), with established atherosclerotic plaques. Mice treated with anti-miR33 for 4 weeks showed an increase in circulating HDL levels and enhanced reverse cholesterol transport to the plasma, liver, and feces. Consistent with this, anti-miR33-treated mice showed reductions in plaque size and lipid content, increased markers of plaque stability, and decreased inflammatory gene expression. Notably, in addition to raising ABCA1 levels in the liver, anti-miR33 oligonucleotides directly targeted the plaque macrophages, in which they enhanced ABCA1 expression and cholesterol removal. These studies establish that raising HDL levels by anti-miR33 oligonucleotide treatment promotes reverse cholesterol transport and atherosclerosis regression and suggest that it may be a promising strategy to treat atherosclerotic vascular disease.

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.

Niemann-Pick C1-like 1 (NPC1L1) is required for cholesterol absorption. Intestinal NPC1L1 appears to be a target of ezetimibe, a cholesterol absorption inhibitor that effectively lowers plasma LDL-cholesterol in humans. However, human liver also expresses NPC1L1. Hepatic function of NPC1L1 was previously unknown, but we recently discovered that NPC1L1 localizes to the canalicular membrane of primate hepatocytes and that NPC1L1 facilitates cholesterol uptake in hepatoma cells. Based upon these findings, we hypothesized that hepatic NPC1L1 allows the retention of biliary cholesterol by hepatocytes and that ezetimibe disrupts hepatic function of NPC1L1. To test this hypothesis, transgenic mice expressing human NPC1L1 in hepatocytes (L1-Tg mice) were created. Hepatic overexpression of NPC1L1 resulted in a 10-to 20-fold decrease in biliary cholesterol concentration, but not phospholipid and bile acid concentrations. This decrease was associated with a 30%-60% increase in plasma cholesterol, mainly because of the accumulation of apoE-rich HDL. Biliary and plasma cholesterol concentrations in these animals were virtually returned to normal with ezetimibe treatment. These findings suggest that in humans, ezetimibe may reduce plasma cholesterol by inhibiting NPC1L1 function in both intestine and liver, and hepatic NPC1L1 may have evolved to protect the body from excessive biliary loss of cholesterol.

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.

The class B, type I scavenger receptor, SR-BI, binds high density lipoprotein (HDL) and can mediate selective uptake of HDL cholesteryl esters by cultured cells. The high levels of expression of SR-BI in steroidogenic tissues and the importance of selective uptake from HDL as a source of cholesterol for steroidogenesis raised the possibility that SR-BI may participate in cholesterol delivery to steroidogenic tissues in vivo. We have used immunoblotting and immunohistochemical methods to show that SR-BI is specifically expressed in a distinctive pattern on the surfaces of steroid-producing cells in the murine adrenal gland's cortex and that its expression in vivo is induced by adrenocorticotropic hormone and suppressed by glucocorticoids. Thus, expression of SR-BI protein is coordinately regulated with adrenal steroidogenesis. These data provide strong support for the hypothesis that SR-BI is a physiologically relevant HDL receptor that provides substrate cholesterol for steroid hormone synthesis.The intercellular transport of lipids, including cholesteryl esters and triglycerides, through the blood involves their packaging into water-soluble lipoproteins and the targeted delivery of lipoprotein lipids to cells via receptor-mediated processes (1). The best understood of the lipoprotein transport systems is the LDL 1 receptor pathway of receptor-mediated endocytosis. LDL binds to its surface receptor, is internalized via coated pits, and the entire lipoprotein particle is subsequently degraded in lysosomes to release free cholesterol to the cell. HDL also delivers cholesterol to cells through a less well defined process called selective cholesterol uptake (2-12). HDL particles bind to surface receptors on target cells, their cholesteryl esters are selectively transferred into the cells without the degradation of the lipoprotein particle, and the lipid-depleted particles including their two major apolipoproteins, apoA-I and apoA-II, are released from the cells, a process that is fundamentally different from the endocytic uptake of lipoproteins mediated by members of the LDL receptor family.The HDL-cholesteryl ester selective uptake pathway occurs in cultured hepatocytes and in the liver (3, 4, 7-9, 11, 12), where it may contribute to the clearance of plasma cholesteryl ester in the terminal stage of reverse cholesterol transport (13,14). The HDL selective uptake pathway plays a prominent role in cholesterol delivery to steroidogenic cells of mice and rats where it appears to be responsible for the overwhelming majority of the uptake of HDL cholesterol (3,4,6,8,12). Indeed, HDL is significantly more effective than LDL in supplying cholesterol for cholesteryl ester accumulation and corticosterone production in adrenal glands of rats and mice (5,(15)(16)(17). Using gene knockout mice, Plump et al. (18) showed that apoA-I deficiency caused an almost complete failure to accumulate cholesteryl ester in steroidogenic cells of the adrenal gland, ovary, and testis, a result illustrating the importance of HDL-apoA-I, presum...