Cholesterol metabolism is tightly regulated at the cellular level. Here we show that miR-33, an intronic microRNA (miRNA) located within the gene encoding sterol-regulatory element–binding factor–2 (SREBF-2), a transcriptional regulator of cholesterol synthesis, modulates the expression of genes involved in cellular cholesterol transport. In mouse and human cells, miR-33 inhibits the expression of the adenosine triphosphate–binding cassette (ABC) transporter, ABCA1, thereby attenuating cholesterol efflux to apolipoprotein A1. In mouse macrophages, miR-33 also targets ABCG1, reducing cholesterol efflux to nascent high-density lipoprotein (HDL). Lentiviral delivery of miR-33 to mice represses ABCA1 expression in the liver, reducing circulating HDL levels. Conversely, silencing of miR-33 in vivo increases hepatic expression of ABCA1 and plasma HDL levels. Thus, miR-33 appears to regulate both HDL biogenesis in the liver and cellular cholesterol efflux.

Cellular imbalances of cholesterol and fatty acid metabolism result in pathological processes, including atherosclerosis and metabolic syndrome. Recent work from our group and others has shown that the intronic microRNAs hsa-miR-33a and hsa-miR-33b are located within the sterol regulatory element-binding protein-2 and -1 genes, respectively, and regulate cholesterol homeostasis in concert with their host genes. Here, we show that miR-33a and -b also regulate genes involved in fatty acid metabolism and insulin signaling. miR-33a and -b target key enzymes involved in the regulation of fatty acid oxidation, including carnitine O-octaniltransferase, carnitine palmitoyltransferase 1A, hydroxyacyl-CoAdehydrogenase, Sirtuin 6 (SIRT6), and AMP kinase subunit-α. Moreover, miR-33a and -b also target the insulin receptor substrate 2, an essential component of the insulin-signaling pathway in the liver. Overexpression of miR-33a and -b reduces both fatty acid oxidation and insulin signaling in hepatic cell lines, whereas inhibition of endogenous miR-33a and -b increases these two metabolic pathways. Together, these data establish that miR-33a and -b regulate pathways controlling three of the risk factors of metabolic syndrome, namely levels of HDL, triglycerides, and insulin signaling, and suggest that inhibitors of miR-33a and -b may be useful in the treatment of this growing health concern.lipid homeostasis | posttranscriptional regulation | cardiovascular disease

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.

Abstract-Dicer is a key enzyme involved in the maturation of microRNAS (miRNAs). miRNAs have been shown to be regulators of gene expression participating in the control of a wide range of physiological pathways. To assess the role of Dicer and consequently the importance of miRNAs in the biology and functions of human endothelial cells (EC) during angiogenesis, we globally reduced miRNAs in ECs by specific silencing Dicer using siRNA and examined the effects on EC phenotypes in vitro. The knockdown of Dicer in ECs altered the expression (mRNA and/or protein) of several key regulators of endothelial biology and angiogenesis, such as TEK/Tie-2, KDR/VEGFR2, Tie-1, endothelial nitric oxide synthase and IL-8. Although, Dicer knockdown increased activation of the endothelial nitric oxide synthase pathway it reduced proliferation and cord formation of EC in vitro. The miRNA expression profile of EC revealed 25 highly expressed miRNAs in human EC and using miRNA mimicry, miR-222/221 regulates endothelial nitric oxide synthase protein levels after Dicer silencing. Collectively, these results indicate that maintenance and regulation of endogenous miRNA levels via Dicer mediated processing is critical for EC gene expression and functions in vitro. (Circ Res. 2007;100:1164-1173.)Key Words: endothelium Ⅲ Dicer Ⅲ miRNA Ⅲ angiogenesis M icroRNAs (miRNAs) are short noncoding RNAs that have been identified in a variety of organisms and have been shown to regulate gene expression. 1,2 In mammalian cells, these small RNAs (Ϸ22 nt) are transcribed as parts of longer molecules that are processed in the nucleus into hairpins RNAs by the protein Drosha. 3,4 These premiRNAs are then transported to the cytoplasm, via an exportin 5-dependent mechanism, where they are digested by a second, doubled-stranded specific ribonuclease called Dicer. 5,6 The mature miRNAs are incorporated into a ribonucleoprotein complex 7,8 or RISC complex,9 that mediates the downregulation of target gene activity by translational inhibition or target mRNA degradation, resulting in reduced levels of the corresponding protein or transcript, respectively. 7,10,11 miRNAs have been implicated in the control of a wide range of physiological pathways 12,13 such as development, differentiation, growth and metabolism. 14 -17 Moreover, tissue-specific patterns of miRNAs are providing insights into their possible functions. Many miRNAs exhibit striking organ specific expression patterns, or even expression restricted to single tissue layer within an organ 18 and different miRNAs have been specifically cloned from heart, brain, embryonic stem cells and pancreatic islet cells. 19 -23 To dissect the significance of miRNAs in mammalian biology, several groups have disrupted the Dicer gene in mice 24,25 and the loss of Dicer resulted in embryonic lethality, demonstrating that Dicer is necessary for normal mouse development. Other reports using conditional knockout approaches have demonstrated that Dicer plays essential roles in the maintenance of hair follicles, 26 lung epi...

Posttranscriptional gene regulation by microRNAs (miRNAs) is important for many aspects of development, homeostasis, and disease. Here, we show that reduction of endothelial miRNAs by cell-specific inactivation of Dicer, the terminal endonuclease responsible for the generation of miRNAs, reduces postnatal angiogenic response to a variety of stimuli, including exogenous VEGF, tumors, limb ischemia, and wound healing. Furthermore, VEGF regulated the expression of several miRNAs, including the upregulation of components of the c-Myc oncogenic cluster miR-17-92. Transfection of endothelial cells with components of the miR-17-92 cluster, induced by VEGF treatment, rescued the induced expression of thrombospondin-1 and the defect in endothelial cell proliferation and morphogenesis initiated by the loss of Dicer. Thus, endothelial miRNAs regulate postnatal angiogenesis and VEGF induces the expression of miRNAs implicated in the regulation of an integrated angiogenic response.endothelium ͉ VEGF M icroRNAs (miRNAs) are short (Ϸ22 nt) noncoding RNAs derived from long primary transcripts through sequential processing by the enzymes Drosha and Dicer. Dicer-generated miRNAs are incorporated into the RNA-induced silencing complex that mediates miRNA-dependent translational suppression or in some instances cleavage of respective mRNA targets or translational activation (1, 2). The significance of miRNAs in mammalian biology has been dissected by Dicer gene disruption in mice. Mutant and disrupted Dicer alleles caused embryonic lethality associated with a loss of pluripotent stem cells (3) and defective blood vessel formation (4). Tissue-specific inactivation of Dicer has led to the conclusion that Dicer is essential for several processes, for example, limb, lung, and skin morphogenesis, the maintenance of hair follicles, T cell development/ differentiation, and neuronal survival (5-11).The growth of blood vessels is essential for organ growth and tissue repair. During adulthood, most blood vessels remain quiescent to fulfill their main function of conducting nutritive blood flow to organs; however, during pathological events such as tissue ischemia, inflammation, and tumor progression, endothelial cells (ECs) become activated and angiogenesis ensues to provide conduits for blood flow (12). An imbalance in the growth of blood vessels contributes to the pathogenesis of numerous disorders (13), and the growth of vessels is a complex process, requiring a finely tuned balance between numerous stimulatory and inhibitory signals (14). VEGF has been identified as a central mediator of angiogenesis (15). We (16) and others (17) have recently shown that reduction of miRNA levels via Dicer silencing strongly impacts EC functions in vitro, suggesting a critical role for miRNAs in angiogenesis. The role of Dicer-regulated miRNAs in ovarian angiogenesis is suggested by data obtained in mice expressing a global hypomorphic Dicer1 allele, where female mice are infertile because of corpus luteum insufficiency and defective ovarian angiogenesis...