Carlos Fernández-Hernando

MiR-33 contributes to the regulation of cholesterol homeostasis.

miR-33 in Cholesterol Control With the well-established link between serum cholesterol levels and cardiovascular disease and the availability of effective cholesterol-lowering drugs, cholesterol screening has rapidly become a routine part of health care. Yet, much remains to be learned about how cholesterol levels are regulated at the cellular level (see the Perspective by Brown et al.). Now, Najafi-Shoushtari et al. (p. 1566, published online 13 May) and Rayner et al. (p. 1570, published online 13 May) have discovered a new molecular player in cholesterol control—a small noncoding RNA that, intriguingly, is embedded within the genes coding for sterol regulatory element-binding proteins (SREBPs), transcription factors already known to regulate cholesterol levels. This microRNA, called miR-33, represses expression of the adenosine triphosphate–binding cassette transporter A1, a protein that regulates synthesis of high-density lipoprotein (HDL, or “good” cholesterol) and that helps to remove “bad” cholesterol from the blood. Reducing the levels of miR-33 in mice boosted serum HDL levels, suggesting that manipulation of this regulatory circuit might be therapeutically useful. A small noncoding RNA helps regulate cholesterol levels in mice. 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.

Dicer Dependent MicroRNAs Regulate Gene Expression and Functions in Human Endothelial Cells

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.

Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides

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.

Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis

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.

miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling

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-CoA-dehydrogenase, 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.

Dicer-dependent endothelial microRNAs are necessary for postnatal angiogenesis

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 up-regulation 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.

MicroRNAs in lipid metabolism.

Purpose of review Although the role for microRNAs (miRNAs) in regulating multiple physiological processes including apoptosis, cell differentiation, and cancer is well established, the importance of these tiny RNAs in regulating lipid metabolism has only recently been uncovered. This review summarizes the evidence for a critical role of miRNAs in regulating lipid metabolism. Recent findings Lipid metabolism is tightly regulated at the cellular level. In addition to classic transcriptional regulation of cholesterol metabolism (e.g. by SREBP and LXR), members of a class of noncoding RNAs termed miRNAs have now been identified to be potent post-transcriptional regulators of lipid metabolism genes involved in cholesterol homeostasis and fatty acid oxidation. Several reports have recently shown that miR-33 regulates cholesterol efflux and HDL biogenesis by downregulating the expression of the ABC transporters, ABCA1 and ABCG1. In addition, miR-33 also inhibits the translation of several transcripts encoding proteins involved in fatty acid β-oxidation including CPT1a, CROT, and HADHB, thereby reducing fatty acid degradation. Other miRNAs including miR-122, miR-370, miR-335, and miR-378/378*, miR-27 and miR-125a-5p have been implicated in regulating cholesterol homeostasis, fatty acid metabolism and lipogenesis. Summary Recent advances in the understanding of the regulation of lipid metabolism indicate that miRNAs play major roles in regulating cholesterol and fatty acid metabolism. These new findings may open new avenues for the treatment of dyslipidemias.

MicroRNA-758 Regulates Cholesterol Efflux Through Posttranscriptional Repression of ATP-Binding Cassette Transporter A1

Objective—The ATP-binding cassette transporter A1 (ABCA1) is a major regulator of macrophage cholesterol efflux and protects cells from excess intracellular cholesterol accumulation; however, the mechanism involved in posttranscriptional regulation of ABCA1 is poorly understood. We previously showed that microRNA-33 (miR-33) is 1 regulator. Here, we investigated the potential contribution of other microRNAs (miRNAs) to posttranscriptional regulation of ABCA1 and macrophage cholesterol efflux. Methods and Results—We performed a bioinformatic analysis for identifying miRNA target prediction sites in ABCA1 gene and an unbiased genome-wide screen to identify miRNAs modulated by cholesterol excess in mouse peritoneal macrophages. Quantitative real-time reverse transcription–polymerase chain reaction confirmed that miR-758 is repressed in cholesterol-loaded macrophages. Under physiological conditions, high dietary fat excess in mice repressed miR-758 both in peritoneal macrophages and, to a lesser extent, in the liver. In mouse and human cells in vitro, miR-758 repressed the expression of ABCA1, and conversely, the inhibition of this miRNA by using anti-miR-758 increased ABCA1 expression. In mouse cells, miR-758 reduced cellular cholesterol efflux to apolipoprotein A1 (apoA1), and anti-miR-758 increased it. miR-758 directly targets the 3′-untranslated region of Abca1 as assessed by 3′-untranslated region luciferase reporter assays. Interestingly, miR-758 is highly expressed in the brain, where it also targets several genes involved in neurological functions, including Slc38a1, Ntm, Epha7, and Mytl1. Conclusion—We identified miR-758 as a novel miRNA that posttranscriptionally controls ABCA1 levels in different cells and regulates macrophage cellular cholesterol efflux to apoA1, opening new avenues to increase apoA1 and raise high-density lipoprotein levels.

MicroRNA-16 and MicroRNA-424 Regulate Cell-Autonomous Angiogenic Functions in Endothelial Cells via Targeting Vascular Endothelial Growth Factor Receptor-2 and Fibroblast Growth Factor Receptor-1

Objective—MicroRNAs play key roles in modulating a variety of cellular processes by posttranscriptional regulation of their target genes. Vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR2), and fibroblast growth factor receptor-1 (FGFR1) were identified by bioinformatic approaches and subsequently validated as targets of microRNA (miR)-16 and miR-424 in endothelial cells (ECs). Methods and Results—Mimetics of these microRNAs reduced VEGF, VEGFR2, and FGFR1 expression, whereas specific antagonists enhanced their expression. Expression of mature miR-16 and miR-424 was upregulated on VEGF or basic fibroblast growth factor (bFGF) treatment. This upregulation was accompanied by a parallel increase in primary transcript (pri-miR)-16-1 and pri-miR-16-2 but not in pri-miR-424 levels, indicating a VEGF/bFGF-dependent transcriptional and posttranscriptional regulation of miR-16 and miR-424, respectively. Reduced expression of VEGFR2 and FGFR1 by miR-16 or miR-424 overexpression regulated VEGF and bFGF signaling through these receptors, thereby affecting the activity of downstream components of the pathways. Functionally, miR-16 or miR-424 overexpression reduced proliferation, migration, and cord formation of ECs in vitro, and lentiviral overexpression of miR-16 reduced the ability of ECs to form blood vessels in vivo. Conclusion—We conclude that these miRNAs fine-tune the expression of selected endothelial angiogenic mediators in response to these growth factors. Altogether, these findings suggest that miR-16 and miR-424 play important roles in regulating cell-intrinsic angiogenic activity of ECs.

MicroRNAs in Metabolic Disease

Alterations in the metabolic control of lipid and glucose homeostasis predispose an individual to develop cardiometabolic diseases, such as type 2-diabetes mellitus and atherosclerosis. Work over the last years has suggested that microRNAs (miRNAs) play an important role in regulating these physiological processes. The contribution of miRNAs in regulating metabolism is exemplified by miR-33, an intronic miRNA encoded in the Srebp genes. miR-33 controls cellular cholesterol export and fatty acid degradation, whereas its host genes stimulate cholesterol and fatty acid synthesis. Other miRNAs, such as miR-122, also play a critical role in regulating lipid homeostasis by controlling cholesterol synthesis and lipoprotein secretion in the liver. This review article summarizes the recent findings in the field, highlighting the contribution of miRNAs in regulating lipid and glucose metabolism. We will also discuss how the modulation of specific miRNAs may be a promising strategy to treat metabolic diseases.