Inhibition Of miR-33 Research Articles

Abstract 624: MiR-33 Antagonism Prevents the Progression of Atherosclerosis by Promoting an M2 Macrophage Phenotype

MicroRNA-33 (miR-33) plays a central role in regulating HDL biogenesis and reverse cholesterol transport (RCT) via post-transcriptional repression of cholesterol efflux genes (ABCA1, ABCG1, NPC1). Delivery of miR-33 inhibitors to mice and non-human primates fed a chow diet raises plasma HDL cholesterol (HDL-C) and increases RCT, suggesting miR-33 as a promising therapeutic target for the treatment of atherosclerosis. However, recent studies in mice have shown that the HDL-raising effects of anti-miR33 are blunted during Western diet feeding, and its efficacy in reducing atherosclerosis progression under these conditions remains controversial. To further test the effects of miR-33 inhibition during atherogenesis, we treated Ldlr −/− mice with control or anti-miR33 oligonucleotides for 8 weeks coincident with Western diet feeding. Morphometric analyses revealed that anti-miR33 reduced atherosclerosis progression by 40% in both the aortic root and the aorta en face without altering plasma total or HDL cholesterol. Because anti-miR33 accumulates in plaque macrophages where it may directly alter gene expression, we performed Nanostring profiling of plaque macrophages isolated by laser capture microdissection. We found a marked increase in markers of alternatively activated M2 macrophages (Arg1, Fizz1, Cd206, Ym1) in anti-miR33-treated mice compared to controls. Moreover, in vitro transfection of peritoneal macrophages with anti-miR33 reduced the expression of M1 markers (IL-1, TNFα) and increased the expression of M2 markers (Arg1, IL-10, Fizz1), suggesting that miR-33 directly controls the inflammatory polarization of macrophages. This regulation of macrophage polarization by miR-33 was found to be independent of its effects on ABCA1, but dependent on the targeting of AMP Kinase, a key integrator of cellular energy homeostasis. Notably, inhibitors of AMPK blocked the anti-inflammatory effects of miR-33 inhibition in vitro and in vivo . Collectively, these results identify a novel role for miR-33 in the regulation of macrophage inflammation and show that antagonism of miR-33 prevents atherogenesis, in part, by reducing plaque inflammation through the polarization of macrophages from a pro-inflammatory M1 to a reparative M2 state.

Abstract 262: Anti-miR33 Therapy Promotes PGC1α Expression, Mitochondrial Function and Immunometabolic Pathways in Macrophages in Atherosclerosis

Macrophage foam cells are central to atherosclerotic plaque formation and therapies that increase macrophage cholesterol efflux remain an attractive yet elusive target. Therapeutically inhibiting microRNA-33, a critical regulator of lipid metabolism, increases circulating HDL and reduces atherosclerosis. However, the precise molecular mechanisms that regulate these beneficial effects are unknown. Thus, we sought to uncover novel gene networks regulated by miR33 that may contribute to atherosclerosis. Results: Microarray analysis of macrophages from anti-miR33 treated Ldlr-/- mice revealed several novel miR33 target genes that were de-repressed. Among these genes was PPARγ coactivator 1α (PGC1α), a crucial gene that controls cellular energy homeostasis and mitochondrial metabolism. 3’UTR luciferase assays verified that PGC1α is a specific and direct target of miR33. Macrophages transfected with miR33 mimics or anti-miR33 had decreased or increased PGC1α mRNA and protein respectively. Bioinformatic pathway analysis predicted that in addition to PGC1α, miR33 also regulates multiple mitochondrial proteins, such as pyruvate dehydrogenase kinase 4 (PDK4), solute carrier 25 A25 (SLC25A25) and Acyl-CoA Dehydrogenase (ACADSB). Anti-miR33 treatment of macrophages increases PDK4, SLC25A25 and ACADSB mRNA expression, validating these genes as miR33 targets. Seahorse XF Flux Analyzer studies show that macrophages treated with anti-miR33 have increased basal and maximal oxygen consumption rates, confirming that anti-miR33 therapy impacts positively on mitochondrial respiration and function. miR33 inhibition increased macrophage cholesterol efflux, which was abolished by oligomycin- a mitochondrial respiration inhibitor. This suggests that miR33 regulates cholesterol efflux at least in part by regulating mitochondrial function. Lastly, western-diet-fed apoE-/- mice treated with anti-miR33 injections (10mg/kg) for 8 weeks had a marked reduction in aortic sinus lesion area relative to control (p≤0.05), despite no change in HDL levels. Conclusion: Anti-miR33 therapy can positively impact on mitochondrial function, promoting macrophage cholesterol efflux and may contribute to HDL-independent anti-atherosclerotic effects.

Abstract 18676: Anti-miR33 Therapy Induces PGC1a Expression to Regulate Immunometabolic Pathways in Atherosclerosis and Obesity

Atherosclerosis and obesity are epidemic causes of morbidity and mortality, and immunometabolic mechanisms are at the intersection of both diseases. Recently, microRNA 33 (miR33) was identified as a novel and critical regulator of lipid metabolism and a therapeutic target for reducing atherosclerosis. However, the exact mechanism(s) by which anti-miR33 exerts these beneficial effects are unclear. Comprehensive microarray profiling of mRNA from atherosclerotic plaque macrophages of anti-miR33 treated mice identified PPARγ coactivator 1α (PGC1α) as a novel miR33 target gene that is markedly de-repressed. PGC1α is a key regulator of mitochondrial function and energy metabolism, and promoting its activity improves metabolic syndrome (e.g. insulin resistance, obesity, inflammation). Here, we determine if anti-miR33 therapy upregulates PGC1α and affects energy metabolism in atherosclerosis. Results: miR33 overexpression reduced the 3’UTR activity of PGC1α by 40% (p≤0.05), confirming it was a direct and specific target of miR33. Transfection of macrophages with either miR33 mimics or anti-miR33 in vitro resulted in a decrease or increase of Pgc1α mRNA and protein respectively. Using the Seahorse XF Flux Analyzer, we showed that anti-miR33 treatment boosted maximal mitochondrial oxygen consumption rate, confirming that anti-miR33 impacts positively on mitochondrial respiration. Anti-miR33 treated macrophages also increased the expression of known functional mitochondrial genes (e.g. Slc25a25, Nrf1, Bid and Mtch2), implying miR33 controls several genes in the mitochondrial metabolic pathway. Inhibition of miR33 in vivo in Ldlr-/- mice upregulated Pgc1α mRNA expression in adipose tissue, liver and macrophages. Adipose tissue from anti-miR33 treated obese mice also showed upregulation of so-called “browning” genes (e.g. Ucp-1, Elovl3) that contribute to increased energy utilization of white adipose tissue, possibly improving whole-body metabolism. Conclusion: Anti-miR33 therapy specifically targets PGC1α in macrophages and adipose tissue and improves mitochondrial function, all of which can positively regulate inflammatory and energy utilization processes in cardiometabolic diseases (eg. atherosclerosis and obesity).

Antisense oligonucleotide-mediated inhibition of miR-33 in cultured neurons, astrocytes and microglia: Effects on ABCA1 expression, APOE lipidation, cellular cholesterol and beta-amyloid neurotoxicity

Antisense oligonucleotide-mediated inhibition of miR-33 in cultured neurons, astrocytes and microglia: Effects on ABCA1 expression, APOE lipidation, cellular cholesterol and beta-amyloid neurotoxicity

Nuclear Receptors and microRNA-144 Coordinately Regulate Cholesterol Efflux

The ATP-binding cassette transporter A1 (ABCA1) is a key mediator of cellular cholesterol efflux and HDL maturation. ABCA1 mRNA has an unusually long 3’ untranslated region, which makes it highly susceptible to microRNA (miRNA) targeting and repression. As such, multiple miRNAs have been reported to directly target ABCA1, including miR-33a/b, miR-26, miR-106b, and miR-758. Many of these miRNAs participate in feed-forward or feedback networks in controlling cholesterol and lipid homeostasis. Antisense oligonucleotide-based inhibition of miR-33 was found to increase HDL-C levels and regress atherosclerosis in mice and non-human primates. In this edition of Circulation Research, two separate studies identified novel miRNA networks driven by nuclear receptor induced miR-144 targeting of ABCA1 and cholesterol efflux. The first study reports that miR-144 serves to buffer uncontrolled ABCA1 activation in response to high cholesterol states and liver X receptor (LXR) activation in macrophages and liver. The second study highlights the role of miR-144 and ABCA1 in promotion of bile acid secretion in response to farensoid X receptor (FXR) activation in the liver. These studies suggest that anti-miR-144, like anti-miR-33, could be a novel approach to targeting HDL and reverse cholesterol transport.

Abstract 78: Inhibition of Mir-33 Overcomes the Deleterious Effects of Diabetes on Atherosclerosis Regression.

Objective Diabetes increases the risk of cardiovascular morbidity and mortality which remains elevated with conventional therapies, such as statins. We have shown that diabetes also impairs plaque regression following LDL reduction in mice, as evidenced by a higher macrophage (CD68+ cells) plaque content and a higher inflammatory state of these cells compared to non-diabetic mice. Type-2 diabetics have dyslipidemia characterized by elevated triglyceride levels and low HDL, the latter is thought to contribute to their risk, based on epidemiologic studies. In non-diabetic atherosclerotic mice, inhibition of miR-33, an intronic micro-RNA located within the SREBF2 gene, increases the plasma level of apoAI and HDL-cholesterol (HDL-C) and promotes plaque regression. We hypothesize that elevation of HDL through the inhibition of miR-33 will overcome the deleterious effect of diabetes on macrophage content and inflammatory phenotype in plaques in a regression evironment. Methods and Results Diabetic (STZ-injected) and non-diabetic Reversa (LdLr-/-Apob100/100Mttpfl/flMx1-Cre+/+) mice, a model in which diet-induced hypercholesterolemia can be quickly and efficiently reversed, were fed a western diet for 16 weeks and then treated with anti-miR-33 oligonucleotide or control anti-miR for 4 weeks. Treatment with anti-miR-33 increased HDL-cholesterol in both diabetic (17%) and non-diabetic mice (30%). Anti-miR-33 treated diabetic mice showed an improvement in plaque regression as evidenced by a 26% reduction in CD68 content compared to diabetic mice receiving control anti-miR, and an enrichment in anti-inflammatory M2 macrophages (assessed by MR 1 and YM1). Monocyte tracking with latex beads suggests that the reduction of macrophage content is due to a decrease in monocyte recruitment to the plaque, rather than an increase in macrophage egress. This reduction in recruitment correlated with a decrease in the monocytosis associated with diabetes in the anti-miR-33 treated diabetic mice. Conclusion These findings suggest that miR-33 inhibition is able to overcome the deleterious effects of diabetes on atherosclerosis regression, by decreasing monocytosis, monocyte recruitment and improving the inflammatory status of plaque macrophages.

Abstract 45: Antiatherosclerotic Effects of miR-33 Inhibition: Increased Reverse Cholesterol Transport and Alternative-Activation (M2) of Macrophages

Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL and exploit its atheroprotective effects have remained elusive. Recent studies identified miR-33 as an intronic microRNA, located within the SREBF2 gene, that suppresses expression of the cholesterol transporter ABC transporter A1 (ABCA1) and lowers HDL levels. Conversely, mechanisms that inhibit miR-33 increase ABCA1 and plasma HDL, suggesting that antagonism of miR-33 may be atheroprotective. We hypothesized that systemic delivery of an oligonucleotide inhibitor of miR-33 would increase plasma HDL and promote reverse cholesterol transport (RCT), and therefore have a beneficial impact on atherosclerosis. To test this, we treated Ldlr -/- mice with established atherosclerotic plaques with anti-miR33 or a control anti-miR for 4 weeks. Treatment with anti-miR-33 increased circulating HDL levels by 37% and enhanced RCT to the plasma, liver, and feces by up to 80%. Consistent with this, anti-miR33-treated mice showed a marked reduction in plaque size and lipid content, as well as an increase in indicators of plaque stability. Laser capture microdissection of lesional CD68+ cells demonstrated that anti-miR33 oligonucleotides directly targeted the plaque macrophages, where they enhanced ABCA1 expression and cholesterol removal. Moreover, macrophages from anti-miR33-treated mice showed an enrichment in anti-inflammatory M2 markers (Arg1, Il10) and reduced expression of proinflammatory M1 markers (iNos and Tnfa). Notably, overexpression of miR-33 in pMφ in vitro decreases markers of the M2 phenotype, Arg1 and Il-4, and increases the expression of inflammatory cytokines such as Tnfa and Il-1b. In contrast, anti-miR-33 polarizes pMφ to an M2 phenotype (Arg1, Fizz1, Il-10 and Il-4), with an associated downregulation of inflammatory genes (Tnfa, Il-1b). Overall, these results indicate that anti-miR33 has multiple beneficial effects on atherosclerosis, including increasing HDL and RCT, and reducing lesional inflammation by promoting macrophage polarization to the reparative M2 state, highlighting the promise for anti-miR33 therapy for the treatment of cardiovascular disease.

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.

MicroRNAs in metabolism and metabolic diseases.

Aberrant cholesterol/lipid homeostasis is linked to a number of diseases prevalent in the developed world, including metabolic syndrome, type II diabetes, and cardiovascular disease. We have previously uncovered gene regulatory mechanisms of the sterol regulatory element-binding protein (SREBP) family of transcription factors, which control the expression of genes involved in cholesterol and lipid biosynthesis and uptake. Intriguingly, we recently discovered conserved microRNAs (miR-33a/b) embedded within intronic sequences of the human SREBF genes that act in a concerted manner with their host gene products to regulate cholesterol/lipid homeostasis. Indeed, miR-33a/b control the levels of ATP-binding cassette (ABC) transporter ABCA1, a cholesterol efflux pump critical for high-density lipoprotein (HDL) synthesis and reverse cholesterol transport from peripheral tissues. Importantly, antisense inhibition of miR-33 in mice results in elevated HDL and decreased atherosclerosis. Interestingly, miR-33a/b also act in the fatty acid/lipid homeostasis pathway by controlling the fatty acid β-oxidation genes carnitine O-octanoyltransferase (CROT), hydroxyacyl-coenzyme A-dehydrogenase (HADHB), and carnitine palmitoyltransferase 1A (CPT1A), as well as the energy sensor AMP-activated protein kinase (AMPKα1), the NAD(+)-dependent sirtuin SIRT6, and the insulin signaling intermediate IRS2, key regulators of glucose and lipid metabolism. These results have revealed a highly integrated microRNA (miRNA)-host gene circuit governing cholesterol/lipid metabolism and energy homeostasis in mammals that may have important therapeutic implications for the treatment of cardiometabolic disorders.